THE ION CHANNEL FactsBook I
Extracellular Ligand-Gated Channels
THE ION CHANNEL FactsBook I
Extracellular Ligand-Gated Channels
Other books in the FactsBook Series:
A. Neil Barclay, Albertus D. Beyers, Marian L. Birkeland, Marion H. Brown, Simon J. Davis, Chamorro Somoza and Alan F. Williams The Leucocyte Antigen FactsBook Robin Callard and Andy Gearing The Cytokine FactsBook Steve Watson and Steve Arkinstall The G-Protein Linked Receptor FactsBook Rod Pigott and Christine Power The Adhesion Molecule FactsBook Shirley Ayad, Ray Boot-Handford, Martin J. Humphries, Karl E. Kadler and C. Adrian Shuttleworth The Extracellular Matrix FactsBook Robin Hesketh The Oncogene FactsBook Grahame Hardie and Steven Hanks The Protein Kinase Factsbook
THE ION CHANNEL
FactsBook 1 Extracellular Ligand-Gated Channels Edward C. Conley Molecular Pathology, c/o Ion Channel/Gene Expression University of Leicester/Medical Research Council Centre for Mechanisms of Human Toxicity, UK with contributions from
William J. Brammar Department of Biochemistry, University of Leicester, UK
Academic Press Harcourt Brace & Company, Publishers LONDON SAN D I E G O NEW YORK BOSTON SYDNEY TOKYO TORONTO
This book is printed on acid-free paper ACADEMIC PRESS LIMITED 24-28 Oval Road LONDON NW1 7DX
United States Edition Published by ACADEMIC PRESS INC. San Diego, CA 92101 Copyright 9 1996 by ACADEMIC PRESS LIMITED
All rights reserved No part of this book may be reproduced in any form by photostat, microfilm, or by any other means, without written permission from the publishers A catalogue record for this book is available from the British Library ISBN 0-12-184450-1
Typeset by Alden Multimedia, Oxford and Northampton Printed and bound in Great Britain by WBC, Bridgend, Mid Glam.
Cumulative tables of contents for Volumes 1 to 4 (entry 01) Acknowledgements Introduction and layout of entries (entry 02) How to use The Ion Channel FactsBook Guide to the placement criteria for each field
Abbreviations (entry 03)
VIII XII XIII XV XVII XXXIX
VOLUME I
ELG Key facts (entry 04)
Extracellular ligand-gated receptor-channels - key facts References
3 11
ELG C A T 5-HT3 (entry 05)
Extracellular 5-hydroxytryptamine-gated integral receptor-channels Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
12 12 14 18 19 23 27 31 33
ELG CAT ATP (entry 06)
Extracellular ATP-gated receptor-channels (P2xR) Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
36 36 40 47 49 53 63 70 72
ELG C A T GLU AMPA/KAIN (entry 07)
AMPA / kainate-selective (non-NMDA) glutamate receptor-channels Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
75 75 86 99 105 113 122 129 136
m
Contents
entry 01
(entry 08) N-Methyl-D-aspartate (NMDA)-selective glutamate receptor-channels ELG C A T GLU N M D A
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 09) Nicotinic acetylcholine-gated integral receptor-channels
140 140 146 170 172 191 198 220 226
ELG C A T nAChR
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 10) Inhibitory receptor-channels gated by extracellular gamma-aminobutyric acid
234 234 238 248 256 269 274 284 288
ELG CI GABAA
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
(entry 11) Inhibitory receptor-channels gated by extracellular glycine
293 293 299 309 310 321 329 350 361
ELG CI GLY
Nomenclatures Expression Sequence analyses Structure & functions Electrophysiology Pharmacology Information retrieval References
Feedback and access to the Cell-Signalling Network (entry 12) Feedback Guidelines on the types of feedback required The Cell-Signalling Network
m
366 366 371 378 380 388 391 394 397 403 403 404 404
entry 01
Rubrics (entry 13) Entry number rubric Field number rubric
Index
417 417 419 420
Note: A set of supporting appendices (Resources) and a cumulative subject index for Volumes I to IV appears at the end of Volume IV. An on-line glossary of terms marked with the dagger symbol (t) will be accessible from the Cell-Signalling Network 'home page' from mid-1996.
VII
Cumulative table of contents for Volumes I to IV Contents Cumulative table of contents for Volumes I to IV (entry 01) Acknowledgements Introduction and layout of entries (entry 02) How to use The Ion Channel FactsBook Guide to the placement criteria for each field Abbreviations (entry 03)
ELG Key facts (entry 04)
ELG CAT nAChR (entry 09)
Extracellular ligand-gated receptorchannels- key facts
Nicotinic acetylcholine-gated integral receptor-channels
ELG CAT 5-HT3 (entry 05)
ELG C1 GABAA (entry 10)
Extracellular 5-hydroxytryptaminegated integral receptor-channels ELG C A T ATP (entry 06)
Extracellular ATP-gated receptorchannels (P2xR) ELG CA T GLUAMPA/KAIN (entry 07)
AMPA / kainate-selective (nonNMDA) glutamate receptor-channels ELG C A T GLU NMDA (entry 08)
Inhibitory receptor-channels gated by extracellular gamma-aminobutyric acid ELG C! GL Y (entry 11)
Inhibitory receptor-channels gated by extracellular glycine Feedback and access to the CellSignalling Network (entry 12)
N-Methyl-D-aspartate (NMDA)selective glutamate receptor-channels
Rubrics (entry 13) Entry and field number rubrics
ILG K e y facts (entry 14)
ILG Ca Ca RyR-Caf (entry 17)
The intracellular ligand-gated channel group- key facts
Caffeine-sensitive Ca2+-release channels (ryanodine receptors,
ILG Ca AA-LTCa [native] (entry 15)
Native Ca ).+ channels gated by the arachidonic acid metabolite leukotriene C4 incorporating general properties of ion channel regulation by arachidonate metabolites
RyRI ILG Ca CSRC [native] (entry 18)
Candidate native intracellularligand-gated Ca)+-store repletion channels
ILG Ca Ca InsP4S [native] (entry 16)
ILG Ca InsP3 (entry 19)
Native Ca )`+ channels sensitive to inositol 1,3,4,5-tetrakisphosphate (InsPa)
Inositol 1,4,5-trisphosphatesensitive Ca~+-release channels (InsP~R)
entry O1
ILG CAT Ca [native] (entry 20) Native calcium-activated nonselective cation channels (NSca) ILG CAT cAMP (entry 21) Cation channels activated in situ by intracellular cAMP ILG CAT cGMP (entry 22) Cation channels activated in situ by intracellular cGMP
Cumulative contents
ILG CI Ca [native] (entry 25) Native calcium-activated chloride channels (C1ca) ILG K AA [native] (entry 26) Native potassium channels activated by arachidonic acid (KAA) incorporating general properties of ion channel regulation by free fatty acids
ILG CI ABC-CF (entry 23) ATP-binding and phosphorylationdependent C1-channels (CFTR)
ILG K Ca (entry 27) Intracellular calcium-activated K§ channels (Kca)
ILG C1 ABC-MDR/PG (entry 24) Volume-regulated C1--channels (multidrug-resistance P-glycoprotein)
ILG K Na [native] (entry 28) Native intracellular sodium-activated K§ channels (KNa)
INR K Key facts (entry 29) Inwardly-rectifying K* channelskey facts
JUN [connexins] (entry 35) Intercellular gap junction channels formed by connexin proteins
INR K A TP-i [native] (entry 30) Properties of intracellular ATPinhibited K§ channels in native cells INR K G/A Ch [native] (entry 31) Properties of muscarinic-activated K§ channels underlying /KACh in native cells INR K [native] (entry 32) Properties of 'classical' inward rectifyer K* channels in native cells (excluding types covered in entries 30 & 31) INR K [subunits] (entry 33) Comparative properties of protein subunits forming inwardlyrectifying K+ channels (heterologously-expressed cDNAs of the KIR family) INR K/Na I~q [native] (entry 34) Hyperpolarization-activated cation channels underlying the inward currents if,/h, iq
MEC [mechanosensitive] (entry 36) Ion channels activated by mechanical stimuli MIT [mitochondrial] (entry 37) Survey of ioni channel types expressed in mitochondrial membranes NUC [nuclear] (entry 38) Survey of ion channel types expressed in nuclear membranes OSM [aquaporins] (entry 39) The vertebrate aquaporin (water channel) family SYN [vesicular] (entry 40) Channel-forming proteins expressed in synaptic vesicle membranes (synaptophysin)
IX
Cumulative contents
VOLUME IV
VOLTAGE-GATED CHANNELS
VLG K e y facts (entry 41) Voltage-gated c h a n n e l s - key facts VLG Ca (entry 42) Voltage-gated Ca ~+ channels VLG CI (entry 43) Voltage-gated chloride channels VLG K A - T (entry 44) Properties of native A-type (transient outward)potassium channels in native cells VLG K DR (entry 45) Properties of native delayed rectifier potassium channels in native cells VLG K eag
(entry 46)
Vertebrate K § channel subunits related to Drosophila ether-~-go-
go (eag) VLG K Kv-beta
(entry 47)
Beta subunits associated with voltage-gated K+ channels VLG K K v l - S h a k
(entry 48)
Vertebrate K§ channel subunits related to Drosophila Shaker (subfamily 1) incorporating general
features of Kv channel expression in heterologous cells
Resource A
(entry 56)
G protein-linked receptors regulating ion channel activities
(alphabetical listing) Resource B
(entry 57)
'Generalized' electrical effects of endogeneous receptor agonists Resource C
(entry 58)
Compounds and proteins used in ion channel research
II
entry 01
VLG K Kv2-Shab
(entry 49)
Vertebrate K § channel subunits related to Drosophila Shab (subfamily 2) VLG K K v 3 - S h a w
(entry 50)
Vertebrate K§ channel subunits related to Drosophila Shaw (subfamily 3) VLG K Kv4-Shal (entry 51) Vertebrate K § channel subunits related to Drosophila Shal (subfamily 4) VLG K K v x (Kv5.1/Kv6.1)
(entry 52)
Features of the 'non-expressible' cDNAs 1K8 and K13 VLG K M-i [native]
(entry 53)
Properties of native 'muscarinicinhibited' K§ channels underlying IM VLG K m i n K
(entry 54)
'Minimal' subunits forming slowactivating voltage-gated K § channels VLG Na
(entry 55)
Voltage-gated Na § channels
Resource D
(entry 59)
'Diagnostic' tests Resource E
(entry 60)
Ion channel book references (sorted by year of publication) Resource F
(entry 61)
Supplementary ion channel reviews (listed by subject)
1
entry O1
Resource G (entry 62) Reported 'consensus sites' and 'motifs' in primary sequence of ion channels Resource H (entry 63) Listings of cell types Resource I (entry 64) Framework of cell-signalling molecule types (preliminary listing)
Resource J (entry 65) Search criteria & CSN development Resource K (entry 66) Framework for a multidisciplinary glossary Cumulative page index (for volumes
My)
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the coverage/contents can be sent to the e-mail feedback file
[email protected]. (see field 57 of most entries for
further details)
Ui
Acknowledgements Thanks are due to the following people for their time and help during compilation of the manuscripts: Professors Peter Stanfield, Nick Standen and Gordon Roberts (Leicester), and Ole Petersen (Liverpool)for advice; to Allan Winter, Angela Baxter, Shelly Hundal, Phil Shelton and Sue Robinson for help with photocopying, to Chris Hankins and Richard Mobbs of the Leicester University Computer Centre, and to Dr Tessa Picknett and Chris Gibson of Academic Press for their enthusiasm and patience. Gratitude is also expressed to all of the anonymous manuscript readers who supplied much constructive feedback, as well as the following who provided advice, information and encouragement: Stephen Ashcroft (Oxford), Eric Barnard (London), Dale Benos (Harvard), William Catterall (Washington), K. George Chandy (UC Irvine), Peter Cobbold (Liverpool), David Clapham (Mayo Foundation), Noel Davies (Leicester), Dario DiFrancesco (Milano), Ian Forsythe (Leicester), Sidney Fleischer (Vanderbilt), George Gutman (UC Irvine), Richard Haugland (Molecular Probes, Inc.), Bertil Hille (Washington), Michael Hollmann (G6ttingen), Anthony Hope (Dundee), Benjamin Kaupp (Jtilich), Jeremy Lambert (Dundee), Shigetada Nakanishi (Kyoto), Alan North (Glaxo Institute for Molecular Biology), John Peters (Dundee), Olaf Pongs (Hamburg), David Spray (Yeshiva), Kent Springer (Institute for Scientific Information), Steve Watson (Oxford), Paul Van Houlte (I.R.I.S.)and Steven Wertheim (Harvard). Thanks are also due to the Department of Pathology at the University of Leicester, Harcourt Brace, the Medical Research Council and Zeneca Pharmaceuticals, for providing generous sponsorship, equipment and facilities. We would like to acknowledge the authors of all those papers and reviews which in the interest of completeness we have quoted, but have not had space to cite directly. ECC would like to thank Professors Denis Noble in Oxford and Anthony Campbell in Cardiff, Tony Buzan in Winton, Dorset and Richard Gregory in Bristol for help and inspiration, and would like to dedicate his contributions to Paula, Rebecca and Katharine for all their love and support over the past four years.
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~
Left: Edward Conley, Right: William Brammer
XII
Edward C. Conley
Entry 02
The Ion Channels FactsBook is intended to provide a 'summary of molecular properties' for all known types of ion channel protein in a cross-referenced and 'computer-updatable' format. Today, the subject of ion channel biology is an extraordinarily complex one, linking several disciplines and technologies, each adding its own contribution to the knowledge base. This diversity of approaches has left a need for accessible information sources, especially for those reading outside their own field. By presenting 'facts' within a systematic framework, the FactsBook aims to provide a 'logical place to look' for specific information when the need arises. For students and researchers entering the field, the weight of the existing literature, and the rate of new discoveries, makes it difficult to gain an overview. For these readers, The Ion Channels FactsBook is written as a directory, designed to identify similarities and differences between ion channel types, while being able to accommodate new types of data within the framework. The main advantages of a systematic format is that it can speed up identification of functional links between any 'facts' already in the database and maybe provide a raison d'etre for specific experiments where information is not known. Although such 'facts' may not go out-of-date, interpretations based on them may change considerably in the light of additional, more direct evidence. This is particularly true for the explosion of new information that is occurring as a direct consequence of the molecular cloning of ion channel genes. It can be anticipated that many more ion channel genes will be cloned in the near future, and it is also likely that their functional diversity will continue to exceed expectations based on pharmacological or physiological criteria alone.
A n e m p h a s i s on properties e m e r g e n t from ion c h a n n e l m o l e c u l a r f u n c t i o n s Understanding how the interplay of currents through many specific ion channel molecules determines complex electrophysiological behaviour of cells remains a significant scientific challenge. The approach of the FactsBook is to associate and relate this complex cell phenotypic behaviour (e.g. its physiology and pharmacology) to ion channel gene expression-control wherever possible even where the specific gene has not yet been cloned. Thus the ion channel molecule becomes the central organizer, and accordingly arbitrates whether information or topics are included, emphasized, sketched-over or excluded. In keeping with this, ion channel characteristics are described in relation to known structural or genetic features wherever possible (or where they are ultimately molecular characteristics). Invariably, this relies on the availability of sequence data for a given channel or group of channels. However, a number of channel types exist which have not yet been sequenced, or display characteristics in the native form which are not precisely matched by existing clones expressed in heterologous cells (or are otherwise ambiguously classified). To accommodate these channel types, summaries of characteristics are included in the standard entry field format, with inappropriate fieldnames omitted. Thus the present 'working arrangement' of entries and fields is broad enough to include both the 'cloned' and 'uncloned' channel types, but in due course will be gradually supplanted by a comprehensive classification based on gene locus, structure, and relatedness of primary sequences. In all cases, the scope of the FactsBook entries is limited to those proteins forming (or predicted to form) membrane-bound, integral ionic channels
Introduction
entry 02
by folding and association of their primary protein sequences. Activation or suppression of the channel current by a specified ligand or voltage step is generally included as part of the channel description or name (see below). Thus an emphasis is made throughout the book on intrinsic features of channel molecule itself and not on those of separately encoded, co-expressed proteins. In the present edition, there is a bias towards descriptions of vertebrate ion channels as they express the full range of channel types which resemble characteristics found in most eukaryotes.
Anticipated development of the dataset - Integration of functional information around molecular types Further understanding of complex cellular electrical and pharmacological behaviour will not come from a mere catalogue of protein properties alone. This book therefore begins a process of specific cross-referencing of molecular properties within a functional framework. This process can be extended to the interrelationships of ion channels and other classes of cell-signalling molecules and their functional properties. Retaining protein molecules (i.e. gene products) as 'fundamental units of classification' should also provide a framework for understanding complex physiological behaviour resulting from co-expressed sets of proteins. Significantly, many pathophysiological phenotypes can also be linked to selective molecular 'dysfunction' within this type of framework. Finally, the anticipated growth of raw sequence information from the human genome project may reveal hitherto unexpected classes and subtypes of cell-signalling c o m p o n e n t s - in this case the task t h e n will be to integrate these into what is already known (see also
description of Field number 06: Subtype classifications and Field number 05: Gene family). The Cell-Signalling Network (CSN) From the foregoing discussion, it can be seen that establishment and consolidation of an integrated 'consensus database' for the many diverse classes of cell signalling molecules (including, for example, receptors, G proteins, ion channels, ion pumps, etc.) remains a worthwhile goal. Such a resource would provide a focus for identifying unresolved issues and may avoid unnecessary duplication of research effort. Work has begun on a prototype cell-signalling molecule database cooperatively maintained and supported by contributions from specialist groups world-wide: The Cell-Signalling Network (CSN) operating from mid-1996 under the World Wide Webt of the Internett has been designed to disseminate consensus properties of a wide range of molecules involved in cell signal transduction. While it may take some time (and much good-will)to establish a comprehensive network, the many advantages of such a co-operative structure are already apparent. Immediately, these include an 'open' mechanism for consolidation and verification of the dataset, so that it holds a 'consensus' or 'validated' set of information about what is known about each molecule and practical considerations such as nomenclature recommendations (see, for example, the IUPHAR nomenclature sections under the CSN 'home page'). The CSN also allows unlimited cross-referencing by pointing to related information sets, even where these are held in multiple centres around the world. On-line support for technical terms (glossary items, indicated by dagger symbols (t) throughout the
I entry 02
text) and reference to explanatory appendices (e.g. on associated signalling components such as G proteint-linked receptorst) are already supported for use with this book. Eventually, benefits could include (for instance) direct 'look-up' of graphical resources for protein structure, in situ and developmental gene expression atlasest, interactive molecular models for structure/function analysis, DNA/protein sequences linked to feature tables, gene mapping resources and other pictorial data. These developments (not all are presently supported)will use interactive electronic media for efficient browsing and maintenance. For a brief account of the Cell-Signalling Network, see Feedback & CSN access, entry 12. For a full specification, see Resource J - Search criteria & CSN development, entry 65.
l O W TO USE THE I O N C H A N N E L F A C T S B O 0 ~ C o m m o n formats within the entries A proposed organizational hierarchy for i n f o r m a t i o n about ion c h a n n e l molecules Information on named channel types is grouped in entries under common headings which repeat in a fixed order - e.g. for ion channel molecules which have been sequenced, there are broad sections entitled NOMENCLATURES, EXPRESSION, SEQUENCE ANALYSES, STRUCTURE & FUNCTIONS, ELECTROPHYSIOLOGY, PHARMACOLOGY, INFORMATION RETRIEVAL and REFERENCES, in that order. Within each section, related fieldnames are listed, always in alphabetical order and indexed by a field number (see below), which makes electronic crossreferencing and 'manual' comparisons easier. While the sections and fields are not rigid categories, an attempt has been made to remain consistent, so that corresponding information for two different channels can be looked up and compared directly. If a field does not appear, either the information was not known or was not found during the compilation period. Pertinent information which has been published but is absent from entries would be gratefully received and will be added to the 'entry updates' sections within the CSN (see Feedback & CSN access, entry 12). Establishment of this 'field' format has been designed so that every available 'fact' should have its logical 'place'. In the future, this arrangement may help to establish 'universally accepted' or 'consensus' properties of any given ion channel or other cell-signalling molecule. This validation process critically depends on user feedback to contributing authors. The CSN (above) establishes an efficient electronic mechanism to do this, for continual refinement of entry contents.
I n d e p e n d e n t presentation of 'facts' and c o n v e n t i o n s for cross-referencing The FactsBook departs from a traditional review format by presenting its information in related groups, each under a broader heading. Entries are not designed or intended to be read 'from beginning to end', but each 'fact' is presented independently under the most pertinent fieldname. Independent citation of 'facts' may sometimes result in some repetition (redundancy)of general
Introduction
entry 02
1
principles between fields, but if this is the case some effort has been made to 'rephrase' these for clarity (suggested improvements for presentation of any 'fact' are welcome - see Field number 57: Feedback). For readers unfamiliar with the more general aspects of ion channel biology, some introductory information applicable to whole groups of ion channel molecules is needed, and this is incorporated into the 'key facts' sections preceding the relevant set of entries. These sections, coupled with the 'electronically updated' glossary items (available on-line, and indicated by the daggert symbol, see below)provide a basic overview of principles associated with detailed information in the main entries of the book. Extensive cross-referencing is a feature of the book. For example, cross-references between fields of the same entry are of the format (see Fieldname, xx-yy). Crossreferences between fields of different channel type entries are generally of the format see fieldname under SORTCODE, xx-yy; for e x a m p l e - see m R N A distribution, under ELG C1 GABAA, 10-13. This alphabetical 'sortcode' and numerical 'entry numbers' (printed in the header to each page) are simply devices to make crossreferencing more compact and to arrange the entries in an approximate runnin~ order based on physiological features such as mode of gatingr, ionic selectivityr, and agonistt specificity. A 'sort order' based on physiological features was judged to be more intuitive for a wider readership than one based on gene structure alone, and enables 'cloned' and 'uncloned' ion channel types to be listed together. The use and criteria for sortcode designations are described under the subheading Derivation of the sortcode (see Field number 02: Category (sortcode)). Entry 'running order' is mainly of importance in book-form publications. New entries (or mergers/subdivisions between existing entries)will use serial entry numbers as 'electronic pointers' to appropriate files. Cross-references are frequently made to an on-line index of glossary items by dagger symbolst wherever they might assist someone with technical terms and concepts when reading outside their own field. The glossary is designed to be used side-byside with the FactsBook entries and is accessible in updated form over the Internett/World Wide Webt with suitable browsingt software (for details, see Feedback & CSN access, entry 12).
C o n t e x t u a l m a r k e r s and styles e m p l o y e d within the entries Throughout the books, a six-figure index number (xx-yy-zz, e.g. 19-44-01: ) separates groups of facts about different aspects of the channel molecule, and carries information about channel type/entry number (e.g. 19- ~ InsP3 receptor-channels), information type/field number (e.g. -44-, Channel modulation) and running paragraph number (datatype) (e.g. -01). This simple 'punctate' style has been adopted for m a x i m u m flexibility of updating (both error-correction and consolidation with new information), cross-referencing and multi-authoring. The CSN specification (see entry 65) includes longer term plans to structure field-based information into convenient data-types which will be indexed by a zz numerical designation. Italicized subheadings are employed to organize the facts into related topics where a field has a lot of information associated with it. Specific illustrated points or features within a field are referenced to adjacent figures. Usage of abbreviations and common
Introduction
entry 02
symbols are defined in context and/or within the main abbreviations index at the front of each book. Abbreviated chemical names and those of proprietary pharmaceutical compounds are listed within the electronically updated Resource C Compounds & proteins, also available via the 'home page' of the Cell-Signalling Network. Generally, highlighting of related subtopics emergent from the molecular properties ('facts') associated with the ion channel under description are indicated within a field by lettering in bold. All subtopics are cross-referenced by means of a large cumulative subject index (entry 66), which can permit retrieval of information by topic without requiring prior knowledge of ion channel properties. Throughout the main text, italics draw attention to special cases, caveats, hypotheses and exceptions. The 'Note:' prefix has been used to indicate supplemental or comparative information of significance to the quoted data in context.
Special considerations for integrating properties derived from 'cloned' and 'native' channels While a certain amount of introductory material is given to set the context, the emphasis on molecular properties means the treatment of many important biological processes or phenomena is reduced to a bare outline. References given in the Related sources and reviews field and the electronically updated Resource F - Supplementary ion channel reviews accessible via the CSN (see Feedback & CSN access, entry 12) are intended to address this imbalance. For summaries of key molecular features, a central channel 'protein domain topography model' is presented. Individual features that are illustrated on the protein domain topography model are identified within the text by the symbol
IPDTMI. Wherever molecular subtype-specific data are quoted {such as the particular behaviour of a ion channel gene familyt member or i s o f o r m t ) a convention of using the underlined trivial or systematic name as a prefix has been a d o p t e d - e.g. mIRKI: ; RCKI: ; Kv3.1: etc.
GUIDE
TO THE PLACEMENT
Criteria for N O M E N C L A T U R E S
CRITERIA
FOR EACH FIELD
sections
This section should bring together for comparison present and previous names of ion channels or currents, with brief distinctions between similar terms. Where systematic names have already been suggested or adopted by published convention, they should be included and used in parallel to trivial names.
Field number 01" Abstract/general description: This field should provide a summary of the most important functional characteristics associated with the channel type. Field number 02: Category (sortcode): The alphabetical 'sortcode' should be used for providing a logical running order for the individual entries which make up the book. It is not intended to be a rigorous channel classification, which is under discussion,
XVII
Introduction
entry 02
]
but rather a practical index for finding and cross-referencing information, in conjunction with the six-figure index number (see above). The Category (sortcode) field also lists a designated electronic retrieval code (unique embedded identifier or UEI) for 'tagging' of new articles of relevance to the contents of the entry. For further details on the use and implementation of UEIs, see the description for Resource J (in this entry) and for a full description, see Resource J - Search criteria & CSN development, entry 65.
Derivation of the sortcode: Although we do not yet have a complete knowledge of all ion channel primaryt structures, knowledge of ion channel gene familyt and superfamilyt structure allows a working sort order to be established. To take an example, the extracellular ligand-gated (ELG)receptor-channels share many structural features, which reflects the likely duplication and divergent evolution of an ancestral gene. The present-day forms of such channels reflect the changes that have occurred through adaptive radiation t of the ancestral type, particularly for gatingt mechanism and ionic selectivityt determinants. Thus, the entry running order (alphabetical, via the sortcode)of the FactsBook entries should depend primarily on these two features. The sortcode therefore consists of several groups of letters, each denoting a characteristic of the channel molecule: Entries are sorted first on the principal means for channel gatingt (first three letters), whether this is by an extracellular ligandt (ELG), small intracellular ligandt (ILG)or transmembrane voltage (VLG). For convenience, the ILG entries also include certain channels which are obligately dependent on both ligand binding and hydrolysis for their activatione.g. channels of the ATP-binding cassette (ABC)superfamily. Other channel types may be subject to direct mechanical gating (MEC) or sensitive to changes in osmolarity ( O S M ) - see the Cumulative tables of contents and the first page of each entry for descriptions and scope. Due to their unusual gating characteristics, a separate category (INR) has been created for inward rectifier-type channels. The second sort (the next three letters of the sortcode) should be on the basis of the principal permeant ions, and may therefore indicate high selectivity for single ions (e.g. Ca, C1, K, Na) or multiple ions of a specified charge (e.g. cations - CAT). Indefinite sortcode extensions can be assigned to the sortcode if it is necessary to distinguish similar but separately encoded groups of channels (e.g. compare ELG C] GABAA, entry 10 and ELG C] GLY, entry 11).
Field number 03: Channel designation: This field should contain a shorthand designation for the ion channel m o l e c u l e - mostly of the form Xy or Xtv 1 where X denotes the major ionic permeabilitiest (e.g. K, Ca, cation) and Y denotes the principal mechanism of gatingt where this acts directly on the channel molecule itself (e.g. cGMP, voltage, calcium, etc.). Otherwise, this field contains a shorthand designation for the channel which is used in the entry itself. Field number 04: Current designation: This field should contain a shorthand designation for ionic currents conducted by the channel molecule, which is mostly of the form/x(v), /x,Y or/x-Y where X and Y are defined as above. Field number 05: Gene family: This field should indicate the known molecular relationships to other ion channels or groups of ion channels at the level of
entry 02
amino acid primary sequence homologyt, within gene familiest or gene superfamiliest. Where multiple channel subunits are encoded by separate genes, a summary of their principal features should be tabulated for comparison. Where the gene family is particularly large/or cannot be easily described by functional variation, a gene family treet derived by a primary sequence alignment algorithmt (see Resource D - 'Diagnostic' tests, entry 59) may be included as a figure in this field.
Field number 06: Subtype classifications: This field should include supplementary information about any schemes of classification that have been suggested in the literature. Generally, the most robust schemes are those based on complete knowledge of gene familyt relationships (see above) and this method can identify similarities that are not easily discernible by pharmacological or electrophysiological criteria a l o n e - see, for example, the entries JUN (connexins), entry 35, and INR K (subunits), entry 33. Note, however, that some nativet channel types are more conveniently 'classified' by functional or cell-type expression parameters which take into account interactions of channels with other co-expressed proteins (see, for example, discussion pertaining to the cyclic nucleotide-gated (CNG-) channel family in the entries ILG Key facts, entry 14, ILG C A T cAMP, entry 21, and ILG C A T cGMP, entry 22. Debate on the 'best' or 'most appropriate' channel classification schemes is likely to continue for some time, and it is reasonable to suppose that alternative subtype classifications may be applied and used by different workers for different purposes. Since the 'running order' of the FactsBook categories depends on inherent molecular properties of channel cDNAst, genest or the expressed proteins, future editions will gradually move to classification on the basis of separable gene locit. Thus multiple channel protein variants resulting from processes of alternative RNA splicingt but encoded by a single gene locust will only ever warrant one 'channel-type' entry (e.g. see BKc~ variants under ILG K Ca, entry 27). Distinct proteins resulting from transcription, of separable gene loci, for example in the case of different gene family members, will (ultimately)warrant separate entries. For the time being, there is insufficient knowledge about the precise phenotypict roles of many 'separable' gene family members to justify separate entries (as in the case of the VLG K Kv series entries). Classification by gene locus designation (see Field n u m b e r 18: Chromosomal location) can encompass all structural and functional variation, while being 'compatible' with efforts directed to identifying phenotypic and pathophysiologicalt roles of individual gene products (e.g. by gene-knockoutt, locus replacementt or disease-linked gene mappingt procedures - see Resource D - 'Diagnostic' tests, entry 59). Subtype classifications based on gene locus control can also incorporate the marked developmental changes which pertain to many ion channel genes (see Field n u m b e r 11: Developmental regulation) and can be implemented when the 'logic' underlying gene expression-controlt for each family member is fully appreciated. A 'genome-based' classification of FactsBook entries may also help comprehend and integrate equivalent information based for other ('non-channel') cell-signalling molecules (see Resources G, H and l, entries 62, 63 and 64).
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Field number 07: Trivial names: This field should list commonly used names for the ion channel (or its conductancet). Often a channel will be (unsystematically)named by its tissue location or unusual pharmacological/physiological properties, and these are also listed in this field. While unsystematic names do not indicate molecular relatedness, they are often more useful for comparative/descriptive purposes. For these and historical reasons, trivial names (e.g. clone/isolate names for K§ channel isoforms) are used side-by-side with systematic names, where these exist. A standardized nomenclature for ion channels is under discussion, e.g. see the series of articles by Pongs, Edwards, Weston, Chandy, Gutman, Spedding and Vanhoutte in Trends Pharmacol Sci (1993) 14: 433-6. Future recommendations on standardized nomenclature will appear in files accessible under the IUPHAR entry of the CellSignalling Network (see Feedback & CSN access, entry 12). Criteria for E X P R E S S I O N s e c t i o n s This section should bring together information on expression patterns of the ion channel gene, indicating functional roles of specific channels in the cell type or organism. The complex and profound roles of ionic currents in vertebrate development (linking plasma membrane signalling and genome activation) are also emphasized within the fields of this section.
Field number 08: Cell-type expression index: Comprehensive systems relating the expression of specified molecular components to specified anatomical and developmental loci ('expression atlases') are being developed in a number of centres and in due course will form a superior organizational framework for this type information (see discussion below). In the meantime, the range of cell-type expression should be indicated in this field in the form of alphabetized listings. Notably, there is a substantial literature concerned with the electrophysiology of ion channels where the tissue or cell type forms the main focus of the work. In some cases, this has resulted in detailed 'expression surveys', revealing properties of interacting sets of ion channels, pumps, transporters and associated receptors. Such review-type information is of importance when discussing the contribution of individual ion channel molecules to a complex electrophysiological phenotypet and/or overall function of the cell. For further references to 'cell-type-selective' reviews, see Resource H - Listings of cell types, entry 63 accessible via the CSN (see Feedback & CSN access, entry 12).
Problems and opportunities in listing ion channel molecules by cell type: Understanding the roles which individual ionic channels play in the complex electrophysiological phenotypes of nativet cells remains a significant challenge. The overwhelming range of studies covering aspects of ion channel expression in vertebrate cells offers unique problems when compiling a representative overview. Certainly the linking of specific ion channel gene expression to cell type is a first step towards a more comprehensive indexing, and towards this goal, cell-typeselective studies are useful for a number of reasons. First, they can help visualize the whole range of channel expression by providing an inventory of conductancest observed. Secondly, these studies generally define the experimental conditions required to observe a given conductance. Thirdly, they include much information directly relating specified ionic conductances to the functions of the cell type
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Introduction
concerned. Collated information such as this should be of increasing utility in showing the relationship of electrophysiological phenotype to mechanistic information on their gene structure and expression-control (which largely correlates with cell-type lineage). At this time it is difficult to build a definitive catalogue of ion channel gene expression patterns mapped to cell type, not only because the determinants of gene expression are scarcely explored, but also because there remain many unavoidable ambiguities in phenotype definition. Some of these problems are discussed below. Problems of uneven coverage~omissions: Certain cell preparations have been intensely
studied for ion channel expression while others have received very little attention for technical, anatomical or other reasons. Furthermore, a large number of nativet ionic currents can be induced or inhibited by agonistst that bind to co-expressed G proteint-coupled receptorst. Thus a difficulty arises in deciding whether channel currents can be unambiguously defined in terms of action at a separately encoded receptor protein. While it is valid to report that an a~onist-sensitive current is expressed in a defined cell type, the factors of crosstalk! and receptor-transducert subtype specificities in signalling systems are complex and may produce an ambiguous classification. Receptor-coupled agonist-sensitivities are an important factor contributing to cell-pharmacological and -electrical phenotypet, but the treatment here has been limited to a number of tabular summaries of ion channel regulation through coupling to G protein-linked effectort molecules (see Resource A - G protein-linked receptors, entry 56). As stated earlier, the entries are not sorted on agonist specificity except where the underlying ion channel protein sequence would be expected to form an integral ionic channel whose gatingt mechanism is also part of the assembled protein complex. Cell preparation m e t h o d s are variable: A further problem inherent in classifying ion channels by their patterns of expression is that the choice of tissue or cell preparation method may influence phenotypet. The behaviour of channel-mediated ionic currents can be measured in nativet cells, e.g. in the tissue slice, which has the advantages of extracellular ionic control, mechanical stability, preserved anatomical location, lack of requirement for anaesthetics and largely undisturbed intercellular communication. Cell-culture techniques show similar advantages, with the important exceptions that normal developmental context, anatomical organization and synaptic arrangements are lost and (possibly as a consequence) the 'expression profile' of receptor and channel types might change. Cultured cell preparations may also be affected by 'de-differentiationt' processes and (by definition) cell linest are uncoupled from normal processes of cell proliferationf, differentiationt and apoptosist. Acutely dissociated cells from nativet tissue may provide cell-type-specific expression data without anomalies introduced by intercellular (gap junctional) conductances, but the enzymatic or dispersive treatments used may also affect responses in an unknown way. Verbal descriptions of cell-type expression divisions are arbitrary and are not rigorous:
Definitive mapping of specific ion channel subtype expression patterns has many variables. Localization of specific gene products are most informative when in situ localizations are linked to the regulatory factors controlling their expression (see glossary entry on Gene expression-controllT). The complexity of this task can extend to processes controlling, for example, developmental regulation, co-expressed protein subunit stoichiometries and subcellular localizations.
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Complete integration of all structural, anatomical, co-expression and modulatory data for ion channels could eventually be accommodated within interactive graphical databases which are capable of providing 'overlays' of separately collected in situ expression data linked to functional properties of the molecules. By these methods, new data can be mathematically transformed to superimpose on fixed tissue or cell co-ordinates for comparison with existing database information. Software development efforts focused on the acquisition, analysis and exchange of complex datasets in neuroscience and mouse development have been described, and the next few years should hopefully see their implementation. For further information, see Baldock, R., Bard, J., Kaufman, M. and Davidson, D. (1992) A real mouse for your computer, Bioessays 14" 501-2 Bloom, F. (1992) Brain Browser, v 2.0. Academic Press (Software). Kaufman, M. (1992)The Atlas of Mouse Development, Academic Press Wertheim, S. and Sidman, R. (1991) Databases for Neuroscience, Nature 354:88-9 To help rationalize the choices available for selection of these 'prototype' classifications, see Resource H - Listings of cell types, entry 63. These listings may also have some practical use for sorting the subject matter of journal articles into functionally related groups. A proposed integration of information resources relating different aspects of cell-signalling molecule gene expression is illustrated in Fig. 4 of the section headed Feedback & CSN access, entry 12. Field number 09: Channel density: This field should contain information about estimated numbers of channel molecules per unit area of membrane in a specified preparation. This field lists information derived from local patch-clamp 'sampling' or autoradiographic detection in membranes using anti-channel antibodies. The field should also describe unusually high densities of ion channels ('clustering') in specified membranes where these are of functional interest. Field number 10: Cloning resource: This field should refer to cell preparations relatively 'rich' in channel-specific mRNA (although it should be noted that many ion channel mRNAs are of low abundancet). Otherwise, this field defines a 'positive control' preparation likely to contain messengerf RNAt encoding the channel. Preparations may express only specific subtypes of the channel and therefore T r eprobes l a(especially t e d PCR probes) may not work. Alternatively, a genomict cloning resource may be cited. Field number 11: Developmental regulation: This field should contain descriptions of ion channel genes demonstrated (or expected to be) subject to developmental gene regulation - e.g. where hormonal, chemical, second messengert or other environmental stimuli appear to induce (or repress) ion channel mRNA or protein expression in nativet tissues (or by other experimental interventions). Protein factors in transt or DNA structural motifst in cist which influence transcriptional activationt, transcriptional enhancementt or transcriptional silencingt should also be listed under this fieldname. Information about the timing of onset for expression should also be included if available, together with evidence for ion channel activity influencing gene activationt or patterningt during vertebrate development.
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Introduction
Field number 12: Isolation probe: This field should include information on probes used to relate distinct gene products by isolation of novel clones following lowstringency cross-hybridization screenst. The development of oligonucleotidet sets which have been used to unambiguously detect subtype-specific sequences by PCRt , RT-PCRt or in situ hybridizationt should be identified with source publication. Both types of sequence m a y be able to serve as unique gene isolation probes, dependent upon the libraryt size, target abundancet, screening stringencyt and other factors. Field number 13: m R N A distribution: This field should report either quantitative/ semi-quantitative or presence/absence (+) descriptions of specific channel mRNAs in defined tissues or cell types. This type of information is generally derived from Northern hybridizationt, RNAase protectiont analysis, RT-PCRt or in situ expression assays. See also notes on expression at]ases under Field number 08: Cell-type expression index. Field number 14: Phenotypic expression: This field should include information on the proposed phenotypet or biological roles of specified ion channels where these are discernible from expression studies of native$ (wild-type) genes. Phenotypict consequences of naturally occurring (spontaneous) mutationsT in ion channel genes are included where these have been defined, predicted or interpreted (see also Fields 26-32 of the S T R U C T U R E & F U N C T I O N S section for interpretation of site-directed mutagenesist procedures as well as Resource D - 'Diagnostic' tests, entry 59). Associations of ion channels with pathological states, or where molecular 'defects' could be 'causatory' or contribute to the progression of disease should be listed in this field (for links with established cellular and molecular pathology databases, see Fig. 4 of Feedback & CSN access, entry 12). The Phenotypic expression field may include references to mutations in other ('nonchannel') genes which affect channel function when the proteins are co-expressed. It is also used to link descriptions of specific (cloned)molecular components to native cell-electrophysiological phenotypes. In due course, this field will be used to hold information on phenotypict effects of transgenict manipulations of ion channel genes including those based on gene knockoutt or gene locust replacementt protocols. Field number 15: Protein distribution: This field should report results of expression patterns determined with probes such as antibodies raised to channel primaryt sequences or radiolabelled affinity ligandst. Field number 16: Subcellular locations: This field should describe any notable arrangements or intracellular locations related to the functional role of the channel molecule, e.g. when the channel is inserted into a specified subcellular membrane system or is expressed on one pole of the cell only (e.g. the basolateral$ or apicalt face). Field number 17: Transcript size: This field should list the main RNA transcriptt sizes estimated (in numbers of ribonucleotides) by Northernt hybridization analysis. Multiple transcript sizes may indicate (i) alternative processing
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('splicingt') of a primary transcriptt, (ii)the use of alternative transcriptional start sitest, or (iii) the presence of 'pre-spliced' or 'incompletely spliced' transcripts identified with homologous nucleotide probest in total cell mRNAt populations. Note that probes can be chosen selectively to identify each of these categories; 'full-length' coding sequencer (exonict)probes are the most likely to identify all variants, while probes based on intronict sequences (where appropriate)will identify 'pre-splice' variants.
Criteria for S E Q U E N C E A N A L Y S E S s e c t i o ns This section should bring together data and interpretations derived from the nucleic acid or protein sequence of the channel molecule. The symbol [PDTM] denotes an illustrated feature on the channel monomer protein domain topography model, which is presented as a central figure in some entries for sequenced ion channels. These models are only intended to visualize the relative lengths and positions of features on the whole molecule (see the description for field number 30, Predicted protein topography). The PDTMs as presented are highly d i a g r a m m a t i c - the actual protein structure will depend on patterns of folding, compact packing and multi-subunit associations. In particular, the relative positions of motifs, domain shapes and sizes are subject to re-interpretation in the light of better structural data. Links to information resources for protein and nucleic acid sequence data are described in the Database listings field towards the end of each entry.
Field number 18: Chromosomal location: This field should provide a chromosomal locust designation (chromosome number, arm, position)for channel gene(s)in specified organisms, where this is known. Notes on interactive linking to gene mapping database resources appear under an option of the Cell-Signalling Network 'home page' (see Feedback & CSN access, entry 12).
Field number 19: Encoding: This field should report open reading framer lengths as numbers of nucleotides or amino acid residues encoding monomeric channel proteins (i.e. spanning the first A of the ATG translational start codont to the last base of the translational termination codont). The field should report and compare any channel protein length variants in different tissues or organisms. If considered especially relevant or informative, selected primaryt sequence alignments of different gene family members may appear under this field. Field number 20: Gene organization: This field should describe known introntand exont junctions within or outside the protein coding sequence, together with positional information on gene expression-controlt elements and polyadenylationt sites where known. Note: Functional changes as a result of gene expressioncontrol should be listed under the Developmental regulation field.
Field number 21: Homologous isoforms: This field should indicate independently isolated and sequenced forms of entire channels which either show virtual identity or of such high homologyt that they can be considered equivalent should also appear in this field (but see note on percenta~ge conservation values under Field number 28: Domain conservation). Isoformst* of a channel protein can exist
entry 02
between closely related species or between different tissues of the same species (i.e. the same gene may be expressed in two or more different tissues, sequenced by two groups but named independently). Some tissue-specific variation may also result from alternative splicingt, yielding subtly distinct forms of channel protein. Since small numbers of amino acid changes may. exist from individual-to-individual (as a result of normal sequence polymorphismT in populations) separate isolates may yield sequence isoforms which can be shown to be 'equivalent' by Southern hybridizationt procedures (see Field number 25: Southerns). "Note: In the entries of this book a restrictive definition of molecular identity (or near identity) is used to define an isoformt. In this restricted sense, 'isoforms' would be expected to be the product of the same genet (or gene variant produced by, for example, alternative splicingt), and therefore have very similar or identical molecular constitutions and functional roles in specified cell tyl~es of closely related species. Comparative information on different gene familyl members or multiple variants affecting particular protein domainst may also be included under the Gene family and Domain conservation fields respectively. Field number 22: Protein molecular weight (purified): This field should state reported molecular weights estimated from relative protein mobilities using SDSPAGEt methods (e.g. following affinityt purification from nativet or heterologoust cell membranes). Data derived from nativet preparations generally includes the weight contribution f~om oligosaccharidet chains added durin~ post-translational protein glycosylation. In general, extracellular saccharide! components of glycoproteinst may contribute 1-85% by weight, ranging from a few to several hundred oligosaccharide chains per glycoprotein molecule. Field number 23: Protein molecular weight (calc.): This field should list the molecular weight of monomeric channel proteins equivalent to the summated (calculated) molecular weights of constituent amino acids in the reported sequence (e.g. derived from open reading framest of cDNAt sequences). If 'calculated' molecular weights are less than 'purified' molecular weights (previous field) this may indicate the existence of post-translational glycosylationt on nativet expressed protein subunits in vivo. Field number 24: Sequence motifs: This field should report the position of putative regulatory sites as deduced from the protein or nucleic acid primaryt sequence (with the exception of potential phosphorylation sites for protein kinasest, which are listed under Field number 32: Protein phosphorylation). Positions of sequence motifst illustrated on the monomer protein domain topography model are denoted by the symbol [PDTM]. Typical consensust sites include those for enzymes such as glycosyl transferasest, ligandt-binding sites, transcription factort-binding sitest etc. N-glycosylationt motifs are sometimes indicated using the shorthand designation N-gly:. Signal peptide cleavage sites (sometimes designated by Sig:) can be derived by comparing sizes of the signal peptidet and the mature chaint. Field number 25: Southerns: This field should include information which reports the existence of closely related DNA sequences in the genomet or reports the copy numbert of individual genes via Southern hybridizationt procedures. Note that
entry 02
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nativet diploid somatict cells will generally maintain two copies of a given ion channel gene locust, but stablet heterologoust expression procedures may result in multiple locus insertiont. Multiple locus insertion can be quantitated in SouthernT hybridization procedures using two probes of similar length and hybridization affinityt, one specific for a native locus (which will identify two copies) and one for the heterologous gene (which will yield a hybridization signal proportional to the copy number). Note also that the copy number parameter can not be equated to the physiological expression level of the recombinantt protein unless locus control regions are incorporated as part of the channel expression construct (for details, see the section entitled Gene copy number under Resource D - 'Diagnostic' tests, entry 59, and the section describing heterologous ion channel gene expression under Resource H - Listings of ceil types, entry 63).
Criteria for S T R U C T U R E & F U N C T I O N S sections This section should bring together information based on functional analysis or interpretation of ion channel structural elements. This section includes data derived from functional studies following site-directed mutagenesist of ion channel genes and molecular modelling studies at atomic scale. Future developments linking on-line information resources for protein structure to 'functional datasets' are illustrated in Fig. 5 of Feedback & CSN access, entry 12, and in Resource J Search criteria & CSN development, entry 65.
Field number 26: Amino acid composition: This field should include information on channel protein hydrophilicityt or hydrophobicityt where this is of structural or functional significance. Similarities to other related proteins should be emphasized.
