Current Topics in Membranes and Transport, Volume 33: Molecular Biology of Ionic Channels

Current Topics in Membranes and Transport VOLUME 33

Molecular Biology of Ionic Channels

Advisory Board

G.Blobel E Camjbli J: S. Cook D. Louvard

Current Topics in Membranes and Transport Edited by Jowph F. Hoffman

Gerhard Giebirch

Deparhent of CeNUlor and Molmhr physiology Yale University School of Medicine New Haven, Connecticut

Deportment of Celluhr and Molecub physiology Yale University School of Medicine New Haven, Connecticut

VOLUME 33

Molecular Biology of Ionic Channels Guest Editors Wllllam S. Agnew, Tonio Claudio, and Frederick J. Sigworth Depllrtnrnt of Cellub and Molaculnr physiology Yale University S c h I of Medicine New Haven. Connecticut

Volume 33 is part of the series from the Yale Department of Physiology

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I

Contents Preface, xiii Yale Membrane Transport Processes Volumes, xvii

1. ion Channels of Patameclum, Yeast, and Eschotfchla coll

CHAPTER

YOSHIRO SAIMI AND CHING KUNG Paramecium, 1 Yeast, 4 111. Escherichia coli, 6 IV. Conclusion, 7 References, 9 I. 11.

PART I.

CHAPTER

GRAMlClDlN

2. Gramicldin: Conclusions Based on the Kinetic Data S. B. HLADKY

Introduction, 15 Gramicidin Forms Pores, 16 Selectivity, 16 IV. The Evidence That the Pore Is a Dimer, 17 V. Structural Inferences from the Kinetic Data, 18 VI. Ion Conduction through the Pore, 19 VII. Conclusions, 29 References, 30 I. 11. 111.

V

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CONTENTS

C H A ~ 3. R

Gramicidin, a “Simple” ion Channel B. A. WALLACE

Text, 35 References, 48

CHAPTER

4. ion interactions with the Gnmicldin A Transmembrane Channel: Cesium433 and Calcium43 NMR Studies DAN W. URRY, NAIJIE JING, TINA L. TRAPANE, CHI-HA0 LUAN, AND MARSHA WALLER

I. 11. 111. IV.

Introduction, 52 Cesium Ion ’Ransport Studies, 63 Calcium Ion Interaction Studies, 75 Appendix, 82 References, 86

CHAPTER

5. ion Transport through Transmembrane Channels: Ab /n/f/0 Perspectives PETER C. JORDAN

I. Introduction, 91 11. Theoretical Approaches, 92 111. Ab Znifio Methods-General Considerations, 94 IV. Oramicidin, 95 V. Applications, 102 VI. Summary, 107 References. 108

CHAPIXR

6. Rapid Gatlng Events and Current Fluctuations

In Gramicidin A Channels

F. J. SIGWORTH AND S. SHENKEL I.

Introduction, 113 Methods, 114 111. Properties of Gaps in Channel Currents, 116 IV. Open-Channel Noise, 122 V. Conclusions, 128 References, 129 11.

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CONTENTS

PART 11.

CHAFER

NICOTINIC ACETYLCHOLINE RECEPTORS

7. Function of Mammalian Nicotinic Acetylcholine Receptors: Agonisl Concentratlon Dependence of Single Channel Current Kinetics STEVEN M. SINE AND JOE HENRY STEINBACH

I. 11. 111.

Introduction, 134 Methods, 134 Results and Discussion, 135 References, 144

CHAPTER

8. Regulation of the Nicotinic Acetylchoilne Receptor Channel by Protein Phosphorylation RICHARD L. HUGANIR

I. 11.

Protein phosphorylation, 147 The Nicotinic Acetylcholine Receptor, 149 111. Protein Phosphorylation of the Nicotinic Acetylcholine Receptor, 150 IV. Conclusions, 159 References, 160

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CHAFER

9. Synthetic Peptides in the Study of the Nicotinic Acetylcholine Receptor EDWARD HAWRCYT', KIMBERLY L. COLSON, THOMAS L. LENTZ, AND PAUL T. WILSON

Overview, 165 Synthetic Peptides in the Application of Inmunochemical Tests of AChR Structure, 167 111. Functional Activities Associated with Synthetic Peptides, 177 IV. Determination of the Solution Conformation of Small Synthetic Peptides Relevant to the Ligand-Binding Site of the AChR, 187 References, 192 I. 11.

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CHAPTER

10. Expressfon of Acetyichoiine Receptor Subunits

In Sacchmmps cersWs/8e (bast) MELODY T. SWEET, JON LINDSTROM, NORIHISA FUJITA, KATHRIN JANSEN, CHURL K. MIN, TONI CLAUDIO, NATHAN NELSON, THOMAS D. FOX, AND GEORGE P. HESS Text. 197 References, 214

CHAPTER

11.

Establishing a Stable Expression System for Studies of Acetylcholine Receptors TONI CLAUDIO, HENRY L. PAULSON, DEBORAH HARTMAN, STEVEN SINE,AND F. J. SIGWORTH

I. 11. 111.

IV.

Introduction, 220 Materials and Methods, 222 Results, 229 Discussion, 242 References, 243

PART 111.

CHAFIBR

VOLTAGE-SENSITIVE SODIUM CHANNELS

12. Molecular Cheracteristlcs of Sodium Channels

In Skeletal Muscle ROBERT L. BARCHI I. 11. 111.

IV. V. VI. VII.

Introduction, 251 Biochemistry of Skeletal Muscle Sodium Channels, 252 Functional Reconstitution of the Purified Sodium Channel, 255 Channel Primary Sequence, 259 Probing Channel Topography, 260 Sodium Channel Subtypes, 265 Summary, 267 References, 268

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CONTENTS CHAPTER

13.

Electrlcal Recordings from Cloned Sodium Channels Expressed In Xenopus Oocytes WALTER STUHMER

Text, 271 References, 275

CHAPTER

14.

Tissue-Specific Expnmlon of Genes Encoding the Rat Voltage-Gated Sodium Channel SHELLEY A. GRUBMAN, SHARON S. COOPERMAN, MARY P. BEGLEY, JOSHUA L. WEINTRAUB, RICHARD H. GOODMAN, AND GAIL MANDEL

I. 11.

111. IV.

Introduction, 277 Ontogeny of Sodium Channel m e I and '&pe I1 in Rat Brain, 278 Tissue-Specific Expression of Sodium Channel 5 p e 11, 279 Discussion, 284 References, 287

CHAITER

15.

A Model Relating the Structure ol the Sodlum Channel to Its Function H. ROBERT GUY

I. Introduction, 289 11. Model of Sodium Channel Bansmembrane Segments, 294 111. Tertiary Structure, 295 IV. Experimental Tests, 305 V. Conclusions, 306 References, 306

CHAPTER

16.

Sodium Channels in Llpld Blleyers: Have W8 Learned Anything yet3 CHRISTOPHER MILLER AND SARAH S. GARBER

I. 11. 111. IV.

V.

Introduction, 309 The Method, 310 Interaction of Guanidinium Toxins, 311 Fixed Surface Charge, 316 lkto Not Totally Speculative Proposals, 324 References, 326

CONTENTS

X C H A ~ R 17.

Voltage-SensitiveSodium Channels: Molecular Structure and Function WILLIAM S. AGNEW, EDWARD C. COOPER, WILLIAM M. JAMES, SALLY A. TOMIKO, ROBERT L. ROSENBERG, MARK C. EMERICK, ANNA M.CORREA, AND JU YING ZHOU

I. 11. 111. IV. V.

Na Channels as Proteins, 329 Protein Structure and Channel Gating, 339 Function of the Purified Protein, 342 Chemical Modifications That Alter Regulation of Ion Conductance, 350 Conclusion, 361 References, 361

PART IV.

CHAPTER

CALCIUM CHANNELS

18. Molecular Properties of Voitage-Sensitive Calcium Channels WILLIAM A. CATTERALL, MICHAEL J. SEAGAR, MASAMI TAKAHASHI, AND BENSON M. CURTIS

I. 11. 111. IV. V. VI.

Introduction, 370 Identification and Purification of Calcium Channels from Skeletal Muscle, 370 Functional Properties of the Purified Calcium Antagonist Receptor in Phospholipid Vesicles, 377 Subunit Structure of Dihydropyridine-Sensitive Calcium Channel, 379 Immunospecific Identification of Calcium Channel Components in Other Tissues, 386 Conclusion, 387 References, 388

CHAPTER

19. Cardiac Calcium Channels: Pore Size and Symmetry of Energy Profile R. L. ROSENBERG, E. W. McCLESKEY, P. HESS, AND R. W. TSIEN

I. 11. 111. IV. V.

Introduction, 393 A Model for the Ca Channel: A Single-File Pore with l’wo Binding Sites, 395 Recordings in Intact Cells to Estimate Pore Size, 397 Recordings in Planar Bilayers, 402 Discussion, 407 References, 410

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CONTENTS

PART V.

CHAPTER

CONCLUSION

20. Aonotoxlns and VoltagaSensltlve Calcium Channel Subtypes LOURDES J. CRUZ, DAVID S. JOHNSON, JULITA S. IMPERIAL, DAVID GRIFFIN, GARTH W. LeCHEMINANT, GEORGE P. MILJANICH, AND BALDOMERO M. OLIVERA

I. 11. 111.

Introduction, 417 The o-Conotoxins and Their Receptor Targets, 419 Discussion, 426 References, 428

Index, 431 Contents of Recent Volumes, 447

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Preface One of the great accomplishments in physiology has been the linking of electrical events, includingthose involved in synaptic transmission and impulse propagation, to discrete membrane components, the ion channels. Recent progress in biophysics, biochemistry, and molecular biology has revealed ion channels to be integral membrane proteins which form ion-selective conductance pathways through the lipid bilayer, often regulated by the binding of ligands or by changes in the transmembrane electric field. A variety of information will be required to definitively relate the structure of channel proteins to their functions. The articles in this volume illustrate the insights gained from protein biochemistry, recombinant DNA technology, macromolecular structure determinations, molecular dynamics calculations, reconstitution, toxicology, and cell biology. They are organized around discussions of four examples of ion channels, gramicidin A, nicotinic acetylcholine receptors, voltage-activated sodium channels, and voltageactivated calcium channels. Each of these is amenable to different methods; each represents a different stage in our understanding. By investigating the structure of ion channels directly we can learn much about their possible mechanisms of function. We also gain information about their precise identities, their diversity, and evolutionary relationships. During the preparation of this volume, new studies emerged demonstratingthat ligandoperated channels and voltage-sensing proteins fall into families related in evolutionary origin and in probable mechanisms of action. Thus, the findings discussed in this book are likely to have broad implications as the list of well-characterized proteins grows. In the 1970s the gramicidin channel was much in the news as the first channel protein to have a three-dimensional structure proposed (by D. Urry) and to have its mechanism of ion permeation scrutinized by methods of singlechannel recording (starting with the work in the laboratory of D. Haydon). By 1980 much was known about this channel, but one wondered whether its remarkable properties-multiple ion-binding sites, ion-ion interactions in the permeation process-might just be esoteric curiosities. However, it was soon found that these multiple ion behaviors (summarized by S. Hladky in this volume) also appear in potassium channels and, very dramatically, in calcium channels, vindicating this simple channel as an important model system in channel biophysics. Further, its small size has allowed it to be studied recently by a number of physical approaches that have yet to be applied successfully xiii

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PREFACE

to other channel peptides. In this volume Dr. Wallace describes her progress in structural studies on the gramicidin dimer, while Dr. Urry summarizes the use of ion NMR to obtain the binding kinetics of ions to the channel. Gramicidin’s small size also makes feasible the direct computer calculation of the electrostaticsof ion transport as described by Dr. Jordan; in the future, similar calculations on the energetics of conformationalchanges may be able to explain the kinetic fine structure observed by Drs. Sigworth and Shenkel. Nicotinic acetylcholine receptors from the electric organs of strongly electric fish (Torpedo and Electmphorus) were the first postsynaptic neurotransmitter receptor proteins to be identified, isolated, and functionally reconstituted and to be cloned and studied in transient expression systems. These receptors are proteins of 250,000 Da, formed of five peptides of four types. The subunits, a ,0, y, and 6 , appear on gels to be 40,000,50,000, 6O,OOO, and 65,000 Da, respectively. The assembly, a20 y 6, contains two binding sites for acetylcholine(and a-bungarotoxin) and forms a water-filled nonselective cation channel when activated. Ordinarily, binding of acetylcholine results in channel activation, but extended exposure to the agonist can result in desensitization. Fast desensitization inactivates the channel and response to acetylcholine, while slow desensitization increases markedly the binding affiity for the ligand and may muire seconds or longer for recovery when the agonist is removed. Single channel recording methods have revealed details about the regulation of conductance stages by agonist binding. Drs. Sine and Steinbach discuss a sophisticatedanalysis of the effects of ligand concentration on conductance states for nicotinic receptors in cultured cells. In addition, Dr. Huganir describes the direct effects on desensitization of nicotinic receptors by enzymatic phosphorylations of specific sites on the receptor, both in vitm and in vivo. Because of the availabilityof the amino acid sequence of the alpha subunits, which form the mechanistically important agonist binding sites, considerable attention has been given to the peptide segments which directly interact with the ligands. Dr. Hawrot and co-workersdescribe the use of synthetic peptides to test observations from chemical binding and mutagenesis studies. At present, more than twenty types of acetylcholinereceptor subunits have been cloned from different species and from tissues at different stages of differentiation. These clones permit the characterization of functional receptors either in transient or stable expression systems. Dr. Hess and co-workers describe the expression of acetylcholine receptor subunits in the yeast Sacchummyces cerevisiae. This readily manipulated organism offers distinct opportunities for investigating assembly and conformationalmechanisms by site-directed mutagenesis. In addition, Dr. Claudio and co-workers describe progress in establishing stable expression of acetylcholine receptors from Brpedo in transfected cells. This approach yields immortal cell lines carrying