Field number 27: Domain arrangement: This field should describe the predicted number and arrangement of protein domainst when folded in the membrane as determined by hydropathicity analysist of the primaryt sequence. Note that structural predictions of transmembrane domainst on the basis of hydrophobicityt plots may be misleading and prematurely conclusive. For example, high resolution (~9 A)structural studies of the nicotinic acetylcholine receptor (nA ChR, see ELG CAT nAChR, entry 09) predict that only one membrane-spanning c~-helixt (likely to be M2, a pore-lining domain) is present per subunit, with the other hydrophobic regions being present as fl-sheetst (see Unwin, J Mol Biol (1993)229: 1101-24). By contrast, extracellular ligand-gated (ELG)channels such as the nAChR display four predicted membrane-spanning regions (MI-M4) on the basis of hydrophobicity plots. From the foregoing it must be emphasized that all assignments given for the number or arrangement of 'predicted' domains in this field are tentative. Field number 28: Domain conservation: This field should point out known structural and/or functional motift sequences which have been conserved as protein subregions of ion channel primaryt sequences during their evolution (such as those encoding a particular type of protein domain?). Cross-references should be made to functionally related domains conserved in different proteins including 'non ion channel' proteins. Note that 'percentage conservation' values are not absolute as they depend on which particular subregions of channel sequences are aligned, the numbers and availability of samples, and/or which sequence alignment algorithms~ are used.
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Field number 29: Domain functions (predicted): This field should indicate predicted functions of channel molecular subregions based on structural or functional d a t a e.g. regions affecting properties such as voltage-sensitivity, ionic selectivityt, channel gatingt or agonistibinding. Field number 30: Predicted protein topography: This field should include information on the stoichiometrict assemblytpatterns of protein subunits derived from the same or different genes. This field indicates whether channel monomers are likely to form homomultimers~, heteromultimerst or both, and lists estimated physical dimensions of the protein if these have been published. Note: 'topography' is a convenient term borrowed from cartography which when applied to proteins, implies a 'map' at a level of detail or scale intermediate between that of an amino acid sequence and a larger-scale representation such as a protein multimeric complex. Topographic maps (or 'models')are therefore particularly useful for displaying selected sets of (inter-related)datatypes within a single 'visual framework'. The protein domain topography models (symbolized by [PDTM] throughout the entries)provide prototypes for this form of data representation. The considerable scope for further development of 'shared' topographical models which interactively report and illustrate many different features in the text are described in Search Criteria & CSN Development (Resource J). The terms 'protein topography' and 'protein topology' are often used interchangeably (sic), but the latter should be reserved for those physical or abstract properties of a molecule which are retained when it is subjected to 'deformation'. Field number 31: Protein interactions: This field should report well-documented examples of the channel protein working directly in consort with separate proteins in its normal cellular role(s). The 'protein interactions' described need not involve physical contact between the proteins (generall referred to as 'protein-protein' interactions), but may involve a messenger molecule. The scope of this field therefore includes notable examples of protein co-localization or functional interaction. For instance, reproduction of nativet channel properties in heterologoust cell expression systems may require accessory subunit expression (e.g. see VLG K Kv-beta, entry 47). Common channelreceptor or G protein-channel interactions are described in principle under Resource A - G protein-linked receptors, Receptor~transducer interactions.
entry 56 and Field n u m b e r
49:
Field number 32: Protein phosphorylation: This field should describe examples of experimentally determined 'phosphomodulation' of ion channel proteins, and if possible list sites and positions of phosphorylation motifst within the channel sequence. Only those consensus sitest explicitly reported in the literature are shown, and these may not be a complete description and may not be based on functional studies. Examples of primaryt sequence motifst for in vitro phosphorylation by several kinasest are listed in Resource C - Compounds & proteins, entry 58 and Resource G - Reported 'Consensus sites' and 'motifs', entry 62 (both updatable via the CSN). Abbreviations used within this field for various enzyme motifst (e.g. Phos/PKA) are listed in Abbreviations, entry 03. Electrophysiological or pharmacological effects of channel protein phosphorylation in vitro by use of purified protein kinasest should also be described or cross-referenced in this field.
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Criteria for ELECTROPHYSIOLOGY sections This section should bring together information concerning the electrical characteristics of ion channel molecules - how currents are turned on and off, which ions carry them, their sensitivity to applied membrane voltage or agonists, and how individual molecules contribute to total membrane conductance in specified cell types.
Field number 33: Activation: This field should contain information on experimental conditions or factors which activate (open)the channel, such as the binding of ligandst, membrane potential changes or mechanical stimulation. Descriptions of characteristic gatingt behaviour such as flickeringt, burstingt, activation latencyt or thresholdt of opening are also included. Applicable models of activation and the time course of current flow are briefly described here or referred to Field number 38: Kinetic model. Field number 34: Current type: Where clarification is required, this field should contain general descriptive information on the type, shape, size and direction of ionic current. Field number 35: Current-voltage relation: This field should report the behaviour of the channel current passed in response to a series of specified membrane potential shifts from a holding potentialt under a specified recording configurationt. For ligandt-gated channels (i.e. those with sortcodes beginning ELG and ILG)entries should report the current evoked by specific concentrations of agonistt applied at various holding potentials. This field should attempt to illustrate channel behaviour by listing a range of parameters such as slope conductance t, reversal potentialst and steepnesst of rectifyingt (non-ohmict) behaviour. The conventions used for labelling the axes of I-V relations for different charge carrierst are outlined in the on-line glossary. Field number 36: Dose-response: This field should contain information relating activator 'dose' (e.g. concentration) to channel 'response' parameters (e.g. open timer, open probabilityt) and whether there are maxima or minima in the response. Agonistt dose-response experiments are used to derive parameters such as the Hill coefficientt and Equilibrium dissociation constantt. Field number 37: Inactivation: This field should describe any inactivationt behaviour of the channel in the continued presence of activating stimulus. The field includes information on voltage- and agonistt-dependence, with indications of time course and treatments which extend or remove the inactivation response. Where known, this field will distinguish channel inactivation from receptor desensitizationt processes, which are of particular significance for the extracellular ligandt-gated (ELG)channel types (see ELG Key facts, entry 04).
Field number 38: Kinetic model: This field should contain references to major theoretical and functional studies on the kinetic behaviour of selected ion channels. The field contents is limited to a simple description of parameters, terms and fundamental equations.
Introduction
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Field number 39: Rundown: This field should collate information on channel 'rundownt' ('washout')phenomena observed during whole-cellt voltage clampt/ cytoplasm dialysist or patch-clampt experiments. Conditions known to accelerate or retard the development of rundown should also be listed. Field number 40: Selectivity: This field should report data on relative ionic permeabilitiest under stated conditions by means of permeability ratiot and/or selectivitY,T ratiot parameters. The field may also corn?are measured reversal potentials in response to ionic equilibrium potentials with specified charge carriers under physiological conditions. This field also lists estimated physical dimensions of ionic selectivity filterst where derived from ion permeationt or electron micrographic studies. Field number 41: Single-channel data: This field should report exarnples of singlechannel current amplitudes and single-channel conductancest measured under stated conditions. In the absence of authentic single-channel data, estimates of channel conductancest derived from whole-cell recordingt and fluctuation analysist may be listed. Field number 42: Voltage sensitivity: This field should describe the behaviour of the channel in terms of parameters (e.g. P,,pent) which are directly dependent upon applied membrane voltage. A distinction should be made between 'voltage sensitivity' resulting from intrinsic voltage-gatingt phenomena (i.e. applicable to channels possessing integral voltage sensorst) and indirect effects of applied membrane voltage influencing general physical parameters such as electrochemical driving forcer. Criteria for P H A R M A C O L O G Y
sections
This section should bring together information concerning pharmacological or endogenous modulators of ion channel molecule activity. Regulatory cascades in cells may simultaneously activate or inhibit many different effector proteins, including ion channels. Analysis of patterns of sensitivity to messengerst and exogenous compounds can help elucidate the molecular signalling pathway in the context of defined cell types. Field number 43: Blockers: This field should list compounds which reduce or eliminate an ionic current by physical blockade of the conductancet pathway. The field should include notes on specificity, sidedness and/or voltage sensitivity of block, together with effective concentrations and resistance to classes of blockers where appropriate. Where sites of block have been determined by site-directed mutagenesisT, these should be cross-referenced to Domain functions, field 29. Field number 44: Channel modulation: This field should summarize information on effects of important pharmacological or endogenous modulators, including descriptions of extracellular or intracellular processes known to modify channel behaviour. Loci of modulatory sites on the channel protein primaryt sequence (as determined by site-directed mutagenesist procedures)should be cross-referenced to Domain functions, field 29.
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J
Field number 45: Equilibrium dissociation constant: This field should list published values of Kd for agents whose concentration affects the rate of a specified process. See also on-line glossary entry for equilibrium dissociation constantt. Field number 46: Hill coefficient: This field records calculated Hill coefficientst of ligandt-activated processes. The Hill coefficient (n) generally estimates the m i n i m u m number of binding/activating ligands although the actual number could be larger. For example, a Hill coefficient reported as n >/3 suggests that complete channel activation requires co-operative binding of at least four ligand molecules (e.g. see ILG CAT cGMP, entry 22). See also Field number 36: Dose-response. Field number 47: Ligands: This field should include principal high-affinity radioligandst which have been used to investigate receptor-channel function and that are commercially available. Note that numbers of ligandt-binding sites cannot be equated to functional receptors because they only indicate the presence of a ligand-binding entity that may not necessarily be linked to an effectort moietyt. Field number 48: Openers: This field should list compounds (or other factors)which increase the open probabilityt (Popen)or open timer of the channel in nativet tissues. Field number 49: Receptor~transducer interactions: This field should briefly discuss known links to discrete (i.e. separately encoded)receptor and G protein molecules (see also Resource A - G protein-linked receptors, entry 56, accessible via the CSN). Types of 'receptor/transducer~channel' interactions account for many of the physiological responses of ion channel molecules within complex signalling systems. Note: Many pharmacological agents acting at receptor or transducer proteins (beyond the scope of these entries, but see Watson, S. and Arkinstall, S. (1994) The G-Protein Linked Receptor FactsBook. Academic Press, London) partially exert their biological effects because these receptor/transducers have ion channel molecules as an ultimate effectort protein. Field number 50: Receptor agonists (selective): For the extracellular ligandt-gated (ELG) receptor-channels, this field should list compounds which selectively bind to the ligand receptor portion of the molecule and thereby increase the open timer, open probability! or conductancet of the integral channel. Anta~onistst should be categorized as competitivet, non-competitiveT: or uncompetitivJ where this has been determined. Field number 51: Receptor antagonists (selective): This field should list agents that selectively bind to the ligandt receptor portion of integral receptor-channel molecules but do not activate a response. Field number 52: Receptor inverse agonists (selective): This field should list compounds which selectively bind (extracellular ligand-gated)receptor-channels but which initiate an opposite response to that of an agonistt, i.e. tending to reduce the open timer, open probabilityt or conductancet of the integral channel.
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Criteria for I N F O R M A T I O N
RETRIEVAL sections
This section should provide links to other sources of information about the ion channel type, particularly accession to sequence database, gene expression, structure-function and bibliographic resources operating over the Internett or available on CD-ROM. A full discussion of the potential scope for integration of these resources with molecular-based entries appears in Resource J - Search criteria & C S N development, entry 65. Brief details are given in Feedback & CSN access, entry 12, in each volume.
Field number 53: D a t a b a s e listings~primary sequence discussion: This field should tabulate separately listed items of relevance to the channel type and may include 'retrieval strings' such as locus names, accession numbers, keyword-containing identifiers and other miscellaneous information. Note that terms used by databases are often abbreviated (e.g. K for potassium, Na for sodium etc., therefore only specific identifiers (such as the accession numbers, locus and author names) should be used for retrieval. The actual names and numbers quoted have been sourced from NCBI-GenBank" (prefixed gb:)or EMBL (prefixed em:). Since there is now a high concordance between the contents of the EMBL and NCBI-GenBank" nucleic acid databases, the NCBI-GenBank" accession numbers given should retrieve the information from either database. Note that in all of the Database listings sections, the lower case prefixes are not part of the locus name or accession number, but merely indicate the relevant database. Sources of pre-translated protein sequences are indicated by references to the following databases (given in alphabetical order following the NCBI-GenBank" nucleic acid reference): SWISSPROT (prefixed sp:), Protein Identification Resource (prefixed pit:). The journal-scanning component of GenBank uses the NCBI 'Backbone' database (prefixed bbs: for backbone sequence, composed of several individual sequence segments; bbm: for backbone molecule) - these are maintained by the NCBIt (National Center of Biotechnology Information). General notes on sequence retrievals: Updating and error-correction procedures for public domain databases may modify a protein or nucleic acid sequence (retrievable by a given accession number) between releases of a database. Thus, two users performing an analysis on a given database record may come to different conclusions depending upon which release was used. Note also that (i) accession numbers sometimes disappear with no indication of whether a new record has replaced the old one, (ii)multiple databases sometimes each give a different accession number to a single record, and (iii) some databases do not respect the ranges of accession numbers 'reserved' by other databases. Although the 'traditional' format of accession numbers has been a letter followed by five digits (with a m a x i m u m space of 2.6 million identifiers), the rapid rate of sequence accumulation will eventually force a different format to be used. Because of these problems, the NCBI now uses unique integer identifiers (UIDs) to identify sequence records and encourages their use as the 'real' accession numbers for sequence records. Reference numbers prefixed 'gim' can be read from CD-ROM media, but only refer to a 'GenInfo Import ID' - a temporary identifier unique only to a given release of the CD-ROM compilation (such as a numbered release of Entrez - see below). Should a sequence supplied by a database change, the record
Introduction
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]
will usually be allocated a new 'gim' number, but the old one will still be available under its UID from the ID database. Because of the transient nature of 'gim' identifiers, they are not recommended as search/retrieval parameters and are generally not listed in the Database listings field (except where an accession number proper has not been found). In compiling The Ion Channels FactsBook, extensive listings of aligned protein or nucleic acids to show sequence relatedness have been avoided (as these were judged to be best served by development of on-line data resources specializing in sequence alignments - for a prototype, see Hardison et al. (1994) Genomics 21: 344-53). See also entry 65. Alternatively or in addition, alignments can be performed according to need by dedicated sequence-manipulation software. Presently available compilations of sequences (e.g. the Entrez CD-ROM set or online equivalent, for example)can perform powerful 'neighbouringt analyses' based on pre-computed alignments of any sequence against the remainder of the existing database. Establishment of homologous alignmentst can proceed by finding a match between the query sequence and any member of the 'neighbouring set'. In practice, comprehensive retrievals can be performed interactively by just one or two rounds of neighbouring analysis. As indicated at the beginning of each Database listings field, the range of accession numbers provided can be used to initiate relevant searches, but following on from this, neighbouring analysis is strongly recommended to identify newly reported and related sequences. Descriptions of features based on primaryt sequence data listed within fields of the SEQUENCE ANALYSES or STRUCTURE & FUNCTIONS sections can be more readily interpreted if an interactive sequence analysis program is available. Electronic mail serverst at the NCBI can receive specially formatted e-mailt queries, process these queries, and return the search results to the address from which the message was sent out. No specific password or account is needed for these, only the ability to send e-mail to an Internett site. For local searches, alignment programs such as BLAST can also be retrieved by anonymous filetransfer protocolt or FTP. Detailed information on interactive linking to remote nucleic acid and protein database resources will appear under an option of the Cell-Signalling Network 'home page' (see Feedback & CSN access, entry 12). Accession numbers can be issued for newly submitted sequences (normally within 24 hours) by remote Internet connection or by formatting/submission software (e.g. Seqwin, obtainable from the NCBI using an anonymoust FTPt). NCBIGenBank" can also be accessed over the World Wide Webt (http:// www.ncbi.nlm.nih.gov).
Sample retrievals in the absence of a CD-ROM resource: For a nucleic acid sequence from the EMBL database, use the e-mailt address below exactly as shown, specifying the appropriate accession number (nnnnnn) by the GET NUC command. For example, a database entry can be automatically e-mailed to you by the EMBL
[email protected] GET NUC:nnnnnn An analogous procedure can be used to retrieve protein sequences from the SWISSPROT database, substituting the GET NUC: command with GET PROT:.
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Nucleic acid sequences from NCBI-GenBank 'I can be retrieved using the servert at the NCBI. In this case, send an e-mailt message to the service (address below) specifying the name of the database, the command BEGIN and the accession numbers or key words. A sample request is shown below for an accession number /~/f/n F//~/F/"
[email protected] DATALIB genbank BEGINnnnnnn Protein sequences from the Protein Identification Resource (pir:)can be obtained using an e-mailt request containing the command GET followed by the database code. The database code is distinct from the accession number but can be obtained by typing the command ACCESSION and then the number. For example, to specify a request for an entry of database code X X X X containing the accession number nnnnnn, you would send an e-mail message as follows:
[email protected] G ET X X X X ACCESSION nnnnnn General information on using these file serverst can be obtained using the above emailt addresses followed by the single command HELP. The Database listings tables contain short-form references to original research articles which have discussed features of the channel protein and/or nucleic acid primaryt sequence(s). Sequences are retrievable with the specified accession number or the author name shown in the short-form reference.
Field number 54: Gene mapping locus designation: This field should list references to human gene mapping loci t using terms defined by a human genome mapping workshop (HGMW)T convention where possible. Notes on interactive linking to gene mapping database resources appears under an option of the Cell-Signalling Network 'home page' (see Fig. 4 of Feedback & CSN access, entry 12). The opportunities for linking to a wide range of genetic information resources are discussed in Resource J - Search criteria & CSN development, entry 65.
Field number 55: Miscellaneous information: This is a 'catch-all' field used within the entry to reference relevant peripheral information or perspectives on the channel molecule or its function. This field also should be used to contain information about ion channels showing partial functional relatedness to those in the main entry, but which also possess some features indicating the expression of a distinct genet (for example, description of potassium-selective ligand-gatedt channels within an entry describing non-selective cation channels gated by the same ligandt, or vice versa). Normally, ion channels with distinct properties are covered in 'their own' entry whenever there is sufficient information available to make a clear set of 'defining characteristics'; the Miscellaneous information field therefore encompasses those channels which either have been infrequently reported, show only minor variations with the channel type under description, or are otherwise beyond the scope of the (present) collection of (largely)vertebrate
channel-type entries.
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Field number 56: Related sources reviews: For reasons of space, the FactsBook cannot provide citations for every 'fact' within individual entries. Citations within this field should provide a starting point for locating key data through major reviews and other primaryt sources where these have been quoted extensively within the entry. A full discussion of how future entries could be linked to established on-line bibliographic resources appears in entries 12 and 65.
Field number 57: Feedback: Information supplementary to the entries but appearing after the publication deadlines will be accessible from the CMHT servert over the Internett using a World Wide Webt utility from mid-1996 (see below). An aim in compiling this book is that the scope and arrangement of the information should, in time, be refined towards containing what is most useful, authoritative and upto-date: Feedback from individual users is an essential part of this process. The Feedback field identifies the appropriate address for e-mailt feedback of significant corrections, omissions and updates for the contents of a specified entry and fieldname. Comments regarding new or modified field categories (or supplementary reference-type material for incorporation into entries and appendices)would also be most welcome from users (for details on accessing entry updates via the CellSignalling Network, see Feedback e,) CSN access, entry 12).
Field number ## (inserted at appropriate points): In-press updates: This field has been used occasionally (at the most relevant points in the printed versions of the book) to index publications containing important (direct) evidence which may significantly alter several statements or conclusions in the 'finalized' entry as sent to the publishers. It is acknowledged that no 'book-form' information index can ever be completely up-to-date, and it is in the nature of scientific progress that 'interpretations' based on reported 'facts' may change considerably in the light of additional or more direct experimental approaches to a problem. The scope of the Cell-Signalling Network means that users (especially 'non-specialists')can be directed towards citations containing the 'latest' interpretations (or important 'additional facts'). The pace of change across all of the fields touched-on by the FactsBook means that 'specialists' in a given area can help 'speed-up' this indexing process by e-mailt notification where 're-interpretation' is justified (see Feedback & CSN access, entry 12, and Resource L entry 65). According to the original aims and 'philosophy' of the project, the entries will probably never be 'complete' as such. More appropriately, the framework will continue to evolve towards one which is hopefully more useful, authoritative, and able to comprehensively relate 'consensus' knowledge on ion channel molecular signalling.
Criteria for R E F E R E N C E S s e c t i o n s This section should contain 'short-form' references for numbered citations within the entry. For textbook coverage, refer to the Book references listed under Related sources and Reviews (field 56), Resource E - Ion channel book references, entry 60, Resource F - Supplementary ion channel reviews, entry 61 and Resource H - Listings of cell types, entry 63. Plans for 'hyperlinking' to full bibliographic databases within the CSN framework are described in Resource J - Search criteria & CSN development, entry 65.
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Criteria used for compilation of supporting computer-updatable resources The following reference appendices are referred to within the text and figures of the main entries. Updated versions of these files will be accessible via the 'home page' of the Cell-Signalling Network from mid-1996 - for further details, see Feedback & CSN access, entry 12 and Resource J - Search criteria & CSN development, entry 65. Resource A - G protein-linked receptors: A large number of ion channels are regulated as part of signalling cascades initiated by activation of G protein-coupled receptor proteins. This appendix should describe the basic principles associated with this type of regulation, limiting descriptions to those most relevant to ion channels. Tabulations of known receptort and G protein t molecules should form a framework of possible regulatory mechanisms based on specific protein subtypes. The entry may clarify or suggest likely interactions between receptors, transducerst (e.g. G proteins) and ion channel molecules described under the fieldnames Devel-
opmental regulation, field 11, Protein interactions, field 31, Protein phosphorylation, field 32, Channel modulation, field 44, and Receptor~transducer interactions, field 49. Resource B - "Generalized' electrical effects of endogenous receptor agonists: This resource should present a tabulated summary of genera] patterns of agonisttinduced ionic current fluxes that have been reported across a large number of studies, predominantly in the central nervous system. The table may help to indicate whether receptort agonists tend to act in an excitatoryt or inhibitoryt fashion 'or both'. Resource C - Compounds & proteins: Compounds and proteins mentioned in the entries which are commonly used to investigate ion channel function and modulation should be listed, including those used to analyse interactions with other cell-signalling molecules. In general, only frequently reported compounds which are commercially available are described in this appendix. Resource D - "Diagnostic' tests: This appendix is intended to be an alphabetical listing of common experimental manipulations used to 'implicate or exclude' the contribution of a given signalling component or phenomenon associated with ion channel signalling. For the most part, these approaches use the pharmacological tools listed under Resource C, but may also include sections describing common molecular biological and electrophysiological 'diagnostic' procedures. Resource E -
Ion channel book references: This appendix should list details of
published books which have addressed themes in ion channel biology or closely related topics. These references complement those of the main entries, which are almost entirely based on citations from scientific journals. Resource F - Supplementary ion channel reviews: The ion channel literature contains a large number of useful 'minireviews' which summarize the development of defined subjects and which do not necessarily fall into a single
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Introduction
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channel 'molecular type' category. This appendix should therefore list these 'supplementary' sources, indexed by topic. Updated 're-writes' of subject reviews covering similar areas may replace earlier listings. Note: Subject reviews dedicated to aspects of an ion channel type or family can usually be found under the Related sources & reviews field of appropriate entries. 'Topic-based' reviews making reference to the basic properties in the 'molecular type' entries are planned for expansion within the CSN framework (for details, see entry 65). Resource G - Reported 'Consensus sites' and "motifs': Based on extensive analysis of primaryt sequences and determination of substrate specificities for various enzymes~ a number of 'consensus' recognition sequences for post-translational modification of proteins (including ion channels)have been determined. While these sites are not absolute, they can be highly conserved across whole families of ion channel proteins and in many cases (e.g. following phosphorylation) can lead to profound changes in ion channel function. However, the presence of 'consensus' sites or motifst (or even demonstrations of substrate specificity in vitro) does not necessarily prove that such modifications operate in vivo. This appendix should list 'consensus' motifs that are well-characterized, giving examples of 'authentic' sites for comparison. This appendix also contains a subset of consensust sites from genomic DNA sequences associated with mechanisms of ion channel gene expression-controlt (e.g. in transt protein factors which act at DNA structural motifs_ t in cist, influencing transcriptional activationt, transcriptional enhancementt or transcriptional silencingt of ion channel genes#). Resource H - Listings of cell types: Studies of ion channels within the context of celltype function often reflect 'recruitment' of selected genes from the genomet in a celldevelopmental lineaget. Because of this, similar 'sets' of ion channel molecules can often be observed in cell types with broadly similar functions. This appendix should describe a framework for describing how integrated sets of ion channel molecules (and their associated signalling components) have co-evolved for specific functions in terminally differentiatedt cell-types. To begin with, a tentative classification of functional cell types should be employed, used to cross-reference 'surveys' of ion channel expression wherever possible. This appendix should also contain available information pertaining to efficient and appropriate heterologous expression of ion channel genes in selected cell types, as this is often a limiting factor in biophysical characterization of clonedt ion channel cDNAt or gene products. Resource I Framework of cell-signalling molecule types: The flow of information into, within and between cells (signal transduction) generally depends on a multiplicity of co-expressed cell-signalling molecules which provide 'measured' responses to stimuli. Communication between different cellular compartments (e.g. between the cytoplasm and the nucleus)often requires 'interconversion' or 'transduction' of chemical, electrical (ionic), metabolic and enzymatic signals, with receptors and ion channels playing key roles in transducing such stimuli. For example, the 'activation' of signal transduction molecules such as kinasest or tcanS~rivPtiltngfaCtorl~tg na~egrt tg 'sense' 'activated' conditions which resembles a i phenomena commonly observed for ion channels. These modes of protein activationt probably have many features in common, and understanding their
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interrelationship has important consequences for comprehending fundamental links between receptor signalling, cell activation and gene expression. To facilitate integration of information between these diverse fields of study, this appendix should provide a preliminary listing of signal transduction molecules, with some consideration of their inter-dependency in the 'activated' state. By making a ratidnal 'connection' between activation of receptors, ion channels, enzymes and other effectort proteins, it is hoped that some general principles will emerge on the electrical- and ligandt-control of complex cell phenotypest (such as those affecting the cell cycler, cell proliferationt, cell differentiationt and apoptosist). The importance of ion channel activation (and activation of receptor/G protein transducerst which modulate ion channel activity)in other fundamental cell processes such as signal transmission/amplification, secretion (multiple forms), muscular contraction, endocytosist (and other cellular 'uptake' phenomena), sensory transduction (all types), cell volume control/osmotic responses, mechanotransduction (various forms), membrane potential control (multiple modes)and developmental compartment formation are well-documented and multiple examples appear in several fields, notably Developmental regulation, field 11, Phenotypic expression, field 14, Domain functions, field 29, Protein interactions, field 31, Protein phosphorylation, field 32 and Channel modulation, field 44.
Resource J - Search criteria & CSN development. The framework of database entries which form the basis of The Ion Channel FactsBook were derived by 'scanning' primary research articles and reviews appearing in a set list of 'principal' journals dealing with ion channel and receptor signalling. A disadvantage of 'journal scanning' by 'keyword' is that search terms used are often ambiguous, and contextual or unconventional grammatical usage of keyword terms within articles often results in failure of specific retrieval. To circumvent this problem, this appendix should suggest new unique embedded identifiers (UEIs)which when specified by authors in the keywords section of submitted articles should ensure appropriate electronic retrieval from the primary literature. The adoption of finalized 'UEI' codes should be open to debate. Their implementation outside the context of the CSN will be difficult unless contributing authors and journal editors acknowledge the benefits. If an alternative system is proposed and accepted by field consensus, then the CSN will move to adopt the system in the interests of simplifying search criteria on specific molecules or topics. The central principle of unique embedded identifiers is that they can 'automatically' find articles on topics of interest (in for example weekly literature scans). Coupling to an 'expansion' section with further search terms in a conventional order will help enormously in data compilation/consolidation processes on strictly defined subjects within 'validated' databases. Finally, Resource J should act as a forum for discussing limitations of data representation when comparing ion channel properties and suggest improved methods for facilitating information exchange (including graphical resources), diagnostic conventions, resolution of 'controversial' results, and identification of areas or highly focused topics requiring consolidation/extension of knowledge. The importance of standardized computer software compatible with Internett-mediated
entry 02
communication should be emphasized (see also Feedback & CSN access, entry 12). Contents organization within each 'specialist' field of the FactsBook gives further opportunities for comparative data analysis. In due course, the -zz term of the xxyy-zz index number will be used to indicate such structured information.
Criteria used for selection of on-line glossary and index items Consolidated versions of the FactsBook support glossary (i.e. extensions, updates and corrected items) are accessible from the Cell-Signalling Network 'home page' (see Feedback & CSN access, entry 12). Entry 65 contains a full specification of the CSN.
Index of on-line glossary items [ t]: To avoid unnecessary duplication of definitions within the text and to provide assistance to readers unfamiliar with a field, the online glossary should provide short introductions to technical terms and concepts. Throughout the text, cross-references to the on-line glossary items are shown by means of a dagger symbolt.
Cumulative subject index for The Ion Channel FactsBook, volumes I to IV. For the most part, The Ion Channels FactsBook should be 'self-indexing': 1. Locate the channel 'molecular type' by sortcode, or table of contents 2. Go to the appropriate section (NOMENCLATURES, EXPRESSION, SEQUENCE ANALYSES, STRUCTURE & FUNCTIONS, ELECTROPHYSIOLOGY, PHARMACOLOGY, INFORMATION RETRIEVAL or REFERENCES). 3. Look under the most appropriate fieldname (as described by the criteria above). Further 'structuring' will arise in due course, when more data are entered (see previous section). For location of information on ion channel molecules by miscellaneous related topics, the cumulative subject index should comprehensively list pertinent functional characteristics, concepts, compounds and proteins including those shown in bold text under the fieldnames, relating the topic to the six-figure index number. The subject index should also allow the initial location of entries through alternative names of channels, associated signalling phenomena or commonly reported properties. Electronic cross-relation of topics is intended to be a development focus of the CSN, exploiting the principle of hyperlinking between database files stored in 'addressible' loci. For further details on how this might be achieved, see Resource J - Search criteria & CSN development, entry 65.
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to this introduction can be sent to the email feedback file
[email protected]. (see field 57 of most entries for
further details)
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For most abbreviations of compound names in use, refer to the Resource C Compounds & proteins, entry 58, as well as the FactsBook entries. Abbreviations for ion channel currents are listed under the Current designation field of each entry. Terms marked with a dagger symbol appear in the on-line glossary section 0 C a 2+
5-HT 7TD A aa
AHP AP APD AV AVN
Ca2+-free solution 5-hydroxytryptamine; serotonin 7 transmembrane domains amperet amino acidt afterhyperpolarizationt action potentialt action potential durationt atrio-ventricular atrio-ventricular nodet (of heart)
BP bp
large ('big')-conductance calcium-activated K§ channels blood pressure base pairst
C C-terminal C/A or C-A Ca(mech) or Camech Cav cds CF CICR CI(Ca) or C1ca CNG CNS COOH CRC cRNA CTK Cx or Cxn
coulombt carboxylt terminalt (of protein) cell-attached~ (recgrding configuration) mechanosensitiveT Ca 2§ channel voltage-gatedt Ca 2+ channels codingt sequence (used in GenBankt ~"~'entries) cystic fibrosist calcium-induced-calcium-release calcium-activated chloride channel cyclic-nucleotide-gated Ichannels) central nervous system carboxyl groupt calcium release channels complementaryt RNA cytoplasmic tyrosine kinaset (cf. RTK) connexin
Da
DHPR DMD DPSP
daltons putative (consensust)site for dephosphorylationt by a specified enzyme, e.g. Dephos/PP-l: endogenous protein phosphatase-1; Dephos/PP-2A: protein phosphatase-2A dihydropyridine receptor Duchenne muscular dystrophyt depolarizing post-synaptic potentialt
E EAA E-C
potential differencet, inside relative to outside excitatory amino acidt excitation-contractiont
BKCa
Dephos/enzyme
entry 03
Erev
50% effective concentration equilibrium potentialt for K§ ions (analogous nomenclature for other ions) extracellular ligandt-gated (as used in FactsBook sortcode) membrane potentialt European Molecular Biology Laboratoryt electromotive forcet endiPlttet ~otentialt e c'ta ory post-synaptic potentialt endoplasmic reticulum reversal potentialt
F F fS
faradt Faraday's constantt femtosiemens (10-is Siemenst)
G g G/Gmax gb: gj, Gj or G(j)
conductancet conductance (unit- Siemenst, formerly reciprocal ohmst or mhot) peak conductancet designation for GenBank c"~accession numbert gap-junctional conductancet
HGMW HH h.p. HVA
Human Gene Mapping Workshopt after Hodgkint-Huxleyt holding potential high-voltage-activated Ca 2§ channels
I
IPSC IPSP
currentt subscript abbreviation for intracellular peakt currentt inside-outt (patcht, recording configurationt) concentration which gives 50% of maximal inhibition effect in a dose-inhibition response curvet. intracellular ligandt-gated (as used in FactsBook sortcodest) maximal currentt collective abbreviation for inositol polyphosphatest e.g. InsP3, InsP4 inositol 1,4,5,-trisphosphate-sensitive receptorchannel inhibitoryt post-synaptic currentt inhibitoryt post-synaptic potentialt
JCC
junctional channel complex
k KA or K(A) kb KCa, Kca or K(Ca)
Boltzmann's constantt A-typet I~§ channels kilobases (kbp - kilobase pairs or bp x1031 calcium-activated K* channels
EC5o EK ELG Em EMBL EMF EPP EPSP ER
i
I/Imax I/O or I-O ICso ILG max
InsPlx1or InsPx
-
InsP3R or IP3R
]
entry 03
Kv
equilibrium dissociation constantt kilodaltonst (daltons • equilibrium dissociation constantt for an inhibitor for KATP channels, the ATP concentration (/~M) that produces half-maximal inhibition of channel activity inhibition constantt at zero voltaget inward rectifiert-type K§ channels shorthand designation for mechanosensitive K§ channels voltage-gated K§ channels (generally delayed rectifierst)
LTD LTP LVA
long-term depressiont long-term potentiationt low-voltaget-activated Ca ~§ channels
mAChR MARCKS Mb MEPC MDa MH
muscarinic acetylcholine receptor myristoylatedt, alanine-rich C-kinase substrate megabasest (Mbp - megabase pairs) miniature endplate currents megadaltonst (daltons • malignant hyperthermia relative molecular masst messenger RNAt millivolt (10 -3 V)
KD
kDa KI Ki ATP
/;i{01 KIR or K(IR) K(mech) or Kmech
Mr
mRNA mV N //
nAChR Nav N-gly: NH2 NSA NSC NSC(Ca) nt N-terminal O
O-gly OHC O/O or O-O PCa PCAP PDE
number of functional channels a l s o - Avogadro's numbert Hill coefficientt nicotinic acetylcholine receptor-channel shorthand designation for voltage-gated Na § channels predicted sites for N-linked glycosylationt (e.g. Ngly: aa122, specifying amino acid number 122 from known glycosylaset substrates) aminot group non-selective anion (channel) non-selective cation (channel) non-selective cation channels (calcium-activated) nucleotides amino-terminal (of protein) subscript abbreviation for extracellular O-linked glycosylationt outer hair cells outside-outt (patcht, recording configurationt) permeabilityt of Ca ~§ ions (analogous nomenclature for other ions, e.g. PK, PNa, PRB etc.) pituitary adenylyl cyclase-activating polypeptide phosphodiesteraset
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pS PSC PSP PSS
protein domain topology model; within the text, use of the abbreviation in square brackets denotes a positional feature illustrated on the model intracellular pHt Putativet (consensust) site for phosphorylationt by a specified enzyme, e.g. Phos/CaM kinase II- multifunctional (Ca2+/calmodulin)-dependent protein kinase II; Phos/CaseKII: casein kinase II; Phos/ GPK: glycogen phosphorylase kinase; Phos/MLCK: myosin light-chain kinase; Phos/PKA: cAMPdependent protein kinase (PKA); Phos/PKC: protein kinase C (PKC); Phos/PKG: cGMPdependent protein kinase; Phos/TyrK: tyrosine kinase (TyrK)subtypes post-injection Protein Identification Resourcet (protein sequence database) designation for Protein Identification Resourcet accession numberst peripheral nervous system t polyadenylation t (site) polyadenylated~ (mRNA).fraction of total cellular RNA channel open probability~ picosiemens (10-12 Siemenst) post-synaptic currentt post-synaptic potentialt porcine stress syndrome
Qlo
coefficientt for a ten-degree change in temperature
R
receptor resistancet (unit- ohmt), reciprocal of conductancet resting potentialt ribosomal RNAt receptor tyrosine kinaset (at plasma membrane, cf. CTK) ryanodine receptor-channel
[PDTM] pHi Phos/enzyme
p.i.
PIR pir: PNS poly(A) poly(A)+ Popen or Po
R r.p. rRNA RTK RyR
SAN SAPs s.c.a. s.c.c.
s.c.p. SCR SD SDS-PAGE SEM
Siemenst (unit of conductancet; reciprocal ohm t or mhot) sino-atrial nodet (of heart) signal-activated phospholipasest single-channel amplitudet single-channel conductancet (symbol, 7) single-channel permeabilityt single-channel recordingt standard deviation sodium dodecyl sulphate-polyacrylamide gel electrophoresis t (i) standard errort of the meanst or (ii)scanning electron microscopyt
entry 03
spike frequency adaptation t indicates the range of amino acids which form the signal peptide of a precursor protein (e.g. Sig: aal26); alternatively, the abbreviation indicates the actual cleavage sitet forming the signal peptidet and mature chain~ from the precursort protein substance P designation for SWISSPROT protein sequence database accession numbert sarcoplasmic reticulumt disulphide bondt; in sequence database entries, the S-S: symbol is sometimes used to denote positions of a known disulphide bond linkaget or motift between two residues on a protein molecule, e.g. an experimentally determined link between residues 154 and 182 on the same chain would be written as S-S: 154-bond- 182.
SFA Sig:
SP sp: SR S-S"
TM Tm
Tm TPeA+ TT
transmembrane melting temperaturet upper limit to the amount of material that carriermediated transport can move across a membrane tetrapentylammonium ions transverse tubulet
VDAC VDCC
voltt voltaget voltage-activated calcium channels; analogous nomenclature for other channels, e.g. VAC1C, VAKC, VANaC, VDAC voltage-dependentt anion channel voltage-dependentt calcium channel
W/C or W-C WCR
whole-cellt (recording configuration) whole-cellt recording
YAC
yeast artificial chromosomet
Y yj or ylj) ~A f~ co-CgTx
unitary (single-channel) conductance single-channel junctional conductancet microamp (10 -6 Amperes) ohmt, unit of electrical resistance t; reciprocal of conductance t omega-conotoxin
V V VACaC
Feedback: Comments and suggestions regarding the scope, arrangement and other matters relating to the abbreviations section can be sent to the e-mail feedback file
[email protected]. (see field 57 of most entries
for further details)
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EXTRACELLULAR LIGAND-GATED CHANNELS (ELG)
i/
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Edward C . Conley
n
Entry 04
N o t c Thc. 'Key facts' srPu!ronsnrc ~ r ~ t ~ n {or c l rrellrlrrs ~f unfamiliar w t h thc morc $encral rlIL' W T hE ~ O W L ' I / ~inI ~l u ~ ~mejRhbn"rjllRt lt oaalyds rolrtrnr7\ (for (Ic\r.rrpt ;on. \rrp !hr II(i!rr/?n\t~I ~ i t l r ~Iir,Jrl x ~ In tllc / I ? ! rr)(Iuct 1011 CJ I i i i ~ i r t o/ ( T r ~ ! ~ i ~ Jm\ , t rt7 02) For ( T ( I ~ T I ~\ ()l!( ~ / t ,~,( R ! ~ i *of~ J v .r rtn %-\j,rrit,\ vrrrrrrrl!i or rr~lr~tc,d grxnr, /onlllrlt n~r,lnhrbr\ crm rcnrlllv r:r-c*c'\w7rlh v onr. ox two T O ~ I ~ I01~ \~ ~ r ' i ~ h ! y r I)I R~( ~I / Vf \,I n, (which ~~ rlrr hriwd o r ] prr,-r.o~npt~t or1 nlrenmrw! r pcrlorlnrd 11wn.y 1 F ~ r u I I I ~ A S T(ilgr~rr ~ i hrn I,v thr, N C I ~ ~ TIFI$ ' ) flbcltt!rr 1 9 1710\t ~:\cfl:I kw rctr~r~vrrl of zcprprnt.r, runrrlrfs d~,po\i!ed rrT d ( i l ~ ~ ! > rlt~/t,r ~ w \ t l x ~ tihoc(, ~ 11\fr(!/w~lowTh11+,,rLJj~r(,\cntr1trvr ~ n r m / r r , rol ~ known uxr/lrr,nrra ~ ( I I I I O I O S ~gI r o l ~ p ~ n ,itrt7 y ~ II\IC(I to pcrrr~lt lnrtro/ drrcct rt.!rltvrnE~ h r r nuururc~on ~?trml?r'r,rI?~thor/rcf~~rerm or ~~c~rnrncfr~rrrre Follow~ng -rI~rcc! rnut.ras5~r~r~, howr,vrr, - -- n - c ~ ~ h h o ~ r rc~rlr~ly\~s r r ~ ~- -t .. i\ r!ror?~ly - ~ cc t ~ ~ ~ ~ r n c- nrrrFcnll/y f.r-, r- l ! o--n-m--d yrr~l~orr -ran-s~nrE - rrlr~!r~~~~cqt~criucr. I
Nnmcnclatiirc 5-HTI rcccptor prccurcnr: 5HTIR-AI ('long form'l
Specicc;, DNA
Acccwon
sourcc
Origrnnl ~solatc
Mousc ncurohlnstoma ccll cxprcssron tthrary
487 ;la; ~ g : gh M74425 23 nn ( 4 h i , plr: PLqYJ9 inatlire procltc: protctn) crr7 I'S0023h
FI"jTM1 55966 13.1
(from cl3Nh)
~
r
p
ScqucnccJ discussinn
Mnr~cq, Sclrmcc [ 19Y 1 1 254. 4.32-7.
cntry 05
Nomcnclaturc S-MTAreceptor precursor: 5HTxR-A? (’short form‘)
Spccics, DNA so~irccu
Original
Rat superinr cervical gang1inn library
461 aa
Acccssion
1solatc
Scqucnccl
discussion not found
Likely rat variant of mousc S-
Fohnson, Soc Neurosci Rhs
(l9UZ) 18: 11815.
I-ITqR-A,
5-HT,?rcccptor precursor: 5 HT.1R - As [‘short form’)
Rat superior
partiat cDNA
cervical gang1ion library
not fnund
IsenhcrR, NeuroR epor z [199,715: 1214.
460 aa
rcccptor Mousc ncuroprccursor: 5hlastoma ccll MT3R-Aq lint NlE-115 (‘short form’]
sig: 23 aa 53 17R Da [from eDNA)
gb: X7L395
Hope, E m 1 1%o rm o cnl (1993) 245: 187-92.
Related sources & reviews 05-56-01: Maior sources t h a t includc suhtypt dcfinitians”.“ ’.*’**,’; nthcr snurccs with a rcvicw clcmcnt includc - thc cstahlished and potential
therapeutic uses for S-HTa reccptor antagonists; hchavioural pharmacnlogy of S-HT\%rcccptor antagonists7’ thc ncumcndocrinc pharmacohm of scrw toncrgic (5-HT) ncuronmAd;molcculnr cloning and functinnal cxprcssion’; advances in clectmphysiological characterization of 5 H T 1 receptnrs’. ?.’; soluhi 1ization and physico-chcm ical cl~amctcrizatinnof S-FIT,?rcccptor-hind ing sitcs (Miqucl el at., lYR3, ~ e ~ I Rh Nenmsczcnccs, ~ s Vol. 1 1 - see Xesourcc E - Ion channel hook refcrenucs. m i r y 601.
’‘;
Rook references 05-56-02:
Andrews, P.L.R.and Sanger, G.J. (cds) (19921Erne in Anti-Cancer Therapy. Chapman and Mall, London. Fozard, J.R. [cd.j I19893 The Penpl~ernlActions nf 5-Hydroxytryprarnine. Oxford Wnivcrsity Press, Oxford. Frrzard, J.R. and Saxena, P.R. (cds) (1941) Serotonin: Molecuhr Biology, Receprors nnd Ftinctional Effects. Rirkhauscr, Bawl. Hamon, M. [cd.)(19923 Cenrrrzl and I’crrpheml S-HT,?Ileceprars. Acadcmic Press, London. Joncs, R.J., King, E. and Sanger, G.J. ( e d s ) 1994). 5-HT.7 Receptor dnfa,enniqts. CRC Press, Roca Raton. Saxcna, P.R., Kluwcr D., Wallis, D.I.,W(iuters, W.and Bcvan P., [cds] [I9901 Cnrdinvmscu /or P l ~ i ~ ~ m i ~ o ?of o g y5-Hydroxyt ryptnmincr Prospective TherrrpuuIic Applicntrons. Kluwcr Acadcmic, Dordrecht. Stonc, T.W. (utl.) [ 199 13 Aspccls of Synapric TmnSI??iSSz#n. LTI’, Gokanin, I1piord.s. Aritonomic and 5-HT. T ~ y l o and r Francis, London, New Ynrk.
cntry 05
-
Feedback Error-correct ions, cnhr~rrccmentond ex!ensjons 05-57-01: I'lcnsc notity spcc~flccsrnrs, c i m ~ ~ s i o nupdatcs s, ant1 commcnts on thrs entry hy c o n t r ~ h u t ~to n ~~ t se-mail feedhack file (for dr~trrzls,sce RCFOIFTCL' I, SP(IT(-JII;1rj!(,r111 ~4) CSN I l c v ~ l ( y ~ n ~ r lFor l t ) .this entry' scnd cmail rnrssagcs TI):
[email protected], inrlicat~ngthc nppropriatc paragraph hy cntcring its six-figure index number (xx-yy-zz or other idcnt~flcr]intn thc Subject: flcld nf thc mcssagc ( c . ~ Suhjcct: . OK-SO-O?]. I'lcasc fcctlhack m only one specified paragraph or f i g ~ ~ rper c message, n o r ~ n ~ l hy l y s c n d i n ~a corrected-rtplwcrnent ~ c c o r r l ~ton ~zhu gui~Eclincsin Frpdl>ack c+) CCSN ~ c r c 5 r. E n l ~ ~ ~ n c c n ~ant! c n t oxtcnslcins s can also hc sumcstcrl hy thlr rovtc ( i h r d ) . Not1f1t.d changcs wlll hc. rntlcxcd via ' I ~ o t l ~ n k sfrom ' the CSN 'Homc' page (http://www.lc.ac.uk/~xnJ) frcm mid-1996.
Entry suppnrt
group^
nnd e-mrril newsluttcrs
05-57-02: Authors who have cxpcrtise in nnc or marc ficlds of t h ~ cntry s [and arc willing tn P ~ ~ F V Iuditonnl ~ C or othcr support for developing its cantcnts) can join its suppnrt group: In thiy cnsu, sand a rncssagc Tn: CSNOSkWe.ac.uk, ( c n t e r i n ~thc worrls "suppnrt grr~up"in thc Suhlcct: ficldl. Fn thc mcssagc, plcesc ~ndicntcprtnc~pal inturcsts (sut. ficldnr~rnecritrrm In the Intrl)dl1c'tlon for i.ov('r~;p,) t o ~ c t l l ~with r any rclcvnnt http://www site links (ustahlishcd or proposctl) ant! d c t a ~ l cs ~ any f othcr pclrsihlc cnntrthut~nns. In tluu coursc, support group n~crnhcrs w ~ l l (optionally] rccclvc e-mail new~lettrrs1ntundc.J to ca-ordinate and devclnp thc prcscnt (tcxt-hasurll cntry/f~cldnanic framuwork.; into :I 'Irhrsry' of intcrllnkctl rustiurccs covcrlng Ion clrsnnul s i K n n l l i n ~ Othcr (morc gcncral) ~nformation of intcrcqt t o cntry contr~hutrlrsmay alst~hc sent to thc ;ihovc ;~CIIJTCSS.for group distri2~utionant! fccrlh~ck.
Ilr / l)hnrn~ot.o/ ( 1957) T 2: 32.2-8. M:~r~cq, Sclr5nt*r, (199 1 ) 254: 4.12-7. " Hopc, Fur I I'l~rirn~r~cnl (19931 245: I X7-92 Ht~mphrtsy,Tr,,r~dcI'hnrnmnrol Sri [lYY.7\ 14: 23.1-6. ' IuIiub, Annt~Sicv NL'IITOCL-I II0F)IF 14: .135--00. "" I'croutka, Nt,rrror.hcn~(19BRl hR 408-1 h. R~chardson,Trr.nrl~ Nc~iro';cl(1WhJ9 : 424-6. 19crkacl1, Noflrri>1 1989) 339: ?Oh-'3 I)C~CTF, Trerdc I'hr~rr?rrrr.olS ~ ( II YXY) 10: 172-5 '" c ~ l t , I%vvcrol Lorlrl [ 19Xq 41 I : 2.57-69 *' NLamhcrt, Hr / I)h(lrnlncol {IYH9l 97: 2 7 4 0 . l2 YakcI, Ijroln H r j r (lY901 533 46-52, II Ysng, l'h~.c;olS.oncl'ot~1 l W 2 ] 448 237-56. " F i ~ r ~ t k a w[;Nr~,rophycrt>l ~, (19921 67: X I 2-19. S h ~ n , N ~ ~ , ~ o p h ~( I' Vc Y~I o) (15: l 630-X. ''l 7 T~cota,I'rr~t.Nrrtl Autrrl Sr, I I S A (19'9.3) 90 1430-4. Wcrncr, .lot. N i ~ ~ ~A!)< r i ( ~I V9ltagc-g,atucl calciuin channcls on thc c c l l wrfncc to thcir firing thrcsholdt . I’C12 ccll ATP-Ratcd influx sites arc v:iriahlc in ccll hociics hut niort. hrmmgcncous in Krowth cont‘s5’.
entry 06
Local depolarization affecring ATP ngonist release synaptic contact
~t
regions of
06-16-02: P2, purinoccptnrs are present at both nerve terminals and cell bodies of peripheral and central ncurones. Local K ' depolarrzation of thc cntls of coeliac ncuritcs of the guinea-pig evoke single-channcl currcnts charactcristic of PzxR in outsidc-out patchcs whcn patchcs arc positioncd near thc rcgion of apparcnt synaptic contact. This cffcct is not nhscrvcd when patches arc positioncd at remotc regions5".
Transcript size 06-17-01: See mRNA distribution. 06-13.
Table 3. PZxR1 (QRF 472 nu) mRNA distrfhntinn:sornrnary of data derived from Northern andyses" [From 06-23-02)
Prcpara t ion
-2 kb transcript
Adrenal
+ +
Brain Heart Intestine, large Intestine, small Kidney
Liver
{-I)'
+ +
If1 Efl
Lung
I4
Ovary
I*!
Ncuroncs, supcnor ccrvical ganglia
PC 12 cclls Pituitary Skclctal mwsclc Spinal cord Spl ccn Testis Urinary hladdcr Vas deferens
+ +" +++++ + t + + +"
I-?
++
(%I
4-
+
f+++
"The'relative abundance' of the various transcripts are shown on an arbitrary scalc I+to + + + + + and (&I or (-3 for low or zero detectable expression in singlc trials) as judged from the published data'"'. ?he absence of transcripts in these tissues {where oativc PIX rcccptars have bccn chnractcri~cd"-~~) plrohnhly indicates the 472 aa P2xRl isofnrm docs not underlie responses in these preparations. 'ATP-gated inn channels have heen characterized in sensorJ ganglia". "Although 'striing cxprcssion' of mRNA cncoding this isofmm has hccn demonstrated in both the intermediate and antcrior lobes of thc pituitary a [hnth ncumsecretory cclls and stellate support CCIIS, cited in physiological role fnr cxtracelrular ATP has not been described in thcsc ccll 'types to date.
Table 4.
Functional roles of PPxR-channeh in varinus prcpurations (From Oh-14-01)
Prcpara tion
Fcaturcs/functionaI roles of punnoccptor-c hanncls
Refs
Hcart, neurotransmission
Extracellular ATP-activated cation channels in smooth musclc gcncrally producc contractile [cxciltatciry]rcsponscs by dircct admission of Ca7,+.In cardiac neurnnes these channels may contrihute to nnn-adreneqic non-chnlinergic (NANC1 neurotran sm issi on and mediate, in part, the vagal innervation of the mammalian heart
."
Caz'-influx thmugh ATP-activated channels in ~ ~ cells 1 (hut 2 not voltage-gated Ca2* channels) cnntrrhute to ATP-evoked noradrenaline release and at the samc timc inactivatc thc Ca2'-selcctive channels in thew cclls. Thc PC12 lATPnr rcscmhlcs the PlxR expressed in scnsory ncurnnes. A number of ATP analogues arc effective in stimulating catechohminc rclcasc, and thc receptor antagnnists suramin and Reactive blue 2 inhihit the nuclcotidc-induceti naradrcnalinc release [see Rrceptor r z n t ~ ~ ~ o n r s06-5 r s , I , nnd Receptor ngnnistq,
"
Phacochrnmoc ytoma PC12 cells
26
''
06 - 50)
Coeliac ganglinn ncuroncs
ATP acts as a ncurntransmittcr at scvcral junctions between autonomic nerves and visceral musclc. Feature? of excitatrrry lunctiont currents in the coeliac ganglion, c.g. rcvcrsal potential+, time cnursc and I-V rclationt Ins dmm 3n Current-voltage relotion, 00-.75)can he mimicked by gpplicatinn nf cxo~cnousATP Synaptic currents measured i n s 3 t pr~sscss ~ similar current-voltagc rclatianships ta currcnts produccd by ATP, arc incrcased tn frequency hy K ' depnlarizatinn (ina '0"-dependent' manncr), ant! arc rcdWCd hy ATP antagonists (see Reccptor nntngoni7ts, Oh-s1 )
Hippocam pal nuiironcs
In cultiircd hippocnmpal ncuroncs, ATP directly activates small sustained currents, and indirectly induces the transient currents by cvnking glutamatc rclcasc
35
Table 4.
Continued
Preparation
Fcatures/~unctionalroles of purinoceptor-channels
Refs
Smooth musclc
ATP acts as a (co-)transmitterat several junctions hctwecn autonomic ncrvcs and vascular smooth muscle. The ATP-activatcd channcls provide a distinct mechanism for excitatory synaptic current and Caz+-entryin smooth muscle. P2x receptors mediate
32
sympathetic vasoconstriction in small arteiics and a r t c r i n h . ATP may also initiate smooth muscle relaxation (vasodilation]hy
5tn52
indirect agonism at endothclial cell ATP receptors coupled to second messenger systems (see Appendix A, entry 56) Smooth muscle, hladder, non-human
The bladder of most non-human species receives dual purinergic and cholinergic , excitatory inncrvat ion. Activation of P purinoccptors dcpolarizcs thc cclls, incrcascs thc spike frcqucncy and causcs contraction. Addition of agnnists rapidly activatcs nonsclcctivc catirm channcls, which underlie the excitatory lunction potentials seen on stimulation of the intrinsic ncrves
.39
53
’’
Skeletal muscle fihres, adult rat
Extracellular ATP ( 5 0 - 1 O o p M ) has heen shown to activate junctional and cxtralunctional currents similar to those of acetylcholine receptor-channels in isolated adult rat skeletal muscle fibres, hut exhihit a shorter open time
Liver, hepatoma cells
Calcium-permeable channels expressed in rat hepatoma cells are activated by extracellular nucleotides
Parotid acinar cells lPzzR channels1
In rat parotid acinar cells, cxtracellular ATP increases influx of Ca2’ across the plasma rncmhranc, in contrast to rcccptor-mcdiatcd rcsponscs to carbachol (which also clcvatcs ICa’+],-d-fi-fnld, hut primarily by release of Ca” from intraccllular storcs).Within 10 s, ATP [ 1 m M ) and carhachol ( 2 0 p ~reduce ) the cellular CI m n t c n t by 39-50% and cell volvme hy 1525%. Both stimuli significantly reducc (hy -57(15%) the cytosolic K ’ content of the parotid acinar cell through multiple types of K’pcrmcablc channols. ATP anti carbachol also stimulatc thc rapid entry of Na’ into the parotid cell, and elevate the intracellular Na’ content to -4.4 and 2.6 times the normal level, respectively - part of this flux is due to PZZR-channek (we alco Receptor anfngonists, 06-5 I )
entry 06
Encoding I'redrcred sizcs of pmlrfnx unundud hy P7xR genes 06-19-01: Thu prcdicturl prntcin encoded by thc P l x R cDNA svnthcs~zcdfrom vas dcfcrcns ~ R N A hns ' nn opcn r c a d ~ frarnct n~ of 399 a ~ n ~ acids n o (-45 kDa withnut ~lycosylationi1; thu opcn rcading framc of thc P7xR cDNA dcrivurl from mRNA fmm phac(~chrr~rnocytc~mn cclls2 prctlicts a protein of 472 arnlnr) acads (-52.5 kDa withnut glycnsylnticlnt\.
Gene organization Evidencc for K N A splicing within thc protein c ~ d i region n~ of PpxR gene? 06-20-01: P2,R [QRF 399 a,]: T h u cxistuncu of scvcral 'high molecular w u ~ g h t hantls' on ~ r ~ r ~ h c r n t l ~ v h r l d l z a t( ~~ocnrnllNd cs dlctril,trrlon, 00-1,7) and thc ohwrvation of ~rnproc~ssed Fnrrns nf thc PlxR gcnc ropruscntctl by isnlatc
RP-2" ncvuloprr~cntrrl ~ i ~ ~ ~ l lon, l r n !Oh-1 1 ) arc intlicativc of sn R N A splicing mechanism w ~ t h ~t nh t protein coding rcgion of thc sctlucncc ciicotI\ng thc ,399 an PJXRisoform. (z.cpr,
HomoIngnus isoforms
U
00-21-01: Scc thc scction ~ n a t c h ~ nn;ltlvc r: tissuu-spccific prnpcrtlcs of P?xR wit I1 prt ~pcrticsof 'clonctl' 1'1~11 untlur C c l l - ~ v l cxl>rcj.rcror~ ~r ~ncJr.u,O()-OX.
Protein molecular weight (purified) 06-22-01: Scc 'I'tr l ? l r h I ~lni!r,rC;i,llru ir~znrIy..Ill,-05.
Protein mo1ecular weight (calc.) Oh-23-01: Srr Trrhlc I urlrler I:i?nr Tcrmtly,Oh-0.5.
Sequence motifs ATI1-hindin'q site m o t i f r 06-24-01: A mot16 ~ i m r l r ~ tro thr Walker type A phosphate-hinding site c ; ( x ~ ~ G K ( x ~ ) ( Iis/ vfound ) " ~ hctwccn rcqiducs I,?] and 144 of thc I)2x pi~xinoccPtor-channrI1. Wnlkcr typc A motifs5' have also hccn indicatcd rln thc rcportctl pr1m:iry scqilcncc clf thu cI3NA cncoding thc P,xRI ~ ~ r r f o n n ~ s o l a t t dfrom rat PC12 cclls2 (setu I?rIc)w). Thc cxtmcclluEnr location.: c ~ f thcsc consensus clcmcnt.; a r t ~~rc.;urncrl to form p;lrt of the AT]'-hindinp, s1tr ( ~ ( /lvl'rA4/, ~ 0 Fi.y
f).
Typlcal form of "Cys-Cya bop" motif
Walker Type-A ATP-blndlng rnotrt (aa 131.144)
Consensus H-glycosyrrtlon sltes (actual podtlon8: aa 153, 194, 210, 284, 300))
(diagram rnatlc)
Proposmd P-Ilk.
'pon-formlngn
or HHS-likO domaln forming the Inner linlng (narrowest part) of the artracelrular hall of the h I c pow
Extracellular Not.: Tho multimerlc topography (I.& subunlt numbem per homo-multlmerle channel cornprmx and the exlatenee and/or stolchlornetry of hetero-rnultlmer8) 'have not yet been determined l o r thls class of channel.
- .
Monomeric domains _ _
Channel 8ymbOl
Na,K,Cs N H 2 (na 1)
(aa 599)
ATP
7 ~.
-I
V
Figure 1. Monomeric protein domain topography [PDTM] model for the rat extracellular ATP-gated receptor-channe] (Pp&) exemplified for the 399 amino acid isoform rsolated from vas deferens. Note: All relative positrons of m o t h , domain shapes and sizes are diagrommatic ond are subject to re-interpretorion.(From 06-2441)
entry 06
N-Glycosylation sites cind putative 'Cys-Cvs loop' motifs
06-24-02: PzxR .-- [ORF -399 __ aal': The N287 aa hydrophrlr (extracellular) region hctwcen the two hydrophobic (putativc memhranc-spnnning:)domains in this isnform shows five potential sitcs for N-linked glycosylationt (10 cysteine residucsl. PzxRl (ORF 472 sal': The -270 aa cxtraccllular region of this isoforrn dlsplays thrce potential N-linked glycosylation motifs and scvural rcgularly spaced cystcinc residues which resernhle 'Cys-Cys loop' motifst fnund in the nAChR and other members of the extraccllular ligand-gated channcl family. Note: Cys-Cys loop motifs are proposed to he important in stabilizing the stnrcture nf extracellular ligand-binding pockets (see o h [I'DTMj. F i g . I ond Protein phnsphorylfltron. 06-32), I
Apparent lnck of secretory leader (signal) peptrde Oh-24-03:PlxR (ORF,799 aa)': Thc prcsence of chargcd rcaduw in the first 28 ~
amino acids of thc open reading framef of thc prntntypc P2,R suggest the ahscncc of a sccrction leader. This supports a model In which both thu Na n d C-tcrmini arc in the cytnplasm of thc ccll (see [PDTM], F i g 1).
Amino acid composition I'rcdiction of L] novel strucriire for P2xR-chonnel subunits
06-2(3-01: R y hydrnpathicityt analysis, PlX receptors cxhihit only two
hydrophohict segments 'sufficiently long' to cxist as transmcmbrane domains (see /PDTA4/. F A R 1). Thusc hydrophobic scgmcnts are separated by n largc hydrtlphilicf Scgtncnt nf -270 aa [for thc 4J2 aa PlxRI') or -287 3.1 [for the .39scn~itzvilv, 06-42)
R ( I I PS O J PPxlidcsen F it tzn I ion crnd 're wns it IziI r ion' 06-37-02: In smooth inusclu cells {if rdt vas dcfcrcns, the ATP-induccd current rlisappcars within 2 min cvcn in thc continuous prcscncc of ATP7-'; ccllr of the same prcpnmtirm rcctiver frnm dcscnsitization in the nhscncc of A T P with a resensitization h n l ~ - t ~ mr icb 2 rnin.". ATP-activated currents In dcirsal root ganglion ncurrmcs from rat< and hullfrngs are similar, cxccpt that ciirrunts in rat ncuroncs dvscnsitizc ;It ;I faster wtc" In singIc ccIIs iwlatcd from guinea-pig iirinarv hladdcr, thc tiino course of rapid dcscnsitizntmn 1s a function of thc ATP conccntration and can hc frttcd hy t w o cxponcn t ials"".
M m o r effeciq nf P Z x mRnnism on relense of Cn2' from inttocelhlar st nrcs 06-37-03: In the rat nciirosccrttory phacochrnmocytoma (PC12)cell 1 ine, ATP evokes a risc in ICa"], which mpirlly inact~vates'~. The minority of thc total rcsponsc to ATP in thcsc cclls [
Internti1 Cn" ion hlock of
rcccptnrs
06-43-02: Thc amplitude of inward channel currcnts cihtaincd with 150 mM cxtcrnal Na' nsc rcducctl h y incrcasctl internal C d ' rn thc insidc-nut patchclamp configuratinn. This reduction is cihscrvctl at hwur concentrations than that hy cxtcrnal Ca". Internal h'' and Cdv+ ~nrliicc similar r d t t c t i m s tn ciirrcnt amplitiitk". A qimplc nnc-lmdin~-situmodcl with symmetric energy 'harriers I$ inwfficicnt to explain I~~drrcctic>nal Ca' ' hlock in 1'C 12 cL.lls22( w e r 1 l c o V O h I p ~cran.5it ivrtv, M-421.
Ionic hlockrrs rd A TI'-,qritud channels
06-43-03: An cffccttvc t h c k e r of thc ATP-stimrrlatcd Ca" cnnductance in rat hcpatoma cells is gadolinium inn2". Voltagc-gatcrl calcium channcl blockcrs such as nifcdipinc ;ind vcrapamil fail to inhibit "'Ca'' uptaku in thcsc cclls". High conccntrations of zinc ions rcducc and p i d o n g ATP-activated currcnts in rat syrnpathctic ncuronm (crmsistent with open-channel Mock)
whilc lnwrr (micrnmnlar] conccntratirms potcntiatc I,,, Chnnncl modiilntion. 06-44]
I,hr detoiE7,
we
P hn r m acnlocqicnlblockers
06-43-04: In thc prcscncc nf thc non-sclcctwc hlockcr (+-J-tuhocurarinc,
maximal rcsprinscs of rat phncochromocytarna (PCI1) cclls to ATP arc dccrcnscd, hut ATP cnnccntratirms pmducing half-maxima! rcsponses a f v unchsngcd25. Narc: Thc blocking action of (+j-tuhocurarine affccts influx through othcr c x t r a c c l l u h Iigmd-gntcd channcls and vdtagc-gated channcls powcssing dtstinct stmct~ircs.( F o r cxnrnple, wr' ELG CAT 5-HT,7, critry 0 5 . F I A ; CAT n A C h R . r n t r y 09, nnd V l X C(i, enfry 421.
Scpnrahle PPx purinergic and nicotinic receptor-channel responses
06-43-05: ATP-activated and nicotinc-activated influx currents in ncrvc gmwth factor (NGFJ-trcntcd rat phaeochromocytoma (PC12) cclls show many similar propcrtics, and tentativcly, the possihility that thc channels . inward underlying thc currents wcrc idenricnl was ~ f f e r e d ' ~Hnwcvcr, currcnts mediated through ATP-nctivatcd channcls in thcsc cclls can hc sc~cctivclyantaganizcd hy suramin7* (we Receptor nntrljymists, ~ t l - 5 1 ) . Furthcrmnre, ATP-ptcd currcnt IS not affected hy -100 I I M hirsutine (an alkaloid that praduccs a potcnt ganglian hlocking cffcct by potently Hocking nicotinic rcccptnr channels and partially inhihiting voltagc-Katd Ca" and K' ~ h a n n e l s ~ ~ ' .
Channel modulation Positive and negotive modulation of native PZxX channels h y Zn2' ions
06-44-01: Two distinct modulatory sites of action for Zn2+ions have bccn proposed for ATP-activated channels in rat sympathetic neurones4'. First, therc is cvidcncc tor a positively acting allosteric site that enhances current amplitude (-hc-fold with micromolar Zn''1". Ry modulation a t this site, Zn" ions can increase rncmbranc dcpnlarization and action potential firing elicited hy ATP a~onists"'. Thus low concentrations of extracellular Zn' rapidly and rcvcrsihly potcntiotc both I AI I,nr and the introccllular Ca'" rise. The potentiation hy 10 (IM Zn" is dependent on agonist concentration"'. Zn" ions increasc thc sensitivity nf activatmn without potentiatmg thc maximum rcsponsc (i.c. possihly by increasing thc affinity of PzxIi for a g m i ~ t " " ' ~ ' )Sccnndly, . there is evidence for a negatively acting modulatory site for Zn2' (pcissihly within the pow) that blocks cnntluctancc through the I'2xR-channc14".
Positive modulation
of cloned P,,R expressed in m c y t e s 06-44-02 PzxRl (ORF 472 aa): Addit'Lolon of lil pu Zn2+to thc bathing solution shifts the EC5{)fnr ATP from 60 p~to 15 p d .
Dopnminergic modulation
06-44-03: In pfraeochromocytnma [PC12]cells, ATP-activntcd channcl currcnts are enhanccd by dopaminergic mechanisms, although this motlulatinn has not hccn attributed tr) any single class of dopamine receptorsR'. in these cells, 10 JIM dopamine enhances a n inward currcnt nctivntcd by 100 pu ATP. Similar cnhancements are produced by 10 I ~ M apornorphine, a non-sclcctivc dnpaminc rcccptor agonist, 10 I ~ M(+J-SKF38393 4a sclective dopamine n, receptor agonist), and 10 JIM (-)guinpirnle (a sclectivc dopamine DI receptor agonist). Morcnvcr, 30 I ~ M/+)-SCH-23390 [a dopamine Dl reccptor antagonist, and 30 IIM (-)-sulpiride (a dopnminu LJ7. rcccptor antagonist] also cnhancc thc ATP-activatcd currcnt".
Mechanism of dopominergic modulatr'nn of P2xH-chmnels
06-44-04: In PC12 cclls, thc 'dopamine effect' (see rilrwel has hcen shown to shift the dupcnrlcncc of activation rate canstantst on thc conccntration of
ATP toward a Irwcr cnncentrattnn rangc hy approximately two-fddz'. Dopaminc also x c c l c r ~ t c s thc inactivation and thc deactivation [as dctermincd from thc currcnt dccay upon washout of ATP). T h u s dopaminc augments thc ATP-activatcd inward currvnt by facilitating association d ATP tn its binding site. This augmcntation may hc mcdiatcd thmugh some protein kinasc which is diffcrcnt from cyclic-nucleatidc-dependentprntein kinnsos o r protcin kinasc C2'.
Comparative note: ATP rrs
pmtcins
fl
multiple modulator of other celhlnr
06-44-05: In addition to dircct and indircct ( G protein-linked) gating of ion channels, ~nimcellolmrATP is n candiciatc for multiple modulation 4311 other ccll-signalling moTvculcs ( f mexornplc, see ILc; Ca Ca Ryix subtype receptorxhnnncl of rabbit car artery smooth muscle cells is unaffected hy SKWF 96365 ( a novel inhibitor of reccptnr-mediated calcium entry7uvR").Ry contrast, this compound reduces 6 protein-mediated ATPstimulated currents by ahout 80% in human neutroyhils"".
Receptor agonists ATP evokes inward currents through PPx receptors with short latency and fnst rise time 06-5041: ATP, as well as the rclatcd molecules s,fl-rnethylene-ATP, 2-
rnethyltbioATP and ADP evoke inward cuncnts with shnrt latency+ (typically < 2 rns minimum) and fast rise timc (twically < 10 ms for 10-90% rise) following application. Thc PIXrcccptors display variable desensitizationt kinetics with ATP and ATP-dcrivcd agonists [with some voltage-depcndencel, dependent on thc preparation and (prcsumahly) molecular suhtypc (see Inactivntion, 06-37, Voltage sensitjvrty, 06-42, and Table 7).
Apnisrn nt clnned Ppx receptors expressed in oocytes and HEK-293 ceIIs - comparisons with native P2,K 06-50-02 PzxR (ORF 399 aa): 10 pu ATP, x,/Lmethylene-ATP, 2-
methylthioATP and ADP evoke 'typical' inward currcnts with latency+ of c 2 rns and a rise timc of -7 ms'. For this isofnrm the order of agonist potency is 2-mcthylthioATP 2 ATF > x,P-methylene-ATP >> ADP. F2xltl (ORF 472 aa]: ATP, ATP-7-S and 2-rnethylthioATP arc equipotcnt as aganists, whcrcas z,/bmcthylcnc-ATP and P,;.-methylene-ATP are inactive as sgonists or antagonists. Note: Thc 472 aa isofclrm displays agonist sensitivity that resemhlcs nativc PIxR a n PC12 and ccrtain sensory and
entry Oh
P2 receptor aRonism by 2-methylthioATP on cardiac sympathetic neurnnal P,,R
ATP rcceptnr4mm-wls cfcscrihcd in rat cardiac 21r syrnpathctlc ncumncs show an cirdcr of agcmist potcncy of 2-incthylthinATP = ATP > ADP 7 AMP I, adcnminc = z,/f-rncrhylcnc-ATP > /i,;.-methylcnc-ATP (a sequcncc alsn consistent with the C, prritcin-linked Pzv rcccptnr suhtypc). ATP and AMP are a n ! n ~ o n i s ! snf thc PIT rcccptnr channel suhtypc expressed nn platclcts {.we Sriluype cl~icsrfmtions.Oh-QhI.Note. ATPvvokctlcurrtnts in thispreparatirin Arc nrrcnrrnreclr by r,/i-rncthylcnc-ATP lICGcF10 p ~see , hchwl snrl rcvcrsihly inhihitecl in a dosc-dcpcndcnt manner hy Rcactivc h l w 2 (KLI= 1 MI R9 x,/l-Mcthylcnc-ATP is a mcta‘holically stahie ATI’ ~na111gw that charsctcristicallv activates then dewnsitizcs PgXrccceptors. Arylazidnarninopropumyl ATP(ANAAPP.3)covalcntlyhinds tp Pzx rvcvptors fo!lowin~irrdiaticin, and ISa l w capahlc ot inrlucing rcccptw activation then hlockadc Fast-synaptic currt‘nts within much1 hahcnula 2R ccntrAl neuroncs (part of A wcll-charactcrizcd chcilincrgic pathway which arc hlockcd hy suramin descmitize fnllowing application of .x,/~-int.tt~ylenc-ATP. Miniaturc post-synaptic currents ohscrvcd following spontancnus rclcasc of trmsrnittcr Tn this preparation arc alsti rlcsunsitizccl hv this ngcinist Lhcnsitization hy r,/l-mcthylcnu-ATP also hlr1ck.i the rcspnnsc to ATP in single cells isolated from guinea-pig urinary hladdcr. rJ-Mcthylcnc-ATP i s -50-100 tirncs marc pnttnt than ATP at u l i c i t i n ~a ctintaactilc rcsponsc of strips nf dctrtisor smooth musclc. 1 Similar ricscnsittzatson hchaviour t(i ATP [ 2 1 I I M ) is chstrvuti in cells cxprcssing rccornhinant PlxR ( O R F ,199 aal PlxRl _ _(ORF _ 472 aal: Arnnng scvcral nucleotide and nuclcosicfc dcrrvativcs cxarnincd, only CTP ant1 dATP clicit small hut detectable current responses from this isofnrm In cxcitatnry synaptic trammissinn hetwecn ccwliac neurtincs of thc guincn-pig, ATP cvrkcs rnward currents with greatcr pntcncyt and clficacyt than acetvlcholinc IACh)
-
.x,/l-Methylene-ATP and ANAPP3
Induction of duscnsitizntian by r,/l-methylene-ATP on ncurnnal P l x R
Inductinn of dcscnsitization by r,/l-methylene- ATP o n smooth musclc b X R
CTP and dATP
ATP [cxamplc of tissue-dcpcndcnt, cndogcnous agon25t rcspnnses
1
autonomic neurnnc~"*~'.~"~' (this pattern differs from that ohsewed fnr P2xR on vascular smooth muscle, vas defercns and some CNS ncuroncs, where r,/f-methylcnc-ATP acts as a potent agonist"'"').
Multiplicity of modulntory and ngonist roIcs of ATP
06-50-03: ATP has multiple actions on nthcr proteins, including ( I ) indirect gating of ion channels through G protein-linked receptors (see Kesorirce A C; prcjrein-linked receptors. entry 5h) and ( i i ] as a modulatory factor on sssociatcd signalling mmponcnts, including ion channcls x t i v a t c d hy othcr ncumtransmittcrs or second messengers (see, f o r exnmple, I L G C n Ca KyRCaf. entry 17, nnd ILG Cn I n ~ l ' \entry ~ , 191. Partly hccause of this multiplicity of targets, highly sclcctivc agonists for PZx rcccptors arc cuncntly not
svailahlc. Tahlc 7 lists m m c applications of thosc presently in
USC.
Receptor antagonists 06-51-01: The [non-suhtype-selective)ATP receptor antagonists Reactive blue 2 (RB2) and suramin reversibly block hinding of ATP to P2 receptors Id.[+)tubocuradne, a potent antagonist of acetylcholine- and serotonin-gated channcls acts as a non-sclcctivc hlockcr of ion permeability through the ATP-activated channel - sec Rlockcrs, 06-43),RA2 and mramin can
distinguish PzxR responses from othcr extracellular lipnd-gated channels''. Comparative studies of clcctrophysiological effects of the ahovc compounds show all thrcc of these compounds inhthilt ATP-gated current in rat phactv chromocytoma (PC121 cells in a concentration-dcpcndcnt manncr [ordcr of potency RR2 > suramin > ~+]-tuhocurarinc]25. Unlikc for surainin or RR2, hlnckadc indiiccd hy (-t)-tuhocurarinc is not rcvcrscd after a 5-min washout Furthcr characteristics of thcsc antaRonists arc Iistcd in Tahlc 8. In gcncral, thcru is a nccd fnr morc sclcctivc antagonists actinK at PIXpurinoccptors.
Antagonism at cloned PPxR expressed in oocytes and HEK-293 ceJls
06-51-02: PTxR [ORF399 aa): Cutrcnts cvokcd by ATP, x,/bmcthylcnc-ATP, 2methylthinATP and ADP arc rcvcrsihly blocked by summin (3-100 J I M )and by
~ytidoxalphosphatc-T,-sxophcnyl-2',4'-di~ulpho~i~ acid (FPADS, 1oL30 puJ hut not hy arnilnride (100 p ~ ) T. h e w propertics scrved to idcntify thc cxpresscd rcccptors as of thc PIX purinoccptor subtype'*'. PzxRl (ORF 472 aa]: - Both suramin and Reactivc blue 2 reversihly antagnnize ATFcmkcd rcsponscs of this isoform by > 95%; [+j-tuhocvrarinc only partially blocks (-50%) ATP-evoked responses of this isoform'.
Relative potency for P2-rnedioted release of nnrndrendrne from IT12 cells
06-51-03: The relativc potcncy of ATP and a number of analogues for cliciting nnmrircnnlinc rclcasc from rat phavochnimocytnma [ PC 121 cells in thc presence d cxtraccllular Ca" has hcun shown to follow thc nrdcr adenosine 5"-0-(3-thint~iphnspRate)> ATP > ndenosinc S'-O-[1-thiotnphosphatcl = 2mcthylthioatfcnosi!ic S'-triphnsphatc [MUSATPI > 2'- nnd 3'-Q-(4-hcnzoylhcnzt1yl)ATP (RzATP) > hDP > ~-nd~nylylimidndiphosphatc2~.
Table 8. Chnmctmstics r d AT{’ nntngonists (From Oh-SI-OIJ Antagonist
Rcactivc hluc 2
Suram in
Application [cxamplcsl
Refs
Thc ATP-actwnrcd channel in rat hepatoma cells is inhihrtcd in thc prcscncc of Reactive Blue 2 (RR21, suggesting that channel activation is dcpcncicnt nn purincrgict receptor interaction, ~n coeliac neurnnes of the guinea-pig, the antagonists Reactkvc hlut 2 (and suramin, see helow! rcduce the effects of ATP [1Ci0- l - l O p ~ Fhut not acctylcholinc agcinists. RB2 is a slowly-acting antagnnist (if t h c ATP-Ratcd channel. In rat phaeochromocytoma (PC12) cells noradrenaline release is inhibited hv RR2 [ I C 5 0 - l - l Q ~ ~ ~ F
2o
5o
24
lri The ATP rcccptor antagonist surarnin lacks sclectivlty for 112 purinoreceptor suhtypes, hut can discriminatc hctwccn P*x responses and those of othcr fast ncurotransmitters. Suramin is a competitive hlrnckcr of hoth cndogcnous transmittcr and ATP-cvokcd cumcnts in coeliac ganglion preparations (see Cell-tvw exprmsmn 1 R d C X . Oh-OX). Fn rat phacochromocytoma (PCl21 cclls, noradrcnalinc release is also Inhibited hy suramin (IC5r! 30 prw)
’‘
-
Stilhcnc Scvcrnl stilbene isothiocyanate a n a l n p e s nf the calcium-activatcd chloride current inhihitor DIDS isothiocyanata analogues at Pzz [dihydm-DIDS, SITS hut not DNDS - we purinnccptors Appendix C. cntrv S8F can hlock hoth the binding (comprirrrt I ve m t c of I ”PI-ATP t o intact parotid cell? and thc only) , rurinaceytclr-channcl~~. activation nf thc P T h c pntency nf thc stilhene diwlphonates is rehtcd to the numher of i ~ o t h i o ~ v a n aErnups te on cach cnmpound ( c g . DIDS, Ic5(,35 p ~ S ,I T S ICGr, 125p ~ DNDS ; lacks isothincyanatc (SEN 1 groupS]. Eosin-5-isnthincyinate (ElTC) and fluoroscein-5-isothiocya~ate (FITC1, nanstilhcnc isothiocyanatc compounds wsth single SCN groups, also hlock thc response to ATP but arc less potcnt than DlDS. TrinitrophenylATP (TNP-ATPJ,an ATP clcrivativc that is not an cfftctivc agnnist of thc parotid acinar cell PIZR, hlocks thc covalcnt hinding of DIDS to the plasma mcrnhranu, suwestiny: t h a t ATP and UIDS hind to thc same sitc. Thc drstilhcnc DIDS and 2‘,?’-dinlduhyde-ATP irrever4ihlv inhibit the skclctal rnuwlc ATP-gatcd channcl. DJDS also irrevcrsihlv hlocks ATP-induccd Ca”-entry in parotid x i n a r cclls of rat
-
+
’’
’‘
’‘
entry 06
Table 8. continued An tagonis t
Application (examples)
Refs
Adenosine derivatives without aRonist activity
These types of compound usually act as weak competitive inhibitors of PzxR (c.g. adenosine 5'-1P,;.-dichloromethylene~~ziphosphanate, ICqo-21 pu at neuronal PlxR)
RR
rJ-Me thyleneATP
a,fl-Methylene-ATP possesses agonist activity in some preparations (see Receptor agonfsts, Qh-5QJ and may therefore act via a dcscnsitization mechanism. rx,/f-Methylcne-ATP inhibits agonism by ATP in parasympathetic neuronal and cardiac atrial preparations, hut not in vas deferens, skeletal muscle nr sensory ncumncs
26,
37B 77. Rh
Effect af ATP nnnlo,ques on P ~ X Rexpressed i n smooth muscle from rat VQS deferens
06-51-04: In smooth muscle cells isolated from the rat vas deferens, the analogues 9,P-mcthylene-ATP and AMP-FNP (lt,y-irnido ATPJeach produce a small, relatively sustained inward currcnt (not resembling the ATP current\. The analogue AMP-PCP (I{,;-mcthylcnc-ATP] has little or no effect in this preparation9'.
Agonism b y other adenosine derivatives and ATP-y-S 06-51-05: Ry definition, PI receptors arc sclcctivcly agonized by ATP over
adenosine. ADP is a weak agonist (seF above) and CTP may elicit some currcnt in ncumncs", hut GTP and UTP are nnt effectiveas agonists. ATP-7-5 (adenosine 5'-0-&thiotriphosphate, see above) i s apparently equipotent with ATP in PC 12 cells", cardiac muscle", skeletal muscleR', and neurones2'.
inactive agonists nt cloned PZx receptors
06-51-06: PzxR (ORE 399 aa): UTP ( ~ O O ~ U ]GTP , (100pu3,acetylcholine ( 100p ~ and ) 5-hydmxytryptarnine (SOJIM) are ineffective as agonists'. PIxRI (ORF 4J2 aa): ADP, AMP, 5'-adenylylamido-diphosphate,adenosine, GTP, UTP, CAMP, cGMP, acctylcholine, glutamate, glycine, 7-aminohutyric acid (GARA] and 5-hydroxytryptamine (serotonin] do not activate this isoform when expressed in oocytes'.
~
~
~
~
~
@
l
~
~
~
Database listings/primary sequence discussion
1
06-53-01: The relevtint dntnlinsc is tndrcnted hy the lower cast' prclix ( e . g gh:,, which .sliould not 1 7 ~typed (.wc Introduction el 1ctvotJt of entries. e n t r y 02). D a t a h e 10ctr.5 ri~fniustinil ~ ~ C C P S Sntrnlhfrs ~ O ~ immetiioteh follow the colon. Note t h t a comprehensive listing of iill nvnilnhlc accession numhcrs is superfluous {or location o{ re!eviint s e q u r n c r in
~
~
cntry 06
GenRnnk" rrsnurcrc, whrch ore now ov(rrlnble wit11 pnwerlul in-/milt neighhflaringi onnly.;is routinrs (Tor d~vcriptron,FCC the Dotnhnse l i s t r n ~ s {irlrl In rhr Introduction nnd l o ~ o u ! oT cntrles, entry 02). For exnmple. sequcnccs of C~OFF-SPCCIC.C Iwrrc:nf\ or IL'J(JIL'II gene (om11 rn~rnherccon he rcr~dilvrrcccr..;sed t>y one o r two rn1:nd.s oi nrighbnuring +ana[vsiq (wl~rch art. /~oscrJ on prc-comp!rted o b ~ n m r n t cprrfnrmrd using the R L A S T ~ o l ~ o n r h r n h y tlac NGNITj. Thrc ienltlrr! mnqt slwiul Cor retrrevol of sequrpncc cntrrev dt.povit~'~j rn dlltr~l>(t~i~c Imt~rthmn thnce livted Irelnw. Thus, rr>prcr;rntutrv{> mrmhrrs ot Irnown wquunce homology grnupin~srrrc Jrsr cd to pcrrnlt ~ n lrr11 t d ~ r r c t rrvrrievr~lq hy at-cr w n n number, nurhorl reletcnr*i3 or nomrnclulrrrr Fnllomng rirrcct ncccssion. J~owevcr. neighhour_lngf --unnlj~eiq i.s qtrmgly rrcornrnrndtd t o ~rientrty newly Efyc~rtir/nnrfrclrrtcrl scsltlpnocq. --- -
Y
Nomcnclaturc
Speclcs, DNA sourcc
Orig~nal isol:~tc
Accession
Scquuncul discussion
P2xR
Rar, vas
ORF 399 aa
gh- XH0477
Valcm, Nnrure
bxR1
Isolatc RP-2 (partla1 cUNA scqucncc)
dcfcrcn~
(1994)371:
cDNA library
516-19.
Rat, phacochrnmocytoma (I'C 1 21 cDNA l i h r ~ r y Dtr~vurPfrom a suhtmct~vcf hyhndlzatlonf library
ORF 472 aa
gh: U 14444
Rrakc, Nflrure ( 19'141 371:
5 19-23. Originally
dcscribcd as an 'apoptosis~nduccd protein' - see
gh: MR0602
Owcns, Mnl CPII Rrnl (1991111: 41 77-88,
Ilcvclo/?mental rcgr11ntlnn. O(I-I I
1
Related sources and reviews 06-56-01: Pharmacalogical and clcctrophysiological charactcristics of ATPactivated inn channelrs'; ATP-activatcd channcls in cxcitahlc cc11s7 and vascular smooth musclc ~ o l l s " ~ATP ~ ~ ;as a co-transmatter with noradrenaline In thc sympathetic nervous systcm's~"P;ATP rtccpzor classif~catirjnsnntl: n ~ r n e n c l a t u r e ~ 'Ecatures ~~? nf clnncd PzxR ~sofnrms'~'.
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1
2
3 4 5
d
7 R 9 10 11 12 1.7
14 15 16
17 IR IY
2n 21
22 27 24
25
26
Valcra, Nnturc (1994) 371: 5 16-1 9.
Brake, Nntrire ( 1 994)371: 519-21. Clwcns, Mnl CPII R i d (19911 11: 4177-88. Rn, J N z d Chem (1942) 2h7: 1 JSKI-7. himstock, Ann NY Acnd Sci (1990)h03: 1-17, Iran~l~ c n r q "(Fmm 07- 78-01 )
Glult-A gunc homclloguc C;luHI (- 97'Xbtduntlty with rat GIURI 1 G1ul.t-13 gunc h n m o l o g ~ ~ u GluR-C genc homologuc GluR-I3 gcnc homologuc GluKS gcnc h r ~ m o l o ~ u t . HRC;III '
H 11~;~ 2 " H Rc: ~ 2 '
Human chrr,mnsolnc 5 Human chn,mosomc fiq,Mh
Human chromosome 4q.32-.33' Huinan chromosome X Human chrr~mt~soinc 1 1'' Human chromnsornc 2 142 1.1-22.2" Human chromosome fiq,? 1.,3,2,1.,3 ( f r o n ~ (:cnHrrnkH entry) Human chrr,mosrlme 5~1.11..7-.1,7.3w Hum:~nchrrimoso~nc4q25-.34..7"
"Erir dctcrminatlon of chromosotnal 1oc;uions of AMI'A siihunit gcncs, suc rcf '". "AS dctcrmtncd by fluorcsccncc In s f l l r hybridization [FISH\. ' T h ~ location s cxcli~rlt..;Cli~li-!< as a wnditlatu gcnu involvcd in Huntingtcln's rliscasu. ,I T h e rt'xlon containing thc G l u R - n gcnc has hccn assnciatcd with linkage tn schiznphrcn~aand rnnior dcprcssinn. ' T h c CluRS Rcnc is In thc vlcinitv of thc Rvno linked t c ~familial amyotrophic latcral scl~ro.;i.;""~~. 'Isoletc nainc, . ; r ~Drrt(rhrr+c I~sr~n$.c, 177-53
Encoding 07-19-01: For prcdictcd clpcn reading framct lcngths dctcrrnincd from =DNA scrlucnccs, scc. the rcspcctive suhunit gcnc namc in Td-rlcs 1 , 3 rlnder Ckne {(i~njly,[17-05
Gene organization GluR-channel diversity vja the alternative splice variants ‘flip’ ond ‘flop
07-20-01: AMPA: In the GluR-A t~ GluR-D family, a 115 hp segment preceding thc gcne region encoding the predicted M4 transmembrane domain (see IPDTM], Fig. 3) has been shown t~ exist in two versions with
different amino acid scqucnces. These 38 amino acid modules, designated ’flip’ and ’flop’, arc encoded by adiaccnt cxonst of thc rcccptor gems and impart ditltcrcnt pharmacological and kinetic propcrtics on currents evoked hy t-glutamate or AMPA, hut not thosc cvokcd by kainatc (for dctnrk see Receptor ngonfsfs,07-50), ‘Flip’ and ‘flop’ ditkr in only a fcw (9-11) of the 38 amino acids. A pentapeptide occurring at a comparable position in each suhtypc is consistcntly diffcrent in the ’flip’ and ’flop‘ versions (see Fig. I, and /I’DTM], Frg. 3).
Comparison of cDNA and genomic sequences encoding AMPA receptors 07-20-02: The switched versions of ’flip’ and ’flop’ are generated hy altemativc
aplicinft ot the two regions which are on adjacent e x c ~ n s ~separated ~‘ by an intron of -900 hp (see Fig 1). For each receptor, the alternatively spliced rnRNAst show distinct expression patterns in rat hrain, particularly En thc C A I and CA3 ficlds of thc hippocampus (see m R N A dnrrihution, 07-13). Thus, thc flipJflap modules cnahle functional properties of glutamatcactivated currents to hc controlled hy alternative splicingt events. (For the phcnotypic conscquenccs of flip and flop modules, see Inactivatron, 07-37.)
A note on splice variont nomenclature 07-20-03:The names ‘flip‘ and ’flop’ may imply that thc alternative cassettes arc in ’reverse orientation’ to each other, hut this is not thc case (see FiR. I for clnrjficntionl.
Existence of further nlternative splice vnrinnts
07-20-04: A third typc of transcript (GluRQcflop) derived from the GluR4 gene hy differential RNA pmcessingt has been isolated fnllowing screening a rat ccrchullar cDNA !ibray‘2. GluR4c transcript encodes a protein with a
‘ f f ~ p ’module hctwccn transmcmhranc regions 3 and 4, but with a C-
tcrminal segment of 36 amino acids different from the previously descrihed GluR4 tlip/floy, cDNAs. Transcripts synthesized in vrtro from GluR4c flop” form kainate/AMPA-activated channels showing strong inward rcctiticationt whcn expressed in Xenopus oocytes.
Alternntive splicing in the GluRS gene
07-20-05:Alternative splicct variants of thc high-affinity kainatc receptor subunit G h R 5 h a w a l w hccn dcscrihd‘. Thc longtr GluRS-I variant has an open reading frarnct (ORE] of 920 38 and dcrivcs Imrn inscrtion of a 45 nucluotidc sequence within thc first third of the N-terminal {cxtracellwlar) glutamatc rcccptor domain. A full open reading frame1 tor the shorter splice variant (GluR5-2, lacking thc 45 base inscrt for a prcdictcd 905 aa ORF] was not d a t e d in the original study4.
-Region
b
'Flop'
exon
Linear n?pmw?tat/onof part or the gene encoding the GIuR subtypes A-D
-38- aa
1
i
! \,
encoding putatrve transmembrane d m i n s I to II'
in trnn (*YO0 h p ) I
-
-
'Flip' exon ,38 . - .aa t
---. .
,
1
\
Figure 1. 'Flip' and 'flop' alternatjve splice voriantr in AMP.4 receptor genes. (a) Sequencing o f cDNAs for the four subtypes of she AMPA receptor revealed o 1 1 5 hose sequence encoding n 38 oa segment which existed in two sequence versionF designated 'flip' and 'flop' Ib) Sequencing I R the reginn of genomic DNA precedinx that cncodmg domain IV revealed the 'fhp'and 'flop' segments to be encoded hv two separate exom. flanking OR mtron, as shown. Alternative use of 'flip' and 'flop' exon sequences in AMPA receptor genes confers different kinetic properires of currents evoked b y glutemote or A M P A on G h R - A to GluR-D subunits. For details, we Gene organization, 07-20. and Receptor ogonists, 07-50,Within the alternative 38 aa flip/flop module (occupying equivalent residue positrons En the cDNAs), flop versions of the Tubunits contoin a conserved SGGGD motif, while flip versions are variabte ot single residue (nrrowed) in the subunits. (From 07-20-02)
GluR subunit sfmcturnl changes via I2 NA editing mechanisms
07-20-0(,: A further possible mechanism for generating hctcrngcncity in thc channel coding regions nf G h R transcriptst is RNA editingt. In thc case of
thu GluR-R gcnc, i t was found that there had been a nuclcntide change [an adcnosinc-to-guanosinc transitiont 1 ohscrvcd hctwcen the scqucnccs ohtaincd from gcnornict DNA and O N A t sources" (note. normally thcrc is perfcct agrccmcnt of scqucncc). It has bccn propnscd that ‘editing’ of An adenosine to inosine givcs risc to an Arg [R in singlc-lcttcr mdu) mstcad of a Gln (9)rcsirluc a t the critical QIR site (for the consequences of rhis chnnge, see Domain functions, 07‘-29).
Subunit R N A transcript selectivity for editing ~ T O G C S S E S
07-20-07: A ~ t h o u hthe GPuR-R suhunit transcript appears to undergo editing In > 99% of isolates found, the GluR-A, -C and -D subunit transcripts [sharing identical domain M2 scqucnccs) dn not appear to be edited. In transcripts for GluR5 and GluR6, cdited and nnn-cditcd transcripts are found at different The principle nf RNA cditingt is illlistrated in Fig. 2. Note: R N A editing has been shown not to he a RenerclI cellular mechanism for GluR diversity (see mRNA distribution under ELG CAT GLU NMDA. 08-13].