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PREFACE

xv

normal or mutagenized receptor components for electrophysiological studies of channel gating and biochemical investigations of processes involved in subunit assembly, insertion at the cell surface, and control of the surface clustering which accompanies synapse formation. The picture presented by voltage-sensitive sodium channels is in many respects distinct from that of the nicotinic receptors. Physiologically these channels are regulated by the transmembrane electric field rather than by the binding of chemical effectors. Nevertheless, toxins and anesthetics can bind at susceptible sites to force the protein into one or another conducting state. Structurally, unlike the nicotinic receptor, the principal functional unit appears to be a single peptide of 208,OOO-220,OOODa. This peptide appears to fold into four “pseudosubunits,” perhaps forming a rosette analogous to that of the five acetylcholine receptor subunits. Attached to this peptide is a large arborization of carbohydrate, up to 85,000 Da. In the case of the electroplax protein, extended oligosaccharide chains may reach 10-20 nm into the extracellular environment. In brain and muscle sodium channels, one or two smaller heavily glycosylated subunits, whose functions are not yet known, also are evident. The articles by Drs. Barchi, Miller and Garber, and by Agnew et ul. discuss the biochemistry of sodium channels from skeletal muscle and eel electric organ and the behavior of brain sodium channels incorporated into artificial membranes. Dr. Guy presents a theoretical model for the conformation adopted by the large sodium channel glycopeptides and a discussion of the relationship of these predictions to models of gating and conductance. Calcium channels represent a diverse family of channels, often responsible for triggering intraceIlular processes such as neurosecretion, mechanochemical contraction, and synaptic facilitation. This diversity, together with a model for ion permeation, is discussed by Dr. ’Men and co-workers. The protein biochemistry of voltage-sensitivecalcium channels, a rapidly advancing area of research, is described by Dr. Catterall and co-workers. A wide range of channels and cellular functions remains to be studied. The use of single-celled organisms for the genetic manipulation of ion channels is discussed by Drs. Kung and Saimi. Much of what has so far been established about receptors and voltage-activated channels discussed here began with the identification of specific biochemical markers, often neurotoxins. aBungarotoxin and tetrodotoxin, for example, were indispensible in the isolation of nicotinic receptors and sodium channels. An example of an arsenal of peptide neurotoxins from the venoms of cone snails, as yet largely unexploited, is described in a discussion by Dr. Olivera and colleagues. This volume and the conference from which it was drawn were organized to afford a range of perspectives on the current state of knowledge of ion channels. The editors thank the contributors for their enthusiastic partfcipation. In addition, we wish to acknowledge and sincerely thank the following

xvi

PREFACE

organizations for generously providing funds for the conference: Abbott Laboratories, Ciba-Geigy, The Grass Foundation, Hoffman-LaRoche, G.D. Searle & Co., ICI Americas, Inc., Merck & Co., Miles Laboratories, Smith Kline Beckman, Inc., and The Upjohn Co. WILLIAMS. AGNEW TONICLAUDIO J . SIGWORTH FREDERICK

Yale Membrane Transport Processes Volumes Emile L. Boulpaep (ed.). (1980). “Cellular Mechanisms of Renal Tubular Ion Transport”: Volume 13 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. William H. Miller (ed.). (1981). “Molecular Mechanisms of Photoreceptor Transduction”: Volume 15 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. Clifford L. Slayman (ed.). (1982). “Electrogenic Ion Pumps”: Volume 16 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Joseph F. Hoffman and Bliss Forbush I11 (eds.). (1983). “Structure, Mechanism, and Function of the Na/K Pump”: Volume 19 of Current Topics in Membranes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, New York. James B. Wade and Simon A. Lewis (eds.). (1984). “Molecular Approaches to Epithelial Transport”: Volume 20 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, New York. Edward A. Adelberg and Carolyn W. Slayman (eds.). (1985). “Genes and Membranes: Transport Proteins and Receptors”: Volume 23 of Current Topics in Membranes and Transport (F.Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. Peter S. Aronson and Walter F. Boron (eds.). (1986). ‘“a+-H’ Exchange, Intracellular pH, and Cell Function”: Volume 26 of Current Topics in Membranes and Transport (A. Kleinzeller and F. Bronner, eds.). Academic Press, Orlando. Gerhard Giebisch (ed.). (1987). “Potassium Transport: Physiology and Pathophysiology”: Volume 28 of Current Topics in Membmnes and Transport (F. Bronner and A. Kleinzeller, eds.). Academic Press, Orlando. William S. Agnew, Toni Claudio, and Frederick J. Sigworth (eds.). (1988). “Molecular Biology of Ionic Channels”: Volume 33 of Current Topics in Membmnes and livlnsport (J. F. Hoffman and G. Giebisch, eds.). Academic Press, San Diego.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 33

Chapter I Ion Channels of Paramecium, Yeast, and Escherichia coli YOSHIRO SAIMI AND CHING KUNG Laboratory of Molecular Biology and Department of Genetics University of Wisconsin Madison, Wisconsin 53706

I. Prrrcrmecirrm A. Physiology B. Genetics C. Chemistry 11. Yeast 111. Escherichiu coli IV. Conclusion

References

The importance of ion channels in the heart and brain is reflected in the concentration of research on them, as evident from this volume. However, what about ion channels of life forms with no heart or brain? Green plants also have ion channels (Moran et al., 1984; Schroeder et al., 1984). This leads naturally to the question of whether the mouse channels and the tree channels evolved independently or derived from some primordial channels in primitive organisms. Could channels have evolved very early like rhodopsin, adenylate cyclase, or DNA? The answer seems to be yes, because we find ion channels in unicellular microorganisms such as Puramecium, yeast, and even Escherichia coli. Microbes with proved advantages in biochemical and genetic experiments should add to the studies of ion channels. 1.

Paramecium

Paramecium is a ciliated protozoan. It is eukaryotic, has membraneenclosed nuclei, mitochondria, 80 S ribosomes, and carries out mitosis 1 Copyright B l 19x8 hy Acddemlc Pre% Inc All right\ ot reprodurtion in m y form rewrved

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YOSHIRO SAlMl AND CHING KUNG

and meiosis. It is motile and has an action potential. Its growth requires vitamins and the 10 essential amino acids. much as in humans. A.

Physiology

In 1964, Kinosita and co-workers (Kinosita et al., 1964) showed that Pqramecium can generate action potentials. The action potentials were found to cause a reversal of the beat direction of the cilia. The ionic mechanisms of the action potentials and touch-receptor potentials were further analyzed (Naitoh, 1974; Eckert et al., 1976). The application of the voltage clamp to the study of paramecia (Oertel et al., 1977) allowed us to sort out the various currents through its plasma membrane. Under voltage clamp, depolarization of the wild-type membrane induces a Ca" inward current rapidly (milliseconds) followed by a rapid outward K' current (the delayed rectifier). Ca2+,which carries the fast inward current, activates a slow inward Na' current (tens to hundreds of milliseconds) which, in turn, is followed by a Ca2+-activatedK+ outward current. We believe that these four currents constitute the action potential for Paramecium in its natural environment (Saimi et al., 1983). Membrane hyperpolarizations activate a separate set of ion channels. B. Genetics

Behavioral mutants were isolated and found to be defective in single genes and in their action potentials (Kung et al., 1975). There are at least seven complementation groups @awns and CNRs; Haga et al., 1983)having mutations which turn down the Ca" current. There is one complementation group (Dancer) of mutations which strengthens the Ca2+current by preventing normal inactivation [a process through which the Ca" channel arrives at a less-activatable state (Eckert and Chad, 1984)l.There are probably more than two complementation groups (pantophobiacs) of mutations which turn down the Ca2+-dependentK' current. There is at least one complementation group (tetraethylammonium insensitive) of mutations which appears to turn up this current through a rapid activation. Similarly, there are mutations that turn the Ca"-dependent Na' current down Cfast-2)or up (paranoiacs). The biophysical properties of the wildtype and mutant currents through these channels have been periodically reviewed (Kung and Saimi, 1982, 1985; Hinrichsen et al., 1985; Ramanathan et al., 1988). Patch clamp methods (Hamill et al., 1981) have been applied to Paramecium channel recording, and several ion channels have been identified (Martinac et al., 1986; Table I). Mutations have been used to isolate most of these currents as have conventional pharmacological or voltage maneuvers. Note that mutations

1. ION CHANNELS OF UNICELLULAR ORGANISMS

3

are very specific and have u priori targets unlike the pharmacological agents. Mutations have also been used in a search for the gene products, i.e., the channel proteins or other proteins that regulate channel activities. Although attempts to detect differences in membrane proteins from the wild type and various mutants were confounded by physiological variations and the results were not conclusive (Adoutte et a / . , 1983), some of the gene products have been purified or enriched using a functional assay of restoring normal phenotypes in mutants as described below. C. Chemistry

The behavioral peculiarities of some of these mutants can be corrected by microinjection of wild-type cytoplasm. Typically, an injection of 10% cell volume of the wild-type cytoplasm is enough to restore normal behavior. The restoration typically begins within I hr and lasts 2 days and can take place in the presence of protein synthesis inhibitors. Preparative quantities of wild-type cytoplasm can then be fractionated through differential centrifugations and ammonium sulfate precipitations followed by various column chromatographies. The presence of the restoration activity is assayed by injecting the fractions into the mutant, and measuring the return of the normal behavior and the normal membrane current. This method, though relatively difficult, ensures that the wild-type substance purified or enriched is a functionally active one. In the case of one of the pantophobiac mutants, pntA, which is missing the Ca’+-dependent K + current, the restoration factor was found to be a small, soluble, acidic, heat-stable protein. In various gel electrophoresis conditions, it migrates differently depending on whether Ca” is present. All these are characteristics of calmodulin, the well-known Ca”-binding protein which is highly conserved among eukaryotes. Calmodulin has four Ca”-binding domains arranged in such a way that a Ca”-binding loop is located between two perpendicular helices (E-F hand configuration; Kretsinger, 1980). The wild-type calmodulin from Purumecium has been completely sequenced through protease cleavage and sequential Edman degradation (Schaefer et al., 1987b). It consists of 148 amino acids and has a molecular weight of approximately 16,000. The calmodulin from the p n f A mutant has also been sequenced. The pntA mutation caused a single substitution of serine with phenylalanine at position 101 (Schaefer et al., 1987a). Serine at position 101, located in the third Ca”-binding loop, is conserved from yeast and Paramecium to mammals. Structural models indicate that the oxygen of this serine is one of the six oxygens in the loop which might form bonds with the Ca” ion. It is therefore likely that the third domain of the pnrA calmodulin binds Ca” poorly or not at all. pntA mutant is the first to have a known molecular defect among all the