Rnxe-poired intron-exon sequences are required for GlnR-R RNA editinx
07-20-08: Low RNA editing efficiency is observed in GluR-R gcnc constnicts rnodificd in sequences at the proximal part of the intmnt downstream of the
.-
-
dr RNA-spsEifre adcnoslne dsamlness (see Gens o r g e n h f l o n )
(3) In edited RNA,
adenosine ( A ) deeminntes to an
inosine (1) mwmmmmal CfG cA[Is m m w m m m i
(4) In unedifed RNA, adenosine (A) is unchanged
mmmmmmi (5) Resultant
R
Q
in pore-llning domain M2 {see the PDTMJ (R = Arg; 0
CM -3
Q
.nmml
Q
= Gh)
Figure 2. R N A e d i t i q RS n mechnnism for Renerating heseropcriirs of KA- I IKA-2 suhuniis wilhin heturornuric ctrmplcxes 07-27-03:K A I N : Atthnugh thc Kh-I or KA-2 subunits f c ~ i lto f o r r functlclnal chnnncls w % cxprcssutl a< h r t m t ~ m u l t i r n c r s ' ~ ~thcy ' ~ , do cxhihit a highabfinitv for kalnatc. ~ u t u r o r n c r l c i cxprussmn c ~ fKA-2 with thc distantly rclatcrl G l ~ ~ l ior f r G l i ~ l i hs u l ~ ~ ~ nIcatEs t t s t c ~forlnatlc~nof functional channcls with novel p r o p ~ r t i ~-sc . ~AMPA . actlvatcs C;Iulll,/KA-2 channclq hut docs no! activ;~tchoint~nlcricCiluRC, rcccptorsr4.
Domain conservation Conservnrion of xicynml sequences nncJ s r ~ k r l n ~armn s t i t l ~ t i 111 r l nscvcraf s rcclons (.wr II'DTMI, FIX.,3 rind Grnr clrgrrnlz(lrlon. 07-21)). T h c 'flip' and 'ilopr forms (if the A M P h rcccptnr s ~ ~ h u ~ iarc i t s gcilurattd hv alternative sFlicingi hctwccn rcgrons cncoding trnn~i~lclnhranc t l r t r n a ~ nM.3 ~ anrl M4. Variant s u h u n ~ ttcirmr ;llri) d c r ~ v c ~ l frt~nialtcrnat~vusplicrng1 u ~ i In ~ tthc N-tcrrninal dornaln (c.g. the G I U R S ~ and thc NR- I suhun~t.;l.(For rhc phcnotypiu ronrrqrirnccs of C I I ~rin~lF IJop nrodlrlr~r.scc 11111~tivot1on. 117-371. Seqrience hornolqyy het ween !he iGluR nnd ~ l u t n m i n e - h ~ n d t n , ~ prof eins 07-28-04: Thc isolates C h R - K 2 ant1 G h R - K . l (ccirrcspclncEing tn the 'flip' vcrsion of GluR-H ancl GluR-C) havc hccn rcpc.~rtcdov as showing sign~ficant sctlilcncu con.;crvation with the ~lc~tnlninc-hintl~ng component nf thc glutamine petmcasc of F i.oli
Kcrlncitu-A M P A suhunir srrnilr~rirics 03-28-85: KAIN: RA- I , thc first high-affinrty kalnatc suhunit to hc cSoncdn5 I I ; I ~ ;I ;30'X, scclucncc sirn~laritywith thc AMPA ruccptor s u h u n ~ t sGluR-A to -1). Not(' Thc frca ;~ntl c h ~ c k brain k a ~ n a t c - h ~ n d i nprtitclns g [scr
P u M m glutannH-hmdlnp donulnm brrd an p.riW to pmkmyolk prrrramlnr mnrpurrers
Fllp / Flop module Cm'
. nomalogy
, '." 3 U t
QrpaOiut'onl
w4,ri
-
Lw?;
--. r-z'l-
y3u4
y3 H i Mb
Extracellular
Psntamarlc arrangemsnt (pu ta tl va)
(a) Monomsrfc dornalns FntraceEIuler
-
o l l g o ~ r l ca t w m
(prramr-seemeiwds
-8?enpememd
,,
-coon \
deiemlnmtc
0-R .It.
( m n G m orp8nhilm and Dmnmlrr htncrlunr # M s ) -a . P uiun-5 Q or a
-u
'
-c
'
Q
'
-D
'
Q
'
R
' 9 '
.
n /OlU.t.rlng
mm
.
QluR-b a.11111-1
bl hi
ol *-'
CQI..nwU.
+
.
Q or R
.
0 - 9 ' ' 9 '
. *
-
Addmmut -them#} mnwn h r r o d u c d In moms to pol^ mod.lm lu n l a h cenmlnnncy wlth Itrueturs/lwncllon data ln Suburg (lee31 Tfmnds Pk.mr8cd Scl 14: 297.303. Comp8n W t h a)l.m.tlva model. wflh larps ( U 3 - M ) Imiwesllulsr h o p and rmdcwIIuNr CODH
-
I8//
ELO CAT
s-H~)
Chmnol aymmbol Wa.K, ICaF
RM
nhlnLvAdtm Pronln p l m @ ~ l I n n )
m
In A Y P W l n l m IWuR r t h c h d b* dlnruthn Iklng wnntm (mOm. arpmnlzaHonJ
NOTE:
All d # v W pOSlt/On6
Of
mOflh,
doomeln Bh8peS #kM ere dfagrarnmatlc and am sub/ecr to re-lntarpretetton
-
'" \
I
I
ELO
__
v
Figure 3. Monomeric protein domain topography model (PDTM] for AMP~lkainate-selectiveronotroprc glutamate receptors (iGluR). In press updates [see criteria under Introduction d layout of entries, entry QZ): A modified 3-irmsmembrane domain model based on N-glycosyletion site togging data has appeared in press. For brief details, see the rnsert before Amino Q C I composition (07-26). For further details, refer to the entry update pages via the CSM. (From 07-28-02)
~
Table 7. Extent of amino acid sequence identity (%) amongst members of the cloned ionotropic GluR subunit family and kainate-binding proteins (From 07-28-01) GluR-A GluR-B GluR-C GluR-D GluR5 GluR6 KBP-f KBP-c KA-1 KA-2 -:
GluR-A
GluR-B
GluR-C
GluR-D
GluR5
GluR6
100
70 100
69 73 100
68 72 73 100
40 40 41 41 100
41 41 42 40 81 100
information not found.
KBPf 38
-
-
42 43 100
KBP-c 37
KA-1 35
-
-
38 40 56 100
42 44 35 34 100
-
-
KA-2
07-53)appear to lack thc first -350 aa nf the GluR-A protein ant! possess only -25% amino acid identity when compared to thc N-turminal half of GluR-A. Dn!nhnsc Irstings.
Domain functions (predicted) The QIR site in the M2 domain is crittcnl for regulntion of ion
permeahiIisy and I-V re?oiionships
,
I
07-29-01: Thc M2 domain scqucncc shows variability at n position known as thc glutarnlnc/argininc sitc or QIR site (see [fie sequence alrprncnt helow and lPnTM/ F F ~ 11. m.wt). GliiR-R possesses an aegininc (R) a t this sitc comparcd tn a glutamine (Q) hcing prcscnt in thc liomologous pcisitmn in GluR-A, GluR-C and GluR-D. Significantly, thc argininc cndcin is not found in thc GluR-R gcne, hut is introduced hy an RNA editing process (see Gene orgnnizoticm, 07-20), The Q / R site also detcrrnincs thc Ca2'-permeabilityt of thc channel (for detarls, sec ScI~ctivity. 07-40'". I"'). Site-dirccted rnutagenesist of the QJR sitc has shown that thc single amino acid difkrunce in the GluR-R subunit also dctermincs thc I-V rclationshipt of hctcromcrict channclsFo2(fordetails, see Fig. 4).GluRh also occurs in two forms with rcspcct tn thc amino acid residue occupying thc Q/R For comparison, the scqucncc alignment helnw also lists thc cquivaknt (aligncd) aminn rcsiducs of thc NMDA rvccptor subunits NRI and NR2A (see ECG CAT GL1J NMDA, entry O X ) .
Scqzicncc ali,ynment-i in rG1riR suhunits surmundrnR the Q I R site in the MZ domI1in
GluR-AFGI
F N S L W F S L G h F M B Q G C 1 I I
S P
GlitR-B F C I
F N S L W F S L G A F M m Q G C D 1
S I'
T L L N S F WI: C Y G A L M
0 C . S E L M P
GluRh
F
KA-2
Y T L C N
11
A T L H S A I
NRI
L T L S S A M W F S W G V L C
N S G I
NRZA
F T I G K A I W L L W G L V F
m N S V P V Q N
S
L W F 1' V C. C. F M
Q G S E I
MI'
W I V Y G A F V ~ Q G G E S S V C E G A
1
Thc Q/R
Fite
Control of Co''-pcrrneohi/jty of koinnte receptors determined b y R N A editing
03-29-02: KAIN: __ Ca2'-pcrmcahility of kainatc rcccptcir-channcls can vary dcpcntling nn 'editing' cif RNks cncoding both M 1 and M2 transmcinbmnc domain sequences".'. In addition tci cditbng a t thc critical QIR sitc in M2 (src rrliove mnd ref."*), scqucnccs forming the GItiR6 putativc transmcmhranc dnmnin MI, arc also divcrsificd by RNA editing (sec /IJ13TME.FIR. 3 rind thr,
wvrn J ~ ~ I rdj!(,(I ~ . ~ ~ ~o r ~~ ~~ ! ~ ~ j nP t ~~111der r j o n vorpin:z~z!~on, U7-20). T h ~ s process can gcncratc c ~ t h c irs o l c ~ i c ~ noru v a l ~ n cin nnc and tymsinu o r cysrcinc in thc other MI ~ l o m ~ iposition. n In GluRh channels thu pruscncc 4)f Q (glutarninc) at thc domain M2 Q / R site forms channc!.: with low ~ a " permcahility [in contmst with AMPA ruccptor-channclsl. An srginino s t this pnqttlon determines a higher ~ a " - p c r ~ n c a h ~of l i tGluRh ~ channuls ~f domain MI IS 'fully-edited'. In thc 'uncditctl' form of C.l~tR6donlam M I , Ca"pcrmcal~illtyis lcss dcpcnrlent cln thc prcscncc ot cithcr ~ I u t a m i n cclr ar inmu In domain MZ'". Thcsc rcs~tltsraise thu possih~litythat RNA utEiting m.ly morlulatu ~Iutnrnatc-activatcti~ a ? ' - i n f l l t~hxr o u ~ hC;luRh rn vlrJo.
P ,
A$oni.~tt-hindinGysire 07-29-03; Thc region prrt.r~din,y pu tat ivc rransmcmhranc scgtncn t M 1 clf GluRs I S wcll-conscrvcd arnnng sithunits and ha4 hccn propc.,scd tn cunstitutc a part c ~ fthc aganisz-hinding.site (see r~lsoothrr FCC: en!riesl. Fnzroihrction nf pmnt mutations Into chargcrl rcs~tluusof thc rnousc AMPAsclcctivc T I suhunit (Glu.7YX Lys,TYX; Lys445 Glu445) arc sssociatud w ~ t hc h a n ~ c sIn thc KG,, valrlcst with dihfcrcnt agclnists, indicating t h c ~ r involvcmcnt for a~r~nist-sulcctivc interactions of the C;EuR ~ h a n n c l ' " ~ .
-
-
Supplerncrr t nry not c for c~hovc t -Glutal~~;ltc, kalnatc a n d AMPA I~lnd tc~ rl~//cri>.rcnr receptor substructures on rccomhinant AMPA rcccptorsfNY' ( ~ 1 . 0 Ecluilihr~um rl~ssoarir!lr~~? c r ) n s r ( i l l f .07-45, r~ndI,EprrrrJs, 07-47)
07-29-04:
Predicted protein topography Common r~sstlmptionsof hydrophobic seqrlencex as slrr~ctz~rnl domains 07-30-01: L ~ k cothcr incinl>ursof tliu cxtr~cullularlignnrl-gntcd [ELG)family, ;111 ~lutalrn~arc rcccptor-channcls display frmr prrrl~c.rrcl rncmhranc-spann~ng hydrt~phohrcrcglolls ( M1-M4l, ; l l t h o u ~ hsomu modcls Ie s.rr~l."l pnjposc an ;iildlti(~n;il'conjcct~iaal'translncrnhrane domain between M,? and M 4 ( w e I I T I T M j , F1.y. 3 ) . Howcvrr, the l;lck clf direct structural data docs nnt vct ;illow ;Inv firm ctmcl~tsrt~n.;to hc inarlc regarding 'trctc' transmcmhranc ~vntuin.tt~pogmphy(.rcr FLC: Kr.v frrr.tr, rntrv 04) Irr-l~russrrprjrr!c~.Sec ~ l o r cr ~ l ~ o v r~c.l(i r 07-2h
Protein interactions Common srrhlrnlt n.sancinrions with GluR-R n7-31-01: AMPA: GhR-R subunits dominatc prtlpcrtics of
I U ~ I C flow In hctcromcrici k l u ~c.c,mPlcxcs5'~'". For cxarnplc, co-cxprcssinn of thc GlnR-R flil>~tnit wtth c k t h ~ rCIuIt-A, GluR-C cir CrlttR-D forms rccomhinant chnnncls which closely match charactcsistics of natwct rcccptnryY, 1lnpPy1111: thnt inclst nntivct xcceptors arc hctcromuttlmcrici and lncovoratu thc C;luK-I< suhttnit. GluK-I< anti GluR-C ~rnrnunclruact~vity has I~ccnd ~ o w ntcl co-localize with thc mctahrttrt~picirnGlulr rccvptor at thc clirnhlng fihrc synnpqc in ccrchel!umHn.
cntry 07
Evidence against crass-nssernhly of 'AM P A -pteferting' nnd 'kainatepreferrinx' suhunits
07-31-02 The 'absolute selectivity' of cyclothiazide and concanavalin A for respective block of fast desensitization1 of AMPA-preferring and kainate-
preferring receptors (see lnflctivation, OJ-37) has hccn used to monitor subunit assembly patterns in functional recombinant iGluRs" . In all CBSCS, asscmhlv of to-cxprcsscd suhunits from the two difkrcnt families suwcst independent assembly of functional AMPA and kainate receptors without any cvizlcnce for cross-family assembly of subunits.
Common functional interfictions at synopses 07-31-03: Diverse classes of extracellular 1igand-gated channcls commonly interact to shape dcpolatizing pnst-synaptic potentials (DPSPsl 1n the CNS. Fnr example, DPSPs of granulc cclls in lthc dcntatc gyms is part-mcdiatcd by AMPA, GARAA and NMDk receptor proteins (see Protein intercrctions under ELG CI GAR&, 10-31, and Fig. 4 under ELG CAT GCW N M D A ) . Postulation of inhihitory proteins associated with native AMPA recepi ors 07-31-04: The existence of an inhibitory protein (with a pnssihlc 'negative rcgulatory' function] has hccn invokcd to explain the observation that AMPA K,I valucs hccomc much lowcr (i.c. AMPA affinity Increases) upon various mcrnhrane trcatmcnts and upon purification".
Protein phosphorylation
.r
jG1uR.Y display multiple putative phos horegdatory motifs
07-32-01: A nurnher of potential consensus phosphorylation sites for protein kinases [e.g. protein kinase A, protein kinasc C, tyrosinc kinase and casein kinase Ill have heen found in AMPA- and kainatc-sclcctivc GluR suhunit cDNA sequcnccs. Only a minnrity of these have hcen shown to hnvc {unctionnl roles to date. Note: Locatinns of cach putative regulatory sitc can he traced by refcrencc tn citations under D n ! a h o ~ eEr'qtinRq, 07-5,7. PO t en t in t inn of Co2+-fluxes 07-32-02 Ca'"-fluxes can pass through non-NMDA glutamate receptorin thc abscncc of channels cornposcd nt the subunits GluR-A and GluR-C [it. GluR-B] (see Selectivity, 07-40), Calcium flux through open KA/AMPA reccptor-channels can he potentiatedt by phosphorylation mediated through protcin kinasc A as part of a CAMP-dcpcndcnt sccond rnessengcr system"'.
Enhancement of native E'GluR rcsponscs thrmqh ptotcin kinuse A phosphoryln tion
07-32-02 Non-NMDA channels cxprcsscd in culturcd hippocampal pyramidal ncumncs arc suhicct to neurnmodulatory rcRulation through thc adenylate cyclase cascadc. Thc wholc-ccll current rcsponsc to glutamate and kainatc 1s cnhanccd hy fnrsknlin [an activator of adcnylatc cyclascl. Singlc-chmncl analysis has shown that rotein kinasc A incrcascs thc opcning frcqucncyt and the mean open time of non-NMDA-typc glutamate rescptor-channcls.
f
Forskolin, actmg through PKA, incrcascs thu amplitiide and dccay time of spcintanacius cxcitatciry pnqt-synaptic c ~ r r c n t ~ ' ' ~ .
htentifltinn of recornhfnrrnt iGhiR r r s p m s c s h y prntejn kinase A
07-32-04: KAIN: Channels cxprcsscd from GluR6 suhunits (when trans~cntly cxpresscd In mammalian cclts) hevc h e m shown to hc dircctly phrw phorylated hy PKA. Appjrcation of intaacellular PKA incrcasc.: thc amplbtudc (if thc glutamatc rcsponsc25. Site-directed rnutagcncsist nf thu scrinc rcsiduc (Scr6H4J rcprcscnting a PKA conscnsuq site complutcly eliminate4 PKA-mcdiatcd phosphnrylation nf this s i t ~a4 wcll as lthc potcniimont of thc glutamatc rcspnnsc25 (FPP [PDTMJ, Frg 3). ~
PnstuJoted frincfionnl roles of rGhR phosphorylntim
07-32-05: Pmtcin phasphorylatinn of glutamatc reccptorq by protein kinase C and CAMP-dcpendcnt protein kinasc has hcen suggested to regulate their functinn in synaptic transmission (see possibly playing a prominent and lonpterrn depressiont "". Far role in long-term potentiationt additional note4 on thc hroad roles of protcin phosphorylation in thc ELC channcl family, sec ELC: Key /ours, critry 04.
'"."'
Activation AMPA-srlectivrr iC3uKs mediate 'fost' excitcItory sign:nolliqq in the CNS
07-33-01: AMPA rucuptors mcdiatc thc most rapid synaptic excitatory neumtransmissinn and conduct mainly Na' currentq. For example, hrief (-1 ms) appIicntions of glutamatc on mcmhranc patches exciscd from ncurrmcs in thc rat visual cnrtcx producc a rapid rcsponsc that mimicks thc time coursc of mrniaturc cxcitatory pnst-synaptic cuncntst (c.g -2.4 ms for AMPA-cvnkcd EPSPS"~). Thc rate nf cmsct of dtscnsitimlti~m1 is much slowcr than thc dccay rate of thc rcsponw (we Inrrctivntinn. 07-37], implying that thc dccay of miniature EPSCst reflects channel closurc Into a statu road1 Iy avaiIahIc for rc-activation"'.
Rrsc times for iGIuR current nctivotron
07-33-02 Rricf pulscs (< 1 ms) of glutamate ( I mM) on AMPAlkainate rcccptors in granulc cclls of dentatc myms and pyramidal cells of CA.7 and CA 1 hippocam pal reEions activatc patch currents which rise and dccay rapidIy"'. Thu 2040% rise timuf of thcsc GluR-mediated currents is typically -0.2-0.6 ms. At -50 mY, peak currents vary from 10 to 500 pA in diffcrcnt pstchcs.
Ka in R t e R Is r) act ivn 1 es 'AMPA - prcferrinK ' recept o r-c hn nn eIs 07-33-03:Kainatc also activates nnn-desensitizing 1 currents q i r n i h r to those clicitcd in CNS ncuroncs through 'AMPA rcccptor-chnnnclt frirrnutl from suhunitq GIuR-A tn -no. lo.
cntry 07
Current-voltage relation Arnctjonnl evidence for heterornultimers based on shapes of F-V re lo t ion s
07-35-01:AMPA: Thc maiority of nativct ncuroncs cxhihit AMPA rcccptormcdiatcrl inward currents with Fincar or riutwarrlly rcctifyytnKt currentvoltage rclatronshipsjY. When Xenripu.5 nocytcs arc inlcctcrl with RNA encoding GluR-A alonc, AMPA agonists cvnkc ;1 smonth inwardly rectifying+ current unhke thc lincar I-V rclatinnship sccn rn V I V D . Howcvcr, whcn thc cnmhinations GluR-AIR or CEuR-RJCarc co-cxprcssurlyy,a nonrectifying inward current ( I c. lincar and ohrnict) is uvcikutl hy kninatc o r AMPA (closcly rcscmhlinR currcnt typcs sccn in nativc ncumnal cclls). In cnrnmtln with similar studics on othcr ELK-typc rcccptcirs re R. see ELC: C? GARAA, cntry 10, and ELG C A T nAChR, cntry 09) thcsc rcsults indicatc the native receptor tn he a hctcrnrnuftirnert of a t kcast twa diffcrcnt suhunits.
Dominant characteristics of C:luR-R subunits
07-35-02:KAIN: Bergmann glial cells display a kainatc-type glutamatc s sigrntiid (doubty-rectifyingt1 current-voltagc r ~ l a t i o n ' ~ and receptor w arc pcrrneahlc to thc dwalont cations Mg' and Ca"' . Note: Rcrgmann Ella1 cells arc unusual in that they do not cxprcss GluR-B subunits"' (see Cell-type expre~sinn Index, 07-08) Hornomeric GluR-R clranncls (or hctcromcric channcls containing CluR-R as dcscsihcd in the prcviaus paraRraph) cxhihit near lincar 1-V relations and havc low clivalcnt catinn pcrmcahili tics.
''-'"'
Lncolizntion of k e y amino ncids determining I-V re?fitionshjp%
07-35-03A single amino acid difference in lthc GluR-R suhunit dctcrmincs thc I-V rclatirinshipt of hctcromerict AMPA-selcctivc NMDA rcccptorchanncls'". Thc putativc transmcmhranc domain M 2 scqucncc is idunticnl in cach of the GluR-A tn -D suhtypcs, with the cxccptiun that Gh1H-B has a pnsitively chargccl argininc (Arg, R) rcsiduc in sa prisition 5x6 (cf. thc ncutral glutaminc [Gln, Q) rcsiduc n t aa 586 in GluR-A, GluR-C and GluRD. Exchangu of Arg58h GlnfiM in GluR-R and a correspnndmg Gln 4 Arg txchangc in GluR-D hy sitc-dircctcd rnutagcncsist rcvcrws thc shapcs of the I-V curvcst cvokcd by glutamatc in thcsc channcls formed by the hnrnomcrict suhunits"' (see Fig. 4 )
-.
Dose-response Routex nf Cn2'-influx dependent Purkin je cells
OR
ogonist mncentrntims in
07-36-01: Inn channcls integral to non-NMDA rcccptors on immaturc Purkinic cells [%tO-day-ald rats) nrc pcrmcahlc to Ca?', Nn" and Cr?' Incruascs in I&* 1, induccd hy rplotivelv Iriwcr oxonis[ conuentrrilrms arc larguly dcpcndcnt on Ca2+-influxthrough voltage-scnsitivc Ca2+channcls, which arc thcrnsclvus nctivatcd hy a largc Na*-influx. Hi&r concenrrritinns of agonists dnsc-depcndcnt!y incrcasc [C?'], (untlcr ctmditions in which activation of vciltagc-dcpcndcnt Ca?' channcls and NMDA channcls arc hlockcd), intlicat mg a Ca' "-influx thrnugh t hu Ron-NMDA rcccptt~r-chnnncl'".
'".
D t f f c r e n t ~ asensitivity l to eihr~ntlldependent on uppllrd cz~onisr concenfrotion 07-36-02: KAIN: Rcspc~nscs prr)rluccrl by low or high conccntrations of kainatc arc diffcrcnt~aflyinhihitctI by acutc cxposuac of kainatc rcccptnrs tn ethanol whcn cxprcsscrl from rat hippoca~npal nlRNA In oocytcs '". For cxamplt, 50 mM ethanol inhih~ts 1L.5 I I M kainatc rcsponscs hy 45% comparcrl to only 15% inhihitinn nf 400 I I M kainatc rcspnnscs. Ry contrast, acutc cthanol cxposurc inhihits response4 stimulatctl by low and h ~ g h cvnccntmtlons o f N-methyl-11-aspartatcto B sirn~lart!cgrccl". Note. For an ~Ilustrationof thc rclativc potentiating effects on GARA,-~ncdiatcd C1 -flux
high [Ca7'l
high I M ~ ' ' ]
-
-
I-
-
Figure 4. Compr~rrsnnn/ elcctrtlphy~E'olo~icnl properties lrom horntlrneriu ~C:I~~K-r.h(lnnelq c-onto~nin.~ Q, R clr N res~ducsat thc Q/I< site. Top row. P h n V ~ P W nl Q / R sl!r positinn m pcntnmcriu nrmn,Tcrncnt o f cllnnnel ~~rhunrts. M ~ d d l c row. Typicr~E I-V selotionship~. Ifottnm rt~w: TyprcoE whole-cell c u r r r n t a c'll'rjtrd h y glntarnatc under t11e strrted ~xtrrrccllu~lor icln~uuonillr~onsIIrrr ,700 m ~ Note: . The experrrnenfr~lf u l ? ~ t r t ~ ~ to?fnon n osporagine (N) In! n t hc gl:~atomine/arginine(Q/R)site (I! AM PA receptor s u k ~ ~ n gcnPrrilec ~r+ chflnnrlc drsplrrying ri crlectrve prrmeohility for ca7' ovrr M9Q% of available receptors are desensitizedt, although thcir affinity to glutamate is much higher than that measured prior to dcsensitizationt lm.Desensitization at AMPAlkainate receptors has been proposcd to contribute tn the Iast decay of excttatoryt synaptic currents.
Desensitization kinesics 07-37-02: Kainate receptors g e n e r d y desensitize$ only 'extremely slowly', whereas AMPA receptors (with rare exceptions, see rcf.'2'\ undergo this transition relatively rapidly and in a concentration-dcpendent manner (,we examples bclow). Note: Kainate has also heen shnwn to cause rapid desensitizationt of hornorncrict channcls cxprcssed from subunit GluR6.
Utility of 'absolute selective block' of mpjd desensitization for AMI'A channels by cyclothiazide 07-37-03:Potentiation by cyclothiazide (CYZl of recomhinant glutamate reccptur rcsponscs via an allosteric Hock of rapid desensitization shows absolute selectivity for GhRlLGluR4 (AMPA] receptors when expressed in X U I Q ~ U Soocytest1.Rapid dcsensitization in HEK-29.7 cells transfected with AMPA receptors is also Mocked hy CYZ, but is nnly weakly attenuated hy concanavalin A (Con A). Conversely, desensitization+ a t kainate receptors IGhRS-GluR7 and KA-l/FCA-21 is hlocked hy Con A hut unaffected by CYZ". Note: Cyclnthiazide is a henzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide.
CYZJCon A-sensitivity phenotypes can report iGluR subunit assembly patterns in viva 07-37-04: Nativet cell types shown to predominantly cxpress kainatepreferring subunits (e.g dorsal root ganglion ncuwnes, mainly expressing GluRS) show an expected Con A-SensitiveJCYZ-iRsensi~ive phenotype". Conversely, nativet cell types which preferentially exprcss AMPApreferring subunits leg. hippocampal ncurones) display a CYZ-sensitive/ Con A-insensitive phenotype. Table R summarizes findings of comparative studies on nativet ccl! preparations,
entry 07
Table 8. Cyclothinzide ( F Y Z ] vrrwq i+oni*nnuvolinA (Con A ) sensifivity phentitypcF /nnr AMPA-ceki*tivr iGlwR-ohonnelc exprcwcd in native neiirtines (From 07-,37-04)
Preparation
Cyclothiazirlt. phcnotypc
Rcfs
Hippocam pal spinv 'mossy cclls' versus aspiny hilar in t u r n curones
A greater sensittvity tn cyclothiazide in hippocampal spiny 'mnwy cells' v e r w s aspiny hilar intcrncuroneq has heen reported [with half-maximal rcmoval of desensitization hcing 90 WIM and 200 mM, rcspectivel yl
122
Hippocam pal SliGUS, glutamcrgic neurmes responding tn glutamatu
Cyclothiazide ( C Y Z )reduces rapid desensitization, enhancsn): thc steady-mtc and peak current pOduGL't! by 1 r n quisqualate ~ with EC5nvalues of 14 and 12 I'M rcspcctivcly. CYZ causcs glutarnatc to induce long h u r w of channel openings, and greatly increases the numher of repcatcd npenrngs. At 110 I'M CYZ docs not have mcasurahle cffects on the fast componcnt of dcactivation nor docs i t h a w statistically significant clfccts a n thc distributirm (if the faster campancnts of glutamate-~nduccdhurst duration
Hippocam pal neuron es rtspnnding tn kainatc
Responses of hnppocampal neurones to kainatc are strongIy pntcntiatcdt (3100%)hy cyclathiazidc, which is cnnsiderahly rnorc effective ['cnmpletc hlock nf descnsitrzatinn') than annracetam in rurlucing dcscnsitization evnked hy glutamate
Dorsal root ganglion neurnnes compared with h ippncampa 1 neurones
Cyclothiazidc cnmplctcly blocks dcscnsitization produccd hv 5-chlnrowillardiine in hippocampal neurnncs and strongly potcntiatcs rcsponscs to kainatc (thc action of aniracctam is similar hut much wcakcr). In DRG ncuroncs, cyclothiazidc and aniracctam has no cffcct on dcsunsitization hut prtduccs wuak Inhibition of rcspcmsc~to kainate
Cultured ccrehcllar glial cclls (aligodcndnicy t L' lincagc, 0 - 2 A progenitors]
T w n receptor populations arc prcsent in thcsc culls, with high and low affinity for kainatu shriwing ditfcrcnt sensitivity for potvntiatien hy concanavalin A and for hlock desensitization of cyclothiazidc
'"
A mechanistic basis for kainate subunit-selective phenotypes of concnnavdin A 07-37-05: Cnncanavalin A (ConA) is a lectin which can hind to glycosylatedt mernhrane pmteins with high affinity. Expression of rccomhinant iGluR subunits which display II higher prnpnrtinn of glycosylatedt N-termini [extra-
cntry 07
cellular\ might bc thcrcfore expected tn hind Con A with grcatcr affinity. Although both AMPA and karnatc cDNAs show N-glycosylation motifs (see Seqiience motifs, Q7-24)treatmcnt of glycosylatcd G l u M [ kainatcprcferring) with N-glycosidase" induces a 13 kDa shift in M, comparcd with a shift of only 5-6 kDa for AMPA-preferring suhunits'"~FZ".Thcsc analyses sugqcst kainate-prcfcrring iGluRs are glycnsylatcd to a grcatur extent than AMPA suhunits, and may thcrcforc explain thc highcr sensitivity of the kainatc suhunits to lectins like Con A". Note: Thc flip/ flnp variants (SEC helmv) show no apparcnt diffcrcnces in sensitivity tn concanavalin A.
Desensitizing effects of cyclothiazide vary in fliplflop splice varinnts
of AMI'A receptors 07-37-06: Although the molccular basis for 'absolute selective hlnck' of rapid
dcscnsitization in AMPA suhunit channels IS unknown, the 'flop' splice variants show much less potentiatinn hy cyclothiazide (22 f 4-fold fnr gkutamatc responses, 4.2 f Q.7-fold for kainatc rcspcmscs) than their 'flip' variants (130 80-fdd for glutamatc responses, 12.4 f 2.7-fold for kainntc rcsponsus). Similar properties are ahserved with tliplflop variants in hctcrorneric comhinatinns (e.g. GluR-A, + GluR-R, V V ~ S U SGluR-A,, + GluRRJ. Thcsc results are consistent with a rolc for thc Fliplflop locus in regulating desensitizationt (see helow nnd rc(-i''* "3.
+
Desensitizatinn plateflux in alternative splice vorionts flip and flop
07-37-07: AMPA: . - -- Upnn fast applicatian, glutamatc clicits currents at AMPA rcccptorqhanncls which exhihit a fast rise timet and then dccay to a plateau value in the continucd prcscnce of agonist. Thc platcnu i s morc prtinounccd with flip- than flop-containing GluRs (see Gene orgnnizatinn, 07-20), Thus thc differing dcsensitization kinctics shown by receptors containing flip and flop mtidulcs a k c t thc 'peak : steady state' cnmponcnt nf iGluR forrncd from AMPA-prcfurring GluK-A-GluR-D suhunm. Note: Kainatc cvokes dcntical nondesensitizingt currents in hnth flip- and Ilnpcontaining GluR rhanncls formcd from thc GluR-A to -D classes.
Moda 10 t ion n/ desensit izn t inn
07-37-08: The nnotropict drng miracetarn, wheat germ agglutinin, and concanavalin k act via separate mechanisms tn rcducc ricscnsitizatinn evnked hy L-glutamntc in rat hippocam pal neurnncs'". Thc dccny of cxcitntciry synnptic currents, and miniaturc excitatory post-synaptic currentst (EPSCsJ cvnkcd hy sucrose arc slowcd 2- ta Xfold hy aniracctarn. Animcctaiii also incrcascs the rnagnitudc of glutamatc-cvokcd EPSCs 1 .Pfold, prnhahly via a posl-svnnpric mcchnnism (if act inn. Aniracctnm increases the hurst lengthf and peak amplitudest of Lglutamatc-activatcd singlc-channel rcsponscs'". Simulations suggcst that sniracctam eithcr sl(iws cntry into a rlcscnsitizcd statct or dccrcascs thc closing rate cnnstantt for ion channcl gating1 . Cornpnrrrtive note: Whcat germ amlutinrn and cnncanavalin A ruduce EPSC smplituJc via a prcFynrrptrc rncchanism (we helow). Diazoxide cnn also rcducc dcsensitizationi of ~iippocnmpa~ AMPA rcccptors to AMPA, glutainatc and q uisq ualatc.
"'
llr(fcrslnt nr~trorrrrl~.r~?ls show tlif/crcn! nrtcs d:.rensSt1zr1tion
o(
rr'crwrry f r r ~ r ? ~
07-37-09: In nativc rlcnt:~tc EyrLls, hippocampal CA.3 and CAI ccll pntchcs a p p l ~ c a t ~ o nofs I m h ~cliitarnato of 10I1 nls r1ur;ltkon sht)w tltnc constants f for dcscnsitization of L1.4 2. 7, *I 1.3 t 2 H, and 9.3 i 2 H t n y ruspcct~vuly'14.
Dcscnsit~zntinntime c o n ~ t a n t s inru only wc:tkly dcpcndcnt on glutamatc conccntratlon (200 /rM ;ind 1 r n ~ lfor thc thrcc ccll tvpcs. Urluhlc pulse :~pplication.;of glutnm:~tcinrllcatc that 1 ms pulse of 1 inM glut;unatc cause part la1 GluR c11;lnnt.l rlcccn5itiz;ltlon~(-h(l'X,). Thu tfmc colrrqc ot rrTcovrrv from rlcscnsitizaticln I S xl(~wcrin rlcnzatc E Y T I ~ Sgran~rlcccll pi~tchcsthan in CA.7 or CAI pyr;~mirlalccll p3tclrcs1t'. N o t r . Sprci311~0tfIGILIRS~nctliatrng cnmplcx auditory information in cochlcnr nciironcq r~lowl. Thc Ilcrnlcatlon pathways of a r:ingc of nuurr,transrn~ttcr-gatutl ion chnnncls h:ls hccn rcv~cwctl"". (rrpr,
TIT(.C J u K - H ~ u h t r n ~c1r)tnirzr~~c.s t propcrtir~ot JonIc tlow 67-40-02: AMPA: -~ t t c r o i n t . r i c t A M PA rcccptor~ cantazniny: thu GluR-ll subunit display law divalent Eon pcrmeahilities. Howcvur, rccrlmhinanti AMI'A roccptorr lacking the GltiR-R subunit are ca2+-permeable n t physiulogical C;J" coilccntratlorlr [ c . ~CiluR-A, . C;luIl-C or GluR-A t CiluRC In ~ ~ > n ~ h ~ i ~ .H ~C 't~ ~C ~c Ol I nO ~~~C~LXI~~F~~~C S~ S )I I.ofI ~ CIZIR-RmRNA prcrnlxcrl In diffcrcnt tnnlar mtios with rnRNAs cncodlng GluIl-A, GluR-C or GluR-12 sliow ;i I,~rxcr;lnglh 01 ~Ii~t;~matc-;~ctivatcrl (:n"-pvrtncnhil~t~cs1llfl .
Srdc-chhirrrlciza ir~ltichr~r,yerrffcct dfvr~Irtr1j?c>rrncrihilityrn rccomhir7ont C:?FIItl,07-F4 onrj /'rot crn p h n ~ p h o r v l r ~on, r 07-,?2
Equilibrium dissociation constant !dish-nffinrtykrrr~lrrtchlnding sites in nolive t i ~ s u c s 07-45-01: KAIN: Kninntc-binding sitcs that d8ffi.r from high-affinity AMPARintlinc, srtcq h ~ v uhccn tduntlticrl hy T~gnnri-hntlings t u d ~ ~'Class~cal' ~ ' ~ ~ . high-affinity kainatc sitrs (K,i 5 anrl 50 n u kalnatc) exist in t l ~ cCA,? arca of the hippocampal forrnatnon. -+
Sirnilflr ngonrsi nffi~lrtics for nrrtrvr! rind recomhinont krilnntc? reueplors 07-45-02: Thc '~~hartnacolngica1proftlc' of cxprcsqcd rccomhinant KA-1 ~dctcrmincttin h i n t l ~ n cxpcrimcnts ~, with I 'HI-kainatc) differs from that of t h c clonctl AMPA rcccptors, hut is sitii~larto thc mammalian high-affinsty rcLcptor (kainatc > quisilualstc > glutamatc b' AMPA, whcrc kainntc K,, ,k.l,n.l,rl 1% -5 nnh5) In t-onip,lristln, thc inhil~itoryconstant ( K , ] v~lzlusfor cliusq~~alatc,I -glut;lmatc ;tnt! AMI'A arc. I X 200 snd 5000 I I M r ~ s ~ ~ . ~ t ~~'$>tr. v ~ . Rccomhinnnt l~". KA-2 s u h u n ~ t sd o not form channcls In h ( ~ ~ n n m u l t i i n c rI. ;~~t uxh~hrt t h ~ g hnfftniby fclr k a i n ~ t u[KLI 15 ~ I M ) " .
-
-
Incroriserl' mgon i f ! cdfinl! ips oh~rrvrdfor purified iGI11li 07-45-03: AMPA KLIY;IIP~CS havc Iwcn ohscrvctl to hccomc ' ~ n u c hIowcr' upon various iiltii~l~r;inu trc;ltmcnts ant1 followlnr: protcin pllrif~cat~on (r;r.r, Prorcin rrit~,ri/r[ion\,07-&?
K,, volrlr's for rlnltrfrvt rocrpIorc 07-45-04: In Xr-t~oprrvI~rain,kainatc- and A M P A - h ~ n d i ns ~ t c co-cxist s En a I : l ratin snrl crrnnot I>c vcprinrt r ~ r lhy physical a n d chcmtcnl f r,rctionatir~ns*',~~ ( S C ~ ,S ~ i h f j , ~pE~~ I S C I ~ C ( I ~ I O I ~(17-0(>). S, In thcsc protcins, AMPA and kainatc arc mutually and fully competitive+, wlth K , valucs idcntlcn! t o the K,, valucs for thc radioligand (AMI'A, 34 nM: kainatc, 1.5 n ~ ]Channcls . rcconst~tutctlrn hilaycrs can clicit currcnts (sirnl1;lr to natlvc non-NMDA GI~IRsFin rcsponsc to low lcvcls o f h M P h o r kainntc.
Cillrtmmr~tc-. AMPA- r~ndkminr~tc-bindingsltcs 07-47-01: From pharrnactdogical considcratinns, L - ~ l u t a m a t c kainatc , and AMPh hind to tirffcrcnt reccptnr suhntructures on rccc~nhinant AMPA rcccPtors'"" Rcccptnr I,~nd~ng/nuzr~rarl~ographic appn)nchcs to chnrnqtcrizat~rlnof cxcltatnry n n ~ i n oscitl rcccptnrs hnvc hccn rcvicwcd"".
cntry 07
Avniln hle radiolignnds 07-47-02 AMPA: [3AI-AMPA and i3H1-CNQX. Use of AMPA or CNQX can define the nnn-specific binding of ["HI-glutamate. Note: CNQX also hinds to thc glycine site and possibly thc NMDA iGhR. Radiotigands for kainatc sitcs include f3H]-kainate and [3H]domoate (but note the hezcrogcncIty sf domoatc-affinity purified products - see SingIe-channeldata. 07-4 11.
Affinity purificatinn of A M P A receptor-channels using /'H]-AMPA and lor0 spider toxin
07-47-03:A glutamate reccptor has hecn pusificd from Triton X-10fl-solubilized hovtne ccrehehm rnemhranes hy affinity chromatography using a spider toxin 1Jorospider toxin; JsTx,irnmohilizcd on a lysinc-agarosc column followed by a Mrmo Q anion exchange columnlr5".The active fraction purifies an AMRAbinding prnteirt of M, 130 kDa. Lineweaver-Rurkt plots indicatc thc protcin to h a w a K,f of 12.7 n M [%]-AMPA in the purificd fraction. In rccnnstituted liposomes, the purified protcin yields a glutarnatc-activated channel which can bc inhibited with JsTx"" (sce Blockers. 07-43),
-
ReceptorJtransducer interastinns Involvement of second messenxers in excitatory nrnjno acid signal transduct ion processes
07-49-01: Excitatory amino acids {EAAJactivate second rncssengcrt systems via rnotabntropict receptors i n nddirion to the dircct gatinx of 'intcgral' Thraugh these (ionotroyict ) rcccptor-channcls (rcvrcwed in ref. 15'). 'indirect' rnetabotmpic pathways, EAAs are capable of activatmg lmth adenylate cyclase and guanylate cyclase and also to induce phosphoinositidet 11'1) turnover (see, for exomple, Fix. 4 rmder ELL' CATULU NMIlA crnd tnhScs in Resource A - G protern-linked receptors. entry (56,
Calmoddin-dependent inhibition of post -synoprfc vnltagc-gnted co2+currents
07-49-02: Glutamate-evokcd Ca2'-influx through both NMDA and nnnNMaA rcceptor-channcls in rat hypothalamic ncurnncs inhibits highvoltage-activated {WAJ Ca2* channels (see VLG ~ n entry , 42) via a ca3modulin-dependcnt mechanism 52. A pee-synaptic glutamate receptor agonist (L-2-aminQ4-phosphonobutyricacid1 and a sclcctive rnctabotropiq agonist (trans-ACPD) arc ineffective in mimicking the W A Ca" current inhibition prumoted hy glutamate. Inhibition IS also dependent on the presence of extracellular Ca2+,and can hc hlocked hy internal perfusinn of thc cclls with BAPTA. The calmodulin antagonists tdfluoperazine and qalrni-
dazolium completely prevent the inhibition'".
Links bet ween Glu R 1 a,qonism and hormone secretion by heterologous gene expression
07-49-03: Co-expressinnf of a plasmid1 construct encoding growth hormone and a plasmid cncoding a non-NMDA glutamate reccptor, GluRI, yields
chmmaffin cells in which Ca"-depcndcnt growth homonc sccrction can ho stimulatcd hy kainate.
entry 07
Receptor agonists (selective) 07-50-01: Nntr. Availability of sclcctivc antagonists ' and agonislts havc heen central to the rccngnition nf receptor suhtypcs underlying nativef iGluR rcspnnws. Thc hasic fcaturcs of thcsc and nthcr agonists are listed in Tahle 10.
Agonist nffiniiies of recombinant GIuRs
07-50-02: Rcccptors generated from thc GluRl to GluR4 c D N A s ~have higher apparent affinitv for AMPA than for kainatc. Whcn homomerici ruccptora o f thc GluRh class arc cxprcssctl: in Xcnopris mcytes, thc rcccptors are activated by kainatc, quisqualatc and L-glutamate, hut not by AMPA. Furthcrmorc, the apparent affinity for kainate IS higher than for
rcccptors from thc GluR I-61uR4 class2.
ECsn vnlues for I G ~ F ngonists R 03-50-03: Typical EC5[, values fnr recornhinantf GluR-AJR receptors cxprcsscd in oocytcs arc 3.,3l S I M (AMPA); 6.16 I I M Ifilatamatc]; 57.5 / I M (kamatc)"'". ECqIFvalucs for rccomhinant GluR-R/D receptors expressed in cincytcs arc 5.01 prvr (AMPA];32.3 (IM (glutamate); 64.6I'M /kainatelfM.
Agonist riffinities and potencies ot GluX-channel splice variants
07-50-04: Gcnc cxpressnon cnntrnl of thc altcmativc splice variants 'flip' and 'Flop' (ser Gene ciyyunirriiion, 07-20] can confer different kinetic properties on thc currcnts cvtikcd hv the aRnnists glutamatc or AMPA, but not on thosc c w k c d hy kainatc. For both '(lip' and 'hp' vcrsions (if GluR-A to GluR-D, kmnatc cvrA-5 a nrm-tlcscnsitrzingt current whereas both glutamate and AMPA C ~ W C :in initi;il bst-duscnsitizingt current followcrl by a steadystate plateau Whuicss glutamate, AMPA .~ndkainatc cvtikc currents cif similar amplitiirlu in 'flip'-cxprcssinK cclls, kainatc-cvokcd currents arc much largcr than thnrc cvokcd hy gI iitamatc or AMI'A in rflop'-cxpress~ng cclls Clutsmatc sctivntcs channels 4-s-timcq mow cffcctivch whcn acting at the 'flrp' vrrsion of thc GluRs2".
Receptor antagonists 07-51-01; Scvcral stutiics havc indicated that antagonism at NMDA receptors (.we E i L CAT GLU N M D A . entry 08) is only partially protective in stimc r n ( i h ! s of local ischaemia and may hc ineffective in global ischaemia (scc Phencuyyiic vxpressrrm. 07-74 nnd disciissjnn m ref 1571. Dcvclopmcnt (if AMI'AJkainatc-sclcct ivc antagonists (particularly NRQX a n d t h n w nf the 2,3-hcnzodiazcprnc class) havc indicated thcir valiuc as 'neumpmtective' and anti-cnnvulsant agents. Thc hnsw propcrtics of these nntnpnists art' listcd in Tnblc 1 1 in coinparison with rjthcr (less-sclcctivc) nnt:igrmists in u s c
Table 10. Common agonists of AMPAlkainate receptors and their features (From 07-50-01) Agonist Features
Principal agonists AMPA Kainate
Willardiines and bromo-/chloroderivatives
For constitution of 'AMPA-preferring' versus 'kainate-preferring' iGluRs, see Gene family, 07-05. For reported activities of AMPA (a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), see paragraphs below this table and other fields prefixed with 'AMPA:' Kainic acid (kainate)is a full, non-desensitizingt agonist at subsets of iGluR assemblies (see 07-05). A functional kainate receptor has been cloned which is insensitive t o quisqualate/AMPA. Conductance responses evoked by kainate at the GluR channels are competitive with those evoked by AMPA and are not additive. Note: Domoate has also been used as a kainate receptor agonist and affinity ligand (eg. see Table 9). For further activities of these agonists, see paragraphs below this table and other fields prefixed with KAIN: The (S)-but not (R)-isomersof the naturally occurring heterocyclic excitatory amino acid willardiine and 5bromowillardiine are potent agonists for AMPA/kainate receptors. Willarhine [( S)-1-( 2-amino-2-carboxyethyl)pyrimidine-2,4-dione] produces rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine(ECS0,1.5 p ~is ) 7 times more potentt than ( R , S)-AMPA(ECS0,11 p ~and ) 30 times more potentt than willardiine (ECS0,45 P M ) ~ ~ 'Note: . Willardiines are the first compounds characterized in which simple substituent changes in molecular structure are associated with marked ddferences in the ability of agonists to produce desensitization of AMPA/kainate receptors L-Glutamate and L-aspartate are mixed agonistst of the AMPA-, kainate- and NMDA-selective receptors and their effects are partially inhibited by all selective antagonists. In dorsal horn neurones of the rat spinal cord, millimolar concentrations of L-proline elicit an inward current that is partially antagonized by strychnine, APV and CNQX. Thus, L-proline is a weak agonist at strychnine-sensitive glycine receptors and at both NMDA and non-NMDA glutamate receptors. The ability of L-proline to stimulate CNQX-sensitive Ca2+-entryfollowing activation of excitatory amino acid receptors implicates L-proline as a potential endogenous excitotoxin Quisqualate (QUIS)was formerly used as a principal agonist for AMPA receptors, but it has also been shown to be an agonist at metabotropict glutamate receptors. N
Mixed agonists L-Glutamate L- Aspartate L-Proline
Quisqualate
Amino toxins of plant origin BOAA
Refs 161
16'
N
(BOAA)is associated with incidence of neuroThe amino acid toxin p-N-oxalylamino-L-alanine lathyrism (a spastic disorder with acute and chronic onset) associated with consumption of the chick pea, Lathyrus sativus. BOAA appears to act as an excitant on spinal neurones via agonist activity at AMPA receptors
163
82,174
rM Compctittvc antagonists with diffurcntml wlcctiviay for rcctmhinant CluIts inclurlc. 6nit r r ~ - ~ - s i i l p h a m c h ~ c n z o - ~ - q r ~ ~ n o x s l i n c - ~ , ~ ~ , - d i o n c INRQX, PA? 7.1 ). Thc potency of NRQX for hlockinr: currcnts mutlintud hy GluR-A/!3 rcccptors c1i;lnRcs dcpcnding [in thc aRoniqt u w d to activatc thc rcccptors (FA7 valucx 7 2-1 1 0 (11 For hlock OF kainntc rcsponscs; 6.78 1 0 02, for hlock of L-gliit;lnintc rcspnnscs; 6.95 ? 11.112 for hlock 0 6 AMI'A rcsponws]. Ihffurcnccs hctwccn sgonists arc. luss inarkud in cells cxprcssing GIuR-R/U rcccptcm (pAJ valucs: 7 2X t 0 01 fnr hlock of karnatc rcsprmscs; 7 -70 t 0 02 for hlock of I--glutamate rtsponsus; 7 3.5 0.01 for hlock of AMPA rcsprmws). NRQX acts a s a potent and sclcctivu antagonist a t rccmnhinant rcccptors, h u t i t 5 action c:m hc ovcrcomc hy increasing a p n i s t conccntratrtm 1i.c. it is. crlmpctrtivct). NHOX has anticonvulsivu propcrtics in scvcral scizurct mrdcls 2nd i s ncuroprtituctivc in brain isch:icmini models. In native mumhrancs NRQX i s 5lHbh)Id inrirc ScIcctivc for A M P A w c r NMl3A ruccptcirx (crmpnrc thc rclntivc nrv-sclcctivitv of CNQX and IFNUX at
+
-5
rcctiiaihin;lnt rcccptors helriw]
T h c homnphthalazinc t / - ]-GYKI-524(16 1 5 a Rcvlcw' highly-sclcctivc AMPAJkainatc antagonist (1C5rl for kainatc 7.51mJ and docs n n t significantly Affect NMDA, n ~ (~ncta~,otropict ~ u glutarnatcl, o r GARA, ruspcinscs. GYKI-52466 hinds t c i hnth opcn and clmcd recuptor-channels, and is vr)ltnRc-indcycndcnt in its antngonistac cfkcts. Thc action of GYK1-5246h cannot hc m e r c n m c h y raising agrmist conccntmtirms [ I . c .i t is noncotnpctitivc I]. GYKI-52466 1s s h a d - s p c c t r u m nntictinvulsnnt. Thc mcthyl-carhamnyl dcrivatlvc GYKI-53655 i s scvcral-fdd more potunt thnn LYICI-52466. Thc 2,3-henzndiazcpinr class of AMPA/kainatc rcccptcir nnncompctltiva antagonists may h a v c therapcutic application.; ~n cpi Icpsy, i rchacniin, ncurorluKcncratmn nnrl Pairkinson's discnw
-
cntry 07
Tahle 11. Continued Antagonist
Lipophilic competitive antogonisis
DDHR and dcnvativcs
InhaInl ionnl anoesthcrios
Enflurane
Rrphenyl clcrivcltive of NDSA Evan4 blue
Features
Rch
In addition to their spccific antagonistic ctfccts 'Oli on neurnnal GluRs, quinoxalinedioncs (c.g. CNQX) have also hccn shown to hhckglutamatcinduccd rcsponscs mediated by recnmhinant A M P A I M rcccptor-channels when expressed in hetcrologoust systcms (c.R.GIuR-A/R and GluRR/D receptors). Antagonism occurs iwcspcctive of the particular subunit composition and displays little sclcctivity hetwccn AMPA and kainate receptors). 6,~-Dini.aroquinoxatine-2,,7-diflnc (DNQXl,a h shows relatively !ow sclcctivity A class of glutamate receptor antagonists that show cornpctitivei action, significant potency at multiple sites, and a high degree of lipophilicizy are the substituted henzazepines. 2,s-DthyrEro2,s-dinxn-3-hydmxy- bI-bcnzazcpinc (DDHR] and three suhstitutcd derivatives, 4-hromo-, 7methyl- and R-rncthyl-DDHR, block thc activation nf non-NMDA rcceptors by kainatc and L-glutamate (see oho Reccptcjr rmto~onrsrs under F I G C A T GL[J N M D A , 08-51) y-Glutamylaminomethyl sulphonate (GAMS) has .". *", been uscd as .a partially selective non-NMUA receptor antagonist which offers pmtcction against audiogcnic seizurcst. Other cornpetitivc antagonists at thc AMPA-binding sitc arc GDEE and DGG Enflunne a t anaesthetic conccntrations ( 1 .X mM) ' 5 7 inh ihits AMPA-, kainate- and NMDA-induccd currcnts exprcssed in oocytcs hy 29-10%, ,1&%3% and 20-27%, rcspcctivcl y. Inhibition by en flurmc is independent of thc cmctntrations of thc agonists [NMDA, AMPA and kainate] or thc NMDA-coagonfst (glycinc]suggesting that enflurane inhihition dmx not result from a compctitivu interaction at glutamate- or glycinc-binding sites 154 Selectivc blockade of A subset Qf AMPAIKA receptors has been rcported for the noncompctitivct antagonist Evans blue"'. This hiphcnyl dcrivativc (if naphtha1 cnc di sulphnn ic acid hlocks ( a t low crmccntrationns) kainatemediated rcsponscs of the snhunits GluR-A, GluRA,R, GluR-A,C, and ChR-R,C cxprcssud in Xenoptls nocytcs hut nni respnscs of GluR-C or GhR6
'"
'"
entry 07
Table I € . Contrnued Antagonist
-
Fca t w rt‘s
Refs
355 nM for thc subunit combination GluR-AJR). The blocking action of Evans hluc is partially revcrsiblc and docs not cnmpete with the kainate for thu agonist-binding site (ICir,
Nnncompetitive nnragonr cz Riluzole
Responscs faom katnic acid-cvokcd currcrtts in Xcnopus oncytes injected with mRNA from rat wholc brain or cortex can hc nan-carnpctitivclyf ’hlockcd hy the anticonvulsant and ‘ncuroprotcctivc’ compound riluzole (ICrrj 167 I‘M cf. CNQX: Kicl 0.21 I‘M an& NRQX: ICio 0.043 /[MI.Riluzolc is marc potent at hlncking rcspnnscs to NMDA [ICW 18.2 JIM cf. lthc crimpctitivc NMDA rcccptor antagonist 2APV: IC5n 6.1 )/MI
-
-
-
Spider nnd wosp venom toxin?
Is’
-
For thc non-sclcctivc actions of certain spider venom toxins [argiotoxin and roto spider toxin) and the damcr wasp toxin philanthotoxm, we Riockers, (I7-43
Database listingslprimary sequence discussion 07-53-01; The relevon! dotnhase IS indicated by the Inwer cnse prefix ( e , ~ . gh:). whrch chntlld not he typed (see introduction & laynut nf entries, entry 02) Datuhnse riccrscjon numllirrq imrnrdintdy follow the cnlnn. Note thrit o comprehensive listin,F of nll avarlnhle accession numbers is superCluous /or Jocntim o f rctevant seqiicnces in GenRank ‘‘ resources, which are nriw r~vnilnhlem t h powerful in-hurlt neighboaringt analysis routincr (!‘or drsrnptron, we the Dcitrihnsr listingc field In the Introductron (inn lavotit of entries. entry 02). For exumple, sequences of cross-specieT VflriflRtA or relnfed g n e /flrnilvT rnernhers con be rendily occesrcd h y one o r two rounds of neitrhhourm.rt nnnlysis (which lire hnsed on precomputed nlignment 7 performed usmg the RLASTt algorithm h y the NCHlt). Thrc featnre I S mosf useful for rctrrcvnl of sequence entries deposited in dntnhnses later than thow listed bclnw. Thus, rcpcscntntIvc rnernhers OT kiinwn scqiwnce hornolrqp proupi~gsnrc listed to permit rniilml direct rcrriewls b y QCCCSS~OTI number, authorlrcfcrencc or nornench t ure. Fnllow~ng direct accession, however, ncighhounngt annlysiq i q strrintyFj7 r e u o r n m m d d to ~ d ~ n t i npwly ly reporred and rClru/pd .wqllenLc~
Nomenclature Species, Enon-systematic] DNA source
Original isolate
Accession
Sequence/ discussion
Rb: S9437I
Gallo, Neiirosci (1992)12: 1 01 i M t 3
GluR-4~flop
Rat Sprague885 aa Dawlcy; alternatively spliced, cerebellum, cDNA
GIuR-A
Rat forebrain 889 aa g h Xt7184 CDNAJ (clone GluRcxprcssionK1 clnned in oocytes. Equivalent to GluR-K1 4n0w renamed GluRl o r GhR-A)
GhRI (human isofnrm)
Human hippocampal cDNA library
HBGR- 1
Full-length human
[human isoform)
GlnH€ I=GluRl homologue)
'Glutamate receptor I'
GIuR-A GlnR-R
G1uR-C
GluR-D
888 aa [mature]
gh: X5R633 gh: S40299
hornofogue of GFuRl [or the flop version of the GIuR-A clonc) Human brain 907 aa gb: M64752 cDNA lihrary; M, 100 kDa chromosome 5 97% homologous to rat GluR-A Mouse, brain 908 aa gb: X5J49J cDNA sp: PL381R
-
Rat brain cDNA library
889 aa
gh: M36418 sp: P19490
Hollmann, Noture (1 989) 342: 643-8. Hallmann, Cold Spring Harbor SYW Qnnnt R i d
(1990)55: 41-55 hticr, DNA Seq (1992)2: 21 1-18 Sun, Proc Natl Acad Sci USA { 1992) 89: 1443-7
Pwckett, Proc Nail Acad Sci USA (1991 88: 7 5 5 7 4
Sakirnura,
Neuron
(1992)8: 267-74
Keinanen, Science
Accussion
Scrlucncc/ discussion
GlrtK-A flclp GlttR-R flop GILIR-C:flop GluR-I3 flop ClttR-A flip GlvR-It flip GlulZ-c fltp GILIR-I3f l ~ p
Flip nnrl flc~p; gh: M.764 18 ccll-spcclflc gh: M,16439 gh: M.76420 tunct~rlnnl switch {vrxr, ~ h M,16421 : ( ; V r l i * or,Lygh: M,?HOhO gh: M38Oh I nizr~tron, 07-20) gh: M,3X062 ~ hM.3 X O A 3
Adl~ltrat forchr;tin cDNA library
~ h MX50,35 :
I3ollltcr,
sp: 1'1949 1
Sc~bncb (1990) 249: 1 &33-7
'Glutamatc
gh: X57498
receptor 2'
sp: P2.3810
Sakirnura, Netlrnn 1 1992) R:
G h R - A to
GluR-D: Flip
and Flop variants
26 7-74
Glull2 [ - C;luK-HI
Rcntmllct"
GluR2
MUF ~ C I F ( : L I / I I S cxt,nt 2 (strazn RhLRJc, s~thspccaus
Kochlcr, M., Komau, H.17. and
~ O I ~ Ci cS l I~ c )
Scchur~,
tnalc liver DNA
P.H. ctnpcthlrshcd 1 19941.
~ u rntlvctllt~s v cxonl 3
C;luR-~tl (stram RALIIlc, K ~ n o m ~ ~ T ' lsithspcctcs dornesl~cuq] male livcr DNA
:[
GIuR2 [ = GluR-R) Rcnnmlct''
gh: L321YO gh: L32 15 1
Mus rnuscr~lus cxonf 1 sntl {stmrn RALRlc, prornotcr sithspcctcs rcgion ~lornc.st~uusl rnalc livcr DNA
~ h L321Y : 1 gh: L72 IS 1
Ki>ch!cr,M., Knrnau, H.C. : ~ n d Scchurg, P.H. unpuhlashcd 119941.
gh: L32 1 HY gh: L32 15 1 gh: W2152
Kochler, M.,
Knrnau, M.C. and Scchurg, P.H. itnpuhlishurl ( I994J.
entry 07
Nomcnclaturc Species, [non-systematic) DNA sourcc GIuR-R GluR-6
to
genomic sequences
QriRinal
Accessinn
isolatc
Sequence/ discussion
Sorn m c r, Rh: MJ64?7 Cell (19911 gb: M7h43R 67: 11-19 Rh: M764d9 gh: M76440 gb: M76441
Mousc gcnnrn1c
GluR-R, genomic GluR-C, gcnomic GluR-D, gcnomic G~uR-5,gennmic GluR-6, gcnornic
gh: L2OX14
HRGRZ (human Human brain glutamate cDNA lihrary receptor 21
Sun, Neororeport (1993)5: 441-4.
orininal gb: M8SQ36 GluR3 (= GluR- Adult rat sp: P19442 forehrain cDNA pulilished CJ library sequence narnc: RATGLWRq3;
Roult cr, Science ( 1990)249: IO.3.3-7.
$189 aa
hGhR3 flip Ihumanl
hGluR3 flip [human)
Stratagem 895 an cDNA lihrarics 9.36205 and 93 (1206
gh: UlO.701
Stratagenc 895 aa cUNA libraries 986205 and
gh: UlOX02
R a t t 11s
gh: MtlfiQ37
itn puht ish cd
p19941.
The long (putatively1 intracellular loop Of
90,3
33
( 1 994).
norvcgr’cur [strain SpragucDawley) N.R. Subcloned nuclentidc for phosphory- coding latian studics scqziencc segments
hmpcrsarl,
Y. ~inpublishcd
936206
GluR4a I=GluR-Dj
Rampersad, V.
Rcttkcr, Neuron
119901 5: 5 an -9 5.
gh: SS6679 g h S56890
Wright, J R ~ c e p lRes (199,?j 13:
653-65
AMPA-sclcctivc
KluR Kim: 4053 12 Morita, M o l IIrnrn Rcs
(1992114: 143-6.
cntry 07
Nomenclature
Species, [non-systcmatic] DNA sourcc
GluR.5 has two splice variants, GhRS-1 (920 aaj and GluR.5-2 (905 aal (see GCRCo r p nization. 07-20)
Original
Accession
Iw1at.c
GluRS-I: gb: M83552 920 aa (a full open reading frame for the shnrtex GluRS-
Sequence/ discussion Rctth,
Neuron (2990)5: 5 88-95.
2 splice variant
WHS
nnt found]
Rat ccrchcllum 884 aa cDNA library
not found
Egcblcrg, Ncrture (1991) 351: 745-8.
GluRJ 61
not fnund
956 aa
not found
Mouse delta-!
1Q09aa
PIR: JH02hh Yamazaki, Rrachcrn Hrophys Res Commun (1992) 183:
GEuR chain precursor
not found
886-92, 52
Mausc delta-2 G h R cham prccursor
72 ( = K h - 2 hornologue)
Mouse GluR 979 Ramma 2 subunit sclcctivc fnr kainate
GIuR-K~
Rat hippo-
v m i m cif
campus and curchral cnrtcx cDNA
(= fhp
GluR21 GIuR-K~ (=flip vcrsinn of GIuR3I
humEAA2
Human hippocampal cDNA library; strucrurally related, though not identical to KA- 1
not
1
found
gh: DO 127.7
gh: XS46SS
R8d aa
gh: X54656
962 aa plus 18 aa signal scqucncc
not fr~und
-
kDa
Sakimrrra, FEERS Lett (T090)272
J3-80.
888 aa
M, 107 176
Lomeli, FERS Lett (l5)93\ 315: 318-22.
Nakanishi, Neuron (t990) 5: S69-8 1.
Karnhoi, Md
Phnrmacol (1992)4 2
10-15.
Nomenclature Species, (non-systematic] DNA source KA-1 I=31
homoleguc 1
KA-2 {=721
KBF-C
KBP-f
Original: isolate
Accession
Sequence1 discussion
Rat brain 956 aa EDNA, highaffinity kainate; homomeric assemblies do not form channels
gh: X5999h
Werner, Nature (1991)351:
979 aa Rat hrain cDNA, highaffinity kamate; homomeric asscmhlies do not form channels
em: X59996 Herb, em: ZI 1581 Neuron gb: X59996 (1992)8: gh: Z11stEl
775-85.
Chick cerebellum cDNA kainatc binding protein
KA binding (channel inactive) 464 aa
not found
Gregor, NCltslrc ( 1 989) 342:
Frog brain
KA binding
not found
cDNA/kainate- lchannel binding protein inactive) 487 aa initially purified hy dornoic acid affinity chromatography
742-4.
684-92.
Wada, Not lire 11989)342:
684-9.
"The complex alternative splicing+ patterns ohserved in the GloR-B gene [Koehler, M., Komau, H-C. and Seehurg, P.H. unpublished, 1994) can be traced from splice site joining data' accompanying certain databasc entries. Importing sequences in a standard file format can he intcsprctcd by some sequence analysis programs and incorporated in an interactive fcaturc tahlc. 'For example, the GluR-R short-splice form entry /gkW2204/gh: J-32151) contains thc dollowing 'joining' protocols: mRNA join (I-32189:1075..8594,LKl190:1 ..141,LA2191: I ..240,~32192:1.. 197, L3219,3: 1 ..54, L32194:1..1h2,LV 195: 1.. 1(18,L32I96:1..lOs,W2197:1__ 1 11, L72 1% 1 ..2O7, L32 199: 1 ..371,L32200:1__ 199,W22O1:1..248,L?2202: 1 ..I 1 5, W2203:2.. 1 15,1..249] and CDS join (L32189: 1507.. 1594,L32190:1 .. 14l,W219 1 :1..24O,LE 192:1__ 197, L321Y.3: I ..S4,L32144:1..162,W219!i:l..lh8,W219~:€..105,L~2197:1 ..111, L32 158: 1 .,207,W2 199:1..37 1,W2200: 1..199,L.3220 1 :I . .24R,L-32202:1 ..1 1 5, L32202:I..1 15,1..2461 which rclatc the contcnts of database entries ('L-nurnhen' in the example above) to desqwted splice points.
Sr~urcesof ~n/orrnnt Inn on orher glrlrrrrnnte rucuprnrs 07-53-02: N o t r . T h c gcncs anrl cl3NAs tahvlatcd ahovu uncodu s ~ ~ h u n l t s f o r i i i ~ n ~ionotrnpict : glutamate rccrptors (iGluRJ. In datahasc scarchcq, tPicsc shoultl not hc ctlnfuscd with thc Fcnc noinunclaturu llsctl to dcscr11,c thc r n r t a h ~ t r n ~ i c tglutarnntc rcccptors such 21s thc n~C:Ftr,-n~C:lu, sCTIC'Sh. 1hd- ~ ( l.h rnGIu rrccptor functions through C, protclns and cnositc~l
turnover'" arc c l i ~ r a c t c r ~ z cby d sclcct~vu actlvatlcln w ~ t h 1 ainzntl-cyclopcntyl-1,,7-d~carhrlxylatc [ACPDF. Ry gatlng of K t currcnts via rnctal,~)trop~c glut:im:~tc rcccptors, excitatory atnino acids can also act as tory tmnsinl t t crrl" (ccrb R r * r * i ~ ~ ~ ~ r ) r / ~ r rrnt ~ nrrrlc*t s d t ~Ionq t-~~r slow ncrtro~nr~d~lln unrJrr I L ( ; K Clr, 27-49) phosphate.
1 Related sources & reviews I
I
07-5h-OE: Maror ql~r~tutlsol~rccs"" 't"~mY; C;lr!Rs
In h~ppc~campnl r~curr~ncs"~; rnnlccular ncurahlolnm of G ~ U R S ' ~'H-ZN; "~ m(11~~11lar h11110gy 06 lonotropic glutarnatc rcccptrlrs In ~ l r r ~ s r ~ ~ h i l rpcrmcation r'~'; pathways of ncurotransmittcr-gateti ion channclsUY; phystc~lngicaland pathophysiological rthcs of cxcltatory amino acids during dcvclopmcnt"; singlcchanncl recording from i ~ l u ~ " ' ; thcrapcut~c pntcntial of sclcct~vc AMPA/kainatc rcccptor antaRon~sts'57;non-NMnA glutamatu rcccptnrs in g l ~ a lcell siLmallinRH";rolcs of GluRs in CNS f u n ~ t i r ~ n ~ ~ ~ .cxcitatory ~"~'"" arnlno aclrl.; a s cnt!r,gcnt*l~s functional ncurotransrnlttcrs"7T; cxcitatory arnlntl A C I ~actlvatlon of sccontl Incsscngcr systcms In a d d ~ t ~ ot on a tTiruct gattng of Ion ~ h n n n c l s ' ~ ' .Srxrj c ~ l r r ) ~ h r 'Ilr5ct~urr*rF. - lor1 rhhrrnnel I ~ o n k r c { ( l r v ~ w i , v tbrlr , rv h0
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Entry slrpport ,yronps otld e-mnrl newslet tets 07-57-02: Authors who havc cxpcrtisc in nnc or rnrjrc flclds nf this untry [and ;Ire willinl: to pnwldc utIitr)tinl or othor quppnrt for dcvclnping its contcntq1 can Iotn its .iupport group: In this casc, scnd a messaRc To: CSN07@?1e.ac.uk, (ontcring thc wortls "support groi~p"in thc Suhlcct: field). In the intsraFc, pluasu intlicatc principal intcrcsts (scu f~cklnnrnrrhrrlurrrz In I /I(' Iri!rorluc!~on(or r.ovrrr~,qc) t o ~ c t h c rw ~ t hany rclcvant http://www site
cntry 07
links [cstahlishcd or proposed] and dctails of any othcr possihlu cnntrihuticins. En duc coursc, support group mcmhcts will (optionnllyl receive e-mail newsletters intcndcd to co-ordinate and develop the present (tcxt-based) entryJlicldnarnc frameworks into a ’library’ of interlinked rcsources covering inn channel signalling. Other (more Rencral) information of intcrcst to cntry contributors may also hc scnt to thc ahovu addrcss for group distribution and fccdhack.
Snrnmer, Trends Pharmacn! Sci 41992) 13: 291-6. Egehierg, Noturc 19911 351: 745-8. ~ n uter, I Science (1990) 249: P 0.3~3-7. Rettler, Neuron (1990)5: SRd-95. Sakimura, Neuron (1992)8: 267-74. Schoepp, Trends PharmacoI Sci (19931 14: 13-20. Gasic, dnnu Rev Physiol { 1992) 54: 507-36. Nakanishi, Science (1492) 258: 597403. Innas, 1 PhysinI (1992) 455: 143-71. I’ Patneau, Neuron 19Y 1 1 6: 78.5-98. ” Partin, Neuron (1993) 11: 1069-X2. Gallo, J Neirmscr’ (1992) 12: 1010-23. 1 3 Kcinancn, Science [ 1990) 249: 5 5 6 6 0 . Hcrh, Nezirnn (1992)R: 77S-R5. Maycr, Prog Neurohiol (19871 28: 197-276. MacDcrmott, Trend.7 N r u r s c i (1987117: 280-4. I’ Wisdcn, Curr Elpin NeurohioI 119543) 3: 29 1-8. Scchurg, Trends PhnrrnncnI SCF(199.37)14: 297-371k7. l9 N ~ C ~~hy-irnI I I , ~ e ( IvW O ) 70:513-65. Rarnard, Trends PhnrrnncoI Scr 11990) 11: 500-7. 21 Henlcy, Proc Nnll Aced Scr USA (1992) R9: 4806-10. 22 Hcnley, New R m l (3989) 1: 15t%H. Mnnycr, Netiron (1991 6: 799-810. Shcn, Riol Chem [ 1994 268: 190JoL5. Raymond, Nature (19931361: 6,3741. Sommer, Science 19903 249: 158CLS. 27 Mdler, Science (1992)256: 1 5 G M . 28 Rettler, Neuron (19921 8: 257-65. z9 Iomcli, FEBS Lett (1992)307: 1,1943. Larnholez, Neumn (1992) 9 : 247-58. Monycr, Scicncc I19923 256: 1217-2 1. Mcgurn, Nature (1992)357:JM. I S ~ ~ I RroI , hem 19931 2h8: Z H ~ W ~ ~ X R4 Mariynshi, Niture (1991)354: 31-7. Comeli, FEBS Lerr (1993) 315: 3 1 R-22. 36 Wisden, Nciirnsci (1994 (citcd ns in prcss in sourccI. 37 Tiillc, Nerrroscr 11991 1 (cittd ns in prcss in source).
’ ’
’’ ‘‘ ‘’ ’‘
’.’
‘’ *‘
”’ ’’
entry 07
".
---
Kuhsc, FEH.7 1.r'tt [I991 ) 283: 7.3-7.
" "no, I I1hysiol (1990)424: 151-65.
Wochct, Nr~rlron(1 9941 12: 38.3-8, Hcstrin, N c f ~ r o(I~ 9 i 0it [I9921302: 21-5. ",' Matutc, llroc N(lr1 A~,clrlSci IlSA (1992)89: .3.39Y-40.3. " Clark, I Nr~r1ro~c.i (ll)921 12: 664-7.3. ".' Wcrncr, Nllturcj (1 9911 351: 742-4. Hsninssakihritto, Nr,llrosci (1 9'1.1)13: 188%-98. Kristcnscn, FEf3.S f.er f J I'-)c).3\ 332: 14-18 . "'Ray, Hic~chr>r~~ Hir)l,l~ysldRc,TrcnrE\ I)hnrmncr)lSr*r ( I 9901 1 1 : 8 1-6. 156 Chapinan, Nrr~rr>rcrLet( (IYHS)55:325-30. L m , FASEII I (IBY,?\7: 479-85. R~cller,Proc ~Zrr~tl A c d Scl I JSA [ 1993) 90: 605-9. "" Dclchano, Errr Phrlrrnr~col(1993) 235: 2K7-9. *"' Jackson, Trenrl~Npurmc.~(1988) 11: 278-83. HolPmsnn, N{~trlrr(1989) 342: 643-8. Patncau, N t ~ t r r o r c( ~1 902) 12: 59.5-606. 86%Hunzt, Mol I)hr~rmnc*oF( 1992) 41 : 70.7-801. P M Tanahu, Neuron (1902) 8: 169-79. 165 Sugiyame, Nrasrrr)n( 1 989) 3: 129-32. "" M ~ l l c r ,Trcnrlc Phnrrnncol Sci ( 1 991) 12: Su~lyama,Nrlturc (19871325: 5.11-3. "" Charpak, Nr~trrrc(I9YO) 347: 765-7. 16'1 Hunnchcrry, IIroossovc 1 1 9921 14: 465-7 I. $70 Ozawn, Ipn I'hyz104 ( 1 9031 43: 14 1 -50. I71 Rutz, Trcrndr !'hrrrmrrt.ol Scr (199.71 14: 428-31 I72 Cull-Candy, Trends I'hrrrmr~r-olSci (1987) R: 218-24. Hvadlvy, Trrndc Phrlrrnrlco! Sr! 119901 1 1 : 205-1 1. '74 R T ~ ~ ~ c Ns~,T I T O I (\1(YS4) 9: 2073-9. "' Part~n,Mol iJliomilir.rrl ( 1 9041 10: 129-AX.
'3R
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Edward C , Conley
Entry 08
Abstral:t/general description 08-01-01: Prc-synaptic release of glutamate produces an excitatory post-
synaptic currentt (EPSCI] which can he: resolvcd into (i) B fast EPSC
component whcrc the onset and decay i s mediated by nnn-NMUA ionotropicj glutamate receptor-channels (see E L 6 CAT GLU AMPAIKAIN, entry 07) and (ii] a slow [or ’Tong-lasting’) EPSC component mediated hy NMDA-gated receptor-channels 1 NM DARsJ. OR-01-02:Responses of the NMDA receptor-channcl are characterized hy a slnw rise and decay, a large Ca2+-permeahilityi,voltage-dependent Mg2’ black and a rc uircment for glycine (nr a glycnnc-like cndngennus rnoleculej as a co-agonist .
9
08-01-03: Genes encoding cation channels selective for the glutamate receptor agonist N-methyl-D-aspartate(referred to as NMDARs in this entry for convenience) foran part of the extracellular ligand-Rated channel gene superfamily ldescrfhed under ELG Key fncts. e n v y 04). Functional NMDA receptors have been shown to be composcd of a Fundamental subunit, encoded hy the NR1 subunit gene (or cquivalcnt name) and its potentiating subunits (cncodcd by thc NR2A-NR2D gene harniFyt ). Whcn hctcrohgoous!y cxprcsscdt, the NR 1 subunits form hnrnomeric receptnr viirui n t s within popiilrt i ions 08-05-05 Minor drfhcnccs in scqucncc hctwcen the NR2A-D suhunits, poqqihly due to polymorphic variatinnt hctwccn diffcrcnt strains {if rat havc hucn rcprwtctl nnd d i w i w d H .
Subtype classificatinns
1
OH-06-01:For a whtypc chsific;ition hascd on similarity nf NMDAR protcin pnimry I ccqucnccs, v w ~;enra~cirnilv,O H - O . ~
Table 1. Distinguishing features of genes encoding NMDA receptor-channels (From 08-Qti-02) Genelsu bun1t (equivalent nomenclature in brackets)"
Description"
NR1 = NMDARl Homolope:
NMDA 'fundamental' or 'key' subunit. Forms homomerid charnels; significantly potentiated+ by co-expression with anv NR2 subunit.Different NR1/NR2 series cornhinationshave different p r ~ p e r t i e (see s ~ ~other fields)
= zeta 1
(A rrcc.ptor f ( ~ n ? i lmHNAs \~ (Frorn OX- I,?-041
NRl NR2A
NR2R
NR2C
NR2D
o{
Expressed ubiquitously (i.c. almost all ncuronal cells in all hrain regionsl'". T h c hypothalamus contains only thc NRI transcript, suggesting the existence of atlrlitional NMI>A receptor subunits" Wirlcly cxprcsscd in many hrain rcgions. Ahi~nrlantin ccrehral cortcx anrl hippocampus, internal granulc layer of the olfactory ht~lh,nntcrior olfactory nuclci, olfactory tuhcrclc, certain thalamic nuclci, infcrior c o l l i c u l u s , p n t i n c nuclci, inferior oliv;iry nuclci and ccrchcllar cortcx . NR2A mRNA distrihution hears the closest rcscmhlancc tcl that of NRI anrl is prcscnt in both forchrain and ccrchcllum". T h e amyjidaloid nuclci cxprcss mRNAs encoding NR2A and NR2R hut not NR2C" Widely cxprcsscrl in many hrain rcgions, hut morc restricted than NMDR2A. Ahundant in cerebral cortcx and hippocampus, tclcncephalic and thalamic rcgions, hut lowly cxprcsscd in the hypothalamus, lower hrainstem anti ccrchcllumx. NK2H is cxprcsscd in forchrain, and has a complcmcntary distribution to NliZC mRNA". T h e amygdaloid nuclci cxprcss lnRNAs cncorlin~ NR2A and NR2R hut not N R ~ C " ,NR2R-specific mRNA is cxprcsscd mainly in granulc cclls" Shows localized mRNA distrihutic)n in comparison to NMDR2A :ind NMDRTR. Prominently cxprcsscd in the ccrchcllar granular 1:iycr. Motlcratc cxprcssion of this mKNA is sccn in the gl(~mcrul;~r and mitral crll layers of thc main olfactory hulh, Also ohscrvcd in some of tho pontinc, thalamic and vcstihular nuclein. NR2C is cxprcsscd at highest levels in thc ccrchcllum, and has a complen~cntaryrlistrihi~tionto NK2R and mRNA". The ;~mygcl:~loid nuclei cxprcss mRNAs encoding NR2A and NR2R I ~ u tIIO!NII2C:. Siniil;~rly,in thc caudate-putaincn, thcrc is no NR2C: mRNA, hut motlcrntc signals arc dctcctctl with NR2A and NR2R l~rohcs".NR2C-specific I ~ I R N Ais cxprcsscd mainly in tilftctl ;~ntlniitml cclls" Highly cxprcsscd in the dicnccphalic and lowcr hrainstcm (suhcortical) rcgions. High in sift1 hybridization signals arc ohscrvcd in the glomcrular laycr of the main olfactory hulh, the ventral pallidurn, thc majority of the thalamic nuclci, the hypothalamus, superior colliculus, suhstantia nigra, vcstihular nuclci, pontine nuclci, and deep ccrcbcllar nuclci. Low cxprcssic~n is ohserved in the central cortical rcgions and the gmnular laycr of the cerchcllumn
Notc: T h e spatial tlistrihution of channel suhunit-specific mRNAs presumahlv reflects a 'functional specialization' in particular cell types. Mapping of cxprcssion patterns is a complex task anti has to take Inany variahlcs into :iccoiint, such a s in s i t t ~localization, dcvclopmcntal regulation, suhunit stoicliiomctry, ;lntl factors regulating overlapping or co-expression. For notes on the integration of computer-l>;iscd information resources ahle to crossreference thcsc diverse fiictors, sot* Fr.ctll,clck cu) (7SN c.ircr.ss, cntrJr12, mnrj A17[lrsrltli.u / - .%,c~rc.llc.ritvric1 0 1 C S N clc*\,cplopn~cnt,rmtr!r 0.5.
overlap in some hrain rcgions hut also show localized expression pattern^'.^. Anatomical distrihuticins which havc hccn dcscrihcd in thc litcraturc arc givcn in Tahlc 3 (sre ihc ori,pinrtl in situ hvhridizntion s t r r r i i e . ~- rr~i.~"*~*'*. ond ~ h (oornotc c tn Tohlr 3 ) .
''
Phenotypic expression NMDA receptors contrihrrte to n lnrgc number of neurophysiolopicol phenoiypes 08-14-01: Thc demonstrated involvcmcnt or 'association' (if NMDA rcccptors t n several neumphysiological and pathophysiological proccsscs has given grcat impctus for thc dcvclopment of sclcctivc pharmacological modulators of thc NMDAR (scr Kcccptor an!osoni.~ts,08-51) and tcir dcfinitinn of thcsc phcnotypcs at the molecular and cellular Icvcl'"."' (Trthlr 4). A morc extcnsivc list of chmnic neurodegenerative diseases in which iGIuR dysfunction or 'ovcrstimulatinn' mnv play 3 role is included in ~ c f . ' ~ .
Table 4. Fllnctionnl nlki1.v o f N M D A R-chnnncls (Frot?~OX- 14-01) Physiological tunction/pnthaphysicilogic~1phcnntypc involving NMDAR-channels
Sclcctcd rcfs
Developmental synaptic p~asticityt (src hcl(~w and I'rnlr-in intrSrric.tir~ns, OH-31) Learning and memory Trans-synaptic gene expression controlt Normal information processing Srr cllso CollingritIgc, IYHV, rrrldcr Rclrttcd sn1lrcr.v csl rrvirws, OX-56 Sensory ncurotransmission including amplification of auditory responses nnrl ainpliticatinn of excitatory and inhihitory visual signals Anxiogenesis 1 ncvclopmcnt and maintcnance of epileptic+ states Dcvcloprnent of olivopon toccrchcllar atrophyl Dcvclopmcnt of motor neurone disorders ~ ~ ~ o ~ l ~ c a cepisodes rnict Post-ischacmic hmin dnmagc: pathophysiology and ccll death C'nrdic~vascularprcssurc alntrol (haror~cc~tiont ant! control of vasomotor tonet] CcntrnE scnsitiz;~tionduring nocicrptiont (pain reception) (SCP r11.w I,clnrv) Activation and maintcnancc of motor rhythms [ c . ~in . rcspiratory control NMDAR hlockadc cnuscs prolonged inspiration)
u t , SO. 5.7..54.6.1-72 .5.1.6rcvcnticin of glutam;~tc toxicity ( ~ r v I'rotr,irr . intrrrrr.iions, O X - ~ ? l ) . Notit: Alth(lugI1 NMDAK nlny hc the p r r ~ r i o r ~ ~ iroiltc ~~rr~ for~ l~ a " - e n t r y a t toxic Icvrls, other routes nlav hc involveti. o t h e r c:~nrlicl;ltc pathwa y s f ( ~ neurotoxic r ~a"-influx includc Land N-type vnltagc-gated Ca" channcls ( s r , r p VLC; (:(I, ctnrrtr 421, nnnN M D A i(;luRs ( t h r r ~ ~ i gAMPA-sclcctivc h iGluR I(rc.kins GluR-1%subunits . s ~ ( E~ l . ( ; ( : A T ( ; I , / , AMl'A/KAlN, rntry 071, influx channels intlircctly opened hy metahotrnpict glutamate receptor stimulation, ~ a * / ~ a " exchangcrs, and non-spccific rncmhrane leak'". Some of these routes arc illustr;ltcd in Fjcy, 4 rrnrlcr I'mtrin intc,rrlrtions, OH-,?I. N c u m p a t l ~ o l o g i u o cl o n d i t i o n s ru.sociatcd with ' o v c r s t i m n l n t i o n ' o f ionot ropic , y l u t r ~ m r ~rteec e p t o r s 08-14-05: The involvc~ncntof iGluR arc wcll-established in the initiation and and in the t ~ ~ n s s i vneuronal c cell deatht that propaga tion of scizurcst "&* follows pcrir~ds of strnkct-inducetl isrhaemial and hypnglycaemiatpR'" (S(Y Iwloiv). ~ I V S ~ I I I I Cof~ g~II~~tiaInI ~ i n c r ~ ipathways c has llccn implicatcd Ithough not sl~r.c.i/ir*r~lIyasst~ciatctllwith the pathogenesis of a numher of nentoclcgcnerativc diseases (sr,r h c k ) ~ rfull , references listed in Alzheimer's diseaset (tentativrl, Thcsc. tiisortlcrs incl utlc cpilcpsyt'", Huntington's discasel, AIIIS encephalnpathy~/dementia complex, amyotrophic lateral stlcrnsisi and lathyris~n .
'"
entry 08
Time-dependent changes i n synaptic transmission failure in models of 'in vitro ischaemia' 08-14-0(,3 Fivc minutes of oxygen and glucosc deprivation ('in vitro ischaemia') causes long-term synaptic transmission failure (LTF) in the CAI region of the rat hippocampal slice. In huffer containing 2.4 mM ~ a " , thc extent of LTF largely depends on thc average levcl of 'exchangeable' cell c a L ' in CAI during this proccdurc',7". In this model, unidirectional ca2+influx during the 'first 24 min' of ischacmia depenris entircly on NMDARchannels; howcvcr the NMDAR antagonist MK-801 has no effect during the 'second 2f min', prnhahly as a result of NMDAR dcphosphorylation. ~schaemia-inducedca2'-influx during the second 21 min of ischacmia can he partially attenuated Ily -25% hy the voltage-gated channel hlockcr nifedipine ( f i o i l ~ and ] hy an adtlitionnl 35% hy the ~ a ' l ~ a exchange " Thc AMPA/kainatc antagonist DNQX inhihitor benzamil ( 1 0 0 ~ 1 ~Note: ). has nn effect on the ca7-'-influx in this modcl'"".
Total cell ca2+ prior t o in vitro ischaemia is adequate to cause complete LTF 08-14-07: In the hippocampal C A I slicc model of in vitrn ischaemia (previous purogruph), pharmacological hlockadc of enhanccd cal'-entry during ischaemia in 2.4 mM Ca?' has no effect on the LTF phenotype'"". Howcver, thc NMDAR antagonist MK-801 strongly protects against LTF when the buffer contains caZ' at concentrations closcr to physiological lcvcls (1.2 m ~ )A. cornhination of hlockcrs for NMDARxhanncls (MK-801) and AMPA receptor-channels (DNQX) prcvunts LTF in huffcr containing 1.2 mM Ca7'. Thus, AMPAikainate receptor ~ctivation makcs some contrihution to ischaemic damage, although this ApDCArS independent of enhanced ~ a ~ + - c n t r y ~ . " .
L o n ~ c rterm NMDAR-associated neurotoxicity 08-14-08: Slow incrense.~in N M D A channel I:,,, may also provide an excitotoxicf mcchanisrn in that ca2*-influxcan increasc markedly in cells suhject to prolonged depolarization'"' (see below nnd Current-voltage rclntion. 0835).
Extended neuron01 depolnrizntion following removal o f glutamate agonist
u
08-14-09: 'Physiological rcsponscs' of hippocampal pyramidal ncuroncs in primary culture to prolonged (-10 min) exposure to 5 0 0 i 1 ~glutamate show that the ncuroncs remain depoladzed (a20 mV from rest) following 'washout'. This depofarization is accompanied hy a -57.8% increase in mcmhrane conductance, initinllv hy NMDA channel opening and suhscquently through othcr ca2'-influx channels. Since depolarization can he maintained for long periods 1.30 min to < 4 hJ, the phcnomcnon has hcen dcscrihcd as extended neuranal depolarization (ENDJ"'. During END, cells retain 170th the ability to fire action potentials and the ability to respond to glutamate, and can cxcludc vital dyest'?,'.
lndircct neurotoxic effects 08-14-10: In retinal ncurnncs. metabolic inhibition results in the loss of
voltage-dependent ~ g ? hlockadc ' of the NMDA receptor-channels, leading to an incrcascd potency of glutamate agonists"" ((or rlgnil~roncr,svr
Hlock~rs.08-4.31. 'Protection mechnnixn~s' for cxcitotoxic phcnotypc.~ 08-14-11: Extracellular 2n2+dccrcascs NMDA receptor-mediated toxicity1"".
This effect is in contrast to its potentiating effect on AMPA reccptormctliatcrl ncurotoxicity (srpe IJhcnotypic expression under ELG CAT GLIJ AMi'AIKAlN. 07-141. Nitric oxide may havc a role in protection of NMDAmciliatcd cxcitotoxicity (for tlctnils, see Chunnrl modulrition. 08-44). ~ n t i s e n s e oligonucleotides ~ to the NRI sequence havc hccn shown to 'protect' cortic;~lncuroncs from 'cxcitotoxic' reactions hy reducing (i) NRI gene expression and (iil the volume of experimentally induced focal ischaemic infarctionsn4.
I'revcntion o f 'cell death phenotypes' in cells expressing NX IINRZA hetrrotncric NMDAH 08-14-12: Co-cxpression of NRI and NR-2A subunits in HEK-29.1 cells rcsults
in cell clc;~tht, hut viahility can he maintained hy including the NMDAR antagonist 11~-2-arnino-5-phosplionopentanoic acid (AP5) in the culture tncdium post-transfcction" ( s c ~Irane'J2~'"". This rclicvcs thc vnltngc-dcpcndcnt mawcsium hlock of the NMDA rcceptorchanncl, allowing calcium to flow into the dendritic spine. A synaptic model of memoryt involving LTP formation in the hippocampus has hccn proposed (reviewed in ref.").
A n overview o f experimenlol models used i n the study of hippocampol long-term potentiation (LTP) 08-14-26: Within the scope dcscrihcd above (pawgraph 08-14-19), Fig. 1 attempts to summarize aspects of NMDAR LTP phenotypest that
ultimately depend on molecular characteristicst of the rcccptor-channels. Supporting information on scveral subtopics can he fnund under relevant fieltlnames of this cntry and the ELC; CAT GLiJ AMPAIKAIN cntry. ( A hrood illustrrrtion of !he 1ype.s of prc- nnd post-synnptic signalljng proteins internctin,~with N M D A receptor-channels is also shown under Protcin interactions. 08-31 . )
Direct experimental evidence for NMDAR-medinred ca2+-influxi n synaptic plusticity 08-14-27: Optical mcasurcments of synaptically induccd c a 2 ' transients
through NMDA rcccptclrs have been ohscrvcd undcr conditions which eliminate activation of dcndritic voltage-sensitive ~ a ? 'channcls in pyramidal cell dcndritcs within hippocampal sliccs (i.c. steady post-synaptic dcpolarization to the synaptic reversal potential)'". ~ a " - i n f l u x through synaptically activated NMDA rcccptclrs arc ciircctly implicated in thc induction of L T P ~ , since the magnitude of LTP is diminislied when induced with the post-synaptic mernhranc hcltl at progrcssively more positive pcltcntials (with cnmplctc suppression at pcltcntials near $100 ~vI'"~ Note: . Induction of LTP can he blockcti hy injection of intracellular ca2+chelatorst - c.g. microinjcction of EGTA into post-synaptic ncuroncs blocks the induction of LTP'"" and furthcr implicates a dircct rolc for c a L ' signalling in the induction proccss.
'Auqmentntion' o f NMDAR-associated calcium transients and other Caz'' sources 08-14-28: The ~ a * + - ~ e r m e a h i l of i t ~NMDAR-channels t (sec Selectivity. 0840) has Icd to a common nssumption that NMDA receptors play a dircct rolc in potentiation of synaptic rcsponscs. However, vnltage-gated ca2+entry (src VLC: Cn. entry 42) anil ~a?-'-relcascmediatctl hy neuronal ryanodine receptor-channels (scc ILG Ca Cn RyR-Cof, entry 17) and neuronal InsPl receptor-channels (sec ILG CN lnsl'?, entry 19) appear to
OVERVIEW OF LTP INDUCTION
a
I
PERSISTENT ('long-term') INCREASE ('potentiation') of SYNAPTIC STRENGTH
Signal transduction
post-synaptic NMDAR
I mechanisms 1)
I
b
I
EXPERIMENTAL MODELS
Brlel trelns of high-frequency stlmuletlon
I
(i) Typical 'non-decremental' response (LTP)
(ii) Typical 'decremental' response (STP)
Excitatory connections (e.g. perlorent path appllceble to other hippocampal excltstory pathways)
text)
granule cell
synaptlc transmission" see B~ISS, (1973) J Phys101292:331-356
............ .....,,...,,....,,........
Time, hours
c
TIME COURSE OF LTP-INDUCTION AND PERSISTENCE
induction phase: In vivo and In vitro generally
[lor d.scrlptlon of experlmentel meeeuramants, see text; for further dellnsatlon of 'decrementel responses: see Malenka and NIcoN (1993). Trends Nauroacl, 16: 521-5271.
I hour or longer 'Long-term potentiation' Ssnsitlve to protein kinase lnhlbltors, nduclng persistence to 30-60 mln sansitlvo to decraarlng number of stlmuN In tetanus Sensltlva to the degree of post-synaptic NMDAR actlvetion Sensitive to the magnltude and tlmlng of post-synaptlc depolarization
.
/ drstmgu~sh~ng features Subd~v~slons
Durat~on 1
POST-TETANIC POTENTIATION (PTP) Saturation of PTP prevents lnductton of chemically-~nduced potentlatlon' - see note 4
I
NMDARindependent I LTP components i n area C A I e.g. 'Mossy fibre LTP' see note 5 and I mGluRl under Prote~n 1 rnteracbons, below.