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YOSHIRO SAlMl AND CHING KUNG

behavioral mutants with electrophysiological defects in Paramecium or Drosophila. Caz+ activates a large number of enzymes, such as cyclic nucleotide phosphodiesterase and myosin light-chain kinase. Ca2+first binds to calmodulin; the Ca"-calmodulin complex, in turn, binds to and activates the enzymes. The pnrA mutation has revealed a surprising connection between calmodulin and an ion channel which is gated by Ca2+.The nature of this connection is not clear. Among other possibilities, calmodulin may be a subunit of the channel (Ca*'-gating mechanism?; but see Levitan and Levitan, 1986), or it may activate a protein kinase to phosphorylate the channel to an open form (Ewald et al., 1985). Note that calmodulin is a key protein of vital importance. Disruption of the calmodulin gene by large insertions or deletions is lethal in yeast (Davis et al., 1986). The pnrA mutant calmodulin probably retains most of its function since it shows Ca"-dependent shifts in electrophoretic migrations on sodium dodecyl sulfate (SDS) gels and can still fully activate brain phosphodiesterase (R. D. Hinrichsen, A. Burgess-Cassler, and C. Kung, unpublished observations). There are other pantophobiac mutants, most likely of different complementation groups. They also have behavioral and electrophysiological defects similar to pntA. Wild-type Paramecium calmodulin has a trimethylated lysine at 115 and a dimethylated lysine at 13 (Schaefer et u l . , 1987b). One pantophobiac mutant, pntD, has a calmodulin which appears to be undermethylated (M. Wallen-Friedman, J. Colquhoun, and C. Kung, unpublished observations) and gives electrophoretic migration pattern on a native gel different from those of the wild-type or pnrA calmodulins (Wallen-Friedman et al., 1986). How the lysine methylations of calmodulin may relate to the specific ion-channel function is not yet clear. Cytoplasm fractionation and microinjection assay have also been used to enrich a factor which restores the normal behavior in a different mutant, cnrC, which is missing its Ca2+current. The factor(s) has been enriched almost 600-fold over the original soluble cytoplasmic fractions. It is most likely a soluble, acidic protein of less than 30,000 Da. It is not heat stable, does not stimulate brain phosphodiesterase, and apparently does not bind Ca", and is, therefore, not calmodulin (Haga et al., 1984). II. YEAST

Baker's yeast, Saccharomyces cerevisiae, though not green, is a plantlike eukaryote with a cell wall, a vacuole, and a strong H + pump, but has no Na+/K+pump. Removal of the yeast cell wall with zymolyase yields

1. ION CHANNELS OF UNICELLULAR ORGANISMS

5

spheroplasts, which can regenerate the wall when given the opportunity. Diploid spheroplasts are about 5-7 Fm in diameter. Their dimension precludes accurate electrical measurements by conventional electrode penetration. However, the surface of the spheroplasts, which is the outer face of the plasma membrane, can form gigaohm seals with patch pipets. Sustained, vigorous suction delivered to the pipet is needed to form gigaohm seals on this surface. Once the seal is formed, one can further choose any of the four recording modes: on-cell, whole-cell, inside-out patch, outsideout patch, depending on the experiments. The prominent ion channels are of the following two types. Cell depolarization activates a set of ion channels with a unit conductance of about 20 pS in 100 mM KCI solutions. Reversal-potential analyses indicate that the channel passes K’ ; experiments using biionic conditions suggest strong selectivity for K’ over Na’. That it is a K’ channel is further supported by the finding that it is blocked by tetraethylammonium or Ba”. The channel is voltage sensitive; it is more likely to open at depolarizing voltages. Single-channel activities show bursts separated by long (second) interbursts, and rapid (millisecond) flickering between open and closed states during bursts. The number of activatable K’ channels per yeast spheroplast is only 10-300. Because the total membrane current is but an ensemble of channel activities in such small number, the wholecell current is not only small but takes on an appearance of rapid, noisy fluctuations. Clear one-step single-channel behavior can be observed after a prolonged depolarization during which most of the channel activity subsides. Single-channel activities can also be observed after most of the channels have been blocked by tetraethylammonium or by excision of patches which happen to contain one channel (Gustin et ul., 1986). A second type of channel opens when a pressure equivalent to a few centimeters of mercury, negative in the on-cell mode or positive in the whole-cell mode, is applied to the yeast plasma membrane. The stronger the suction, the more likely the channels are to be in their open state. Dilution of the solution bathing the spheroplasts, creating an outward osmotic pressure, can also activate these channels in the whole-cell mode. They have a unit conductance of 40 pS. They prefer cations over anions. The selectivity among various cations is not strong (Gustin et al., 1987); they can pass alkali metals, tetraethylammonium, and even Ca” and Mg’+. Although the number of active channels of this type may be larger than that of the K’ channels, no more than 100 per yeast cell have been observed so far. The discovery of these ion channels in yeast raises the question of their physiological roles. These roles may be revealed through patch-clamp examinations of cells at different stages of cell cycle, at different stages during

6

YOSHIRO SAlMl AND CHlNG KUNG

mating, or after growth in different ionic, pH, osmotic, aerobic, and other conditions. These roles may also be revealed by finding mutants defective in channel activities which cosegregate with other phenotypes such as budding and mating abnormalities. The capability to record from the plasma membrane of yeast, the most manipulable eukaryote in terms of molecular biology, should also allow us to examine electrical expression of foreign channel genes after incorporation into the yeast genome. Indeed, Fujita and co-workers (1986a,b) have succeeded in cloning DNA coding for the a- and &subunits of the nicotinic acetylcholine receptor from Torpedo into yeast. They showed that both subunit proteins were expressed and incorporated into the yeast plasma membrane. It remains to be seen whether a complete, pentameric, functional channel can be assembled in yeast membrane when all the subunit genes are integrated. 111.

Escherichla coli

Escherichia coli, is a gram-negative bacterium. Being prokaryotic, it has no mitochondria, no membrane-enclosed nucleus, has 70 S ribosomes, and does not undergo mitosis or meiosis. Since it is only about 0.5 ym in diameter and 2 pm in length, it is too small for direct application with the patch-clamp pipet. However, a method is available to generate giant spheroplasts from E. coli (Ruthe and Adler, 1985). Wild-type E. coli cells grown in the presence of cephalexin, a penicillin analog, fail to form septa when they attempt to divide. As a result, these cells grow into long filaments 50 to 150 ym in length. After these filaments are treated with ethylenediaminetetraacetic acid (EDTA) and lysozyme to digest the peptidoglycan layer (cell wall), they collapse into giant spheroplasts about 5 to 10 ym in diameter. These spheroplasts are large enough for patch-clamp recording. Although the activities of other types of voltage-activated channels have also been seen (B. Martinac, A. H. Delcour, and M. Buechner, unpublished observations), the most prominent channel activities observed so far are of pressure-sensitive ion channels. Suction of a few centimeters of mercury opens these channels. The opening probability of the channel plotted against the suction can be fitted to a curve according to the Boltzmann distribution. These channels are also voltage sensitive. Depolarization shifts the Boltzmann curve to the left, i.e., making the channel more likely to open at a given pressure. Interestingly, pressure exerted in either direction can open the channel. The channel has a large conductance between 650 and 970 pS in recording solution of about 300 mM

1. ION CHANNELS OF UNICELLULAR ORGANISMS

7

salt. The channel has little selectivity, although it favors anions slightly over cations. It passes inorganic ions as well as organic ones, including solutes as large as glutamate. The kinetic behavior of this channel depends strongly on the species of ions in the solution. For example, replacing K' with Na' greatly reduces the opening probability and the mean open time. All permeant ions and protons appear to affect the channel behavior. This finding indicates that the permeant ions interact with the gate. While the discovery of ion channels gated by pressure or voltage is exciting, there is a question concerning their location. A gram-negative bacterium has two membranes: an outer and an inner membrane separated by the periplasmic space where the peptidoglycan lies. This peptidoglycan layer is digested by the lysozyme during spheroplast formation. It is not yet clear at the time of writing whether the gigaohm seal was formed on the outer or the inner membrane. The outer membrane is full of a class of channels known as porins. Porins have been purified and reconstituted onto planar lipid bilayers (Benz et al., 1978; Schindler and Rosenbusch, 1978). These reconstituted porins are different from the pressure-sensitive channels in that all porins reported so far show some voltage dependency of channel closure (outside of + / - 100 mV; Benz, 1986), whereas the pressure-sensitive channels are activated more with depolarization. Furthermore, no pressure sensitivity of porin activities has been reported. The major porins in E. coli are the products of the ompF and the ompC genes. Mutants with both genes deleted have been examined and were found to still have the pressure-sensitive channels (B. Martinac, A . H. Delcour, M. Buechner, J. Adler, and C. Kung, unpublished observations). Given the small volume of a bacterium and the large conductance and lack of ion selectivity of the pressure-sensitive channel, it seems unlikely that such channels serve to regulate membrane potential. As for the function of the pressure-sensitive channels, osmotaxis of E. coli, a behavioral avoidance of an osmolarity gradient, is implicated (C.-Y. Li, C. Kung, and J. Adler, unpublished observations). IV. CONCLUSION

Although the results reviewed here-especially those on the ion channels of yeast and E. coli-are incomplete, the findings bring up several interesting points. The occurrence of mechanically gated ion channels in the wide range of life forms, including E. coli and nonmotile yeast, is surprising. They probably evolved first to deal with changes in osmolarity rather than with touch or vibration. This seems likely since the concentration of water in

8

YOSHIRO SAlMl AND CHING KUNG

the environment should be of universal concern. Mechanical gating, i.e., opening channels by mechanical forces exerted on the channel proteins and/or the surrounding lipids or cytoskeleton, appears to be as old as, if not older than, voltage gating, ligand gating, or Ca’+ gating. The pressure-sensitive channels in both yeast and E. coli are not very selective in ion permeation. The E. coli channel is highly conductive (f id. (1987). 'Positive pressure from the cytoplasmic side. "Pressure in either direction.

should also be useful in the analyses of the sequences or cascades of events including synthesis, assembly, deployment, turnover, mediation, and modulation of ion channels. The extensive physiological and genetic work on Purumecium ion channels cannot presently be fully and easily exploited for molecular information, because some of the current recombinant DNA techniques have not been successfully applied to ciliated protozoa. I n coming years, Parumecium will be best used to study ion-channel regulation by soluble proteins, such as calmodulin, since we have purified proteins or enriched protein fractions as well as antibodies to them, as is evident in this review. ACKNOWLEDGMENTS We thank Drs. M. Gustin and B. Martinac for critical reading of the manuscript. Supported by grants from NIH, GM-22714 and GM-36386. REFERENCES Adoutte. A,, Ling. K.-Y.. Chang, S . , Huang. F., and Kung. C. (1983). Physiological and mutational protein variations in the ciliary membrane of Picririnccirrm. Exp. Cell R1.s. 148,387-404.

10

YOSHIRO SAlMl AND CHING KUNG

Benz, R. (1985). Porin from bacteria and mitochondria1 outer membranes. CRC Crit. Reit. Biochem. 19, 145-190. Benz, R. (1986). Analysis and chemical modification of bacterial porins. In “Ion Channel Reconstruction” (C. Miller, ed.), pp. 553-573. Plenum, New York. Benz, R., Janko, K.. Boos, W., and Lager, P. (1978). Formation of large ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli. Biochirn. Biophvs. Acts 511, 305-319. Brezden, B. L.. and Gardner, D. R. (1986). A potassium-selective channel in isolated Lv/nnuea s~upnulisheart muscle cells. J. Exp. Biol. 123, 175-189. Corey. D. P.,and Hudspeth, A. J. (1979). Ionic basis of the receptor potential in a vertebrate hair cell. Nature (London) 281, 675-677. Davis, T.. Urdea, M. S., Masiarz, F. R., and Thomer, J. (1986). Isolation of yeast calmodulin gene: calmodulin is an essential protein. CeII (Cumbridge. Mass.) 47, 423-431. Deitmer, J. W. (1982).The effects of tetraethylammonium and other agents on the potassium mechanoreceptor current in ciliate Sylonychia. J. Exp. B i d . 96, 239-249. Dwyer, T. M.. Adams, D. J.. and Hille, B. (1980). The permeability of the endplate channel to organic cations in frog muscle. J . G e n . Physiol. 75, 469-492. Eckert, R., and Chad, J. E. (1984). Inactivation of Ca channels. Prog. Biophvs. Mol. B i d . 44, 215-267. Eckert, R., Naitoh, Y., and Machemer, H . (1976). Calcium in the bioelectric and motor functions of Paramecium. In “Calcium in Biological Systems” (C. J . Duncan, ed.), pp. 233-255. Cambridge Univ. Press, London and New York. Ewald, D. A., Williams, A., and Levitan, 1. B. (1985). Modulationof single Ca”-dependent K’-channel activity by protein phosphorylation. Nuture (London) 315, 503-506. Fujita, N.. Nelson, N.. Fox, T. D., Claudio, T.. Lindstrom, J., Riezman, H., and Hess. G. P. ( 1986a). Biosynthesis of the Torpedo c.olifornicu acetylcholine receptor a subunit in yeast. Science 231, 1284-1287. Fujita. N.. Sweet, M. T., Fox, T. D., Nelson. N.. Claudio, T., Lindstrom, J. M.. and Hess, G. (1986b). Expression of cDNA for acetylcholine receptor subunits in yeast cell plasma membrane. Biochein. Soc. Sytnp. 52, 41-56. Guharay. F.. and Sachs. F. (1984). Stretch-activated single ion channel currents in tissuecultured embryonic chick skeletal muscle. J . Physiol. (London) 352, 685-701. Gustin. M. C.. Martinac, B.. Saimi, Y.. Culbertson, M. R., and Kung, C. (1986).Ion channels in yeast. Science 233, 1195-1 197. Gustin. M. C.. Zhou. X.-L., Martinac, B., Culbertson, M. R.. and Kung, C. (1987). Stretchactivated cation channel in yeast. Biophys. J . 51, 251a. (Abstr.) Haga, N.. Saimi, Y., Takahashi, M., and Kung, C. (1983). Intra- and interspecific complementation of membrane-inexcitable mutants of Parumecitrm. J . Cell Biol. 97, 378-382. Haga, N.. Forte, M., Ramanathan, R., Hennessey, T., Takahashi, M.. and Kung, C. ( 1984). Characterization and purification of a soluble protein controlling Ca-channel activity in Parctmecirrm. J . Cell Biol. 39, 71-78. Hamill, 0. P., and Sakmann, B. (1981). Multiple conductance states of single acetylcholine receptor channels in embryonic muscle cells. Nature (London) 294, 462-464. Hamill, 0. P., Marty. A., Neher, E., Sakmann, B., and Sigworth, F. J . (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfuegers Arch. 391, 85-100. Hinrichsen, R. D., Saimi, Y., Ramanathan, R., Burgess-Cassler, A., and Kung, C. (1985). A genetic and biochemical analysis of behavior. In “Sensing and Response in Microorganisms” (M. Eisenbach and M. Balaban, eds.), pp. 145-157. Elsevier, New York. Hudspeth, A. J. (1983). Mechanoelectrical transduction by hair cells in the acousticolateralis sensory system. Annu. R e v . Neurosci. 6, 187-215.