.-
1 Note 4: Agents characterlsed as el~c~tors of chernlcaliy-lnduced
YES (note 2)
Note 1: LTP duratlon 1s several days In non-anaesthetlsedanlrnais Note 2: Proteln synthes~s lnhlbltors affect 'exlstlng mRNAs Note 3: lnd~cattnga requlrement for transcnptlonal acllvatlon
I
I potentlatlon tnclude arach~donlcac~d,metabotroplcglutamate I 1 receptor agonlsts, the potasslum channel blocker TEA, calctum Ions I and G protean activators (e g sod~urnfluor~deI alummlum chloride) I Note 5: Sens~t~ve to calc~urnchannel antagonists requlres stronger II I tetanlc st~mulat~on than NMDAR-dependentLTP
NO (note 2)
LTP-2
YES (note 3)
I
I II
LTP-1
Other
NMDAdependent forms
*
"Non-Hebbian" Long-term potentiation (see references In Related sources and reviews)
and
e.p.s.p.-spike (E-S) form of activity-dependent potentiation (see references In Related sources and revlews)
e
ABSOLUTE DEPENDENCE OF SYNAPTIC POTENTIATION ON 'SUFFICIENT' POST-SYNAPTIC DEPOLARIZATION
Typical experimental arrangement:
Experimental condition
(' )
,
Hz
Schematic synapse on to a 'target' dendrite\
,
Post-synaptic 'Target' locus of post-synaptic NMDARdendr~te channels ,*'
-, Pre-synaptic -'stimulating electrode (depolarizing shocks)
Post-synaptic stimulating 8 recording electrode
LTP phenotype
+ no depolarization of
pre-synaptic stimulation
& LTP induction.
Analogous l o weak i n vivo stimuli (i.a. those activating only a few input fibres) not belng able to approach a 'threshold' of post-synaptic depolarization.
post-synaptic dendrites h h
+ (*)
pre-synaptic -
(3)
Robust LTP-induction; simultaneous depolarlzation (even at low frequency) facilitates LTP (the NMDAR-channel acts as a 'molecular co-incidence detector').
'paired' depolarization of post-synaptic dendrites
-
pre-synaptic stimulation
I
Robust LTP-induction; analogous to 'synaptically-coupled' neurons i n vivo (see sections on temoorel summation of svnaotic inputs by the NMDAR under Activation, be1owj.l
I & LTP induction.
There Is an absolute dependence for 'sufficient' post-synaphc depolarization for LTP induction lor underlying molecular mechanisms, see below.
-
stimulation
experimentally during tetanus
1
f
MOLECULAR MECHANISM FOR NMDAR-DEPENDENT INDUCTION OF LTP
'Sufflclently dcpolarlzed' (Strong tetanic stimuli inducing Mg2+a ~ p u l s i o n and agonlst blnding)
Under 'resting' conditions (or weak stlmull or those produced by synaptic inhibition) ( H y pcrpolarizcd)
(sss Panel
'Inaulilclmnt' glutamale/
Extracellular Ilgands (agonirts) bound with concomitant post-synaptic depolarizing 'threshold' reached
F, above)
"':.
-* N~ LTP-~ *duction
-_..
For further detatls see thts field (Phenotyprcexpresston) and the ffelds Protern tnlenctrons Cumnr-vofiage retatton. InaCbVahOn. Blockers, Receptorhnsducer mteractfons. Receptor agonists and Receptor antagonists. See also the entries ELG CAT GLU AMPAMAIN and VLG Ca
v
[ LTP-induction I
.: NMDAR-channel {unoctuplrd b y mgonlnla)
Ilmgnerlum ion
ion
block rlh (wlthln the pore1
GtUtmm*qm (ag0nl.t)
IGlyeine (co.agonlat)
-
augment t h c ~ a "transient associated with the synaptic activation of NMDAR. Thcsc results, togcthcr with ohscrvationson the inhihitinn of LTPhy dantrolene (SVC l'h~riotj,pic r x p r ~ s ~ i olin(Ier n ILG CIICOK,vK-C(lf,17-141 and the ~ a store " ~ a " - u p t a k e pump inhihitor thapsigargin (sec I'l~cnotypic.(.xl)r(*,ssion ttndcr II,G C11 CSIpcndcnihippoc(imp(i1LTP h y K' c17mnnc~ll>lockors i~nhnncin,q,ylutnrnrrt[j rrlctrsc OR-14-31: In hippocampal CAI, ir transient block of Ic mcdiatcd hy NMDA rcccptors in isnlatcd hippncampal ncuronrs m;iintaincrl in coll culturefHR.
Co-localization and co-activation o f NMDAR find non-NMDAR channels at single synapses 08-16-04: Monosynaptic excitatory post-synaptic potentials (EPSPsJevoked hctween pairs of cultured neurones from either mouse hippocampus or spinal cord have dcmonstrated that two functionally distinct excitatory amino acid receptor-channels can he simultaneously activated hy transmitter release from a single prc-synaptic ncuroncrx.'. Thc co-localization of NMDA and non-NMDA rcccptor-channcls at singlc synapses is important for the tlcvclopmcnt of L T P ~ , which may hc diffcrcntislly cxprcsscd s t cach synapse according to the mix of receptor suhtypcs at that synapsefHJ(see Protein interactions, 08-31). Note: As summarized hy Daw et al.'" NMDA agonists multiply (amplify) synaptic rcsponscs (increasing the slope of the rcsponsc curve), whereas non-NMDA agonists add to them (moving thc rcsponsc c u ~ u c wards). Thc rolcs of postsynaptic calcium in thc induction of LTP have hycn reviewed, e.g. rcfs"4, I", 186 . For cxpcrimcntal protocols of LTP' induction, see Phenotypic expression, 08-14.
P
Transcript size 08-17-01: Sec Tullle 1 under Gene fomily, 08-05,
Note: The symbol [PDTM]denotes nn illustrnted fentore on the channel protein domain topography model (Fig. 2).
n
U
Chromosomal location 08-18-01: The human gcnc cncoding thc NRI suhunit (NR1, zeta I ) has hccn mnppcd to chmmosomc 9q84.,1"-'HY with gcncs encoding potentiating suhunits epsilon 1 and epsilon 3 hcing localizcd to chromosomcs I6pl3 and 1 7q,3S rcspcctivcly lHv.
Encoding 08-19-01: For open reading framet lengths of reported cDNAs, see Datnllc~se
listings, ON-.5,7.
Gene organization Alternative splice vnriants o f the filndamental suhunit 08-20-01: Alternative splicingt generates functionally distinct NMDA r c ~ c p t o r s ' ~ ' ~ Analysis ?. of thc gene structure cncotliny: NR1 rcvcalcd eight splice variants arising from (iJdifferent comhinations of a singlc 5'terminal exon insertion and (iil three different 3'-terminal exon deletion^'^.' (for further details. sce Domain filnctionx, 08-29).
A n t ~ l v s i sof N R I Rcnr ' U ~ ? S ~ Y C ( I sI ~~I ~ ~ ~ c T I c ( ~ s ' 08-20-02: Cloning and scqucncc analysisl"'of a 3.8 kh EroRI fragment of the rat NRI gene incli!dcd .? kh of promoteri and enhancert region, cxonl 1 ant1 a portion of introni 1. NRI posscsscs scqucncc motifs characteristic of a housekeeping genet rcgulatcd hy immediate-early' gene products. Two major transcriptional start sitest were idcntificd at 2 7 6 and -238 from the first nucleot~cicin codonr onc. One C;SC; and two SPI motifs, hut no TATA hoxt or CAAT hoxi cxists in the rcgicln proximal to the transcriptional start sitcsi"".
1
Homologous isoforms
08-21-01: Thc human NMDA receptor cDNA hNRl shares high scqi~cncc homology (--c)YY/, with the rat hrain NMDAI and the mouse zeta I ~ u h u n i t ' " ~ ~The . rodent and human h o m o l o ~ i c s diverge near thc Cterminus, suggesting that they represent alternatively splicedl messages of the same gcnc (scc (;rnc or,ri~nizc~tion, 08-20. and I>otol~itsc1istin.y~.OX-53). Of the 7 of Y,?H amino acids which nrc tliffcrcnt hctwccn the rodent and h u m m scqucnccs, three occur in the region of the signal peptide+ and thc othcrs in the cxtraccllular (N-tcrmin;lll dom;~inpreceding the four putative tr:lnsmcmhrnnc scKmcntsl' ( s c ~ irlso irllrrnnrivi, (tivc t r r ~ n s m r ~ m l ~ n ~ n c (/OIZI(II~I) rrioil~l, v l i o ~ 111 ~ ~ /I'DTM/, r~ Fi,y. 2).
Protein molecular weight (purified)
C;lyco,~ylr~rc~d m o n o m e r i c and o l i < y o m y r ip r o t c i n s 08-22-01: Roth native and hctcrologouslyi exprcsscrl rat hrain NRI suhunit have an appnrcnt molecular mass of 1 I6 k n a dctcrmincd hy SDS- PAGE^"'. Chemical cross-linking of nativc synaptic mcmhranc protcins shows that the Nlil protcin is p:irt of n receptor protein complex with a molecular mass c ~ f7.30 k ~ I 1 ~ 'The . NRI rcccptor protein is heavily g ~ y c o s ~ ~ a t (sc7r rdt l)r,lor4.l.
N-C:lyc~o.J!- ~ l t ( ~ r ni\uB ( 1 t .spli(~lt~,y (*\vbnt,x. , s ( ~/l)/)TA4]F'i$y.2). T h e structi~ral variation in some splicc v:~ri:~ntsis shown in T'tl~lc.5.
'
Prcsuncc o f N R 2 scrirls 'potcnrirtting' suhunirs in h e t e m m e r i c c o m p l ~ ~ x rc~protluoc~ cs nat ivc N M D A R p r o p ~ r ics t 08-29-05: ~ o r n o r n u l t i m c r s t formed from cxprcssion of N R I J n (zeta sul>units exhibit scvcr:~l fc:~tiircsof nativc N M D A receptors. Si~nificantly, thcsc i n c l ~ ~ dpronoilnccrl c ~ a " - ~ c r r n e a h i l i t(~~ r , cSr~1r.c-tivitj,. ~ 08-40), a rnotli~lntory ; ~ c t i o no f glvcine ( s c ~(:iir~nrirpl iiiorlr~lrrtion. OH-441, and n ncg;~tivc slopc cond~tct;lncc of currents in t h e prcscncc of M R ~ +(src C l ~ r r r ~ r i t - i ~ o lrc,Irr/ion. / ( ~ ~ v OX-.?5). Sithitnits NR?A, NIi213 ant1 N10C yield prominent, typical glutnmatc- and NM11A-:lctivatc(l currcnts clnlv when thcv ;Ire in hctcromcrici configurations with N R I . NRl JNK2A nntl N R l / NR2C channels diffcr in Ratingt behavinut and magnesium sensitivity. FJctcronicric N M D A receptor suhtvnes nrohahlv exist in nativc ncuroncs.
I
entry 08
Figure 3. N M D A R n~utntionsriffecting M,F.'' block. ~d'-pcrmcnhi1ityand sensitivity to ~ n ' ' und open-chnnnel blockers in hctcromcric comljinntions ol NMDAR. The figure cornpnrcs phenotypes for (n) thc wild-typet subunit cornhination r2/CI; (h) the rnlttant snhunit comhinntion r2/Cl-N598Q: (c) the mutant srihunit comhinntion 62-NL589Q/ \ i t r ~ c r ~ x i ~ l t - 1 1 5 t / I ! ( t A \ \ 1 I 1, , , , * --,,, Jc -J
---,-,---
I
d
0
Figure 4b. Overview of post-synaptic GluR-linked signal transduction proteins. stimulation; , inhibition. (From 08-31-04)
+,
cntry 08
-
Table 7. Summary of NMDAR-ossociatcd signal transduction components (From 08-31-05) Class and suhtypc of protein
Key rolcs/intcraction
Regulatory functions/notes
Adenylyl cyclases calmodulinsensitive
Increased levels of CAMP have been demonstrated following tctanict stimulation of the Schaffcr collateral pathway in the CAI regionm4
Calmodulin-sensitive adcnylyl cyclasc in this preparation depends on both activation of the NMDAR and incrcascs in IC~?-']~*" (SCC111.so 'CRIUI~I~II channels. I~igll-voltagc activat cd', this
tahk) ca2'/ calmodulindependent prntein kinase (C~MKII)"'
Targeted disruption of the gene cncnding rrCaMKII (i.c. gene-knockoutt) markcdly reduces (hut docs not climinatc) induction of LTP in brain sliceszn5 ~ C ~ M K I T - ~mice U I Iare ~ dcficicnt in both L T P ~ induction and spatial learning hut show no Rross morpholo~ical changes in the hrain
rrCaMKII i s highly exprcsscd in post-synaptic densities. Primary scqucnccs of cloned NMDAR cxhihit conscnsus~ phasphorylstion motifs for CaMKII (see /I'DTM/. Fig 2 nnd Protein phosphorylntion, 0832)
rlCaMKII is also likely to 'positively-modulate' AMPA/kainatc iGluR receptor-channels (see 'Non- NMDA-type. ionotropia glutmn~otil rcccptors', this t a l ~ l rnnd ELG CAT GLU AMPA/ KAIN, cntry 07) Calcium channels, highvoltage activated (scc V1,G Cn, entry
Calcium influx through both NMDA and non-NMDA receptor-channels in c u l t ~ ~ r crat d hypothalamic ncurones activates a calmodulin-dependent inhibition of the high voltage-activated (HVA)~ a " currcnt2""
Compr~rr~tivc notc: NMDA rcccptor activation has hccn shown to incrcasc CAMP lcvcls and thcrchy the fractional open timct of high-threshold ~ a ? + channels in CAI pyramidal 'ccl
cntry 08
n
Table 7. Conrinurd Class and suhtype of protcin Calcium channels, voltage-gated, type, N presynaptic (we VLG Cu, Pntry 42)
Key rolcs/intcmction
NMDA receptor agonists have hcen reported to sclectivcly and effectively depress N-type ca" channels which modulate neurotransmitter release frnm prc-synaptic sites2"' (see VLC: Cn, entry 42). The inhihitory effect is eliminated hv the competitive NMDA antagonist n-2-amino-5phosphonovalcratc (APV) and tlocs not require c a 2 ' cntry intn the cell
Implies a 'negative feedhack' hetween liberation of excitatory transmitter and cntry of c a 2 ' into the cell, modulating prc-synaptic inhibition and regulating synaptic plasticityt '"'
Calcium-store release channels: Rvanodine receptors lsrr 1LC: Cil Co RvR-Cal, cntry 17) ond InsPl receptors (sec 1f.C: Cn InyP,. pntry 19)
Pcrfusion of dantrolene on to In ccrcbcllar gmnulc cclls a major component groups of cclls during thc sustained plateau phase of of hoth K ' - and NMDAinduced elevation of ~ a " the Ica"], response to K' or involves release from NMDA reduces the response intracellular stores2oR. to hoth agents in a concentration-dcpcndcnt The ~ a " - s t o r e dcplctors thapsigargin (which marine?"'. Note: Dantrolcne is uscd as a clinical antidote blocks thc action of the ~ 3 ' 'ATPascl and for ryanodine rcccptorryanodine (src !LC: CN med~atedmalignant R,vR-CoI, cntry 1 7 ) hyperthermia (see [LC: Co display partial additivity Cn R,vK-Cilf, cntry 171 to K t -and NMDAind~iccdresponses, showing that thcsc aRcnts affcct two overlapping hut nonidentical ~ a "pools
Cytoskeletal proteins
A protein interaction hctwcen actin and NMDA channel regulatory proteins can affect the 'nindown' phenotype of NMDAR in native cclls
Scc Rundown. 08-39
-
Table 7. Contin~red Class and suhtypc of protein
Key roles/intcraction
Regulatory functions/notcs
Endonu~leases'~'
Scveral ca2+-activatcd enzymes may contrihute to excitatory amino acid toxicity, which may includc the activation of various cndonucleasest
Regulation of ca2+-influx appcars fundamental to thc 'ordered progression' of gcnc expression; dysrcgulation of ~ a "homeostasis is also likely to have longcr term effects on cell phcnotype or the initiation of apoptosist [see also 'co2+-scnsitivc pro tease.^', this tnhle)
G proteinFor role of GARAR receptors coupled GABA in low-frequency (-. 5 Hz) receptors, induction of LTP (see (SARAR Reccptor/tran.sducer subtypes interactions, 08-49) G proteincoupled glutamate receptors (mctahotropict glutamatc rcccptors, mGluRl
mCluR which activatc protein kinasc C appear to lowcr thc threshold of induction for LTP (see rer~icw,ref.",? and 'lJrotcin kinnsc C', this tahlc). Targctcd disruption of the gene encoding rnCluR, in micc having sevcre dcficits in motor cn-ordination and spatial learning2""
G proteincoupled muscarinic receptors (MI
Muscarinic receptors which See Appendix A - Index of G activatc protcin kinasc C protein-linkcd rcccptors. appear to lower the threshold entry 56 of induction for LTP (seu rcview14-' and 'Protein kinasc C', this tol?lo)
G protein-
14-opioid rcccptor agonistst potcntiatcr NMDARactivatcd currcnts in trigcminal ncurones of rat medullary slices, probably via activation of protcin kinasc C (scc tliis tahlr nnd ref.'10)
coupled opioid receptors, 11subtypes
~ G I ~ R - n u micc l l t have no gross anatomical or hasic clcctrophysiological ahnormalitics in cithcr the cerchcllum or hippocampus, hut show impairct! cerchcllar long-term depressiont and hippocampal mossy fihrc long-term potentiationzop (see Fi,p. Id). See also Rcceptor/tmnsd~rcer intemution.~.08-49
1,-opioid potcntiationt may hc a feature of synaptic plasticity1 ohscrvcd in central gain reception pathways21o (.?PC I'hcnntj~pic oxpressinn, 08-14, mnd Channel modulation, 08-44)
Class and suhtypc of prcltcln
Kcy rolcs/intcraction
GARA* (inhihitoryl receptorchannels (,s(.r, E l . ( : CI
In most cortical ncuroncs t h e nctiv;ltion of t h e NMUARs (and hcncc the induction of LTP1 - srv,
R c ~ u l a t n r yfunctions/notcc
Morph(~logicaldamage and psychotominicticr effects indilccd hy NMDA-active d r u ~ can s he prcventcd hy I'l?r,nr,tj,picc,xy)nb.scion.OH- 14) other drugs which act a t t h e ( ; A H A A .~'rltr!, rcquircs :I concotnit;~nt gnmm:i-;iminohutyric acid 101 rcctuction of ~ ~ 1 3 h c r ~ i c t (C7ARAA)receptor-channel inhihition hy low tloscs of t h e complcx2", indicating a CAIIA* ;tnt:igoni.;t functionnl inter;~ction h i c ~ ~ c n l l i n eThis ~ ~ ' .inriicatcs hrtwccn these channel types in viva Isrr. Rrcrptor th:~tin the ncocortox t h e activation threshold' c ~ the f r~nrr~A-nctirmtcd c u r r c n t s 08-33-01: NMDAR-channels elicit the slowly rising, slowly decaying ( r several hundrcd r n i l l i s ~ o n d sopen state1 1 ctrmpc>ncnt of cxci tstory postsynaptic currents (EPSC:s7 l in rcsponsc to glutamate (sc'c* I t i i ~ c ~ t i ~ ~ o tOXion, 371. In comparison, A M P A receptors niedi;itc the most rapid synaptic cxcit:~tory ncurotransn~ission~ ;lnd conduct mainly Na' currents (src E1.G CA.1' ( : I , l l A h4I'A Ih'AlA'. c,ntrrr 1/71, l n ~ r i n s i cpropcrl ics o f NMIIA R-c.hrlnn(~1.s drt c r n ~ i n cd u r n 1ion of currcnt 08-33-02: Intrinsic NMIIAR channel kinetics dctcrminc the time conrse of NMDA receptor-11icdintc.d synaptic c i ~ r r c n t s " ~ ~For . ~ ~example, ~. kinetic responses of NMDA r e c e p t o r s i n cxcisctl mcml~ranc patches from hippocnmpus nnd supcrior colliculus show simil:~ritics to that of the NMIIA E I I S C ~suggesting , thc time coiirsci of the NMDA EPSCi rcflccts slow NMI)A channel properties in this prcp;ir;~tionJ". ' K c - l ~ i n t l i nof ~ ' ,q1litn1i1ritr l o ~ h Nc M l l A R fro171 thl' s,vn(lplic cleft ~ O P . S1701 OC('1lt
08-33-03: Rricf pulsrs of glut;~mntcapplied to outside-out rncmhr;lnc patches results in openings of NMIJA chnnnels that persist for I~undrcds o f milli.si~i.on(l.s, indicating that glutamate can rrrrlr~in I ~ o u n d for this pcriot12". Current rise' and decayt is mnrkcdly temperature-dependent, intlic:~tingthat changes in rates of free transmitter diffusion cannot ;ilonc ;lccoilnt tor its time
(:Iutarnotc (117ti ~l!~cirii'(1c.t iv(lliP 1 1 1 NMI>AlZ ~ (IS indcpcnrlcrll c o ogonisl s 08-33-04: Occupation of ;i separate, allnsteric 'glycine receptor site' is also an absolute r c q ~ ~ i r e m e nfor t NMDAR activation (vcc*K(;.c:ptor ( ~ ~ o n i s tOX-501. s. T h c c.onc.r,r?trl~tioriof ~ l y c i n cat the synaptic cleft1 is I>c\ocv a saturated level. In trigcminal ncuroncs, cxtcrn:il C:I" contrihutcs to unusunlly high glycine affinities for NMDA receptors (potcntiatic>nt c~ccuning when glycinc sites ;Ire unsatumtcd - scc E r ~ f ~ i ~ i I l r idi<socir~tion tlm coristnnt. 08451. This form of c a 2 + mndlllation may havc a rolc in regulating thc NMIIA receptor-ch:inncl activities during intensive or sustained ncuronal stimul;ition2'". Analysis of NMI3A clisnncl activation kinetics in outsidcout patches of cultilrrd hippocampal ncuroncs (following mpid steps into . Glul has dctcrniincd the high conccntr;~tionso! glut;~niatc,c . ~ -20011~ ;~ctivnt/ontime c o ~ l r s c to ' he concentration-inclcpcnilcnt and limited hv transitions' hctwccn the shut (hut 'fullv-ligantlctl'l state nil the open s t a t c l . Kinetic motlrls which c:ln :Iccount for activation kinetics following glutam;itc concentration jumps havc hccn derived for NMDAR in hippocam pal n c u r o n c ~ ~(vcc, - ' ~ F I X . 51.
'Temporal summntion ' of synaptic currents relieves ~ p - b l ~ hy c k depolarization 08-33-05:Single, brief applications of glutamate arc sufficient to producc an extended activated state of sever01 hundred millisecond^, whosc transition 10 pM Glutamate 2 -
#
MIC A
MIC
of bindins sites
5 pM Glycine
10 pM Glutamate + 10 WM Glycine
I
Figure 5. Mode1.s nccounting for N M D A R nctivot ion kinetics following low agonist concentrations ( e . ~ . ngonist concentrrttion jumps. At rc21r~tively -2-If) I'M ~ltltnmnte)a two glutamate-binding site model can flccollnt for nctivntion kinetics followins glutcrmntr~ conucntrntion itimps (upper panel)2~'7.A two ~lycinc-bindingsite model cnn also he fitted for channel nctivntion in the continuous presence o f glutnmnte (middle pan~1)2". Agonist and co-n~onist l~indin,p kinetics nre better described l7v nn independent co-agonist binding model, rr~therthnn n scqtrentiol hindins niodrl (lowcr pnncll"". Datn points shown represent npproximntelv thr first 30 nin following mgonist nddition, with fitted model predictions indicrrted hv arrows. MIC denotes rtddition o f 2.5 mn4 methoxyindole carboxylic acid used here to ellminnte spurious grltlng rit~e to glyc.ine contr~minr~tion from solutions (see Receptor nnto~onists,08-fil). Note: Thesc studies predict the NMIIAR to he nt lenst tctrorneric, contninin,y four liwnd-binding subunits, oasum in!: a sin,ylc binding site prr sul~trnit"~.(Ilr~procluccriwith prrmission from Clrmrnts mncl Wcsthrook (199 I ) Neuron 7: 605-18.) (From 08-3.3-041
entry OX
-
t o the closed state is independent of the c o n d u c t i ~ gstate of the channelz3'. This modc c ~ gating f cwlscs temporal summatian1 of synaptic inputst (and conscqucntinl: rlcpolarizatirlnt). Notr.: Summation can cxplnin why 'singlc shock' sti~nulationof nffcrcnts is not ns cffcctivc ns natur;~](rcpctitivc] stinii~latioii,since sin& pulses do not 'sufficiently depnlarizc' ncuroncs t o rclicvc t he M ~ ' ' hlockH' ({or siqniiicrlnt.r, srr F ~ R . I ru rirltl I{lor.Kcrs. OX-43).
Nr~rnhc~rs o f N M D A c:hrjnnt.I opcnirtgs rc!cplirrti to ,ycnc>rrrtc
(In
EI'SC~
08-33-06: Rricf applications of ~ c l u t a m a t cto outside-outt patches from
hippclcampal ncuroncs in the prcscncr and ahscncc of the opcn-channel hlockcr MK-801 havc shown that about .3B'Y0 of the L-glutamatc-hound channels arc open at the peak of the ~urrcnt'"~.Tho high prohahility of opening for NMDA rcccptor-channels following stimulation by LRl~~tamntc2"H sumcsts that rolativcly few channels arc required to 'guarantee' a large, Iocnlizerl post-synaptic c;~lciumtransient.
A~onistsprohnhly remain hound d u r i n ~'supc.rc/ustering' 08-33-07: N M D A receptors havc an unusual propcrty of binding ccrtnin
agcmists (inclutiing gliitarnatcl h)r a long period c ~ ftimc. This property may ] a partly explain why I~ricf(-1 ms) ;~pplicntionsof glutamate [ l m ~prtiducc slowry decrying current, the major component of which has a timc constant of -200 ms. ' ~ u ~ c r c l u s t c r i n ~ hchaviour 't ohserved at low i ~ n ~ ON-41) l may correspond glutamate conccntr;~tions(scr S i n ~ y l ~ - r . l ~ i rclilr(!. t o ;I singlc pcrioti during which one or tnnrc mc~lccralcsof glutanintc arc tlound tt, the rcccptc~i4".
-
Current-val tage relation E f f ~ c tosf difkrcnt cxtrclccllr~l~r inns on 1-V relllrinnships closc to E,,., 08-35-01: T h e response nctivatcd hy NMDA sgonistst cxhihits n voltage-
dependent cxtraccllular ~ g "hlock. Ca"-influx is restricted unricr resting cc~ntlitions, hut post-syniiptic membrane depolarization can rcmovc the hl{lck. Furthcriiiorc, thc largcr thc driving forcei tor M ~ ' + to pcnctrntc tho , Illr~ckcrs.08-4.7). nicmhranc, the largcr the t~lock( f o r frlrthc~rr f ~ t r ~ i l ssrc However, in tight-scnlt, wholc-ccllt r c c o r d i n ~ sof cultured spinal cord and hippocnmpal ncuroncs, high concentrations [ 2 0 m ~ of ) ~ n " ant1 Ca'' display linear I-V relationshipst [within +IS mV of the rcversal potcntintl althouxh thry d(i reduce slope contluctanccs. Ry contrast, extracellulnr ~ n ' ' ions produces a strong, voltagc-dcpcntlent block o! responses to NMDA, such that cvcn close to the rcvcrsal potentiali, the NMDA currcnt-vt~ltagc rclationshi is high1y nt~n-lincaP4'.
Tlir. slow r~oltr~~c-depcndc~nce of I', , ,
in ~,y*'-freesolr~tions
08-35-02: Studies of non-lincart wholc-cellt I-V curvest in free solutions hnvc shown that NMDh channels in cxciscd pntchcs rcvrrsihly shift their I),,,,.,, in n vriltaxc-tlcpcnrlcnt ~ n a n n c r(i.c. they cxhihit -,I- to 4folrl Arc;Itcr I),,,,,.,,at positive pc~tcntialsthan at rcstl'?'. Changes in I),,,,,,, nrc ~n:linly ;ittrihutahlc to shifts in openinp: frequencyt: I:,,,,.,, changes over a s c~l>scsvcd - 'vcrv slr)wJ time crlursc 1-2-15 niinl which ~ ~ n i l r r l i c the
entry 08
hysteresisf of whole-cell current-voltagc curvcs ohtnincd undcr noncquilihrium 1i.e. non-steady-statct ) conditions. Thc slow increase in P,,,,, provides a potential cxcitatoxic mechanism in that CA"-influx can increase markedly in cells dcpolarizctl for prt~longctlperiods of time1.".
Quontitotive N M D A R nciivniion models precIiciin,q E,., nnd cap*infltlx n t different v o I t a ~ c s~ n Ic~'"],, d 08-35-03: In accordance with a quantitative model dcvcloped for NMDARchanncls cxprcsscrl in cu!turccl! hippocampal ncurt)ncs2'*, increasing [ ~ a " l , , markedly shifts the reversal potential to positive values and simrrltmneortsiy dccrcascs thc single-channel conductance at potcntials ncgativc to thc rcvcrsnl potential. Using the model, rclativcly s i ~ p l c quantitative descriptions of calcium pcrmcationt anrl channcl hlackl hy calcium ions can account for ohscrvcd channcl hchaviour and accurately predict rcversnl potcntials and mngnitudcs of calcium influx over a widc rnngc of condit~ons'~~.
Dose-response Thrcshold.~for nctivation of ,qlutamatc receptor-chonnel subtypes in mixed populations 08-36-01: In dorsal horn ncuroncs, activation of ~a"'-pcrmcahlc NMDA receptors cvokcs intraccllular c a 2 ' transients that arc largc 1-780 nA], rise at a morlcratc ratc, and maximize amplitudct at NMDA conccntrations of - , 3 0 0 / 1 ~ . Whcrc mixed subtypes of glutamate scccptclrs arc cxprcsscd, glutamate rcsponscs at conccntrations less than 3 1 1 ~arc d ~ l crxclusivrlv to NMDA receptor activation2*". At higher glutamatc conccntrations, intraccllular rcsponscs arc mcdiatcrl hy I~r~thNMDA ant1 non-NMDA rcccpt~rs~~~'.
Inactivation Inactivation parameters shfipe decay times o/ N M D A receptors 08-37-01: T h c characteristically slow decay of NMDA-mediatcd EPSCS~
appcar to he duc t o (i)persistence of bound glutamate and ( i i ) the long open state of the channels. In hippocampal ncuroncs, repetitive stimulation ot glutamate receptors clicits increasingly smaller ionic currents. For example, in thc prcscncc of 2 . 2 n - 1 ~Ica2'],,, repetitive glutamatc applications ( 1 5 episodes of 4 slmin) elicit progressively smaller currents which stahilizc at -45% of their initial peak This 'interepisode inactivation' is cxacerhatcd hy elcvating extracellular ~ a "to I 1 mM, and is attenuated hy reducing extracell~llar ca2' to 0 . 2 2 m ~ . Current decay shown during individual stimuli ('intra-episodeinactivation') is ciepcndcnt on cxtraccllulnr ~ a ? yet ' remains stat~lcduring rcpctitivc stimuhtion. Thus, inter- and intra-cpisodc inactivations of NMDAR currents result from two distinct proccsscs triggered by ~ a " . Thcsc 'modalities' of inactivation may arise from ~ a - "hindiny: cithcr to the - reccptor or to closely associatcil rcgi~latclrv
I
cntry OX
Distinctions hct~vccnNMDA rcccptor-cl~(~nr~c.l rlcscnsitizrltion nnd intlctivntion 08-37-02: T w o distinct mechanisms have hccn s u p ~ y s t c dfor modulation of N M D A receptors by intracellular ~a"'"'. I)c~~c~nsitizc~tir,n nf N M D A rcccptors is inducetl when I>oth ~ntr;lccllul;~r C;I" is increased ant1 NMIJA receptors activated hy ;igonist. Note: T w o types of steady-state desensi-
tization for t h e N M D A i~gonistsaspartntc and glycine 1i;lve hccn shown in isolated rat hippocamp;ll n e i ~ r o n e s ~ ~I n" .c ~ c ~ t i r ~ r ~oft i oNr ~M D A receptors is produced hy increased levels of intracellular C;I" hut docs not require NMLJA rrccptor activation for intli~ction'~' /scsc. l~c~lrnc,).
'IIo~~~-rc:qulr~tior~ o f jlost - s y t ~ t ~ j ~~ (t1i.c" -c1t1trj7 08-37-03: Studies of calcium-dependent inactivation of N M D A channcls in
cultured rat hippocampal ncuroncs h;ivc suggcstctl a mechanism for downregulation of post-svnaptic c a l c i i ~ n ~cntry during sustained synaptic a c t ~ v i t ~ ' ~In~ .norlnal [ ~ a ? ' ] , (, 1 - 2 m ~ land lO/rhr glycinc, macrnscopic currents evoked hy 15 s applications of N M D A (10/rh~linactivate slowly following a n initi;~lpe;lk. At SO rnV in cells huffcred t o I c ~ ' ] , < 10 M with 10 mh.1 EGTA, t h e in;~ctiv;~tion tirnc constant r,,,,,,., is -5 s. Inactivation does r ~ o t occilr ; ~ t~ n c ~ n h r ; ~potenti;lls ~ic ot -140 mV and is ahscnt at IC;~' ' ] ,, 0.2 mhi, s u ~ e s t i n , 1 hJ. Notahly, open-channel hlockers which arc well-tolcmtctl clinically, such as memantine (sc,c /~c,low)leave ~. the channel promptly ( 1 ,,? > 5 s at micromolar c ~ n c c n t r a t i o n s ) ' ~C.ARAAselective drugs h;lvc Iwcn ohscrved to reduce the psychotomimctict symptoms caused hv ketamine. 08-43-07: Memantine, an adamantane, derivative related to the anti-viral dnig amantadine has hccn shown to hc an open-channelt hlockcrt for NMDA receptors. Adamantanc is well-tolcmtctl clinically unlike othcr openchannel hlockcrs like MK-801 (dizocilpine)"". Norct: The anti-viral drug amantadine is also i~scrlin the treatment of Parkinson's tiisease, hut is consirlcrahlv less potent than ~ncrnantincat clinically tolcmtcd tloscs.
Non-selcctivc. suppression o f cxcitntorj~nmino ricid receptor-channel cL1rrcni.v 08-43-08: In oocytcs uxprcssing mouse hrain mRNA, enflurane at an ;in;~csthcticconcentration (1.8 mhrl inhihits NMIIA-, AMI'A- and kainatc-
induccd currcnts by 29-40"/0, 3 6 3 3 % and 20-27%, rcspcctivcly, suggesting that all three glutamate ionotropic receptors arc susceptible to suppression by inhalational anaesthetics2". Inhibition by cnflurane is inkpcndcnt of thc concentrations of the agonists (NMDA, AMPA and kainatc] or the NMDA co-agonist (glycinc), suggesting that cnfluranc inhibition docs nt~r result from a competitive interaction at glutamatc- or ~lycinc-hindingsitcs. Enflumnc also supprcsscs the oscillation and 'apparent dcscnsitization' of NMDA currcnts, suggesting an inhibition of ca2*-influx through the NMDA channc12".
Drlal srlppression o f EL(:-channel cind voltage-~atedchannel currents h y morpllinans 08-43-09: The morphinan dextromethorphan and thc related molcculc
dextrorphan hlock NMDA-inducctl currcnts ant1 voltage-operatcd inwart! currcnts in culturcd cortical ncuroncs and PC12 cells272. Dextromcthorphan is at least 100 times more potent (ICSo 0.55 I ~ M as ) a hlocker of the current induced by NMDA in cortical ncuroncs than for voltage-gated ~ a channds " in the same preparation (ICso 80 I'M). Notably, this class of hlockcrs arc well-toleratetl clinically'2v. The net~roprotectiveeffect of tlcxtromcthorphan (which occurs in a concentration rangc of 10-100 I ~ M )may thcrcforc be due to a complete blockade of the NMDA rcccptor-channcl and a partinl inhibition of voltage-dependent ~ a ?and ' Na' channels"-'.
-
-
Non-selective channel hlock h y TEA 08-43-10: The
organic cation tetraethylammonium (TEA, 1-5 m ~ ) antagonizes NMDA responses in culturcd mouse cortical ncuroncs in a conccntmtion-cicpcndcnt manner. TEA causes voltage-dependent dccrcascs in single-channel conductance and the frcqucncy of channcl cvcnts is also decreased. Thcsc effects arc not accompanied by any change in average channcl open tim~t'~.'.
Channel modulation Protein kinase C modulation o f N M D A R chnnncl currents jn pain reception 08-44-01: Phosphorylation hy protcin kinasc C (PKC] has hccn reported to enhance NMDA receptor-mediated glutamatc rcsponscs in a number of '4.3.210.22" . For example, in the trigeminal subnucleus caudalis, a centre for processing pain-sensory (nociceptive) information from the orofacial arcas, a p-opioid receptor agonist causes a sustained increase in NMDA-activated currcnts hy iictivating intraccllular PKC (see nl.w I'hcnotvpic expression. 08- 14). Protein kinasc C appears t o rcducc thc voltage-dcpcndent ~ g " - b l o c k in wind-up'tY7 (c.g. triggering of a dmmatic increase in discharge following tissuc injury clr rcpctitivc stimulation of small-diameter afferent1 fibres (we P11cnot)pic expression. 08-14). Thus protcin kinasc C potcntiatcs NMDA responses by increasing IJ,,,,,, duc t o reductions in ~g"'-block": A role for metabotropict glutamate receptors in PKC motlulation of NMDA-mediated processes has also hccn tlcscrihcti"" ( S ~ PC I t ~ n n r dcsignr~tion, l 08-03, rind I'rotcin intcrrlc.rions. OH-31).
entry OX
Modnlntion o f LTIJ proc.rlsses h,v protcpin kintlsc (I(-tivation 08-44-02: In t h e post-synaptic cell, hoth activation of calmodrllin and kinase i z~ctivity arc required for t h e grncration of LTI'"'.'-" (SPC I'lli.not,vpic cxpri,ssion, OX-141. Extrc~ccllulr~r application of protein kinase
inhihitors t o t h e hippoca~npnlslice preparation has heen shown t o hlock t h e induction o f LTP. Intrncpllrrlor injection of t h e protein kinasc inhihitor H-7 i n t o C A I pyramidal cclls also hlocks LTI', a s docs inicction C o n t i n u o i ~ sinflux of ~ a ? ' of t h e calmodulin antagonist calmidazoli~rn~~'. through t h e NMDAR c;In h c prcvcntcd hy prior treatment with t h e protein kin:lsc C inhihitor sphingosine"'5. LTI' is also hlockcd hy inicction of synthetic pcptiilcs ch;iractcrizcd :is potent calmodulin antagonists prcsumahlv hv inhil~iting CnM-KII ; u ~ t o -and suh.;tr:~tc phosphorv1;ltion
IJrotcin pho.xp/~ori,/(~ / io11,08-s32). Nc,q,~ativcrind positive modrilntion o f NMDAR r c d u c i n ~nfpn t s
17y
oxidizing rlnd
08-44-03: NMDA responses in several tliffcrcnt nci~ronalpreparations are
'suhstantinlly rlccrcasctIf following exposure t o t h e disulphidc reducingt agent dithiothreitol ( I j T T , 0.1-1 0 in^), while oxidation with 5,s-dithiohis-2nitrohcnzoic acid (IINTR, -500 p h i ) can potcnti:ltcf the magnitude ot t h e responsr~T'. 2-(, . MotIific;~tionc ~ tf h e NMIJA response hy cithcr oxidation or rctluction a t this redox modulatory sitep7' does rlot nppcar t o affect t h e pllclrriiclcoIcl,yic.c~lproperties of the rcccptor-channel complex. Since redox statei of t h e native NMIIAR varies widely among ncuroncs, rcg~llationof NMDA:~ctivatcd ftlncticlns by i,itllcr reduction or oxidation inav operate in r,ir,r,27".?" (SVI'
Ill.~O1 1 ~ 1 0 ~ ) .
M c c l ~ ( ~ n i somf N M D A rrlc*r.prorhloc.kodr1 1 7 ~ 7nitric. oxidr at the redox n?otlrllntory ,sit[' 08-44-04: Clinically iinportant nitroso comporlnds thnt gcncrntr nitric oxide (NO*, whcrc t h e tlot signifies one frrc electron) have hcrn shown t o inliihit responses mctli;itcd hy t h e NMDA-sclectivc g l u t n n ~ a t rreceptor o n rat cortic:~l nci~rtlncs in rfitro"v"77 . It has hccn proposed that free sulphydryl groups o n t h e NMIJAR rcact t o form one o r more S-nitrosothiols in the presence of nitric oxidc. For cxamplc, rcaction of NMDAR free thiol groups w i t h nitroglycerin via S-nitrosylation has hccn s h o w n t o m o d ~ ~ l ; ~protein te function in a n anal!)~ous manner t o protein phnsphorylation' o r ncylatic~nt2". If vicinall thiol groups react in this way they mil? form dis11Iphitlc hands, constitllting t h e rrdox modulatory site f of t h e r c ~ c p t o ? ~ ' . h'otr,: Fommatic~n of tlisulphitlc honds would result in reduction 01 c;I"-influx in rcsponsc t o NMtJA. Thus, rcaction with nitric oxidc appc;irs t o protect cclls from N M D A receptor-mediated ncurotoxicity277. C:onvrrscly, rctli~cingcontiitions favour the formation of f i e thiol I-SH) groups, and this condition is prcscnt following hcing nssociatrtl with hoth a net increase in C a 2 ' - ~ n f l u xthrough NMDAR c h a n n c l ~ ~ ~ "ant1 - " ~hixhcr ~ npp:ircnt n e i ~ r o t o x i c i t ~(srr ~ ~ 'rllso I'hrnotypic r~xpr~~,ssion. OX- I J ) .
entry 08
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'Reversible' s u l p h y d r y l redox m o d u l a t i o n of NMDAR in r a t c o r t i c a l neurones 08-44-05: Reversal of the effects of sulphydrylt oxidizing agentst hy DTT (see cjhove) can occur over a rapid time course (t1/2 0.6 min). Spontaneorislv oxidized receptors can he further oxidized with DTNB and 'fully reduced' with DTT. When the redox modulatory site275 or sites arc alkylated with N-ethylmaleimide (NEM, 300-500 j i ~ )following reduction with DTT, responses hccome 'permanently potentiatedt' and largely insensitive to oxidation hy DTNR. Blocking effects of protons and potcntiatingt actions of glycinc arc unaffected by alkylationt, and Zn" and MRL' ions produce a significantly weaker hlock of the NMDA whole-cell r e s p o n ~ e ~ ~ ~ ' .
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M o d u l a t i o n of N M D A R-channels by (endopenous) z i n c i o n s 08-44-06: Roth pro-convulsantt and depressant{ actions of z n 2 + ions have
hccn reported (sce ref.2""). Zinc ions are potent non-competitive antagonists of NMDA responses in cultured hippocampal neuroncs at relatively high (micromolar) concentrations2"". Unlike ~ g " , the cffcct of z n L ' is not voltage-sensitive hetween -40 and +60 mV, suggesting that ZnL' and Mg" act at distinct sites. z n 2 ' could also motlulate ncuronal cxcitahility hecause it is present at high concentrations in hrain, especially the synaptic vesicles of mossy fibres in the hippocampus and is rclcascd with ncuronal a c t i v i t y . Comparcjtive note: Zn" ions also antagonize responses to the inhihitory transmitter GARA in the hippocampus ( s ( ~E L G C1 GARAA. cntrv 101. N M D A R - p o t e n t i a t i n g effects o f zn2+ions 08-44-07: Zinc ions may also he positive modulators of NMDA receptors in
certain regions of the brain2?'. Analysis of ~ n "modulation of currcnts through homomerict receptors assemhled from different splice variants of NRI suhunit show that, in addition to its well-charactcrizcd inhihitory effect at high concentrations (src ahovc), z n 2 ' potentiates agonist-induced currents at suhmicromolar concentrations (EC5(,= 0.50 /(MI. Potcntiationt is ohserved only with a suhset of NRl splice variants1"". Zn" potc?tiation is mpidly rcvcrsihlc, voltage-indepcndcnt~ and non-compctitivc~ with glutamate or glycine. Zn?' potentiation is mimicked hy cd", c u " and ~ i " , hut not hy ~ n " , co2', ~ e ~~ ' n, "or ~ g ' +lY.' . Note: For a description of the molecular 'loci' affecting ~ g " - b l o c k phenotypes on the NMDAR, see Domoin functions, 08-29, A u t a c o i d m o d u l a t i o n of NMDAR 08-44-08: The ether phospholipid platelet-activating factor (PAF, an cthcr phospholipid autacoidt) induces a stahlc, concentration-clcpendcnt longterm potentiationt (LTP) in hippocampal s l i ~ c s " ~ . I'AF-induced LTP is blocked hy antagonists of the PAF ant! NMIIA receptors, hut the former antagonists do not block LTP induccd hy high-frequency stimulation. Facilitationt induced hy PAF cannot hc reversed hy PAF receptor antagonists2". For an introduction to long-term potentiation phenotypes associated with the NMDAR, see Phenotypic expression. 08-14.
Complex modul~~tion o f N M D A R-channels hy polyumines 08-44-03: Several hiochcmical and clectrophysiological studics (reviewed in ref^.*^'-^^') S U ~ C Sthat ~ a recognition s ~ t ccxists on the NMDAR for
endogenous polyamines (e.g. normal catabolic intcrmcdiates of argininel like putrescine, spermidine anti spermine). The polyamine site is distinct from binding sitcs for ~ l u t a m a t c ,glvcinc, M ~ " , ~ n " , and open-channel blockers. Polyamines increase the hinding of open-channel blockers hut can also increase NMDA-elicited currents in cultured neurones, sumesting they may play a rolc in 'excitotoxict' responses following neuronal damagc. Polyamine segments are also associated with several neurotoxic venoms from invertebrates (see Receptor nntn~onists. 08-51). Spermine and spermidine havc been descrihed as 'sgonists' at thc polyamine recognition site, which may modulate excitatory synaptic t r a n s m l s s ~ o n ~Furthcr ~~. features of polyamine modulation of NMDA receptors are descrihed In Tr~hle9.
Modulation o f N M D A R by the co-a onist glycine 08-44-10: Glyrine-evolted p t e n t i r t i o j of NMDA receptor activity is
accompanied hy reduced desensitizationt2". Dose-response analysis for the glycinc-sensitive activation of NMDA receptors at +60 mV reveals a 3-5fold increase in apparent affinity for glycine In the presence of 1 mM spermine. This increase in affinity for glycinc is accompanred by a 3.3-fold decrease in the rate of development of glycine-sensitive desensitization, and a 2.4-fold decrease in the rate of dissociation of glycine from NMDA receptors (while the rate constant for dissociation of NMDA 1s not red~ced'~1.
Alcohol-inhibition of N M D A R currents 08-44-1 1: Potency of several alcohols for inhibiting NMDA-activated currents
arc cnrrclatcd with thcir intoxicating potencyt, suggesting that rcsponscs to NMDA receptor activation may contrihute to the neural and cognitive impairments associated with NMDA-activated currents are inhibited by ethanol over a concentration range that produces i n t o x i ~ a t i o n ~ ~ Ethanol " ~ ~ ~ " . may inhibit the NMDA-activated ion current hy interaction with a hydrophohic site on the NMDA channel protein'y4. Compart~tivenntr: Ethanol effects a number of receptor-stimulated and voltage-dependent ion fluxes with differing sensitivity. For an illustration of the relative potcntiatinp, effects on CARA,-mediated C1 flux and the rclativc depressive effects on NMDA-, kainate- and voltage-gated ca2+-flux, see Fi,q 5 rlnd~rE L G Cl GARAA.entry 10.
Non-selective inhibition o f NMDAR-mediated calcium influx by dihydropvridines 08-44-12: The dihydropyridinc nitrendipine suppresses NMDAIglycinemediated calcium influx by a rapid and direct interaction with the NMDA
receptor-channel complex2". Thus nitrendipine may exhihit anticonvulsant and neuroprotectant activity via a combined ability to modulate I~ot11 NMDA-associated ion channels and L-type voltage-sensitive calcium channel^'"^ (src Hlocker~r~ndrrVLG Cn, 42-43).
Table 9. Some features of polyamine modulation of NMDA receptors (From 08-44-09) Feature 'Concentrationdependent' effects of spermine modulation
Voltagedependence of spermine potentiation Separate mechanisms of sperrnine potentiation or block
Examples of block by other polyamincs
Spermine modulation of co-agonist affinity
Description
Refs
Spermine can produce hoth potentiation or hlock of ' " NMDA responses by binding to multiple sites in a potential-sensitive manner. Spcrminc potentiates the action of NMDA at micromolar concentrations but is less effective at millimolar~oncentrations~~~. At low concentrations (1-10 /IM) spcrmine enhances NMDA receptor current in cultured cortical neurones by increasing channel opening frequency. At higher concentrations (> 10 //MI,it produces additional voltage-dependent decreases in channel amplitude and average open time which limit its 'enhancing' action Concentration-jump responses to 100 /rM NMDA 2w in the presence of I0 /rm glycine reveals potentiation by 3 r n spermine ~ at a membrane potential of +6O mV, hut depression at -120 mV 290 Potentiation results from both an increase in apparent affinity for glycinc as well as an increase in maximum amplitude of responses to NMDA rccorrlcd in the presence of a saturating conccntration of glycinc. Sperminc produces a voltage-dependent open-channel hlock of NMDA currcnts at high concentrations [> 1 0 0 / 1 ~hut ) has nocffcct on the block by the putative polyamine site competitive antagonist arcaine (see Rlockers, 08-4.7) The polyamincs diethylenetriamine and 1,lO29' diaminodecane produce vol tage-dependent hlock of responses to NMDA, with apparent equilihrium dissociation constants at 0 mV of 0.75 and 2.93 rcspectivcly. Rlock is voltage-dependent, and most likely due to binding of polyamincs to sites within the ion channel (see Voltage sensitivity, 08-42) Sperminc increases the affinity of the NMDAR 2w for glycine
'"
Comparativenote:Polyamine modulation isnot unique to NMDARchannels. As reviewed CIin ref.*"', polyamincs rnodulatc several Cl- conductances, including ~a'*-activated channcls (see ILG' C1 Co, entry 25) and GARAArcccptnr-channcls (see ELC: Cl CARA,, entry 10).Polyamine toxins have also heen shown to rnodulatc n~cotinicacctylcholinc receptor-channels (see ELC: CAT nAChK, entry 09), non-NMDA iGluRs (see ELC: CAT GLU AMPAIKAIN, entry 07) and scvcral suhtypcs of voltage-gatcd calcium channcls (see V L G C ~entrv42) I, and potassium channcls [c:~~iilwl n c u r o n c ~ ~ ~The " ' . cffcct is :)l,scrvcd i ~ 1t ~ 1 t low h ant1 s;~tur;~tcd concentrations of glycinc, and thc ;~ffinitvTof the NMIJAR for glycinc does not chnngc in the prcscncc of tolhutamidc. Although tolln~tamidchas hccn gcncrallv cliar:~ctcrizctl;IS a blocker of KnrF lur,c- I h ~ Kl ~A T / ' - ; , cPrltrtr301, the :~ctionof t o l l ~ ~ ~ t ; ~ mon i t l cthe NMIjA-;~ctivntctlcurrent is r~ol mctliatcd hy KATI, cli;~nnclssince thc cttcct pcrsists in the prcscncc of ~ntr;~ccllular Cs(:l at conccntr;~tionswhich intlucc total 1,lockl of ; ~ l lK' channels. Tolhutarnide may thcrrf(~rc;~dtlitic~n;~lly rnorl~~l;lte intmcellular messengers influencing NMI>Ali-cli;~nncl;~ctivityin this prcl>;~rntion(st-c, r(*l.'"'/.
Equilibrium dissociation constant S i n ~ i I ( /~' rH / - M K - H O ~ llir~dingin nrrtir~ecrlls rlnd hcrcrologoust NR I / NR2A corill?inations 08-45-01: (:ells transiently cr)-cxprcssing NRI ant! NII?A yiclrl ;I 10-foltl Incrc;rsc in the n~rrn1x-rof [ ' ~ I - ~ ~ - ~ ~ l - h sites i n d colnparcd in~ to channcls cxprcsscd froni Iiornomcric ;~sscmhl~cs. Simil;~r;~ftinitics for ['HI-MK-801 h:~vc I~ecn measured in HEK-29.3 cells tr;msicntlv expressing NRlI2A coml>in:~ticin.i;IS in native adult rorlcnt hr:~ins".
Extcrrlnl ~ n , "r.r~n!ril~u!r..s to 'r~n~r.sr~rrl/v 111,r.h'~11~c.inr~ rlffit1itic.sfor NMIIA rc1crJ17t ors 08-45-02; T h e aff~nityof the NMDAR co-;~gonistglycinc l.sr,cJ Ai.tirwtlon. 08,?.I) is scnsitivc to the c ~ t r : ~ c ~ I I i r(1;1'+ l a r conccntr;~tionin trigcminal ncuroncs
entry 08
(the apparent dissociation constant (ECS"] for glycinc dccrcases with increasing cxtcmal ~ a "cclnccntrations, increasing hy about 3.7 times in ~a"-containing solution^]^^^". Kinetic studies of glycinc binding to NMDA receptors indicate that external c a L ' causes a dccrease in the off ratct of the glycine binding, while having no effect on the on ratci2.?' (see also Activntion, 08-33),
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Ligands 08-47-01: Commercially available radioligandst includc I'HJ-AP~ (['3~I-n-2amino-5-phosphopentanoic acid), ['HI-CGS19755 (['HI-cis-4-phosphonomethyl-2-pipcriciinc carhoxylic acid), ('HI-MK-801 ([.3~]-dizocilpinc,5methyl- 10,I1-dihydro-SH-dihenza~n.d]cycloheptcn-5,10-imine),['HI-TCP (I.'H]- 1-1 -12-thicny1)cyclohexylpipcridine)and ['HI-CPP ([.'~]-lcs the 1c;lrning rate in rahhit n>odclsf7' (,see. crlso cplicpc.t.\ rnonoc.loric11 nriril,otlic.s ~.clhic.lltli.splrlcr ~ l y c i n cfrom thc NMDA rrc.cptor ~rntlcrI'hrnol!f[~ic- cpx[~rr.ssion,OX- 14). S r n s i t i v i t , ~of r c c o i n h i n n n t NMl3ARs t o a g o n i s t s - cxomp1r.s 08-50-07: ~ o m o m e r i c lNMI)A receptors (NR I ) expression-cloned in Xcnol~us oocytcs from cIJNA in plaslnitl vcctor pN60 (sc~c~ Iso/(rtion pro/?cp.08-12) tlisplay the following ;~gonistsensitivities (measured as percentage stcadystate current response compared t o 100 I ~ MNMDA in ~ g ? ' - f r e emedia supplemented with 10 1cl.r glvcinc)'": 100 /(M NMDA (controll, 100Yh; 10 /lM I.-glotamate, 212 1 1 So!,; 100 11" ihotenate, 71 ! 211"L; 1 0 0 I ~ Mquisqualate, 43 ! IO":,; 100 1.-homcrcystcatc, KS t 1Y"O; 100 I ~ M NMIIA without glycine (controll, .ZS t 6%. Other :~gonistsare ineffective in cornparison ( - Soh) and include 500 /(hi k;~inate,50 IIAI AMPA (r-amino-.Z-hvdroxy-5methyl-4-isox;1zolcpropion;1tc,s i p c j EL(; CAT G L I I AMPAIKAIN, cBntqr071, 100 !chi IS, .ZR-ACPD ( I I-amino-cyclopcntyl-I,.Z-tlicarhoxylatc, an rnGluR ;~gonist)and 1 rnhl GARA. M i x c d r l ~ o n i s r nncross iG1ull r?7olcc'ul~rsuht)y>c.s 08-50-08: In nati,vc tissr~cs,the dicarhoxylicacids L-glutamateand L-aspartatcarc mixed aRonistsi of the NMIIA-, ktiinatc- and AMPA-sclcctivc receptors and thcir effects :ire parti;~lly inhihitcd hy all sclectivc antagonistst. 13cfinitc incrcases in hoth thc opcn timci and open-state prohahi lity 1 of NMDAopcrntcd channels h;wc hccn shown t o he intluccd hv pro101i+yd application of g l ~ ~ t a n i a to t c hippocampal slices in .sit~l."'. In spinal cord ncuroncs, I.-proline elicits inward current that can he pc~rtic11Iv antagonizctl hy ~-APs.'"'.
Evidcnc-(J for bit-nrl~ontitr ion (1,s (1 c o - f a c t o r i n R S ~ ~ ~ ~ S ~ - ( J C ~ J J C ~ ~ I ~ J C ~ glutclrncltc r7rurotoxicit,v 08-50-09:The ahility of the (exogenous)neurotoxic glutamate agonist RMAA (/IN-mcthylamino-I.-alanine) to opcn NMDA and AMI'A channcls in isolated mernhranc patches is strongly potcntiatcdt hy hi~arhonate.'~'.T h e nenrotoxic nr.-2,4-diami:~ntlneuroexcitatory effects of two stmctural a n a l o n ~ c s oBMAA, t nnhutyrate ; ~ n d!)I.-2,3-diaminopropionate,arc ;~lsopotentiated hy hicarhonatc. Nor(': IjMAA is ;I neurotoxic glut;~matc agonist implicated in ncuronal denenemtion fount1 in the Guam :imyotrophic lateral sclerosis-l'arkinsonismdementia complex (Guam disease) - FCC c l ~ ~ ~ r ~ ~ c t ~ rIistrd i x t i ( in . . srrl.'"?
entry 08
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Inhibition o f pre-synaptic glutamate ogonist relense by riluzole
-
08-50-10: Sodium channel hlockcrs, including the anticonvulsantt and
n e ~ r o ~ r o t e c t i v ecompound t riluzole, hlock rcsponscs to NMIIA (ICscl 18.2 I ( M ) following expression of rat whole brain or cortex mRNA into Xenopus oocytes3"'. Riluzole also hlocks rcsponscs to kainic acid (kainatc, , (ICqn = 0.04.3 MI. 2-APV lCso = 167 I~M), CNQX (ICso = 0.21 p ~ )NRQX (we Receptor anta~onists,08-5 1 ) yields an lCsIl of fi. 1 ,IM in this ~ ~ s t c m ~ ' ~ " . The inhibition by hoth riluzolc and 2-APV is rcvcrsihlc and docs not appear to he use-dependentt, unlike that of the channcl hlockcr MK-801 (dizocilpine). Riluzolc acts in a direct hut non-compctitivcT manner and does not interact with any of the known ligand-recognition sitcs on either the kainate or the NMDA receptor'". Characteristics of riluzolc and other antagonists of glutamatc release have havc hccn rcvicwcd"'.
Synaptic release o f glutamate in hippocampal slices occurs in an 'allor-none' manner 08-50-11: The substantial differences in reported sensitivities of the NMDA
-
and non-NMDA receptors to glutamatc agonist sumest that changes in transmitter concentration in the synaptic cleft can result in differential modulation of thcsc two components of the EPSC~.Howevcr, pharmacological manipulation of pre-synoptic receptors affecting glutamatc release in CAI pyramidal cells of guinea-pig hippocampal slices 1i.e. baclofen antagonism of GABAR receptors and non-sclcctivc agonism of adenosine receptors hy theophylline) result in parallel c h o n ~ e sof NMDA and nonNMlIA receptor-mediated components of EPSCs over a 16-fold range.'"". Induction of long-term potentiationt ( L T P ~ in J this preparation (hy lowfrequency synaptic stimulation in conjunction with depolarization to +30 mV, see Fig. 1) leads to differential rnhrlncemrnt of the non-NMDA receptor-mediated component of the E P S C ~ .Thus L T P ~appears to occur through either modifications of post-synaptic receptors or through prcsynaptic changes involving increased transmitter conccntration in the synaptic cleft""'.
1
Receptor antagonists (selective) Pharmacologically distinct sites for an tagonism of NMDAX responses 08-51-01: The multiple sites for antagonism on thc NMDA receptor complex
havc hccn rc~icwed~'""'~. Conventionally, thcsc havc hccn separated into pharmacologically distinct sites haset! on thc characteristics of hlock and the pattcrns of additivity or overlap in dose-response experiments. These sitcs include (il the transmitter-recognition site, (ii) the ion channel site, (iii) the glycine (co-algonist) site, (iv) thc zinc (modulatory) site, and [vJthe polyamine (modulatory) site. The present 'classification' of sitcs is largely hascd on pharmacological criteria, and further discrete sitcs havc hccn Many studies provide support for a n t a ~ o n i s m at the sitcs rcprcsented schematically in Fig. 6. In due course it may hc possible to resolve some of these discrete sites of antagonist action ti) dcfincd amino
entry 08
Figure h. l'ostlllrrtrrl crrrTs o f rrrltrr.yorlrst rrc.rron m t the NMDA reccptor ~ . o ~ i i p l ~Kcl(~fivr~ x. sizil.r: orlri positrons o f sitrs on protclin doni(~in.sR ~ C shown on (1 /typotl~i,!icr~/ r/ioEyrrlmrtirrticcross-srction througli ( I N M l ) A l < . Thrs sitc posilioris rrrrJ not rr/~.soIrrtr~ (lnri rrrrT {or i//l~ctrtrtionori1t7 (s(>rp trxtl. (Fro117 OX-5 1 - 0 1 1 acid loci ( c . ~ hv . i n a ~ n sof site-tlircctc(l mutagcncsist). T h e figilre is a compilation of sirnilar ones ptthlishcd in scvcrnl reviews '*". '". *'-.
."'
Gcnernl notus on rnodc.s o f mcrion for cornpcritivr versus noncornput i t ivc NMDAII nntrrgonists 08-51-02: Ry definition, cornpetitivet antagonists for receptor agonist sitcs clan potentially inhihit ntlrmal physiological ;acivities in one hrain region nt r Glutamate accumulation conccntmtions which do not affcct o t h ~ rcgions. to high levels within thc synaptic clefti in conditions such as strokei may nlso 'out-cc~mpctc' t h c cffccts of such conlpctitivci nntagonists"". Ry contrast, non-ct~mpctitivcinntngonists acting ; ~ t'rnodulatc~ry' sitcs have a potc*ntinl thvrapcutic advantage in that they arc ahlc t o inhihit cffccts of cxcc.;s ~ l i t t ; i ~ n a In t c ';~ffrctctl'arvas of thc hrnin, with rrI:ltiveIy little direct influcncc on nclrm;jl rcccptor function2". Examples of such 'rnorlnlatnry sites' incl~ttlc. t l i ~ ~ srvc g ~ ~ l : ~ t cI,y( l polyaminesi, redox rcagentsi, channel ions itntl p~'"''. A .;umm;iry ot cc~mpoun(l.i shown t o h l o ~ k n r l ~zinc , ;~nt;lgonizcN M l l A responses is listed in Tahlc 1 0 , c;itcgc~rizcdhy their sitc ot ;lctio~i.For J T I O ~ P ~ . o r ~ ~ ~ ~ r ~ rrat,ic~v~, , ! i ~ ~ r YW i , ~r i~~, f~~ ~. )'i-'i". ' ," ~
Table 10. Known NMDA receptor antagonists a n d their fpnturcs (From 08-51-02) Antagonist
Fcaturcs
~ o m ~ c t i t i v e n-2-Amino-5-phosphopcntanoic t acid (D-APS)was antagonists at the first NMDA rcccptnr-specific antagonist (pA2 ~ l u t a r n n t e - 5.2-5.9)(see Protein interactions. 0831). Similar [~indingsite compounds are dl-2-amino-5-phosphonovalcric n-AP5 acid {2-APVJ,11-2-amino-7-phosphonoheptanoic 2-APV acid [AP7 or i>-AP7)and 3-(2-carhnxypipcrazin-4CPP yl)-propyl-l -phosphonic acid (CPP).Othcr CGS-19755 cxamplcs arc CGS-19755 (cis-4-phosphonomcthylCGP-37849 2-pipcridine carhoxylic acid, pA2 6.0). CGP-37849
Refs 38"
LY-235959
{n~-~E~-2-amino-4-mcthyl-5-phosphono-3-pcntanoic
SDZEAA494 MDL100453 DAA
acid) and longer-chain glutarnatc analogues (c.g. 11n-aminoadipate, D M ) . Generally, thcsc compounds do not possess sippificant channclhlocking activity. Structure-activity relations of thcsc anrl othcr compounds has hccn rcvicwcd"". Fnr molccular 'loci' of agonist-hinding sitcs, see Domnin functions. 08-29
Competitive anta,yonists at multiple sitcs DDHB and suhstituted dcrivativcs
Suhstitutcd hcnzazcpincs arc class of glutamatc receptor antagonists that show compctitivct action, significant potency at mr~ltiplcsitcs, and a high tdcgrcc of lipophilicity. 2,s-Dihydro-2,s-dim3-hydroxy-lH-hcnzazcpinc[DDHB)and thrcc suhstitutcd derivatives, 4-hrorno-, 7-methyl-, and X-methyl-DDHR, inhihit thc activation of NMDA rcccptors at hnth thc NMDA rccomition sitc and thc glycine allostcric sitc
Noncompetitivct (usedependent. uncompetitive) openchnnnel a n t a ~ o n itss Dizocilpine (MK-80 1 ) Ketamine Tiletamine Phencyclidine SKF10047 Dextrorphan Dextrometharphan Desipramine
"W Dissociativc anacsthctics including dizocilpine (MK-801), (tj-5-mcthyl-10,11-dihydro-SH-dihcnzo"" cyclohcptcn-5,lO-iminc malcatc), phencyclidine (PCP, 'angcl dust'), ketamine, tiletamine anct SKF10047. High-affinity dcxtrorphan binding in rat hrain has been localized to non-compctitivc antagonist sites also recognized hy nanomolar 1-(I-(2concentrations of dizocilpinc and TCP (['HIthicnyl~cyclohcxyl]pipcridinc. Dextromethorphan and rernacemide arc wcll-tolcratcrl clinically. Dizocilpinc has hccn uscd to soluhilizc NMDA rcccptors from rat and porcinc hrain and in its tritiatcd form is a common radioligand for rcccptor hinding and distribution studies (scc I'mtein distrihrrtion, 08-15).Thc S-cnnntiomcr of kctarnine is mtwicc as potent as the R-cnantiomer in exhibiting a voltagc- and use-dcpcndent hlockade of NMDA rcccptor currents. Calculated relative
"I"
"'"
Table 10. Continclcpri Refs
Antagonist
Fcatilrcs
Memantine CNS1102 CNS1505 M ~ " Rcrnacemide/ FPL12495 2%'' (high /m) ~ n ? *
forw:lrd and backward rates suggest that cnantiomcr confonnntional diftcrcnccs influence t h e dissoc~~(ltir~n from t h e hinciing sitc more than the ;~ssociationwith it. T h e tricyclic ;~ntideprcssantdesipramine (DMI, 20-50 ~ l h r lis a potent sclcctivc :~ntagonistof responses t o N M D A in m o u s e hippocnmpnl ncuroncs. T h e potency of DM1 a s a n NML3A antagonist is hixhly v o l t n ~ c - d e p e n d e n twith t h e k',, increasing c p - t o l t l per .Zh mV tlcpolariz;ltion. At 60 I ~ V t,h e Iolishcs these responses. Thc cicrivativc TyrO-cr~nantokin-C.has hccn found to cxhihit pcllyaminc-like actions at 7-fold greater potency than spertninc. Argiotoxin-636, a component of the Arginpr spider venom, has a higher affinity for the NMDAR than for kainatc receptors, hlocking the corresponding ion channels in a vo!ta~c-dcpcndcntmanner. Modifications of the polyaminc tail or the terminal argininc rcsiduc clf synthetic argiotoxin-636 strongly reduces the hlocking pntcncy. The hiology of Conrls (cone snniI1 vcnom pcptidcs which act on ion ch;~nncls including the NMlIAR has hcen reviewed."
lR2
-
""'
--
other Biclxanthmccncs of microbial origin (thc ES-242s) rlntrl,yoni.~l,s n t rcpruscnt novel NMDA receptor antagonists which mtlltrplr sitc5 interact with both the neurotransmitter recognition site and the ion channcl domain
.'""
,3.75
"For further details, scu rct~~*~~'"-."".
T h e therapeutic ,significnnceof NMDA receptor ilntcl,yonists
08-51-03: N M D A R antagonists have hcen examincd as candidate neuroprotective agents for pathological ctlnditions associated with acute snd chronic 'overstimuIatinn' of NMDAR and ~~~-NMDAR~~.'~~~""~'.'~'-'~"' (SL'L, (11so ~ E ~ C ' ~ E I I C under CS I'hcnntvpic cxprcssion. 08-14). NMDA channcl hl(lckcrs can provide n 'thcmpcutic window' hv preventing 'cxcitotoxic' calcium influx during globalt or focalt brain ischaemiaf elicited hy conditions such as strnket. Thc ro1c of cxcitatnry amino acid receptors in epilepsy, and the cffcctivcncss of NMDAR antagonists in various in rrirm and in vitro
entry 08
-
models of epileptic seizuret have been revicwedf2'. Effective therapeutic compounds may act to directly antagonize excitatory amino acid agonist hinding to receptors or may modulate uptake of endogenous agonist within the synaptic c ~ e f t f ' ~ ?The chemical structural requirements for the development of potent+ NMDA receptor antagonists havc been discu~sed""~.
General note on the relationship o f antagonist selectivity and their molecular 'targets' 08-51-04: Thc existence of multiple subunit isoformst (and splice variantst) within functional extracellular ligand-gated channel complexes (coupled with known variahilitics in expression patterns within the CNS) raises the possihility that selection of antagonists capahlc of modulating subsets of hrain functions is a rational goalf2".
Antagonist sensitivities for recom hinant NMDARs - examples 08-51-05: ~ o r n o m e r i cNMDA t receptors (NRI ) expression-cloned in Xenopu.~ oocytes from cDNA in plasmid vector pNhO (see Isolntion prohe, 08-12) display the following antagonist sensitivities (measured as percentage reductions of the steady-statc currcnt response compared to a control of ~ (control, without 100 , r ~NMDA in ~ g " - f r e e media'": 100 l t NMDA antagonists), 100% I0 [IM D-APV, 27 f 6%; 1 I ~ MCPP, 47 f 7%; 1 IIM CGS19755, 43 f 5%; 50 I'M 7-CI-Kyn, 2 i 1%; 1 {tM (+)MK-801, 7 f 4%; 100 / l M GAMS, 97 *2%; 100 nM Joro spider toxin, 83 1671,; 100 IIM CNQX, 70 ho/oln. Note: Differential sensitivity to the open-channel hlockcr dizocilpine (MK-801) hy certain NMDAR subunit comhinations has been rcportcd2"' (for detnil.~,see Nlockers, 08-4.3).
*
Effects of N M D A nntngonism in vivo - hehavioural studies 08-51-06: A systematic study of relative potencicst for glutamate receptor antagonists arlministcred in vivo reported MK-801 and CGP-37849 to havc relatively high potcncy (EDso < 1 mg/kg following thrcc different administration proc~dures).'"~.A range of NMDA channel antagonists (including several listed ahovc plus ifenpmdil, f-N-allylnormetazocine and dextrnmethorphan) prnducc impairment of learning and muscle relaxation when administcrcd i.c.v.t in rats.'.?". 1n addition to 'tcmporary psychiatric disorders', systemic administration of allostcrict and isostcrict NMDAR antagonists has also hccn associated with disorders of cardiorespiratory regulation"4", neuronal degeneration*7qfant! reductions in synaptic strengtht4'. NMDA receptor a n t a ~ o t ~ ineuroroxicily st dependent on neuronnl interconnections
u
08-51-07: Certain NMDA antagonists cause neurotoxic side-effects consisting of pathomorphnlogical changes in ncurones of the cingulate and retrosplcnial eerehral corti~cs""~.FolPowin~ low doses these changes appear to hc rcversihlc, hut higher dnscs can cause irrcvcrsihle neuronal necro~is.'~'. The neurotoxic action of MK-801 in adult rat cingulatc cortex is potentiated hy pre-treatment with thc cholincrgicT agc~nistpilocarpine, and can he aholishcd hy co-admini!tration of the cholinergic muscnrinic antagonist scopolamine and agonists' which act at the GARAA reccptor (c.g. diazepam and b a r b i t ~ r a t e s l This ~ ~ ~ .~rcvenriono f NMDA nntoeonist-induced nr1troroxic:itv
----0
-0 -00 -0
Cingulate neuron
k2 GABA
AW
muscerlnlc \receptor
I
Figure 7. NMDA receptor antagonist neurotoulcltv dependent on neuronal lnterconnectlons Glutam~ner~qrc (Glui axon collateruls o f cingulote neurones 11 I feed hack to NMDA receptors (21 expressed at GARAergzc IGARAI neurones (31 to s at MI muintazn toxic inhibitory control orer the release o f acct~-lchollnc(ACht from chollnerglc neuroneu ,I41 and ~ t actionr cxpretred on the clngulate neurone 1tce1f Phormocolo~calblockade o f the ,hr.h4DA receptor (2) muccannlc receptor\ /i,' crhol~\hes the inhll~ltorvcontrol o w r ACh rcler~seand suhiects the clngulutc neuronc to LI ctute o f perrlrtent cholrnergic h-yperstimulotion This hrperctzmulatron hor been proposed as the maln cause o f the pothomorphologicol effectrIn the czngulate ncurone tollo117ngNA4DA receptor-channel hlockclde lsee Receptor clntagon~etc.08-571 licstorutron o f GABAergic tone fe g bv adrnlnIctration o f barblturc~tecopenlng the GABA receptor-channel at /6]. etTenIn the ahrence o f GARA / w c ELG CI GARAA, entrr 10) or brr MI-se1cctlr.e ~lntugonlsmat [5jt hnr been chorvn to protect cingulate neuroncr against the neurotoxic d e - e f f e c t s o f S M D A ontu,qon~$ts(Rased on doto hom Olner- er rl. 11991, Sclencr 254 151.5-18) (From 08-51-07,
entry 08
-
hy M 1-selective anticholinergic and harbituratc drugs has heen explained hy considering the specific set of connections which modulate glutamcric cingulatc ncuronal activity (fora summary o f these inten~ctionx,scr Fig. 7)*12.
Receptor inverse agonists I'utc~tiveinverse axonisis at the polynmine sitc 08-52-01: Voltage-independent, dose-dependent, non-compctitivet antagonism of NMDA-mediated currents hy chloride transport hlockers has hccn shown in cultured spinal cord neuroncs""'. These agents include furosemide (a widely-used loop diuretic), and the relatcil compounds pirctanide, bumetanide, niflumic acid and flufenamic acid. The action of turoscmidc could arise from interaction with the z n L ' inhibitory sitc (as hlockatle of NMDA-induced responses hy furosemidc and ~ n ? 'arc additive). These agents may act as inverse agonists of the polyamine site ant1 their action may explain the 'protective' effect that has hccn shown for some of these drugs in neuronal degeneration3"' (see I'l~rnotvpic ~xpression,08-14). 08-52-02: 1,lO-Diaminodecane (DAIOI acts as an inversct agonistt at the palyamine recognition site of the NMDA receptor.'"'. The cffcct of DAlO is not mediated by action of DAlO at the binding sites for glutamate, glycine, ~ g or~ z n+2 ' hut is attenuated hy diethylenetriamine (DET). In hippocampal ncurones, NMDA-elicited currcnt is decreased hy LJAlO, an cffcct opposite to that of spermine. The effects of spcrminc arc also selectively hlockcd hy DET'"' (scc cllso Chr~nnclmodulntiori. 08-44).
*
),a4:c-,x
-
a
14: I
Database listingslprimary sequence discussion
08-53-01: The relcvant datahase is indicated hj, the lowcr cclsc p r ~ f i x(c.,~. XI':) w1iic.h should not he typed (see Introduction c*) l(lj~o1lto f cntries, cntrv 02). Dattrh~~se locus ntlmes clnd c~cccssionnrlinhcrs immcrlicltcly follorv the colon. Note that a comprrhciisivc listiri~ o f (111 tiv(~il(lhIe~ ~ ~ c e . s , s i o n nun117er.s is superfluous for location o f rcll~r~clntsrcluences in (;cnliaiik" resources, which arc norv avnilclhlc wit11 porverful in-huilt neighhouringt analysis rontincs (for dcscription. sec the Dtrfrlbclsr Iistin~s i~c'lflin thc Introdr~ction el layout o f entries. cntry 02). For cxmniple*, sc.eluer1c.c.s o f y t c(ln 11e nvrldi1,r c-ross-spcrics vorianta or reIntr(j Rent ~ m i ~ n~c~inl)rr,s 17,. one or two mun(is o f nei,qhl,o~~rin,yt c,nc~Ivsi.( ~ v h i c hr~re1)asrei i~c.cc*s.scd on prc-computed ctlignrnents perforrnrcl sins the HLAST~olgorithrn I)y the N C R I ~ ) This . featurr is most 1lscr111for retric~r~cll o f scqtrcncrp entri(*s depositc~tlin dotnhases 1cltcr thon thosc listrti l ~ r l o n Tlitls, ~. r(ppr~s~ntrrtivc 171c1171~7~~rs o f known scrloencc hornolog~~ groupii1,qs rrrr listrti to pcrrnjt initi(11 e1irc~c.t retrierra1.s lw occe1.s,~ionr i ~ ~ i i ~ l v( ~ r ~, t l i o r / r ~ ~ / c ~or r(~i~c(~ -(1ir~~c.f -(~c.c.cssion, horc,c,vc~,nc.icq/~llorrrii~,yt trncll~sis iioriirnc.li~t~~rr. Follo~ting to iclrntify--r~r.rvl\~ rcpportc.ti rlr~tlrc,lotc~isc~luc~ncc,.~. - is stror~,ql!,rr~commer~clc~el --
--
--
-
--
-
-- -
-
- - -
~
-
-- - - - -
-~
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~
~-
entry OX
Nomcnclaturc
Spccics, IINA ornolo~y scrccnlng
9.M nn, A4, 105 SW
ah: 1110028
Yamaznki, FFIj,? /,ctt [ 1 992) 300: .3945.
NMDARl splice variants NR1a ancl NRl h
NRI spllcc See I r o t gh: t , n .6.32 v;~rinntsNIZ 1:1 and ] ? / ~ o ~ p / ~ c ~ n ~ / ( l t iLO1 OX-.??, clnil NRlh / k ~ r r!rifc.rr.ric~e,\, cepen (;/~r~nric./ (.'epnv o r , y ~ r n ~ . - ~ ~~t ~i ~o no .( / ~ ~ / O,Y.4,1 ot~r~n,
Durantl, I'ro~. N ~ J IAc.(lt/ I Sc.1 IISA (19421 89: :~.;c-tl I'CII scrccn NMIJA 'potcnt~.~t~ng suhun~ts'"
I\olatctl hv I'CK ~ ~ l / r hnr nt ~ n cl3NA l~hrarv
gh: MVI 56 l gll: M1)l 5h2 XI>: MU 156.3
Monvcr, ( 1 902) S(.~r,r~c.c* 256: 1217-21.
gh: 111.321 1 gh: 1)1.3212 gh: l>I.321.3
Ishil, I H ~ o l C.'/1(~1711 l99,Zl 268: 28,3643
gh:
1)1.7214 Epsilon 1 suhunit Mouse. Homology- 1464 an (1445 an, I NMI)AK?.AI hnscd PCR .;crccn mnturcl
gim: 405314
Mcguro, Ne~trtrr.(1992) 357: 70-4.
{
Epsilon 2 sc~hanit Mouse. H o m o l o p - 1482 ;la NMDARLIXI h:~sctl I'C1lZ .;crcc.n
gim: 4052
K~~tsuwndn, Nrrtrlrc. ( 1 992) 358: 36-4 1 .
Epsilon 3 sohonit M o ~ ~ stclc~riiologv. 12.19 nn 1 NMI>ARI('\ h;~scilI'CK scrccn
gim: 305.725
Katsuwad;~, Nc~tz~rtp 1 I 0911
-
Nomenclature
Spccics, IlNA
Original isolate
SOLI~CC
Epsilon 4 suhunit M(luse. Homology- 1%12.3 an
hnscd PCR scrccn Ligand-hintiing prclpcrtics not (C;Rl') of NMDA rcpnrtcrl; nn dirwt reccptor complcx cvidcncc of p l r ~four ~, channcl activity o r NMDAR cDNAs NMnA pharmacological spcciftcity
Glutamatebinding pmtein
Pre-synaptic
glutamatc rcccptor-channcl
-
-
-
Molcculnr mass 57 kDa [including signal peptitlcl. No homolorn to any other CluR or kainatc-hintling protein
Accession Sequence/ discussion not found Ikcdn, FEIZS Lcrt (1992) 313: 34-8. not fount1 Kumar, Nrrttrrc (1991) 354: 70-3.
not found Smirnovn, Scicnce ( 1993) 262: 4.30-3.
Notr: T h e above tahle lists genes encoding subunits NMDA-selective ionotropict glutamate receptors {iGluR\. In datahasc searches, these should not he confuscd with thc gcnc nomenclatures uscd to rlcscribc the C, protein-linkctl (mctahntmpictl g1ut;lmate rcccptors in the r n G I ~ ~ - r n ( : I scries t ~ ~ (see Appendix A - Ir~dcxo f C pmtcin11nkc.d rc.r-cptor.s, entrv 50, a n d rhc norrs l~clowtile nwrn t o l ~ l rin 1)armhnsc Irrtings undcpr ELC: CAT G L 1 I AMI'AIKAIN. 07-53).
Related sources & reviews 08-56-01: SEC OISO Ti~hJc4. Ma.Jorquoted sources for this
128~"6"'"M;
NMDA rcccptor physiola~yrc~icws"~'~"~'."~'~"'~"'~~ , functional distinctions of NMDA and non-NMnp, rcccptor~hannels241.255 259 262.263. .'(a. 3~p8-.37n. physiological and pathophysiological roles of cxcitatory amino acids during d c ~ c l o ~ r n c npharinacc~logy t~~; of thc glutamate receptor NMDARs and Hehb-type synaptic pfasticity"'; excitatory amino acid receptors in cpilcpsy"'; cloning of cxcitntury amino acid r c ~ c ~ t o r s ' ~commentary ~"~~; on original descriptions of cloncd NMDARS""; sites for antagonismt on the NMDA receptor complcx~'"; glutamate ncurotoxicity and ncuronal dcRcneration4~~62. ~2,10~.~.~.~-124,374, , pharmacological strategies (including
NMDAR antagonism) aimed at limiting CNS tissue damagc following traumat'z3n,'.'$ cornmcntarics on NMDARs in synaptic excitatinni"7s'"7~; physiological roles of second mcssengcrs in rewlation of NMDAR function5; reviews on rolcs of GluRs in CNS function and memoq~~.'-"~"*74*18"'""; phosphorylation of iGluRs in synaptic plasticityt65; protein kinasc C function in LTI' inductionrq" the glycine site of thc NMDAR.~'~"""'~; polyamine ; as 3 pharmacological target for modulation of the N M D A R " " . ~ ~ ~NMDAR ethanol2*"; in viirn and in vivo ncurochcmical chnrnctcrization of N M U A I ~ " ~ ' NMDAR ; ktimulus-transcription coupling' in neuroncs (it'. trans-synaptic contrt~lof Kcno c ~ ~ r c s s i n n ) ' ~comp~itational ~~'~; implications of NMDA rccuptor-~hanncls'~'.~'~; permeation pathways of neurotrnnsmittcrgstcd ion chnnnuls'"". See r11so rrvfcw.< l ~ s t c du n d r ~ rEi.C: CAT CLO AMI'AI L 'Ion C channel /look rr/crcnccs. erltry 00. - KAIN,e n t r y 07,clnd R I ? S ~ I I E~ -
cntry OX
Rook reierences 08-56-02: Hall, Z.W., Sargent, P. R., Schcllcr, R. H., Kuss, E. M., Kennedy, M. R., Vale, K. n., Ranker, G, Lemkc, C;., Anderson, D. J., Patterson, P. H., Mardcr, E. and Rrcakcficld, X. 0. [ 1992) An Intmduc-tion to Molcculmr Nc~lrolriolo~qy. Sinaucr Associates, Sunderland, Mnssachusctts. Kozik(iwski, A. P. and Itarrionucvo, G. (crlsl (199 I ) Nrnrol~iolo,yyo f the NMDA Rci:cptor: From Chcmisrry to thr Clinic. VCH Vcrlagsgcscllschaft, Germany. Krngsgaard-Larsen, P. and Hanscn, 1. 1. (cds) (1992) Exr.itcltorv A ~ n i n oAcid Rcccptors. Ellis Horwood. L o d ~ c ,D. (cd.) (19811) Excitlitory Amino Acids in Hcnlth r~nd Discrtsc. Riolo~icalCouncil Symposia on Drug Action. John Wilcy & Sons. Plaitakis, A. (1984)In The Olivop)ntoccrrhcIIll~rAtn)pl~ics(eds R.C. Duviosin and A. Plaitakis), pp. 2254.7. Kavcn Press, New York. Watkins, J.C. and Collingrirlgc, G. L. (19891 Thr NMDA Rcccptor. IRL Press.
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Entrv support ~ r o r ~ p ond s e-mriil n r w s l e t t c r s 08-57-02: Authors who havc cxpcrtisc in one or morc ficlds of this cntry (and arc willing to provide editorial or othcr support for developing its contents) can ioin its support group: In this case, send a message To: CSN08dle.ac.uk, (entering the words "support group" in the Suhicct: field). In thr message, plcasc indicate principal interests [see fieldnamc critcrici in t hr. Intrc>drrction for cnvrr~~qrl together with any relevant http://www site links (cstahlishrci or proposed) ;ind details of any othcr pnssihlc contrihutions. In d i ~ rcoitrsc, support group memhcrs will (optionally] receive e-mail newsletters intcnticd to co-ordinate and develop the present (text-based) cntry/ficldnnmc frameworks into a 'library' of intcrlinkcd resources covering ion chnnncl signalling. Othrr (morc gcnerall information of intcrcst to cntry ctintriht~torsnlay also hc scnt to the nhovc address for group rlistrihution ant1 fretihnuk.
entry 08
' Tanahe, Net~ron(1992) 8: 169-79. Sugiyama, Neuron ( 1989) 3: 129-32. Schocpp, Trend.? Phormncol Sci ( 199.3) 14: 1\1-20, Nakanishi, Ncumn (1994)13: 10,31137. "aycr, Trcnds I'l~orrnncol Sci ( 1900) 11: 254-60. " Monycr, Science ( 1 992) 256: 1 2 1 7-2 1. Mcguro, Not t ~ r r(1992) 357: 7 0 4 . Ishii, / Riol Chenl (1993)268: 2836-4.3. ' Stcrn, Pros I-? Soc Lond (Hiol) (1002) 250: 271-7. Moriyoshi, Nature (1991 ) 354: 31-7. " Karp, Riol Chcm (1993)268: 3728-33. l2 Cik, Riochenl /(1993) 296: 877-8.3. Sakimura, Nellron ( 1992) 8: 267-74. I4 Honori., Eur 1 Phormncol(lYX9)172: 239-42. '"amazaki, FERS Lett (1992)300: ,3945. In Hcnley, Proc Not1 Acad Sci ZlSA ( 1 992) 89: 4806-10. " Smirnova, Science (199.7) 262: 430-,1. la Mackler, Mol I'hnrmacol (199.3)44: ,308-15. " Scchurg, Trenc1.q Pl~ormc~cnl Sci (1~19,1)14: 297-.30.3. Tcichhcrg, FASER / (1991) 5: .3086-91. Usowicz, Nature (1989)339: ,380L3. Trcmhlny, Rrain RPS (1988)461: .19.3-6. Wciland, Endocrinok~,qy( 1992) 131 : 662-8. 24 Mati~tc,Proc Nut1 Acrid Sci IJSA ( 1992) 89: 339940,3. liay, Riochem Riophvs Rcs Comnlun (199.7)197: 1475-82. " Plcasurc, / Ne~jrosci(1992)12: 1802-1 5. 27 Rading, Science ( 1 993) 260: 181-6. 2X Armstrong, Anntt Rev Nrorosci ( 1 993) 16: 17-29. 29 Komuro, Science (199,3)260: 95-7. .7n Hack, Neuroscience (199,1157: 9-20. Pcarson, iVetrrosci Lett (1992) 142: 27-30. Morgan, Cell Colcium (1988)9: 30.7-1 1. Lcrea, I Ncurosci (1992) 12: 2973-81. "4 Morgan, Trends Neuro.~ci (1989) 12: 459-(72. Proc Not1 Acnd Sci USA (1988) 85: 7351-55. ""avaron, 3" Spitzer, / Netjrol>iol (199 1 ) 22: 659-73. Rlanton, Proc Not1 Acad Sci U S A (1990)87: 802730. "* Fox, Nature (1991)350: 3 4 2 4 . NCVC,Pmc Not1 Acnd Sci IJSA (1989)86: 4781-4. 40 McDonald, Rroin Res Rev (1990) 15: 41-70. 4' Constantine-Paton, Annu Rcv Nettmsci (1990) 13: 129-54. 42 Kato, /'roc Not1 Acad Sci I JSA (1 99.3) 90: 7 1 14- 1 8. 4" Rcn-Ari, Nclrrosti Lett (1988)94: 88-92. 44 Ibwc, Drv Rrnin Rcs (1990)56: 55-61. 4" McDonald, Exp Neutol (1990)110: 2.77-47. Hcstrin, N N ~ I (1992) J ~ C 357: (186-9. .?
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entry 08
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Cline, I Nc,rrrosc.i (I')Y01 10: I 197-216.
'"Cline, Nrnrrron (19891 3: 41.3-26. 4')
Cline, Nrvrrron ( 1 991 1 6: 259-67. Klcinschmidt, Sc,ir>rlc~. (19871 238: 355-8. " Udin, Sc.ic~nc~r~ ( 1O')Ol 249: 669-72. .'* Lipton, Trc.nels Ncrrro\;c.i(l98'11 12: 265-70. Collingridge, Trr9nr1\I'hrrrn?clc~olS(.i ( 1'1901 1 1: 190-6. , " Rliss, Nofrrrc, (199,Z)301: (3I-Y. Carnlignoto, S(.icar~c,c-lC)C17\ 258: 1007-1 1 . (11)117)08: ,345-'). Agrnon, Nr~rrro/711\.sic11 "'Masu, Nc~trrrc.(1901)349: 760-5. Tkmosc, Hiol C1icpr?i1199.3) 208: 2266,1-71. SV Kutsuwada, Nrrfrrrr, ( 1 9 W ) 358: .36-4 1. Cotman, Trcnrls Ne*rlrosc.i(1')571 10: 2h3-5. Cotman, Anrlr~/(r8rpNcr~rosci(lc)88\ 1 1 : 61-80. "* Lipton, N E q l / Mrd (19941 330: 61*3-22. ".' Tsumoto, N(~frrrc~ (lc)X7)327: 5 1,3-14. '* Malenka, Trr,nt!~ Nclurocc.i ( 1993) 12: 52 1-7. Raymond, Trrnr1s I ~ h o r n ~ rSci ~c~ ( 1~ 9'1.I41 147: 147-53. Collingritlgc, Trc~nr1.sNe.rrrocc.i (19871 10: 2XH-'L3. Schcctz, t:ASEI< 1 (1'1941 8: 745-51. R;u~schcckcr,I)h\,sioI R r ' r ~( I 9'11 l 71: 587-61 5. 6V Tsumotcl, / p r ~/ l'lir~sio1(10901 40: 57.Z-YC3. 70 Rauschecker, N(rtrrrc' ( 1987) 320: 18.3-5. " C l ~ n c /'roc. , Ntrf/ A(.trtl Scr IISA (1987)84: 4.342-5. '-' Gu, l)c~trIy histochemical staining for a ~ c t ~ l c h o l i n c s t c r ~ s ~ .
entry 09
mRNA.7 encoding muscle suhunits in Xenopus 09-13-02: The genes encoding the two Xcnopus muscle a suhunits show different patterns of expression. The rl, mRNA is found in skeletal musc!e at all dcvelopmcntal stages and in oocytes: the ctlh mRNA is also exprcsscd throughout muscle development, hut is not dctectahle in oocytesz3.
Distrihut ion of neuron01 suhunit mR NAs 09-13-03: Neuronal nAChR genes show differences in thcir patterns of expression among hrain regions, suggesting that different genes are exprcsscd hy unique suhscts of neuroncs. The sites of cxpression of the genes encoding thc various suhunits of ncuronal nAChRs are shown in Tahlc 3.
Phenotypic expression Frrnction of nAChR in the electric organ o f mnrine rays 09-14-01: The richest naturally occurring sources of the nACh receptor are
the electric organs of the marine electric rays, such as Torpedo californica or T. mnrmorata, or the electric eel, Elcctrophorua. The tissue of the electric or an consists of parallel stacks of flat cells (clcctrocytes or electroplax ), each ccll heinn inncrvatcci on one face hy a cholinergict nerve. Thc stacks of cclls arc such that current cannot flow from one side of a cell to the other. Simultaneous stimulation of the ncrvcs causes depolarization of the innervated faces of each electroplax, producing a potential difference hetween the extracellular fluid of each ccll in the stack. The transcellular potentials across each ccll in the stack add to produce a large electrical discharge, which in the electric eel can hc hundreds of volts.
P
Functional cxpression o f nAChR ~t the neuromusculmr junction 69-14-02: At the ncuromuscular junctiont, the nAChRs in the plasma mcmhrane of the skeletal musclc cells arc activated hy the ACh rclcascd from the motor neurones. The resulting transient opcning of the nAChR cation channcls produces an influx of Na' ions, causing localized dcpolnrizationt of thc musclc ccll mcmhmnc. This dcpolarization opens voltagc-gated Na' channcls, resulting in further Na'-influx and cnhnnccd clcpolarization. The crlnsctlucnt opcning o f ncighhouring voltage-activatcd Na' channels leads to thc self-propagating dcpolarization of the plasma rnemhranc characteristic of thc action patentiali.