1. ION CHANNELS OF UNICELLULAR ORGANISMS

11

Kinosita. H.. Dryl, S., and Naitoh, Y.(1961). Changes in membrane potential and the responses of stimuli in Purumerium. J. Fuc. Sci. Univ. Tohyo. Sect. 4 10, 291-301. Kretsinger, R. H. (1980). Structure and evolution of calcium-modulated proteins. CRC Cri/. Rev. Biochem. 8, 119-174. Kung. C., and Saimi, Y. (1982). The physiological basis of taxes in Purumeciiirn. Annii. R e ) , .Physiol. 44, 519-534. Kung, C.. and Saimi, Y. (1985). Ca" channels of Purumecium: a multidisciplinary study. Cirrr. Top. Membr. Trump. 23, 45-63. Kung, C.. Chang, S.-Y., Satow, Y.,van Houten, J . , and Hansma, H. (1975). Genetic dissection of behavior in Purumecium. Science 188, 898-904. Levitan, E. S., and Levitan. 1. B. (1986). Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium. J . Membr. B i d . 90, 59-65. Martinac, B., Saimi, Y., Gustin, M. C., and Kung, C. (1986). Single-channel recording in Purumeriitm. Biophys. J. 49, 167. (Abstr.) Martinac. B.. Buechner. M.. Delcour. A. H., Adler. J.. and Kung, C. (1987). A pressuresensitive ion channel in Esrliericlii~icoli. Pror. Null. Arud. Sci. U . S . A . 84, 2297-2301. Moran, N., Ehrenstein, G., Iwasa, K., Bare, C., and Mischke. C. (1984). Ion channels in plasmalemma of wheat protoplasts. Science 226, 835-838. Naitoh. Y. (1974). Bioelectric basis of behavior in protozoa. Am. Z o o / . 14, 883-893. Oertel. D., Shein, S. J., and Kung, C. (1977). Separation of membrane currents using a Purumcriiim mutant. Nutwe (London) 268, 120-1 24. Ohmori, H. (1984). Mechanoelectrical transducer has discrete conductances in the chick vestibular hair cell. Pror. Nu//.Acud. Sci. U . S . A . 81, 1888-1891. Ramanathan. R.. Saimi. Y.. Hinrichsen. R., Burgess-Cassler. A.. and Kung. C. (19x8). A genetic dissection of ion-channel functions in Purumecirrm. In "Paramecium" ( H . D. Gertz. ed.), pp. 236-253. Springer-Verlag. Heidelberg. Ruthe, H.-J.. and Adler, J. (1985). Fusion of bacterial spheroplasts by electric fields. Biochim. Biophys. A C ~ 819, U 105-113. Saimi, Y.,Hinrichsen, R. D., Forte, M., and Kung, C. (1983). Mutant analysis shows that the Ca"-induced K' current shuts off one type of excitation in Purunieciirm. P r o ( , . N ~ t lArud. . Sri. U . S . A . 80, 5112-5116. Salkoff. L., Butler, A., Hiken, M.. Wei. A., Giffen, K., Ifune, C.. and Mandel, G. (1986). A Drosophilo gene with homology to the vertebrate Na' channel. J. Neiirosci. 12, 1512. (Abstr.) Schaefer, W. H.. Hinrichsen. R. D., Burgess-Cassler, A., Kung, C., Blair, 1. A.. and Watterson, D. M. (1987a). A mutant Purumecinrn with a defective calcium-dependent potassium channel has an altered calmodulin: a non-lethal selective alteration in calmodulin regulation. Proc. N d . Acud. Sci. U.S.A. 84, 3931-3935. Schaefer. W. H., Lukas. T. J.. Blair, I. A.. Schultz. J . E.. and Watterson. D. M. (1987b). Amino acid sequence of a novel calmodulin from Purumeciurn tetrurireliu that contains dimethyllysine in the first domain. J . B i d . Cliem. 262, 1025-1029. Schindler. H.. and Rosenbusch. J . (1978). Matrix protein from EscAerichiu coli outer membrane forms voltage-controlled channels in lipid bilayers. Proc. N a t l . Acud. Sri. U . S . A . 75, 3751-3755. Schroeder. J. I.. Hedrich, R., and Fernandez, J . M. (1984). Potassium-selective single channels in guard cell protoplasts of Viciu fubo. Nuture (London) 312, 361-362. Wallen-Friedman, M.. Elbaum, L.. Saimi, Y., Hennessey. T., Hinrichsen. R., BurgessCassler, A.. and Kung, C. (1986). Calmodulin defects are correlated with defects in the calcium-dependent potassium current in several mutants of Purumecirrm tetrciriralici. J. Neiirosci. 12, 558. (Abstr.)

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Part I

Gramicidin

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 31

Chapter 2 Gramicidin: Conchsions Based on the Kinetic Data S. B . HLADKY Department of Pharmacology University of Cambridge Cambridge CB2 2QD England I. II. 111.

IV. V. VI.

VII.

Introduction Gramicidin Forms Pores Selectivity The Evidence That the Pore Is a Dimer Structural Inferences from the Kinetic Data Ion Conduction through the Pore A. The Ion Fluxes Are Not Independent B. Simple Competition C. Ion Interaction in Gramicidin Pores Is More Complex than Simple Competition D. Suggested Explanations for Deviations from Simple Competition E. Second Ion Entry and Double Occupancy Conclusions References

1.

INTRODUCTION

Gramicidin and tyrocidine are obtained from Bacillus hrevis as part of an extract called tyrothricin. They were separated in 1939 and gramicidin was used topically against gram-positive bacteria. It was noted in the early studies that cells affected by gramicidin no longer take up inorganic phosphate but they still continue to respire. Based on this observation, Hotchkiss (1944) suggested that gramicidin causes the dissipation of an immediate product of oxidative phosphorylation. It was shown conclusively in the 1960s that gramicidin is an uncoupler and that it makes liposomes, red blood cells, and mitochondria permeable to small monovalent cations like 15 Copyright 0 19x8 hy Academic b e \ \ . Inc All rights of reproduclion in any form reserved.

16

S.B. HLADKY

Na', K', and H' (Pressman, 1965; Chappell and Crofts, 1965; Henderson et al., 1969). Gramicidin is a family of closely related pentadecapeptides with molecular weights of about 1880. The principal component, valine-gramicidin A, is (Sarges and Witkop, 1965) HCO-L-Val-Gly-L-Ala-o-Leu-Leu-L-Ala-~-Val-L-Val-D-Val-L-Trp-~-LeuL-Trp-u-Leu-L-Trp-u-Leu-L-Trp-N H-CH,-CH,-OH

All of the amino acids are hydrophobic. Furthermore, even the terminal amino and carboxyl groups are blocked with formyl and ethanolamine groups, respectively. Thus there are no free charges. Not surprisingly with so many nonpolar groups and a polar backbone, gramicidin is not soluble in either hydrocarbons or water, but it is strongly surface active (Haydon and Hladky, 1972; Kemp et al., 1972). II. GRAMlClDlN FORMS PORES

The kinetic evidence that gramicidin forms water-filled pores which span bilayer membranes has been reviewed frequently (Haydon and Hladky, 1972; Andersen, 1984; Hladky and Haydon, 1984). Briefly, when small quantities of gramicidin are added to planar bilayer membranes, it is possible to resolve the currents flowing through individual pores (Hladky and Haydon, 1970, 1972) as shown in Fig. 1. The amplitude of these currents is relatively insensitive to the thickness and tension of the membrane in which the pore is embedded. However, the stability of the conducting unit measured by the combination of the decreasing frequency of occurrence and shorter open-time of the pores falls by orders of magnitude for twofold increases in the thickness and tension (Hladky and Haydon, 1972; Neher and Eibl, 1977; Hendry et af., 1978; Rudnev et ul., 1981; Elliott et al., 1983). These properties are just as expected if gramicidin forms a structure of constant properties in these different membranes. Ions cross the membrane by way of the same structure regardless of the thickness, which accounts for the nearly constant conductance. The changing stability is also easy to understand because it depends markedly on the degree of membrane deformation that is required to accommodate the pore. 111.

SELECTIVITY

The pores are permeable only to small monovalent cations and neutral molecules. Anions, divalent ions, and even monovalent cations larger than dimethylammonium or guanidinium do not pass through the channel

17

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

Fici. I . Single-channel currents for gramicidin in a glyceryl monooleate membrane, I M KCI. I00 mV applied potential, 20°C.

+ n-hexadecane

(Hladky and Haydon, 1972; Eisenman et a / . , 1977; Urban, 1978).The size cut-off for neutral molecules is between water and urea (Finkelstein, 1974). Furthermore, the water fluxes prominently display the long pore or singlefile effect which means that water molecules can not pass each other in the pore (Rosenberg and Finkelstein, 1978a). Also, as expected for a long pore, the movements of ions and water are coupled to each other (Rosenberg and Finkelstein, 1978b; Levitt ef d.,1978; Dani and Levitt, 1981).

IV. THE EVIDENCE THAT THE PORE IS A DIMER The generally accepted conclusion that the conducting unit is a dimer is based on several types of evidence. The conductance of the membrane increases more than proportionally with the amount of gramicidin added to the aqueous phases. However, plots of log(conductance) versus log(amount added) from different laboratories or even just from different occasions have yielded different slopes, typically about I .7 to 1.8 (Hladky, 1972, 1974). Presumably this behavior is seen because gramicidin is not soluble. Thus we never achieve genuinely stable equilibrium conductances. In the absence of equilibrium it is necessary to have some sort of model for how the gramicidin enters and leaves the membrane. When gramicidin as a dilute solution in methanol is dispersed through the aqueous phase and a membrane is then formed from fresh lipid, the increasing conductance is proportional to t', accurately so if a little uncertainty in the origin of the time axis is allowed to account for the time needed to make the membrane (Hladky, 1974). With the plausible assumption that during this period gramicidin is arriving at the membrane at a constant rate and none

18

S.8.HLADKY

is leaving, this result implies that the conducting unit is a dimer of whichever form of gramicidin is incorporated into the membrane from the aqueous phase. For pyromellityl gramicidin, which bears three negative charges at the carboxyl end, the conductances also increase over a long time course after membrane formation, but the conductances measured at a fixed time after formation are much more reproducible. These conductances are proportional to the square of the amount added to the aqueous phase (Hendry et al., 1978). The kinetic order of pore formation has been investigated by looking at the increase in conductance that follows a step increase in applied potential. Bamberg and Lauger (1973) and Zingsheim and Neher (1974) found that the time required for this increase gets shorter at higher gramicidin concentrations. The data could be fitted assuming either a dimerization or a trimerization of the nonconducting form of gramicidin in the membrane. Equivalent information was also obtained from noise analysis (Kolb et al., 1975; Zingsheim and Neher, 1974). Yet another type of evidence was provided later by Veatch and Stryer (1977). PABS-gramicidin, a derivative, produces single channels whose amplitude is smaller than that for gramicidin itself. When a mixture was used, the distribution of channel sizes had three clear peaks, pure gramicidin, pure PABS-gramicidin, and a hybrid with an intermediate conductance. Furthermore, the relative frequency of occurrence of these sizes was just as if all three types of channel could be formed with equal ease. Veatch et al. (1975) attempted to show that the structure contains just two gramicidin molecules. They used fluorescence to measure the amount of dansyl-gramicidin C in the membranes. For membranes in which only a small fraction of the dansyl-gramicidin C was conducting, the conductance increased quadratically with the amount. At the other extreme, for the thinnest membranes in which the largest fraction of peptide should have been in the conducting state, they suggest that the number of channels was just half the number of adsorbed molecules. Gramicidin molecules linked covalently end to end also form pores (Bamberg and Janko, 1977; Ivanov and Sychev, 1982). These channels remain open for much longer periods than the normal channels, which suggests that dissociation of a dimer is the mechanism for closing the normal channels. V.

STRUCTURAL INFERENCES FROM THE KINETIC DATA

From the kinetic data and the primary structure it is possible to reach a number of general conclusions about the nature of the pore. First, it is a pore, i.e., there is a hydrophilic hole which spans the hydrophobic core

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

19

of the membrane. Thus we must have a structure with polar groups inside and a hydrophobic band around the outside. From the selectivity, the hole must be at least 0.3 to 0.4 nm in diameter to pass those ions which do cross, and it can not be very much larger or it would not be able to exclude ions that it stops, such as calcium and chloride. The only polar groups in gramicidin are the carbonyls and amides of the peptide bonds. Thus these must line the hole while the side chains come into contact with the chains of the lipids. Two types of structure satisfy these requirements, dimers of single helices joined end to end as proposed by Urry (1971) and by Ramachandran and Chandrasekharan ( 1972) and double helices as proposed by Veatch et al. (1974). Wallace (1987) discusses the evidence that the preferred channel is the head-to-head dimer in Chapter 3 of this vofume. VI.

ION CONDUCTION THROUGH THE PORE

A. The Ion Fluxes Are Not Independent The simplest mechanism for ion conduction through a pore would have the permeant ions diffusing through the pore much as they do through the aqueous phases. In that case both anions and cations would be permeable, there would rarely be any ions in the pore simply because its volume is small. and the conductance would increase linearly with the ion ioncentration. Even the basic selectivity and the earliest conductance data (Fig. 2) demonstrated that simple diffusion is not a good description of gramicidin. The conductances clearly do not continue to increase proportionally with the ion concentrations and thus the movement of one ion through the pore is clearly not independent of the movements of others.

B. Simple Competition The simplest model predicting conductances which stop increasing at high concentrations is what we can call simple competition. By this 1 mean: When one ion is within the pore no other ion can enter and attempted entries have no effect on the ion within the pore: when the pore is empty, the rate at which ions of a particular type attempt to enter is simply proportional to the concentration of that type; and, when an ion leaves, the pore is ready to receive another ion just as if the first had never been there. If ions interact only by simple competition, then I . The unidirectional flux of an ion from left to right can be written as, Ji

=

P : c : X,,

20

S. B. HLADKY

I

I

100

-

80

-

60

-

I

I

8

a

actlvky

FIG.2. Conductances at 50 mV versus molal activity for NaCl (m)-KCI (W, CsCl ( 0 ) . and TIC1 (0). The curves are drawn according to the four-state model using the constants for fit G-a. Experimental points from Neher P I id. (1978) and Urban P I id. (1980). (Reproduced with permission from Hladky and Haydon. 1984.)

where X , is the probability the pore is empty, cl is the concentration of ions of type i on the left, and Pi,the permeability for this process at low concentrations, is the proportionality constant that describes how an ion interacts with a pore that it can enter. At constant potential, the permeability is the same regardless of the presence of other types of ions or of ions on the opposite side because, by hypothesis, these do not affect the

21

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

ion within the pore. The permeabilities for transfer in the two directions are not independent constants because the predicted fluxes must satisfy microscopic reversibility. This condition requires (see, e.g., Urban rf NI., 1980)

P:lP:’ = exp( -z,FVIRT)

(2)

where z,F is the charge on a mole of ions of type i, F is the Faraday, R the gas constant, and T the absolute temperature. 2. The rate of entry of species i from the left, A:L.’& is proportional to X,, its own concentration, L . : , and a constant independent of all concentrations. A similar expression applies for entries from the right. 3. The mean residence time within the channel for ions of species i which enter from the left, T’,, or right, 7:’. is independent of all the concentrations. 4. The fraction of the time the pore is occupied by ions of each species and side of en1ry.f: orf:, is the product of the corresponding rate of entry and mean residence time. 5. The probability the pore is empty is I - Xu‘, + f:). Thus

For the same concentration of ions on the two sides of the membrane, the apparent binding constants are

K,

=

A’,+,

+ A~T:

(4)

Explicit expressions for these apparent binding constants have been derived for a multisite, Eyring model by Lauger (1973). Equations ( I ) to (4) lead directly with no further assumptions to four important testable results. Levitt (1986) has noted that these results follow for one ion channels whose fluxes can be described by expressions based on either Eyring rate theory or the Nernst-Planck equation. I . For each type of ion the current measured with the same concentration on both sides will vary with ion concentration following a simple saturation curve

I = Z,F(P{ - ly)cj(l

+

K,cJ

(5)

where K, is the apparent binding constant for the ion to the pore. 2. The ratio of the unidirectional fluxes of an ion in the two directions will satisfy JJJ; - t

=

(ci/c’-:) exp( - z F V I R T )

22

S.6.HLADKY

3. The reversal potential or zero current potential

V,, = (RTIF) In(Pic:

+ PJcj)/(Pvc'i'+ KC:?

(7)

measured with different mixtures of permeant ions on the two sides, will be unaffected if all the concentrations are changed in proportion. 4. When the proportions of two types of ion are varied at constant total concentration, the current measured with the same solution on both sides will obey

I

=

zF[(PI

- P':)Ci

+ (Pj - P:')Cj]l(l + KiCi + KjCj)

(8)

This relation predicts a monotonic variation of conductance as the proportion of the ions is varied. C. Ion Interaction in Gramicidin Pores Is More Complex than Simple Competition

The data for gramicidin clearly require a more elaborate model for the transport process. Conductances for cesium chloride (Neher et al., 1978; Urban et al., 1980) are shown in Fig. 3. To recall, if any response obeys

(Q/r)/(pS/M)

FIG.3. Eadie-Hofstee plot of conductance at 50 mV versus activity for CsCI. Open data points from Neher et a / . (1978). closed data points from Urban e? NI.(1980). (Reproduced with permission from Hladky and Haydon. 1984.)

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

23

a relation like Eq. ( 5 ) , then if we plot response versus response/c we should get a straight line, the y-intercept is the maximum response (here the maximum current), the x-intercept is the maximum response x the affinity constant, and the slope is - I/K. If we look just at the data for middle concentrations, the line is relatively steep, which implies fairly weak binding. However, if instead we look at low concentrations, the slope is small in magnitude, which indicates binding with substantially higher affinity than we would have guessed from the data in the 0. I M to M range. The apparent dissociation constant from the low concentration data is of the order of 10 mM. Yet, despite the apparent approach of the current to a small limiting value as the concentration is increased in this low range, when the concentration of cesium is increased above 10 mM the current increases further, at least another 10-fold. At the highest concentrations, the conductances fall. The measured ratios of undirectional fluxes also differ markedly from the predictions of simple competition. The flux ratio exponent is defined by

JIJ

=

[(c”/c’)exp(zFV/RT)]”

* t

For gramicidin, Procopio and Andersen (1979) and Schagina et al. (1983) have found values of n that approach 2 for cesium and rubidium, respectively. For every ion pair, except possibly sodium and lithium, the reversal potentials or permeability ratios are not independent of concentration as required by the simple competition hypothesis. With increasing ion concentrations the less permeant ion appears to become even less permeant as if it were selectively excluded from the transport process (see Hladky and Haydon, 1984, for further discussion). Finally, for certain combinations of ions including thallium and sodium (Neher, 1975) and thallium and potassium (Andersen, 1975) the channel conductance displays a marked minimum as the mole fraction is varied. This anomalous mole fraction effect is inconsistent with simple competition. D. Suggested Explanations for Deviations from Simple Competition

I . DIFFUSION LIMITATIONS It has been suggested that diffusion of ions up to and away from the ends of the pore is rate limiting and that, since the rate of this process

24

S. B. HLADKY

will vary differently with ion concentration than the pore process, limitation by external steps might account for the concentration dependence of the permeability ratios. It is likely that access of ions to the pore is partly limited outside the lumen of the pore (Andersen, 1983a). However, for gramicidin, because this process must occur only just outside the mouth of the pore, simple kinetic models cannot distinguish it from steps which occur just inside the mouth (Hladky, 1984). Neither can account for the deviations from simple competition.

2. DOUBLE-LAYER POLARIZATION It has been suggested that polarization occurring in the electrical double layers might account for the measured shape of the conductance-concentration plots at low concentrations. Polarization of the double layers refers to the creation of potential and concentration gradients in the aqueous phases immediately adjacent to the membrane. Such gradients are produced whenever a potential is applied. They increase in size as the ionic strength is reduced. The effects of these gradients were ignored in all published work on gramicidin prior to that of Andersen (l983b). As shown in Fig. 4,double-layer polarization greatly increases the currents at low ionic strengths and high potentials. However, the size of the effect at low potentials is too small to explain the observed deviations of the conductance-concentration relations from simple competition. 3. REGULATORY SITES Simple competition may fail if ions modify the channel by binding to regulatory or allosteric sites. A general kinetic test that can firmly exclude the existence of such sites has not been found. This difficulty is closely analogous to that of trying to decide whether two drugs which compete with each other bind to the same site or to two different but linked sites. Fortunately, for gramicidin the only likely binding sites for ions are either in the lumen or at the mouths of the pore and they are thus part of the transport process, 4. VARIABLE-CONFORMATION PORE

Simple competition could fail if binding of an ion to a site, which is necessary for the transport process, could induce changes in the conformation of the pore that persist long enough after the inducing ion leaves to affect the next ion. Lauger et al. (1980) have introduced a “simple” model which illustrates this effect. It can predict some of the deviations

25

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

C

0.0

-

9

0.8-

3

-

d

58

0.7-

0.6

-

0.5

-

"-1

c

I

0

1

1

200

100

I

1

300

VlmV

FIG.4. The dependence of the conductance ratio on potential and ionic strength for gramicidin and I mM NaCI. The ionic strength was increased by the additions of choline chloride, from the top downward 0, I . 9, and 99 mM. The data are displayed as mean -t standard deviation. The curve for 99 mM choline chloride has been fitted empirically. I t . in effect. tells the theory which transport process is present in the membrane. The curves for 0. I . and 9 mM have been calculated from the Gouy-Chapman theory for double-layer polarization using the capacity of the unmodified membrane. (Reproduced with permiuion from Hainsworth and Hladky, 1987a.l

from simple competition that are observed with gramicidin (Lauger el (11.. 1980; Lauger, 1984; Ciani, 1984) but there are two important exceptions. If we plot log(conductance) versus log(concentration) for cesium or thallium, the observable portion of the curve starts with a slope less than one but then becomes steeper. This change corresponds to the bend in the Eadee-Hofstee plot. The log-log plot then bends over and finally starts to decline. Mathematically, this means that the second derivative, &log G)ld(log c ) ~ is, first negative because the slope must decrease from one below the observable range, then positive, and then negative again as the

26

S. 6.HLADKY

conductance goes through its maximum. The one-ion two-conformation pore can predict a maximum in the conductance-activity relation, but as Eisenman and Dani (1986) have suspected on the basis of examples, the combinations of constants which yield a maximum (Lauger et al., 1980) cannot predict the negative-positive-negative sequence for the second derivative. For G = Ac( I + Be)/[1 + Cc + Dc’] d log Gld I O ~ C = [ I + 2Br - (D - BC)c’]/[(l+

+ CC+ Dc?]

A maximum requires D > BC. For D > BC as the concentration increases, the second derivative can change from negative to positive but it cannot

again become negative. The observed flux ratios for gramicidin also contradict this model. For symmetrical solutions and low potentials, the flux ratio exponent can be calculated from the conductance and unidirectional flux at zero potential,

n = RTG/z2F2J +

The one-ion two-conformation pore predicts (notation of Lauger et al., 1980)

J

=

RTGIz2F2 +

u#”(v’v~ -

vAv”)~

(UU,

+ uk* + u,kE)

v ’v:k*kEa

The last term is positive definite, which means that s 1. Lauger (1984) has actually used this model with particular choices of the rate constants to describe carrier-like behavior of channels. E. Second Ion Entry and Double Occupancy

1 . CONDUCTANCE-CONCENTRATION RELATION

It is now agreed by everyone that a second ion can enter an occupied gramicidin pore. For a single species of ion the simplest possible model allowing double occupancy has four states: the pore containing only water, the pore occupied by an ion on the left, the pore occupied by an ion on the right, and the pore occupied by two ions, one in each half. The model consists of this list and the rules for transitions between the states. Even this simple description involves five rate constants: Entry of an ion into an empty pore, A; transfer of an ion from one end to the other, k ; exit from a singly occupied pore, B; entry into a pore already occupied at the opposite end, D;and exit from doubly occupied pores, E. Under favorable circumstances these can be estimated from the conductance-concentration

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

27

relation at low applied potentials. In oversimplified terms, A can be estimated from the intercept of the plot in Fig. 3 at low concentrations, B from the initial slope, D from the x-intercept of the straight line at higher activities, the reciprocal sum of k and E from the slope, and kE from the downturn at the highest concentrations (Hladky and Haydon. 1984).