Functional expression o f nAChR in neurones 09-14-03 Numcrous ncuronal nAChRs havc hccn rccognizcd by clcctro-
physinlogical methnds, immunological cross-reaction and hiochumical piirificatic~n. The ncuronal channels differ from the Torpccio and mosclc nAChRs by thcir containing only two typcs of suhunit, r ant1 If, and mnst of them arc not sensitive to r-Rgt. Scclllenccs cncodinfi eight ncuronal 2 suhunits and four ncumnal p suhunits havc hccn idcntificd hy molccutnr cloning. In sito l ~ ~ h r i d i z a t i o noft hrain sections with prohcs for diffcrcnt sulwnit mRNAs shows that each r and /I suhunit gene is cxprcssctl in ;1 iliffcrcnt, rc~ionally specific manner. The functions of the diffcrcnt mr>lcculnr spccics of nAChK in the diffcrcnt arcas of thc brain arc ~loorly untlcrstood.
s of Tahle 3. Sitr's oi r>.uprrassionof thc ,ycnc.s c,r~c.ocljr?!: v ( ~ r i o ~ lstrl~units nruronrl! n A C h R s ( F r o n ~09- 1.7-0;3) Suhunit (12
03
(14 0.5
Cell or tissue tvpc
Rcfs 44.44
Literal spirifnrm nllclel~sof chick dicnccphnlon (El l to ncon;~tcl;Ncuroncs d o not cxprcss dctcctnhlc Icvcls of (12 mRNA until cml~ryonicclay I I (El I I, when fihrcs contilining cho1inc'ncctyltr~nsfcr;iscfirst enter thc nuclcus. T h e r;lt 112 prohc highlights ;i s11i~11numhcr of Purkinie cells in the ccrchcll;~rcortcx. It is not known whutlicr this represents ;i distinct su11-popul:itic~nof cclls, or whether ;ill I'urkinie cells c:in tr:insicntly cxprcss the 112- gcnc Chicken ci1i:iry ganglion (EIX: 900 copies mRNA per ".'".47 ncitronc); si~pcriorccrvical ganglion (E10);goldfish rctinnl ganglia I4 Adult chicken ccrchrum, ccrchcllum, optic lohc Chicken ciliary ganglion ( E l % :200-,300 copies mRNA per ncuroncl; human ne~lrnhlastomacell line, IMR .32; Iiunian small cell lung carcinoma cell line, NCI-
"*"
N-592. 11
7
IIX
09
,I2
In . \ i t r ~hyhridizntiont shows that the rat 0 7 gene is
highly cxprcsscd in olfactory regions, the hippocampus, the hypothalamus, the smygdala ;ind the cerehral cortex. Chickcn Ill 3 p;irnsympnthetlc ciliary ganglion I I X O O copies niRNA per nciironc); ;im;lcrinc ;inti ,qi~nglioncells in chick rctin;~cxprcss the 117 suhunir, detected hv immi~nohistochc~mistrv T h c I,% suhunit is the mnior suhtypc in chick retina, wticrc its distribution has hcen stuilictl hv i~nnlunohistochcniistrv (.so(, I'rotc~rr~ cli~tril~rl!ion. 00- 15) Hypnphyseal gland o f the r:it cnihryo at stage El(,: thc m R N h I S restricted t o the pars tuberalis ot thc :icicnoIivpophysis: : ~ l s ofound in the adult r:it pars tiihcmlis, ;it tlic vcntr:il surt;icc ot the median cniincncc. T h c rt'l subunit mRNA is ; ~ l s odctcctnhlc in the E l 6 rat nasal epithelium of the olf;ictory turhinntcs ;inti in t h e skclctal muscle of the tongue of the dcvcloping rat. In s ~ t l rhvhri~lizationof scrinl sections of t h e adult hrain failcd t o ilctcct r,') mRNA. T h e o c ) sul?llnit mRNA can he dctcctrd in r:it cochlea hv RT-PCRi, using 119specific primers' dcsignctl t o span an intron' of the 09 ncnc nntl thus distinguish cDNA from gcnomic LjNA. I n sitrr hvhritlizntion on sections of rnt cochlcn shows that the r t Y gene is cxprcsscd in hoth the inner and outer hair cells of all cochlcnr turns Chick ci1i:iry ganglion ( E l H: 200-.Z00 copies per ncuronc): In the chickcn optic Inhe, expression ot thc .I2 gcnc is incrcasctl xl0-foltl Iwtwccn E 6 ;ind E l l , after which it rapidly declines. I'hc .f1cxprcssion occurs in n rnstrocal~clalgradient and is coincident with invasion of
'."".62
"' "R
"*'
-
Table 3. Continued Suhunit
Ccll or tissuc typc
Rcfs
thc tectum by retinal afferents. Removal of the eye cup at E2 results in >I(]-foltl reduction of expression of i12 compared with controls. This suggcsts that ~j2 cxprcssion in optic lohe neurones is transiently stimulated hy arriving retinal affcrcnts ;j4
Chick ciliary ganglion (E18: 200-300 copies mRNA per neurnne); superior cervical ganglion (EIO);Purkinic cclls of rat cerehcllar cortex
"0.'".49
An autoimmune disease involving antibodies against nAChR 09-14-04: Myasthenia gravis, an autoimmunct discasc charactcrizcd hy muscular weakness and fatipahility, results from a hreakdown in immune tolerance of the nAChR. ~utoantihodiesito the nAChR are found, and immune complexes ( 1 g ~ tand complcmentt) are deposited at the postsynaptic mcmhrancs, causing interfcrencc with and suhsequcnt destruction of the nAChR (rcvicwcd by "1. A numhcr of infcctious agents, including H S V ~ 1" and several cram-ncgativct hactcria".', cncodc molcculcs that immunologically cross-react with thc nAChR r-chain scqucncc and might initiate thc hrcakdown of self-tolerancei. 09-14-05: Immunization of mice with nAChR purified from Torpedo electric
organ causes a disease similar to human myasthenia gravis, tcrmcd experimental autoimmune myasthenia gravis (EAMG), susccptihility to which corrulatcs with thc H-2 liaplotypei. Pepti~lus derived from the murinc musclc nAChR r subunit strongly stimulntu T hclpcri cclls from - immunized H-2d m i ~ c " ~ .
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Protein distribution Distribution o f nACkRs in adult skeletal muscles 09-15-01: Thc nAChR channels at thc ncuromuscular junctionj, localized hy
immunogold clcctron microscopyt, are conccntratcd at thc crests of the postsynaptic folds and immediately surrounding mcmhranc foIding~',~.
Distrihution o f nAChRs in foetal muscle or denervated adult muscle 09-15-02: In foctal skclctal musclc, the nAChR protein is founri throughout
the muscle cell memhrane. If adult skeletal muscle is denervated, nAChRs arc synthcsizcd and appear throughout thc entire surtacc of the musclc. This increase in cell surface receptors is prcccdcd hy an accumulation of nAChR mRNAs in the muscle sf^.
Distribution of neuronal nAChRs 09-15-03: Many of the nAChRs in hrain appcar to he located on ncrvc
-
terminals, whcrc their role is presumed to he the modulation of transmitter rclcasc. Radioligand-hincling studies in the cat visl~alcortex dcmonstratc the prcscncc of pre-synaptic ~ A C ~ R S " ' Immunochemical . studies with
entry 09
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monoclonal antihodics against electric organ nAChRs show labelling of t h e lateral spiriform nucleus (SpL) nntl specific lavers of t h e chicken optic tectum, w h ~ c his t h e principal site of termination of SpL ncuroncs ;lnd contains axonT tcrn1inals with nAChR i ~ n m ~ t n o r c a c t i v i t y ~ .
nACllKs on tlic non-xyntlplic .sur/~luc~.s in lhc optic tccrum 09-15-04: Electron microscopic cx;~min;ationof irnmitnr~lnhcllcdnAChRs in
t h e optic tectum of t h e frog shows that t h e rcccptors arc prcscnt o n t h e non-svnaptic surfaccs of vesicle-hearing prc~filcs"". It is suggcstcd that ACh relcasctl from cholincrgict terminals in t h e nucleus isthmi of the optic tcctum hintls nAChRs on retinal affcrcntst and motlitics their release propertiesx.
Suhunits c.ornhinntion,s in ciliary grrn,yliir 09-15-05: At least five ncuronal nAC:hR gcncs,
7.3, 25, 27, /12 and 14, are exprcssctl in chick ciliary ganglia. ~ m m u n o p u r i f i c d ~nAChR from emhryonic chick ciliary ganglia has hccn shown hy Wcstcrn hlottingt with subunit-specific monoclonal nntihotlics t o contain ~ 3r5, ant1 P4 ~ u h u n i t s ' ~ . Antihodv specific for t h c 7.3 s l ~ h u n i tremoves XO'X, of t h r P4 suhunit ant1 7,Z":, of the 75 suhunit from a mcml,r:anc extract; s i ~ ~ ~ i l a ranti-114 ly, rcmovcs .ZXo:, of the .;uhunit ;~ntl 56"1, of ttic 7.5 suhunit. Sequential immunonffinityi purification ot t h e rcccptors using anti-23 followctl hy anti-114 nntihodics viclds receptors that contain sul,stanti;al a m o u n t s of t h e 75 gcnc p r o d ~ ~ cThese t. findings support t h e conclusion that n significant proportion of t h c receptors fro111 synaptic sitcs in chick ciliary ganglia cont;ain t h e co-;asscmhlcd 7.3, YS and /I4 suhitnits. T h e same n A C h receptors lack t h c 77 suhunit, Ilut this is prcscnt in t h c distingitishahlc, r-Rgt-hiniling nAChII from non-svn;aptic sitcs in chick ciliarv ganRli;~"'.
Ncrironcll ( 1 7 .suhzir?it.s 09-15-00: T h e scrccninv of a chick brain c D N A lihrary with synthetic
oligonuclcc~ticlc~p r o h c s hascd o n N-terminal pcptidc sequences of a n 1hungarotoxin-hinding protein sithunit Icd t o t h e ist~lationof a c D N A clonc encoding ;I novel Y . ; ~ ~ l > i ~tcrmcd nit, 77'".
l)rc\~alcncc~ o f ( 1 7 .sr~l~unit,s ill nAChI1 from rcri~hrlhrm 09-15-07: T h e receptor affinity purificti fro111 chick cerebellum hv hintling t o Y-hung;~rotc~xin contiains ;at least three suhunits c ~ :Ippnrcnt f mol. w t 51000, 5 7 0 0 0 ant1 67000. T h e i ~ s coi monocl(~nalantihotlics specific for t h c 1 7 suhunit ticmonstratcd that 75% of t h e I ~ I ~ I C C I I I C S prcscnt in t h e purified prcpnration ;Ire of t h e 27 suhtvpc nntl that this antihodv lahcls t h e 5 7 0 0 0 I,;~nd in ;I Wcstcrn hloti. Rcconstmction cxpcrimcnts in planar lipid I>il;~versshow t h ; ~ t thi.; 7-llockcd hy (~t)-tnhoc~~rarine"'.
Ilistrihution o f niluronal 08 .suhut?its 09-15-08: Low-stringcncv scrcrniny: of
-
;I chick hrain c D N A library with n cl)NA prohc for t h e 77 suhunit rcvcalcrl ;I second cDNA clonc encotling :I tlistingt~i.;hnhlc 2-rcl;~tedsuhttnit, now tcrlncrl r ~ ' " . f m m u n o p r c c i p i t ~ i o n t ; ~ n d i r n n i i ~ ~ i o h i . ; t o c t ~ ~ n ~ i s li;~vc t r v ' icicntificd n nAChK sithtvpc that
entry 09
contains 28 suhunits, hut not a7 suhunits, as the maior suhtype in chick retina. This suhtype has a lower affinity for 2-Rgt than does the subtype containing only '27 suhunits. The suhtype containing only a7 suhunits compriscs 14% of the a-Rgt-scnsitivc nAChRs in hatchling chick retina. The suhtypc containing r8 suhunits (hut n o 27 suhunits) accounted for 69%, and thc a7z8 suhtypc accountcd for 17%"2. 09-15-09: Amacrine, bipolar, and ganglion cells display r8 suhunit immuno-
reactivity, and a complex pattern of labelling is evident in both the inner and outer plexiform layers. In contrast, only amacrinc and ganglion cells cxhihit 27 suhunit imniunorcactivity, and the pattcrn of r 7 dctcction in thc inner plcxiform layer differs from that of a8 suhunit lahclling. Thcsc dispritics suggest that tho z-Rgt-sensitive nAChR suhunits arc differentially cxprcsscd by different populations of rctinal neurones. In addition, the distribution of sc-Rgt-scnsitivc nAChR suhunit immunorcactivity differs from that of 2-Bgt-insensitive nAChR suhunits".
Suhcellular locations Subcell~rlarlocation o f nAChR in ciliary ,panglion neurones 09-16-01: Two classes of nAChR havc hccn itlcntificrl on chick ciliary ganglion neurones, where they occupy diffcrcnt suhccllular locations. One class is concentrated in post-synaptic membrane and is rcsponsihlc for mediating synaptic transmission through the ganglion. The other, which hinds r-hungarotoxin, is locatcd predominantly in non-synaptic membrane",'.
Transcript size 09-17-01: The sizes of the mRNAs cncndinp. suhunits of the nAChR arc as
f(1llows: z suhunit
/lsuhunit
;. suhunit ii suhunit r 5 suhunit
r 7 suhunit
(T. cnli{ornicn) (mousc) (chickcn) (T. coliiorniorr) (T. ciiliiornica) (T. califomica) (mouscl (human) (chickcn) (human cell line)
2.8 kh' 1.8 kt?' 2.8 khf4 2.0 kh" 2.1 kh" 6.0 kh" 3.3 kh"' 2.7 kh and 2.1 kh2' 7 kh (major)and 3 kh (minor]" 5.9 kh, 2.6 kh and 1 .,? khZry
Chromosomal location Location o f gcnes en cod in,^ n ~ ~ ~ ssuhunits cle in the mouse 09-18-01: The chromosomal locations of the gcncs encoding four muscle
suhunits of the nAChR in thc mousc havc hccn dctcrminctl hy R F L P ~
entry 0 9
n
-
analysis of DNA from crosses hctwccn Mu\. n~l~sc-nlis tiornr*stic.~~s (DRA/2) and MIIS.sprrP!lrs(SPEI. T h e r gene maps to chromosome 17, the If-gcnc to c h m m c ~ s o ~ n11 c and t h r ;* ant1 rF genes arc closely linked on mouse chromosome I '".
Encoding 09-19-01: Sec Cknc f(in?iJy,09-05, for ( I list o f the pmtrlns encoded hv
the ,yenes of thc nAC17R f n n ~ i kin . vnr~oussprcicr Cl(lssificntiono f suhunits
( I S (t
or
.j
09-19-02: Assignment of a neuron;ll subunit to the ' 7 ' class is haset! on the conservation of the adiaccnt Cys residues at the positions h o r n c ~ l o ~ o utos
Cysl92 and Cysl9.3 of the Torpr,tlo Y suhunitl. Subunits lacking the two adiaccnt Cys rcsitlucs arc generally designated P, hilt investigators working with scrlucnccs from chick and goldfish prefer the term 'non-r'
Mrlsclr subunits rc.cluircd for func.tion 09-19-03: Fully functional nAChli ch:lnncls arc cxprcssctl hy the co-injection of t l ~ ctour cRNAs cncclding the 7, / I , ;* ant1 (5 sl~htlnitsof the musclc channel into Xrnopris oocytcs(". When combinations of RNAs cncc~dingonly a s u t ~ s e t
of t h r suhi~nitsarc inicctctl, the Y suhunit is essential for activity, t o ~ c t h c r with either thc ;*or 15 subunit: the /i suhunits are dispcns;lhlc"6~"'. 09-19-04: Six different comhinntions of thrrc or more suhunit RNAs produce
significant n ~ ~ m l ~ eofr sfunction;il channcls. T h e order of combinations yielding the greatest ;imount of current is rk. -. y/hi rrSt: r15;)r. rtS > T;,. T h e extent t o which a channel type with three different subunits is cxprcssctl is highly dependent upon the ratios of RNAs coding for thc diffcrrnt suhunits ;ind is critic:iIIy tlcprndcnt upon the order of inicction of thc K N A S ~ ~ .
-.
Nr~umnol.sul?units rcquircd for chclnnel f~rnction 09-19-05: Thc RNAs encoding only two nruml suhunits, r and non-lx (alsr~ called /!I, arc sufficient t o encodc functional channels In Xcnopus oc~c~tcs'. Eight distinct Y ant1 four diffcrrnt / I suhunits havc hccn itlcntifictl in ncuronal t i s s ~ ~ cNrural s. /{ suhunits can substitute for muscle /I suhunits in
forming functional channcls in Xcnopuq oocytcsl". 09-19-06: At least five ncuronal nAChR genes, 7.3, 75, r?, /l2 and P4, arc
cxprcsscrl in chick ciliary ganglia. ImmunopurificdT nAChR from crn1,ryonic chick ciliary gangli:~has I,ccn shown hy Western blottingt with subunit-specific monoclonal antihotlics to contain 7.3, 25 ant1 P4 s u l ~ i ~ n i t s " ~ /.SPL, I'roti,1n (l~,s!ril?t~! 1011, 09- 15).
Thc~( 1 7, ( 1 X (lnd 0 9 subunits form homo-oli~yort~cric ch(lnnc:ls 09-19-07: Thc chick 77 and 78 ant1 the mt 70 nAChR suhunits asscmhlc into
functional homo-oligomcrid channels, responding t o acetylcholine, whcn thc corresponding cRNAs arc singly inicctcd into Xcnopus oocytes17~"7v2'R.
cntry O'I
-
Most rv-R,qt-bindingproteins in hrain contain the ,r 7 suI?unit 09-19-08:The mature r 7 protein (479residues) has modcratc homology with all other 1 and non-1 nAChR suhunits and prohahly assumes the same transmemhrane topography. A hactcrial fusion protein containing residues 124-2.39 of 17 hinds lahelled z - ~ g t ' ~In. sit11 hybridizationt maps of 17 mRNA closcly rescmhle the pattern of ['2'~1-r-~gt hinding in rat hrain, sug~cstingthat most rRgt-binding proteins in thc tissue contain 1 7 suhunits. The rR suhunits occur less commonly, representing only 15% of the I-Rn-hintling complexes and tcnding to he associated with the morc abundant 17 s~~hun~ts''.
nAChH from insect CNS 09-19-09:A nAChR purified from thc cockroach CNS hy a-Rgt-binding has an ovcmll size of ahout 300 kDa, hut produces a single hand of 65 kDa on denaturing gels"v. Reconstitution into lipid hiIaycrs prnduccs channels that are gatcahlc hy nicotinic agonists and blocked by thc antagonist, (+)-tubocurarine7". Sequences encoding a cockroach suhunit, zL1, havc bccn cloncd ant1 expressed in Xenopus oocytes, where they specify functional channels gated hy nicotine and hlockcd hy a-Rgt and n - ~ g t . "
Subunit composition o f functional neuront~lnAChR channels 09-19-10: The singlc-channel conductancrt and currcnt arnplitudct of ncuronal nAChR can he manipulated hy c h a n ~ i n gthe chargccl rcsidilcs immctliatcly downstrcsm of the M2 region. Changc of E266 to K in the ~4 suhunit rcduccs thc single-channel conductancc nf z4/[11 channels, while change of the analogous residue (K260) in pl to E incrcsscs the singlcchannel conductancc. When a comhination of cDNAs encoding n4E266 and r4K266 is co-injected with cDNA encoding plE260 into Xcnoplls oocytc nuclei, channels with three different amplitudes arc detectcct in insidc-out patchcs7'. This finding is the prediction if the functional nAChR contains two z suhunits.
Evidence for the pentnmeric nature o f neuronr~lnAChR 09-19-11: Similar experiments co-iniccting cDNAs specifying two distinguishnhlc P suhunits plus onc 1 suhunit ([I!, lIlE260 plus 14) result in four distinguishahlc current amplitudes, as predicted if thcrc were three /i suhunits per functional channel. Thus the functionnl ncuronal nAChR is a pcntarncr, of composition z2/i,372.
-
n
09-19-12: When the nAChRs synthesized following injection of chicken r 4 and /{2 mRNAs into Xennpus oocytes arc la!>cllcd with [,3S~]-methic~ninc, 1.46 times morc lahel is found in /I suhunits than in r suhunits, after correction for their methioninc content7-'. This ratio is very close to the value of 1 .S expected for a stoichiomt.tryt of .x2/i,1.
Gene organization Introns and exons in the
tu
subunit genes
-
-
09-20-01:The chickeni4 and human'" 1genes havc nine coding exonst. In the human r gcne, the lengths of the eight intronst arc 4 . 9 kh, 1 1 1 hp, 1.7 kh, 3 . 1 kh, 0.4 kh, 3.4 kh, 1.2 kb and ,324 hp in the 5' tn 3' directinn'".
-
-
-
-
Intron-rxon structure o f the 02, r r 3 09-20-02 T h e chickcn 22,
y.3,
nrlrI
r r 4 suhunit genes
34 and nan-x gcncs and the rat r2 and 33 gcncs
all have six p r o t e ~ n - c n c n d i ncxonsr, ~ the fifth of whtch 1s large and cnccldcs prntcin sequences h o m o l ( i ~ o u sto th(lse s p c c ~ f ~ chy d cxnns 5 through 8 of the r gcnc'J. T h e pnsltmns of the exnn-intmn houndarics in 12-75 anti nx 1 -nrOVL'], cxons Ill and 1V in the xY gcnv nrc ft~.;ctl"~. [ N o t e . T h c mtron-cxon structurc c ~ fthc r 9 gcnc wnr rlctcriil~nctlhy coniparlnK r;lt cI3NA scqucncc wath n>otrscbgcnomic scquuncu2IH , so that the information presently available strictly appllcs only to thc tnotlrc rY gunc.]
Intron-exon structure of the h ond 3 suhunit Kenems 09-20-05: T h e chick gcncs encoding thc ii ant! ;. s u l > ~ ~ n both i t s contain 12 exonsl. Thc hornol(~gouscxons of the t w t ~gcncs arc vcry similnr in sizc and thc splice sites1 arc ~ x a c t l yconserved. The corrcsprlntlinp, intronsi (if the two gcncs differ sharply in length anti ~ c ~ u c n c c T' ~h c. two gcncs arc vcry closuly linkcd in the chickcn genomc, with only 740 hp I>ctwccn the last cod(ln c ~ fthe ii gene and the tmnslation-initiation codon of the ;. gcne. T h e intergenic region cclntnins n single cnnonical polyadenylationt site, 77 hp downstrcatn ot thc 6 gcnc tmnslntinn-tcrininnt~oncodon''. TIlc human ~5 gcnc ;11so contains 12 cxons, :in
f
The transcription-start site for the h genc 09-20-08: Thc transcription-start site for thc mousc (5 gcne has heen mappcd 55 hp upstrcam of thc tmnslation-initiation codon. A scqucncc TAAACCA at positions -33 to -27 rclativc to the transcription-start sitc is presumed to servc as thc TATA hoxi, and a CATTG sequence, complementary to the C A A T C ~ hox, occupics -66 t o -62. CG-rich scquences having homology with known AP-2t-binding sites are centred at positions -55, -180 and -210~~. A sequence in the h-gene promoter necessary for muscle-specfic expression 09-20-09: A 54 hp rcgion of 5'-flanking DNA from the murinu gcnc encoding the ii suhunit, occupying - 148 to -95, is ncccssary and sufficient for musclespecific gene-expression. Deletion of this sequcncc results in a 50-fold rcduction in cxprcssinn in myotuhcs, whilc fusion of the scqucncc upstream of thc c-fos hasal promotcr confers myotuhc-spccific gcnc expressinn on an othcrwisc wcak, non-tissuc-specific promoter. Thc musclc-specific transcription factor MyoDl d c ~ snot hind to the 54 hp rcgion, hut other nuclear proteins from myotuhcs arc ahlc to hind this clument7'.
Consensus sequences for muscle-specific regulatory motifs in the promoters of genes encoding nACI?R suhunits 09-20-10: Thc consensus sequcnccs for nAChR gcne enhancers contain scvcral motifs that havc previously hccn implicated in tissue-spccific gcnc cxprcssion. Thcrc arc two copics of the CANNTG motif prcscnt in t h e MyoDl target scqucncc characteristic of many musclc-specific regulatory regions: thcsc flank n 'M-CAT' motif and an overlapping TGCCTGG scrluencc, both of which havc hecn proposed as muscle-specific regulatory motifs7'* 74. 1
A sequence common to several nAChR suhunit sene promoters 09-20-11: A sequcncc common to the chickcn z gcne and the mousc PI ;-and (1 gcncs has hccn termed the suhunit homologous upstrcam clement (SHUEJ box. The 13 hp SHUE hnx scqucn<e, all four copics of-which contain CCCTGG/C, is locatcd at -15.5 in the mousc ii gene and -75 in the chickcn sr gene7'. The function of thc SHUE hox has not hccn dctcrmincd.
I
Close Iinknge o f the h and 7 suhnnit gencs 09-20-12: Thc penes cncotlinc the ;iand :-subunits arc vcrv closclv linked in
the chickcn, mouse and human gcnomcs. T h e ;, gene lies 740 hp downstrcam of the ;igene in chicken" and 5000 hp downstream in mouse7'. This, t o ~ e t h c r with the high degree of sequence homcllogy and identical exnn-intmn structure, supports the idea that the two Rencs havc undergone a rclativcly recent tandem duplicatinnlR. T h e human ;, and 6 gcncs can he isolated on a single genomic DNA fragment of 15.7 kh, with B upstreamt of the y
Clr~sterin~ of the no,?, 03 r~nd05 genes in chicken 09-20-13: The threc gcncs encoding the nr3, 7.7 and r 5 suhunits arc clustered in thc chicken genome. Gene nr;i lies 5' of r.3 and is tmnscrihcd in the same .rlircction, whcrcas r 5 is located on the %?'sidec ~ x.3 f and is tr;~nscrihedfrom thc c~ppositcDNA strand. T h e 112.3 and 23 genes arc separated hy ahout 5 kh, and 2.3 anti r 5 hy less than I kh'". There is also an 13 gene cluster, containing gcncs encoding P4, 2.3 and 15 snhunits, in the mt genome7.
Homologous isoforms Homology amongst n~usclrsuhnnits 09-21-01: The musclc r, /I, *; and (isuhunits show scqllcncc similarities with
cach other and with suhunits from othcr channcl types, including the mouse serotonin-gated ion channcl and several glvcinc and GARA receptors. T h e {r' suhunits show approximately 42-45"!, scqucncc idcntity, the ii suhunits havc ahout 405, idcntity and the ;, suhunits .l7-40"/n idcntity with the r suhunits.
Homolo~yhetwccn
(111
nAChR subunits
09-21-02: There is strong conservation of amino acid scqucncc amnngst all
known suhunits c ~ fthe nAChR. This conservation is not evenly distrihutcd throughout the polypeptide chains, hcing strongest in the region that includes the transmemhranc domains M I , M2 and M,Z (see Domain ~.c~nscrvrttio~l, 09-2XI.T h e percentages of amino acid idcntity hetwecn the suhunits of the muscle and ncuronnl nAChRs are shown in Tahlc 4.
Xcnopus mrrscle n suhunits 09-21-03: T h e two Xrnol~upmusclc r isoforms are 89'3'0 similar t o cach othcr,
and they show similar levels of similarity (XS-XY%) to the mammalian or T(>rprdcT suhunit2,'.
Protein molecular weight (purified) The nAChR purified from Torpedo electric orgnn 09-22-01: The nAChR purificd from Torpedo electric organ is a 250 kDa pentamer, formed hv four different types of subunits ( r ,1,;., dl with x2P;d stoi-
chic~mctr~".The individual suhunits, cach c ~ fwhich is glycosylatcd~,have apparent molecular masses of 40 (71, 50 (111, 60 (;.I and 6.5 (h) k ~ a ' ' +
Thr: chicken hrnin nAChli 09-22-02: An nAChR i r n r n ~ n o ~ u r i f i e dfrom t chickcn hrain is cnnstitutcd of
subunits of 49 k n a and 59 kDa mcasurcd hy elcctrnphorctic mohility on
Table 4. Amino acid identity (%) between nAChR subunits (From 09-21-02) n
h
36"
n2
03
n4
n5
n6
a7
32'
08
.I
,?2
,'33
,
j4
f
41"
Footnotes quoted are the percentage amino acid identities between different subunits of rat, chicken ('1 or Torpedo ("1 nAChR subunits.
entry 0)'
-
denaturing gelsR2. Antihodics raised against this material arc ahlc to i m m u n t ~ p r c c i ~ i t arcccptctrs t~ containing 49 kiln and 75 kDa components. In cach case, thc l a r ~ c rs ~ t h u n i t sarc affinity lahcllcdt hy ('HI-MRTA and havc N-terminalt sequcnccs homol(~goils to thosc of r suhunitsH'. Subsequent studies havc rcvealud that thc N-terminal scqucncu c ~ fthe 75 kDa hand corrcspnnds to that of thc r 4 suhunitx" and the N-terminal scqucncc of the 4Y kDa hand to that of thc 112 sul,unitH5. T h e 59 kD8 hand prohahly includes hoth 12 anrl 3.7 su huni tsHh. 09-22-03: T h c 2 7 suhunit irnn~unr~pitrifiedtfrom chickcn h r ~ i nhas an apparcnt M , nf 57 k n a r,n rlr-nnturing gr.lsH7.
l'urificd nAChK from c h i c k e n c i l i ( ~ r vg n n ~ l i n 09-22-04: Purification of nAChRs from extracts of chickcn ciliary ganglia yicl(ls a fr;lction showing thrcc componcnts on denaturing gels, at 40, 52 and 60 knaRH.Thcsc havc hccn identified, using subunit-specific antihodics, as the 2.5, if4 and 2.7 suhunits, rcspcctivclv. It has not hccn cstahlishcd that thcsc components asscmhlc into a singlc nAChR.
The nAChR pzrrified from rnt l ~ r r ~ i n 09-22-05: An nAChR immunopurificd from rat hrain with anti-chicken nAChl< mAh 270 contained sul>units of apparent M, 52 and 80 kDaHV.T h c N-terminal scqucncc c ~ the f XO kIla suhunit corresponds to that of ~ 7 and 4 ~ that of the 52 kDa suhunit to the N-terminal scqucncc of the P2 suhunitH5. Antihodics to thcsc two components rcmnvc ; ,90% of the high-affinity ['HI-nicotine- or I . ' ~ 1 - c ~ t i s i n c - h i n d isites n ~ from detergent-soluhilizcd rat hrain cxtracts, sumcsting that nAChRs cnmprised of 74 and P2 suhunits arc the maioritv spccics in mt hrainY1.
Protein molecular weight (calc.) 09-23-01:Srv, r3(1frr I I I TrlJ~lr'I r1ndr.r (;c8nc f(1rn11v.00-05
Southerns Sout h e r n b l o t s o f c h i c k e n g e n o r n i c D N A 09-25-01: Rlots of chicken gcnomic DNA digested with EcoRI, RrlrnHI and ffinti111 showcd singlc hantls hybridizing t o 5- and ;,-specific probes. T h e two prnhes rcvcal the samc-sizcrl EcoR1 hand, hut distinct hands on an EcoR1-Hind111 double-digest, consistent with the two gencs hcing very 09-20) closcl y linkcd on n singlc Er.oRI fragmcn t (sce Cknc r~r~qclniz(~tion. and with thcir hcing untquc in tlir chicken gcnomclR. Southern rlnrn!y51,s of Xcnopus ,pcnornrc D N A 09-25-02: Southern blot4 of Xcnoptr\ gcnomic DNA prohcd with 7 I , and x 1 h prohcs l n d ~ c a t cthe prcscncc c ~ two f dlftcrcnt Rcncs, cach prcscnt in a ~ ~ n g l c copv pcr gcnnmcz'.
entry 09
-
Domain arrangement Transmembrane domains 09-27-01: Each nAChR suhunit contains a large, hydrophilict, extracellular N-terminal domain, four hydrophohict transmcmhrane domains and a short, extraccllular C-terminal region. The scqucnccs of the four strongly hydrophobic scgmcnts (MI-M4) and that of a rcgion that suggests an amphipathict helix (MA) arc strongly conscrvcd amongst different suhunits and across species. Covalent lahclling of all four scgmcnts with photoreactivet phospholipids supports thc contention that the MI-M4 regions arc mcrnhr;~nc-spnnnin~~*.~~'.
Secondary structure of transmembrane domains 09-27-02:There is no direct evidence that MI-M4 arc z-helical and they are at least candidates for fi-sheet-formers according t o secondary structure prediction algorithmsA".
The MI domain is occessihle to open-channel blockers 09-27-03: The proposed open-channel structure of the nAChR s u ~ e s t that s the putative r-helix MI is exposed at the interstices (clefts] hctwccn the M2 helices. This interpretation is supported hy lahclling of MI helices with open-channel hlockcrs such as quinacrine (see relsv".v7).
Domain conservation Conserved Cys residues in all subunits 09-28-01:All the suhunit genes encode proteins with two cysteincs separated hy 1.3 residues that align with C128 ant1 C142 of the muscle z suhunit. Inv~iriantCys I92 and 193 in tr snhunits 09-28-02: All r suhunits from muscle anrl electric organ have adiacent cystcines at positions corresponding to musclc 1 192 and 193. Neuronal suhunits with this feature arc designated as 2 suhunits; those without it arc designated non-T lavian species) or fl (mammalian species). (Note that the desi,r.nation '11' docs not mean that such a subunit most closelv resembles the musclc p subunit. The neuronf~l/Isnhunits reprc.scnt memhers of o hetero*qeneorrs Rrorrp united hy their common Irlck of the two adincent cvsl r i n ~ s ! ) Similarities between mr~scleand ncuronal suhunits 09-28-03: Thcre is approximately 60% identity hctween the musclc and neural .x suhunits over the first 320 residues and in the MA and M4 regions near the C-tcrmini. The ncuml sequences contain an insertion of 60-160 amino acid residues in thc cytoplasmic region hctwccn M3 and MA. The similarity between muscle and ncural non-z sequences is slightly less - (about 44% in the first 350 r c s i d u c ~ ) ~ " .
entry 09
Overall sequence conservntjon 09-28-04:There is striking conservation of sequence amongst all the suhunits of nAChR channels. Amongst the first 320 amino acids, one-third arc conscrvcd. The strclngcst conscrvation is hetween rcsiducs 224 and 320, which includes the M1, M2 and M3 regions, where there is ahout 50% identity. Thcrc is also ah?ut 25% similarity in the first 223 rcsiducs that constitute the N-tcrminali cxtraccllular domain. The conscrvation is much less for the large cytoplasmic domain hetween M3 and ~ 4 ~ " .
An invariant Pro-Cys in M I 09-28-05:Thcrc is an invariant Pro-Cys (221 and 222) in the centre of the MI helices of the 2- and non-r-suhunits of the nAChR. The Pro will introduce a bend of ahout 20 into an z-hclixi, disrupting local hydrogen-hondingt and leaving amidc and carhonyl groups free to interact with water or a permeating iongH. It has heen s u ~ c s t c dthat the Pro may he a focus for conformational changes involving cis-trans isomerizationT of the peptide bondH".
Similarity bet ween nAChR and ryanodine receptor 09-28-06:The region encompassing segments M2 and M.3 of the nAChR show some sequence similarity with the M2-Mnicl.'.'. Thrcc amino acid differences, changes of Clu237 to Ala, Val251 to Thr nnrl the addition of n Pro after residue 2.36, arc sufficient to produce channcls that were 500-folti more sensitive to ACh, are activated by the competitive antagonist dihydro-8-erythroidine (DHPE), n o longer show inward rectificationi of whole cell currents and arc onion-selective. The M I t o M2 spc~cingc~ffccts ion .selectir~ity 09-29-23: Deletion of the extra Pro residue from this mutant r 7 channcl gives functional channels that show the enhanced sensitivity t o ACh, d o not desensitize r;~pidly, arc cation sclcctivc hut do not pass ca'' currents. Inversion of inn selectivity is also achieved hy the addition of cithcr an Ala or a I'rc~ rvsirluc, fc~llowingposition 2.76, to ;I cation-selective 77 mutant. These d;~tapoint to the irnportancc. of the length of the segment spacing M I and M:! in determination of the ion sclectivity'"~'.
entry 09
The tr7 T251 channel shows additional conducting states 09-29-24: The single substitutions, Clu237 to Ala or Val251 to Thr, do not
invert the ion selectivity, but the Thr251 mutation docs change the apparent affinity for ACh, response to DHPE and rcctificationt properties of the channel. Single-channcl recordings from outsidc-out+ patches containing the Thr251 a7 nAChR show multiple conducting states, including one of 54 pS, similar to the wild-type A state, and one of 86.3 pS, corrcspnnding to a desensitized D' state of the nAChR sccn with the Thr247 r n ~ t a n t " ~ .
The CIu237Ala change in ru7 nffects cap'-selectivity 09-29-25: The alteration of Glu217 to Ala in the 27 nAChR results in a
receptor that responds to ACh normally and shows rapid dcscnsitization~. Although this mutant channel is permeahle to cations, it has lost the ahility to conduct Cab '
s
~
.
Conclr~sionsfrom observations with mutnnt channels 09-29-2fk Structural interpretations of the data ohtained from studies with
nAChR channels altered in the pore region indicate that the wide entrance vestibules of thc nAChR pore contain net negative charges which can attract cations and are particularly important in attracting divalent cation^'^.'. Upon channel opening, thc pcrmeant cations rapidly pass through the uncharged, tapering rcgion of the poret, lined hy the M2 sequences from each of the suhunits. The geometry of the M2 segments, influenced hy the M1-M2 spacer region, is crucial in tlctcrmining ion sclcctivity of the channcl. The narrowest rcgion of the open pore, in the rcgion of Thr244.1, is very short (estimated to contain as fcw as six water molcculcs) and is likely to he the only rcgion of the channcl whcrc strong interactions between permeant ions occur^'"^.
Closed structure o f the nAChR channel 09-29-27: The closed structure of the channel is more difficult to invcstigatc,
for ohvious reasons, hut experiments with M2 peptidcs havc suggested that an association to hlock the porct, with thc appropriate stahility, could he achieved hy interactions hctwccn the M2 r-helices of thc suhunits'.'". Thc clcctron microscopic images of the Torpedo AChR suggest that amino acid residues come closest to the axis of the pore just helow thc middle of the hilayer when the channcl is closed. This coincides with the position within M2 of the highly conserved Leu residues that are known to be in the narrow rcgion and to face the lument of the channcl (scc nbovc). It has hccn suggested that the bulky, hydrophobict Lcu side-chains of the five r-helices associate t o create a harricr of limitcd stahility that forms the gate1.'".
il
09-29-28: Thc MA segment, located hetween Ma?and M4, is part of the
cytoplasmic domain ant1 docs not form part of the poret. Deletion of scqucnces encoding part of MA in the r suhunit eliminates channcl function in the Xcnoptrs oocytc system. Whcn MA and the 20 preceding amino acids arc rcmovcd. 3% of the native activitv is ohtaincd'~".
entry OY
St ruct l ~ r m lchong~:s on riesensiljzrltinn 09-29-29: AEtcratir>nsin protein stmcturc prod~rccrlhy binding r)f cholincrgic :lgonists to purificd nACliR rcconstitutcrl into lipid vcsiclcs can he cIctcctcd hy Fourier-tr:lnsform infrarctl spcctroscopy~ and diffcacntial scanning caloriinctryt. Spectral c h n n ~ c sindicate that the exposure of the nAChR to thc agonist carharnvlcholinc, undcr contlitinns which drive the AChR into the desensitizcd state, protluccs nltcmtions in the protcin scct~ndary stmcturc. Uuantitativc estim:ltio~l of thcsc agonist-inrll~ccd alterations rovc;ils nt? significant changts in the percentage of x-hcliit, hut a decrease in /!-shcctk structure, concomitant with an incrcasc in Icss-ordered stnlctitres. A ~ o n i s t hintling :ilst, rcsults in a ct~nccntration-dcpcncIcnt incrc;lsc in thc pmtcin's tlicrni:~l st:ihility, ns inilicatctl hy thc tcmpcratl~re dcpcndcncc c ~ fthe infr;~rctlspcctrurn a ~ l dby calorimetric analysis, further sumcsting that nAC:hR desensitizatiani intlllcctl hy the cholincrgic :~gnnist involves si~nificantrcnrrangcincnts in thc protcin structurc'"'.
Predicted protein topography Lnhellin,p ,studies 09-30-01: Given fnur ~ncmhmnc-spanningpcpt i J c s ( M 1-M4) in cad1 suhunit (SCI' I l o r n i l j z ~~ l r r m ~ ~ ~ 09-27), ~ ~ r l ~t hc ~ ~Nn tand . C-tcrminal regions must hc on
thc snrnc sitic of thc iilci~lhranc. Antibody anrl chemical lahullinR"H~'~7Y specify the N-tcrmin:alt, ~ - t c r n I i n a l tand M2-M,? linker regions as cxtraccllulnr, with t h c srnall MI-M7. ant! largcr M3-M4 linkers intraccllular, This determination s u ~ c s t sthe MA region t11 ho intraccllu!ar, at or near the membrane-cytoplasm intcrfacc.
Elcctron micrnsr:oy?l'cnnrr1ysi.s of channel strr~cturrat 17 A resolrltion 09-30-02:E!cctrt~nmicroscopy and 3-dimensional image-rcct~nstructionthas providcil the strLtctitrc of tllc pnst-synaptic Tr~rpcdomnrrnormtrr nAChR channel to n resolution of 17 A". I'ost-synaptic incmhrancs forrncd into tuIn!l:ir vcsiclcs sitspcndccl in thin films of icc pcrrnittcd the receptors (organizctl into helical :irrays] to hc seen froln all nnglcs, revealing the r c l ; ~ t i o of ~ l the lipid I7ilaycr nntl thu peripheral protcin (Fix. 2). A pcntomeric, /7r1rrel-stnvenrmngement oJ subunits 09-30-03: T h e nAChR is s pseudosymmctric pcntamcr, with the suhunits in a harrcl-stave arrangcrncnt (.we Fi,y. 2). T h c channcl ct~mplexis ahout 120 A long, prt~icctingahout A0 A into the cxtraccllular space and ahaut 20 A into the intraccllular solution. T h e rliamctcr at the cxtraccllular cntl nf the channcl is ahout 813 A, narrowing to ahnut 50 A in tho mcmhranu-spanning reginn. T h e conduction pathway can he soon to consist of a narrow central porcl across the hilnycr, t c r ~ n l n n t i nin~ entrances 213-25 A wide (fnr rrvicw Si,F I ~ J J . ~ ~ ) J .
of the mntlsr muscle nAChK 00-30-04: T h e structure of thc channcl ohtainod by c x p r c s s i n ~cRNAs
Strtzcture
cncndinc, the four mrlusc niusclc subunits in Xenoprrs oclcytcs can he
cntry 0 9
I
L
0
a
;? h -
iI -
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Figure 2. Axial section thro~rghthe cylindrically ~veragedstmcturc of the Torpedo marmorata nAChR. show in,^ details o f the channel in relation to the lipid hilayer and the peripheral 43 kDa protein. ot the hottom o f the figure. (Reproduced with permi.v,virn from Toyoshima (1988) Naturc 336: 247-50.) (From 0930-02) visualized hy atomic force microscopyt. Thc pcntamcric structure with a central porc is ohscwcd on the extraccllular facc of thc memhranc. Thc angle between thc two Isuhunits was 128 and thc unit cell ahout 10 nm diameter'".
Analysis of the Torpedo channel at 9 A resolution
09-30-05: Analysis at higher resolution (9 A] has hccn ohtained hy recording
images at diffcrcnt lcvcls of dcfocus and averaging data using helical diffraction1 mcthorfsidn. This method allows some identification of secondary structure, particularly thc r-helicalt rods within cach suhunit. In the synaptic part of cach suhunit there arc thrcc rods, oricntcd pcrpcndicular to the plane of thc hilaycr. T w o of thc rods line the cntrancc to the channel, with thc third on thc outside. In the region of thc rcccptor that spans the hilaycr, cach suhunit has only onc visihlc rod: sincc this forms thc lining of thc porc it is assumed to he thc M2 transmemhranc helix. This rod kinks and tilts near its midpoint, whcrc it is closest t o thc axis of the outwards on cithcr side. It is flanked on thc lipid-facing sidcs hy a continuous rim of dcnsity that is intcrprctcti to hc P-shcctt.
Position of the M2 domain in the stnrcturc 09-30-06: Alignment hctwt.cn thc thrcc-dimensional dcnsitics and the scqucnce of M2 placcs the charged rcsiducs at thu cnds of M2 symmetrically hcstridc thc hilaycr, and a highly consuwcd Lei1 rcsidl~c(Leu251 of the z suhunit) at the position of the kink. A model is s u ~ c s t c din which the side-chains of thc Lcu rcsiducs at thc kink proicct into the porct to form a hydrophobic ring, closing thc channcl hy ma kin^ a harrier that hydratcd ic~nscannot cross'40 (Fir. 3).
entry Oc)
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/:lunittrom r:lt, ~vhiclili:~s. 7 X 0 : , identity to t l ~ cr7 ; ~ n t lYS suhunits t \ i P ( ,Ilor~ioloyo~rx rxolort?~\.(10-2I1, ;11.;o fortiis ;I homonicric crocytcs2". In channel that 1s pcmic;~hlcto (:;I" wlic*n csprcszctl in .Yrjr~o;~~rs hoth tlicsc c ; ~ \ c s~, n f l u of s c:;I" tringcrs ;I (:;I "-scns~tivcC:l currrnt. Mur(lr?l r , / ~ r l n ~ ~ ttTitll i , l s ir~.rc,cl.cotl~ c ~ l [ ~ . t i/or \ f i r1ir7cllr1i~t t~~ c.rrtio11s 00-40-00: T h e rcpl;lccmcnt of Thr?.-$4 Ilv Asp in thc p i ~ t ; ~ t i vch;~nncl-f(rrnling c ML scgmcnl ot the c l i ~ e kncurcrn;~l 77 nA(:Iill sulitlnit produces ;I m;~rkcd clx~ngcin the .;clcrt~vitv ot the Iic)mo-oligoriicric ion cli;~n~icl protllrcrtl I>v exlircss~onIn .Y(*r~oprr\ crocvtcs. T h e rcl;~tivci o n ~ c~ c r ~ i i e ; ~ l ~of~ wild-tyl)~ litv A to K \ 1 I I.? I 1 I , I , I , 1.4. In ccrntr~lst, c tliscririiin;~ting Iwt\vccn K ' :1ni1 Nn', A(:Iil 100 / I M ) . The high-affinity sites arc present in one copy per receptor pcnt:ltner, with thc low-affinity sitcs hcing 10-,3[)-fold morc
Non-competitive bIockers intetrlct with the open chnnncl
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09-43-02: Lahellinp; of the nAChR hy photoaffinityt derivatives of nnncompetitive hlockers is enhanced by carhamylcholinc and inhihitcd hy histrionicotaxin. The rate of covalent association of chlorpromazine with the high-affinity sitc incrcascs 100-1000-fold (k,,,= 10' M . ' S - ' ) when acctylcholinc is added in the conccnt rat ion range cffectivc for activation of the channcl in vitro. Competitive antagonists hlock this effect. Tlic rate of ( 'HI-chlorpromazine incorpori~tion tlcclincs on prolonged exposure of the channcl to acctylcholinc, with a time course ant1 concentration dependence similar to those of the rapid desensitization1 of the ion-flux responses of the native mcmhrancs of T. crtli~ornicu.These observations sumcst that the non-competitive blockers frccly diffuse to their high-affinity hinding sitcs within thc porct of the open channel'".
entry 09
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..
A -
Spccific a m i n o acids confcrcted h y non-cot71p~titivch1ockc~r.s 09-43-03: T h e amino acids plio~olnhellcdthy ['HI-~hlor~romazine have hcen
irientified hy peptidc-mapping' and sequencing experiments. T h e rcsidircs ~ S c r 2 4 8 ,/ISer254, PLeu257, ;.Thr2Sc3, ;tScr257, ;,Leu260 and (iSer262 arc all lahellcd hy [ 4 ~ ] - c h l o T r o m a z i n cand protcctccl hv phencyclidine. T h e lahcllcd scrines on all suhunits occupy homologous p o ~ i t i o n swithin the putative M2 helix that lines the pore of the ~ A C ~ ~ R ' ~ " ( I . ' I4,)~. .Experiments with alternative non-compctitivc hlockcrs reach very similar conclusionsffn~ "".
l'rocyrstcronr as
(1
hlockrr o f niwronal nACl7R
09-43-04: T h e in;iinr hr;iin nAChR is ;isscmhled from two suhunits tcrmcd 74 CLEFT 7
P
A I L
CMOPLASM
Figure 4. Modc.1 ol the hi,qll-(rffinitysitc for c ~ h l o r p m n ~ c ~ within z i n ~ ~ the nA