2. ALTERNATIVE MEANSFOR ESTIMATING BINDING CONSTANTS a . Current-Voltage Relations. Obviously the kinetic analysis would be much more convincing if there were independent means to estimate some of the rate constants. A number of different methods have been tried. In the first 1 attempted to use the shape of the current-voltage relations to provide an additional combination of the constants (Hladky, 1972, 1974). As a starting point I took an Eyring or absolute rate theory model that describes the pore in terms of two sites and three energy barriers. This approach was very attractive because it allowed calculation of definite values for the ratio of klB from the shape of the current-voltage relation at low activities. However, I soon realized that this simple model did not work because, at higher concentrations, it predicted currents which increased too much at high potentials. That could be fixed by devices such as modeling transfer from end to end as diffusion over a trapezoidal barrier. With this adjustment the values of klB were somewhat less than I . Unfortunately, 1 found that 1 could not even argue that these values were correct because they were very dependent on the precise method of calculation. For instance, if you abandon the assumption that the access and exit steps vary exponentially with the applied potential, or alternatively model these processes as a sequence of two or more steps, then the same data can be fitted even with the extreme assumption that transfer across the middle of the pore is infinitely fast (Hladky, 1987). The shape of the low-concentration current-voltage relations for Cs' (Hainsworth and Hladky, 1987b),Rb', H ', and TI' (Eisenman and Sandblom. 1983; Eisenman et al., 1983) can almost be superimposed, while it is likely that the values of klB for these ion species differ. These observations suggest that for these ions the shape is insensitive to the value of klB, i.e., that klB is large. This suggestion is consistent with the values of klB for Cs' and TI' inferred from the conductance data (Hladky and Haydon. 1984) and with the conclusions that the currents of Cs' (Andersen. 1983a) and of H' (Decker and Levitt, 1986) are at least partly limited by external access steps. For K' and Na' the currents increase more with potential (Eisenman and Sandblom, 1983; Eisenman et al., 1983; Hainsworth and Hladky, 1987a), consistent with values of klB which are no longer much greater than 1.

28

S.6.HLADKY

6 . Permeability Ratios. The next attempt to obtain further estimates of the rate constants (Urban et al., 1980) was based on the concentration dependence of the permeability ratios. But while this effect gives a clear indication of the interaction of two ions within the pore, it is not straightforward to obtain values of the rate constants from these data. The reason is that for a pair of ions there are 13 "rate" constants, 11 of which have an initially unknown potential dependence. A similar difficulty arises in the interpretation of the anomalous mole fraction effect. At least three more successful methods of estimating constants have now been found. c . Flux-Ratio Exponents. If the maximum value of the flux-ratio exponent can be determined, that provides good estimates for k/B and D/ B. Finkelstein and Andersen (1981) have used this method along with the conductance-concentration relation to infer the rate constants for cesium and gramicidin in diphytanoylphosphatidylcholine membranes.

d . Water Permeability. Dani and Levitt (1981) have reasoned that an ion in the pore would stop or at least greatly reduce the flux of water through the pore in response to an osmotic gradient. From the fall in the permeability to water as the ion concentration is increased, they have inferred binding constants that are consistent with those calculated from the ion flux data (Hladky and Haydon, 1984). e. Equilibrium Dialysis. The binding has been measured directly using radiolabeled thallium. To recap, in a tracer binding experiment you need to be able to detect the amount bound. Thus in the final soup that is assayed, there must be more of the test substance bound than free. With a drug binding to specific receptors with an affinity constant of 10' M - ' , that means we must achieve a receptor concentration of I nM, which is not too difficult. However, for sodium ions binding to gramicidin the kinetic data suggest that we would need about 100 mM gramicidin-binding sites, which is not possible. Veatch and Durkin (1980) were able to get the density of sites up to 2 mM in a vesicular suspension by using a short-chain lipid to make thin membranes and adding as much gramicidin as they could. That was adequate to look at the binding of thallium. Their calculations ignored the Donnan effect of the bound ions, but nevertheless the data show that the binding constant to gramicidin at high concentration in dimyristoylphosphatidylcholine membranes is of the order of lo00 M - ' . The values inferred from the kinetic data measured using low concentrations of gramicidin in glyceryl monooleate membranes are similar.

f. Spectral Changes. Another way to measure binding constants is to find some change in the ions or gramicidin that occurs when ions bind and that can be detected spectroscopically. Urry (1987) has pioneered this

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

29

type of measurement using nuclear magnetic resonance (NMR) and gramicidin incubated with lysolecithin (see Chapter 4, this volume). However, the relation between the constants determined under these conditions and those obtained from electrical and flux measurements is still uncertain. At present the four-state model provides explanations for all the ion transport properties of gramicidin. 1 am not aware of any feature of the data that requires the use of models which allow more than two ions to occupy the pore. However, even for pores which can hold at most two ions, this model is only an approximation (Hladky and Haydon. 1984; Hladky, 1987). Levitt (1982, 1986) has compared the predictions of the four-state model with those of a more elaborate model in which the ions can take up a range of different positions within the pore. He reaches the conclusion that, provided the potential dependences of the constants in the four-state model are chosen appropriately, these models cannot be distinguished by the available experimental data.

VII.

CONCLUSIONS

Despite the difficulties in determining the constants, several interesting conclusions have emerged from the kinetic data. The rates of transfer of ions from one end of the pore to the other are fast, for K', Cs', and TI+ approaching values near 10' sec- ' even at low potentials. Andersen and Procopio (1980). Finkelstein and Andersen (1981), and Dani and Levitt (1981) have suggested that ion movements from end to end are limited by the coupled movement of water through the pore. Certainly there cannot be any substantial electrostatic barrier to this movement. At least for cesium and thallium as the concentration is increased the conductance is limited first by the rate of entry into empty pores and then by the rate of entry to occupied pores. These rates are similar and vary only weakly with the applied potential. Both the weak potential dependence and the weak interaction with ions at the opposite end are consistent with a rate-limiting step at or just outside the mouth of the pore. Andersen (1983a) has noted that the effect of sucrose to reduce the rate of entry and a comparison of the ratios of the high potential currents with ratios of diffusion constants suggest an external step which can be limiting at high potentials for all ions. However, based upon flux ratio measurements (Procopio and Andersen, 1979; Andersen, 1984) and conductances (Hladky, 1974; Andersen, 1983a), he concludes that sodium ions cannot enter pores already occupied by another sodium ion (Finkelstein and Andersen, 198I). This apparent inconsistency remains unresolved. By contrast to the rate of entry, at least for cesium and thallium, the

30

S. B. HLADKY

rate of exit can be increased substantially by the presence of a second ion within the pore. Exit of ions, induced by the arrival of another, accounts for most of the observed current for concentrations above 100 mM. REFERENCES Andersen, 0. S. (1975). Ion specificity of gramicidin channels. Int. Biophys. Congr.. Copenhagen p. 369. Andersen. 0. S. (1983a). Ion movement through gramicidin A channels. Studies on the diffusion-controlled association step. Biophys. J . 41, 147-165. Andersen, 0. S. (1983b). Ion movement through gramicidin A channels. Interfacial polarization effects on single-channel current measurements. Biopliys. J . 41, 135-146. Andersen, 0. S. (1984). Gramicidin channels. Annu. R e v . Physiol. 46, 531-548. Andersen. 0. S .. and Procopio, J. (1980). Ion movements through gramicidin A channels. On the importance of aqueous diffusion resistance and ion-water interactions. Actu PliySiOl. Stand.. Sltppl. NO. 481. 27-35. Bamberg, E., and Janko, K. (1977). The action of a carbonsuboxide dimerized gramicidin A on lipid bilayer membranes. Biochim. Biophys. Acta 465, 486-499. Bamberg, E., and Lauger. P. (1973). Channel formation kinetics of gramicidin A in lipid bilayer membranes. J . Membr. Biol. 35, 351-375. Chappell, J. B., and Crofts, A. R. (1965). Gramicidin and ion transport in isolated liver mitochondria. Biochem. J . 95, 393402. Ciani, S. (1984). Coupling between fluxes in one-particle pores with fluctuating energy profiles. A theoretical study. Biophys. J. 46, 249-252. Dani, J. A., and Levitt. D. G. (1981). Binding constants of Li,K and TI in the gramicidin channel determined from water permeability measurements. Biophys. J . 35, 485-499. Decker, E. R., and Levitt, D. G. (1986). Contribution of bulk solution access resistance to the conductance of H’, K’, and Li’ in the gramicidin channel. Biopliys. J. 49, 376a. Eisenman, G., and Dani. J. A. (1986). Characterizing the electrical behavior of an open channel via the energy profile for ion permeation. A prototype using a fluctuating barrier model for the acetylcholine receptor channel. I n “Ionic Channels in Cells and Model Systems’’ (R. Latorre, ed.), pp. 53-87. Plenum, New York. Eisenman. G., and Sandblom, J. P. (1983). Energy barriers in ionic channels: Data for gramicidin A interpreted using a single-file (3B4S”) model having 3 barriers separating 4 sites. I n ”Physical Chemistry of Transmembrane Ion Motions” ( G . Spach. ed.), pp. 329-347. Elsevier, Amsterdam. Eisenman. G., Sandblom, J.. and Neher, E. (1977). Ionic selectivity, saturation, binding and block in the gramicidin A channel: A preliminary report. I n “Metal-Ligand Interactions in Organic Chemistry and Biochemistry” (B. Pullman and N. Goldblum. eds.). pp. 1-36. Reidel. Dordrecht. Netherlands. Eisenman. G., Sandblom, J.. and Hagglund, J. (1983). Electrical behavior of single-filing channels. I n “Structure and Function in Excitable Cells” (W.Chang, I. Tasaki. W. Adelman, and R. Leuchtag, eds.). pp. 383-413. Plenum, New York. Elliott, J. R., Needham. D., Dilger, J. P., and Haydon, D. A. (1983). Gramicidin single channel lifetime: The effects of bilayer thickness and tension. Biochim. Biophys. Acvtr 735, 95-103. Finkelstein, A. (1974). Aqueous pores created in thin lipid membranes by the antibiotics nystatin, amphotericin B and gramicidin A: implications for pores in plasma membranes. I n “Drugs and Transport Processes” (B. A. Callingham, ed.), pp. 241-250. Macmillan. London. Finkelstein, A.. and Andersen, 0. S. (1981). The gramicidin A channel: A review of its

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

31

permeability characteristics with special reference to the single file aspect of transport. J . Memhr. B i d . 59. 155-171.

Hainsworth. A. H.. and Hladky. S. B. (1987a). The effects of double-layer polarization on ion transport. Biop/i.vs. J. 51, 27-36. Hainsworth, A. H.. and Hladky, S. B. (1987b). Gramicidin-mediated currents at very low permeant ion concentrations. Biiipplivs J . 52, 109-1 13. Haydon. D. A., and Hladky. S. B. (1972). Ion transport across thin lipid membranes: A critical discussion of mechanisms in selected systems. Q. Rev. Biophvs. 5, 187-282. Henderson, P. J. F., McGivan. J . D., and Chappell, J. B. (1969). The action of certain antibiotics on mitochondria1 erythrocyte and artificial phospholipid membranes. Biochcvn. J . 111, 521-535. Hendry. B. M.. Urban, B. W.. and Haydon. D. A. (1978). The blockage of the electrical conductance in a pore-containing membrane by the n-alkanes. Biockim. Biopliys. A d t r 513, 106-116.

Hladky. S. B. (1972). The mechanism of ion conduction in thin lipid membranes containing gramicidin A. Ph.D. Thesis. Univ. of Cambridge. Cambridge. England. Hladky. S. B. (1974). Pore or carrier'?Gramicidin A as a simple pore. In "Drugs and Transport Processes" (B. A. Callingham. ed.), pp. 193-210. Macmillan, London. Hladky. S. B. (1984). Ion currents through pores. The roles of diffusion and external access steps in determining the currents through narrow pores. Biopphys. J . 46, 293-297. Hladky. S . B. (1987). Models for ion transport in gramicidin channels. How many sites? In "Ion Transport Through Membranes" ( K . Yagi and B. Pullman, eds.). pp. 213-232. Academic Press, Tokyo. Japan. Hladky. S. B.. and Haydon. D. A. (1970). Discreteness of conductance change in bimolecular lipid membranes in the presence of certain antibiotics. Nurrrre (London) 225, 45 1-453. Hladky. S. B . . and Haydon, D. A. (1972).Ion transfer across lipid membranes in the presence of gramicidin A. I. Studies of the unit conductance channel. Biochim. Biopliys. Ac,rrr 274, 294-312. Hladky. S. B.. and Haydon, D. A. (1984). Ion movements in gramicidin channels. Cirrr. Top. Memhr. Trunsp. 21, 327-372. Hotchkiss, R . D. ( 1944). Gramicidin, tyrocidine and tyrothricin. Adv. Suhj. Biocliein. Enzvmol. ReIui. 4, 153-199. Ivanov. V. T.. and Sychev, S. V. (1982).The gramicidin A story. In "Biopolymer Complexes" ( G . Snatzke and W. Bartmann. eds.), pp. 107-125. Wiley, New York. Kemp. G . . Jacobson, K. A., and Wenner. C. E. (1972). Solution and interfacial properties of gramicidin pertinent to its effect on membranes. Biochirn. Bioplivs. Acru 255, 493501.

Kolb. H.-A,. Lauger, P., and Bamberg, E . (1975). Correlation analysis of electrical noise in lipid bilayer membranes: Kinetics of gramicidin A channels. J . Memhr. B i d . 20, 133154. Lauger. P. ( 1973). Ion transport through pores: A rate theory analysis. Biocliim. Bioph.v.s. Acru 311, 423-441. Lauger. P. ( 1984). Channels with multiple conformational states: Interrelations with carriers and pumps. Cnrr. 7 o p . Memhr. T r u m p . 21, 209-326. Lauger, P.. Stephan, W., and Frehland. E. (1980). Fluctuations of barrier structure in ionic channels. Biocliim. Bioplivs. Aclu 602, 167-180. Levitt, D. G. (1982). Comparison of Nernst-Planck and reaction-rate models for multiply occupied channels. Eiopkvs. J . 37, 575-587. Levitt, D. G . (1986). Interpretation of biological ion channel flux data-Reaction-rate versus continuum theory. Annri. Rev. Biophys. Cliem. 15, 29-57. Levitt, D. G . , Elias, S. R.. and Hautman. J. M .(1978). Number of water molecules coupled

32

S. B. HLADKY

to the transport of sodium, potassium, and hydrogen ions via gramicidin, nonactin or valinomycin. Biochirn. Biophys. Actu 512, 436451. Neher, E. (1975). Ionic specificity of the gramicidin channel and the thallous ion. Biochim. Biophys. Actu 401, 540-544; erratum, 469, 359 (1977). Neher. E.. and Eibl, H. (1977). The influence of phospholipid polar groups on gramicidin channels. Biochirn. Biophys. Actu 464,3744. Neher, E., Sandblom, J., and Eisenman, G . (1978). ionic selectivity, saturation, and block in gramicidin A channels. 11. Saturation behaviour of single channel conductances and evidence for the existence of multiple binding sites in the channel. J. Membr. B i d . 40, 97-1 16. Pressman, B. C. (1965). induced active transport of ions in mitochondria. Proc. Natl. Acud. Sei. U.S.A. 53, 1076-1083. Procopio, J . , and Andersen, 0. S. (1979). ion tracer fluxes through gramicidin A modified lipid bilayers. Biophys. J. 25, 8a. Ramachandran, G. N., and Chandrasekharan, R. (1972). Studies on dipeptide conformation and on peptides with sequences of alternating L and D residues with special reference to antibiotic and ion transport peptides. In "Progress in Peptide Research" (S. Lande, ed.). pp. 195-215. Gordon & Breach, New York. Rosenberg. P. A.. and Finkelstein, A. (1978a). interaction of ions and water in gramicidin A channels. Streaming potentials across lipid bilayer membranes. J . Gen. Physiol. 72, 327-340. Rosenberg. P. A., and Finkelstein, A. (1978b). Water permeability of gramicidin A treated lipid bilayer membranes. J . Gen. Physiol. 72, 341-350. Rudnev, V. S., Ermishkin, L. N., Fonina, L. A., and Rovin, Y. G. (1981). The dependence of the conductance and lifetime of gramicidin channels on the thickness and tension of lipid bilayers. Biochim. Biophys. Actu 642, 196-202. Sarges, R., and Witkop, B. (1%5). Gramicidin A. VI. The synthesis of valine- and isoleucinegramicidin A. J . Am. Chem. Soc. 87, 2020-2027. Schagina, L. V., Grinfeldt, A. E., and Lev, A. A. (1983). Concentration dependence of bidirectional flux ratio as a characteristic of transmembrane ion transporting mechanism. J . Membr. Biol. 73, 203-216. Urban, B. W. (1978).The kinetics of ion movements in the gramicidin channel. Ph.D. Thesis. Univ. of Cambridge. Cambridge, England. Urban, B. W.. Hladky. S. B., and Haydon, D. A. (1980). ion movements in gramicidin pores. An example of single-file transport. Biochim. Biophys. Actu 602, 33 1-354. Urry. D. W. (1971). The gramicidin A transmembrane channel: A proposed mL,, helix. Proc,. N u t / . Al'ud. Sci. U . S . A . 68, 672-676. Urry. D. W. (1987). On the mechanism of ion transport through the gramicidin A transmembrane channel. In "Ion Transport Through Membranes" (K.Yagi and B. Pullman, eds.). pp. 233-254. Academic Press, Tokyo, Japan. Veatch. W., and Durkin, J. T. (1980). Binding of thallium and other cations t o the gramicidin A channel. J. Mol. B i d . 143, 411417. Veatch, W., and Stryer, L. (1977). The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels. J . M d . B i d . 113, 89-102. Veatch. W.. Fossel, E. T., and Blout, E. R. (1974). The conformation of gramicidin A. Biochemistry 13, 5249-5256. Veatch, W.. Mathies, R., Eisenberg, M., and Stryer, L. (1975). Simultaneous fluorescence and conductance studies of planar bilayer membranes containing a highly active and fluorescent analog of gramicidin A. J. Mol. Biol. 99, 75-92.

2. GRAMICIDIN: CONCLUSIONS BASED ON DATA

33

Wallace. 9. A. (1987). The structure of gramicidin A. a transmembrane ion channel. I n "ion Transport Through across Membranes" (K.Yagi and 9. Pullman. eds.). pp. 255-275. Academic Press, Tokyo. Japan. Zingsheim. H. P.. and Neher. E. (1974). The equivalence of fluctuation analysis and chemical relaxation measurements: A kinetic study of ion pore formation in thin lipid membranes. Biopliys. Cliein. 2, 197-207.

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CURRENTTOPICS IN MEMBRANES A N D TRANSPORT. V O L U M E 33

Chapter 3 Gramicidin, a “Simple” Ion Channel B. A . WALLACE Department of Chemistry and Center for Biophysics Rensselaer Polytechnic Institute Troy, New York I2180

The plasma membrane is responsible for the interactions of cells with their environments and neighboring cells. Membrane proteins play a major role in transport processes, cell-cell contact, and signal reception. The biological activities of these molecules are dependent on the conformations they adopt in the hydrophobic environment of the lipid bilayer. Just as detailed studies of soluble protein structures have provided insight into their modes of action and motifs for folding, high-resolution information concerning the structure of proteins which are embedded in lipid bilayers may elucidate their functioning as channels. This information will be especially valuable if information on the biochemistry and physiology of the molecule is available and can be related to its structural properties. For this reason, we have chosen to study the “simple” ion channel gramicidin A, whose conductance properties have been extensively characterized (Finkelstein and Andersen, 1981). and for which there exists a wealth of information on its chemistry. Gramicidin A is a linear polypeptide antibiotic which forms transmembrane channels. These channels are specific for the conduction of monovalent cations (Hladky and Haydon. 1972). The polypeptide consists of 15 amino acids with alternating L- and D- configurations, and has the sequence (Sarges and Witkop, i965): FormyCL-Val-Gly-L-Ala-~-Leu-t-Ala-D-Val-L-Val-u-Val-t.-Trp-

D-Leu-L-Trp-D-Leu-L-Trp-u-Leu-Leu-L-Trp-ethanolarnine

Variants with Phe or Tyr at position I 1 are designated gramicidin B and C, respectively, and cocrystallize with gramicidin A. For the purpose of this paper, “gramicidin” will refer to the natural mix of -85% gramicidin A, 10% gramicidin B, and 5% gramicidin C. Conductance experiments have shown the importance of the N-terminal 35 Copyright (0 19XX hy Ac.idemic Prew Inc All rights of reproduction in any form re\erved

36

8. A. WALLACE

formyl group and the sensitivity of single-channel conductance to the type of amino acid at the N-terminus of the polypeptide, but the relative insensitivity of single-channel conductance and mean channel lifetimes to modifications at the C-terminus (Bradley e t a / . , 1978; Morrow et al., 1979; Wallace et a / . , 1981; Russell et al., 1986). More subtle discriminations between different types of amino acids at other positions along the chain have been attributed to interactions between side chains and steric restrictions to movements of the backbone which permit ion passage, or to electrostatic interactions between the side chains and the permeating ions (Urry et af., 1984; Mazet ef al., 1984). Fluorescence and conductance measurements in black lipid membranes and phospholipid vesicles have demonstrated that the active conducting form of the molecule is a dimer and that essentially all dimers conduct (Veatch et al., 1975; Veatch and Stryer, 1977). However, the molecular nature of the conducting dimer has been the subject of considerable investigation in a number of laboratories, by both experimental and modeling met hods. In an early study based, in part, on circular dichroism (CD) and nuclear magnetic resonance (NMR) studies of the molecule in organic solvents, Urry (197 I ) first modeled gramicidin as a IT^^.^, helical conformation in which two monomers were associated via their N-termini (Fig. la). A T helix has adjacent carbonyl bonds pointing in alternating directions along the helix axis, unlike a-or 3,,-helices, whose C-0 bond vectors all point in the same direction along the helix. Conductance studies in black lipid films formed from monooleate and decane using gramicidins chemically

b

FIG.I. Schematic diagrams indicating the folding motifs of the (a) channel and (b) pore forms of gramicidin.

3. GRAMICIDIN, A “SIMPLE” ION CHANNEL

37

modified at their N-termini (Szabo and Urry, 1979: Baniberg et al., 1977) supported this model and, to some extent, a similar T helical dimer but with its C-termini associated (Bradley ~t ul., 1978). Within this class of folding motifs, it was recognized that different helical pitches could be formed, depending on the number of residues per turn and the hydrogen bonding patterns present. A helix with six residues per turn was later modeled as the most likely candidate for the channel because of its compatibility with the length of the bilayer and the size selectivity of the channel (Urry er al., 1971). Because of the alternating L- and D-amino acids in the sequence, the strong bias for formation of right-handed helices (as found for all L-amino acid chains) does not exist for this polypeptide, so not only were different pitches, but also different-handed helices possible. Modeling studies did not distinguish a bias for either hand of structure. Later, based primarily on infrared and CD spectroscopy of gramicidin in alcohol and dioxane solutions, Veatch et ul. (1974) proposed an alternate family of models, the parallel and antiparallel intertwined TITdouble helices (Fig. lb). These, too, could exist as both left- and right-handed helices, and could potentially form structures of different pitch. Interestingly, although their topologies are entirely different, structures which are both energetically favorable and of very similar length and diameter could be formed by either the helical dimer or double helical types of folding motifs (Fig. 2) (Wallace ~t a / . , 1981; Koeppe and Kimura, 1984). Of prime interest, was to determine the conformation of the conducting channels in membranes. However, none of the studies that lead to the various models had examined the structure of the gramicidin in phospholipid bilayers. NMR studies provided the first direct physical evidence that the predominant structure in phospholipid bilayers is a helical dimer (Weinstein et al., 1979, 1980). Fluorescence imaging studies in lipid bilayers further served to demonstrate that this is the major structure found in membranes (Boni e t al.. 1986). The ambiguity as to helical hand has persisted longer. While NMR studies of ion-induced shifts of gramicidin in lysolecithin-bound specimens suggested that the hand of the dominant channel is left handed (Urry et al., 1983). Arseniev e t a / . (1985b) using two-dimensional N M R studies of gramicidin in SDS micelles, concluded that the channel is right handed. CD studies (Wallace et a / . , 1981) seem to support a left-handed model, but are not conclusive, due to the strong tryptophan exciton interactions which tend to dominate the backbone region of the spectrum. While spectroscopic studies did provide information on the orientation and relative juxtaposition of gramicidin molecules in membranes, they did not provide any information on the detailed structure of the channel, nor could they confirm that the folding motif was actually a T helix, although

B. A. WALLACE

FIG.2. CPK models of the backbone of the (a) helical dimer and (b)double helix forms, showing their overall similarity in dimensions.

such a model was consistent with all available data. They also did not address the helical pitch or side-chain conformation, nor the nature of the binding site. Finally, all these studies examined the predominant conformation in membranes, but could not rule out the presence of a minor amount of another dimer conformation. In fact, some (up to 10%) of the conducting events seen for gramicidin may be of very long duration (J.

3. GRAMICIDIN, A “SIMPLE”ION CHANNEL

39

Durkin, personal communication). They are different from the major component events and may correspond to a different, long-lived dimer structure. Hence, even in bilayers there may be more than one channelforming motif for gramicidin. Because data for gramicidin in different environments and studies using different techniques had resulted in different models for the molecular structure being proposed, it was important to determine if there was “a” gramicidin conformation, or if the molecule could form various of the model conformations, depending on its circumstances. CD spectroscopy was used as a single technique to demonstrate that, indeed, gramicidin does adopt a number of different structures, depending on its environment (Fig. 3) (Wallace, 1983). The dominant structures it forms in phospholipid membranes and in a variety of organic solvents were shown to be distinctly different, even though the CD studies did not uniquely define what those structures were. Furthermore, Veatch et u l . (1974) showed that in organic solvents there exist a number of different interconvertible structures, which have distinct CD spectra. These were attributed to double helical conformations with different hands and pitches (Veatch, 1973). The spectra

10

-20

uva816M Y

FIG.3. Circular dichroism spectra showing the differential effects of ions on the channel and pore structures of gramicidin: gramicidin in dimyristoylphosphatidylcholinemembranes with (----) and without (-) cesium ions present; gramicidin in methanol solution with (--) and without (-----I cesium ions present.

40

8.A. WALLACE

of the dominant organic solvent forms are consistent with right-handed helices, In membranes, spectra obtained for a wide range of gramicidinto-phosphatidylcholine lipid ratios (from I : 15 to 1 : 363) are all very similar. Although there was a report that, at the low ratios which might better correspond to molecular ratios used in conductance measurements, different spectra were obtained (Ovchinnikov and Ivanov, 1983), it was later found to be the consequence of an uncorrected baseline (Wallace. 1986, 1987). The spectrum of the channel form is unlike that of any of the organic solvent species and does not correspond to a linear combination of those spectra (Wallace, 1983). The plethora of spectra observed by CD for gramicidin in these many different environments suggests that perhaps both of the general folding motifs (helical dimer and double helix) originally proposed by Urry (1971) and Veatch et al. (1974) may exist. In this chapter. the two general forms represented by the dominant membrane and organic solvent structures are referred to as the channel and pore forms, respectively (Fig. I). The effect of binding ions on both the channel and pore forms of gramicidin has also been examined by CD spectroscopy (Fig. 3). When the pore form of the polypeptide binds cations, a large change in its spectrum occurs (Wallace, 1983, 1986), corresponding to a major change in conformation of the molecule. Not only is the magnitude at 226 nm increased approximately twofold when saturating amounts of cesium are bound (a consequence of a change in pitch of the helix), but also the sign of the curve (and hence the hand of the helix) is reversed (Kimball and Wallace, 1982). This suggested the ion-bound form was a left-handed helix. Furthermore, the calculated decrease in helix pitch upon binding cesium (as derived from calculations based on these CD data) corresponds well with the differences in helical repeat and subcell dimensions of the molecule in crystals formed with and without cesium ions (Koeppe et al., 1978, 1979; Kimball and Wallace, 1984; Wallace, 19861, and would correspond to a change from around five residues per turn to approximately six residues per turn. The spectrum obtained using saturating amounts of the smaller ion lithium (Wallace, 1986) is also opposite in sign when compared with the spectrum for the polypeptide without ions, but is smaller in magnitude than that for the cesium form, indicating a larger pitch and suggesting that the pore is not as large for the lithium ion (and is more like the size of the ion-free form, but of different hand). The ion-binding sites can be titrated to saturation, although the binding constants for lithium and cesium differ by approximately two orders of magnitude (Wallace, 1986). These results suggest an interesting possible mechanism for ion binding in which, upon binding ions, the molecule foreshortens and widens and, upon releasing the ions, extends to the longer narrower pore. One

3. GRAMICIDIN, A “SIMPLE” ION CHANNEL

41

could envision this as a gating mechanism. However, such differences are not detected in the spectra of the channel form of the molecule in phospholipids upon binding saturating amounts of ions (Wallace, 1983; Shunga et al., 1986), suggesting that no change in pitch or in overall secondary structure occurs in the membrane-bound channels. This is reasonable because a 7~~ channel should be sufficiently large to accommodate even cesium ions. On the other hand, the pore structure may enlarge to accommodate the cesium because the pore also contains solvent (methanol) and counterions (chloride) (Wallace and Hendrickson, 1985). The differential effects of ion binding to the pore and channel forms are another demonstration that these two conformations differ (Wallace, 1983), and provide information on a functionally related aspect of the structure. There is also evidence that detergent- and lipid-bound forms of gramicidin may not be the same. Urry er ul. (1983) have examined the CD spectra of gramicidin in lysolecithin suspensions and Arseniev et al. (1985b) have examined it in SDS micelles. While both samples produce CD spectra similar to that of gramicidin in phospholipid bilayers and so have been argued to represent the channel conformation, neither is identical to it. Furthermore, although no change is seen in the CD spectrum of gramicidin in lipid vesicles upon binding ions (Wallace, 1983; Shunga et al., 1986), some change was seen in the lysolecithin-bound form when TI’ ions are bound (Urry et al., 1983), again suggesting an alteration in detail of the structure from that in membranes, another example of the polymorphism of the molecule. Gramicidin exhibits single-file conductance and has two cation-binding sites per channel. The relative locations of those sites have been suggested by channel profile and chemical modification studies (Mazet et al., 1984) and by NMR studies in lysolecithin suspensions (Urry et al., 1982). The latter provide the most direct evidence, although the altered occupancy of ion sites in the detergent-bound form of the molecule (Shunga c’f ul., 1986) and the difference in the CD spectrum in this environment make the relevance of this data to the native channel structure unclear. The effects of altered lipid structures on the channel have also been investigated. CD studies (Wallace et al., 1981) have shown that, when gramicidin is incorporated into membranes which are thick compared to the polypeptide length (i.e., distearoylphosphatidylcholine),the gramicidin dimer dissociates (Wallace et al., 198 I ) , no longer forming channel structures. This result is in accord with conductance measurements (Kolb and Bamberg, 1977) which indicated decreases in the mean channel lifetimes in thicker membranes. Hence, a structural feature of the channel can be correlated directly with an observed functional property; this feature could be related to the selectivity of target cells for this antibiotic.

42

6.A. WALLACE

As shown, spectroscopic and conductance studies can provide useful information on structural features of the gramicidin molecule. Ultimately, however, one would like detailed molecular information on the polypeptide backbone, side chain, and ion-binding site positions of gramicidin, which spectroscopic studies have not provided. The most suitable method for determining these features at high resolution is X-ray diffraction of wellordered single crystals of the molecule. Crystals of one type (the uncomplexed form prepared from an alcohol solution) were first reported 39 years ago (Hodgkin, 1949). Despite attempts in the intervening years to solve several different crystal forms of this molecule (Cowan and Hodgkin, 1953; Olesen and Szabo, 1959; Veatch, 1973; Veatch et al., 1974; Koeppe et al., 1978, 1979; Koeppe and Schoenborn, 1984), no structure of gramicidin had been solved at the molecular level. This may be because the molecule falls in a difficult size range: too large for direct methods used for small molecule crystallography, but rather small for Multiple Isomorphic Replacement (MIR) phasing methods used for macromolecular crystallography. Problems with the latter method stem from the difficulty in forming isomorphous derivatives. Due to the dependency of the gramicidin conformation on environment and the extreme flexibility of the molecule, when Cs' or Th' is added to ion-free gramicidin crystals, the crystals shatter (B. A. Wallace, unpublished observations). As an alternative, crystals can be grown in the presence and absence of these ions. However, a large difference in unit cell dimensions (as well as space group) (Table I) is found for the crystals grown with and without heavy atoms (Koeppe et al., 1979; Kimball and Wallace, 1984; Wallace, 1986); these crystals are, therefore, not suitable for MIR studies. In addition, monovalent ions tend to bind to similar sites within the channel, while the hydrophobic side chains and blocked N- and C-termini provide very limited opportunities for reaction of the polypeptide with heavy atom compounds to form independent derivatives. Finally, the low solvent content in these crystals (generally nce 200, 435436. Cowan. P. M.. and Hodgkin. D. C. (1953). Proc. R . Soc. London. Ser. E 141, 89-92. Finkelstein. A., and Andersen, 0. S. (1981). J . Membr. B i d . 59, 155-171. Hendrickson, W. A., and Teeter, M. M. (1981). N u t w e (London) 290, 107-1 13. Hladky, S . 8.. and Haydon. D. A. (1972). Biochim. Eiophys. Actu 274, 294-312. Hladky, S. 5.. Urban, B. W., and Haydon, D. A. (1979). I n "Ion Permeation Through Membrane Channels" (C. Stevens and R. W. Tsien, eds.), pp. 89-104. Raven Press, New York. Hodgkin. D. C. (1949). C o f d Spring Hurbor Symp. Qrrunf. B i d . 14, 65-78. Kimball, M. R., and Wallace, B. A. (1982). Eiophys. J . 37, 318a. Kimball, M. R.. and Wallace, B. A. (1984). Ann. N. Y. Acud. Sci. 435, 55 1-554. Koeppe. R. E., and Kimura, M. (1984). Eiopol.vmers 23, 23-38. Koeppe. R. E., and Schoenborn. B. P. (1984). E i o p k y s . J . 45, 503-508. Koeppe. R. E.. Hodgson. K. 0..and Stryer, L. (1978). J . Mof. B i d . 121, 41-54. Koeppe. R. E.. Berg, J. M.. Hodgson, K . O., and Stryer, L. (1979). Nutrtre (London) 279, 723-725. Kolb. H. A.. and Bamberg. E. (1977). Biochim. Eiophys. Aciu 464, 127-141. Mazet, J . L., Andersen. D. S., and Koeppe, R. E. (1984). Eioplivs. J. 45, 263-276. Morrow. J. S.. Veatch. W. R., and Stryer, L. (1979). J . Ma/. B i d . 132, 733-738. Myers, V. B.. and Haydon, D. A. (1972). Biochirn. Eiophys. Actu 27, 313-322. Naik. V. M.. and Krimm, S. (1984). Biochem. Biophys. Res. Cornmrrn. 125, 919-925. Olesen. P. E.. and Szabo, L. (1959). Ntrrrrrc, (London) 183, 749-750. Ovchinnikov. Y. A.. and Ivanov. V. T. (1983).1n "Conformation in Biology" (R. Srinivasan and R. H. Sarma. eds.). pp. 155-174. Russell, E. W. B.. Weiss. L. B., Navetta, F. I., Koeppe. R. E., and Andersen. 0. S. (1986). Biophys. J . 49, 673-686. Sarges, R . , and Witkop, B. (1965). J . Am. Chem. Soc. 87, 2011-2020. Short, K. W.. Wallace, B. A., Myers, R. A.. Fodor, S. P. A,, and Dunker. A. K. (1987). Biochemistry 26, 557-562. Shunga, D. C . . Hinton, J . F., Koeppe, R. E.. and Millett, F. S. (1986). Biochemistry 25, 6 103-6 108. Szabo. G., and Urry. D. W. (1979). Science 203, 55-57. Urry, D. W. (1971). Proc. N u t / . Acud. Sei. U . S . A . 68, 672-676. Urry. D. W.. Goodall, M. C., Glickson, J. D., and Mayers, D. F. (1971). Pro(..Nurl. Actrd. Sci. U.S.A. 68, 672-676. Urry. D. W.. Prasad. K. U., and Trapane. T. L. (1982). Proc. N a f l . Acud. Sci. U . S . A . 79, 390-394. Urry, D. W.. Trapane. T . L.. and Prasad, K. U. (1983). Science 221, 1064-1067. Urry. D. W.. Alonso-Romanowski. S . . Venkatachalam. C . M., Trapane, T. L., and Prasad, K. U. (1984). Eiophys. J. 46, 259-265. Veatch. W. R. (1973). Ph.D. Thesis, Harvard Univ.. Cambridge, Massachusetts. Veatch, W. R., and Stryer, L. (1977). J . Mid. B i d . 113, 89-102. Veatch. W. R.. Fossel, E. T.. and Blout, E. R. (1974). Biocfientistry 13. 5240-5256. Veatch. W. R.. Mathies. R.. Eisenberg, M., and Stryer. L. (1975). J. M o l . E i o f . 99, 75-92. Wallace, B. A. (1983). Biopo1vmer.s 22, 397402. Wallace, B. A. (1986). Eiophys. J . 49, 295-306.

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Wallace. B. A. (1987). I n "Ion Transport Through Membranes" (K. Yagi and B. Pullman, eds.), pp. 255-275. Academic Press, Orlando. Florida. Wallace, B. A. (1989). J. Mol. B i d . . in press. Wallace, B. A.. and Hendrickson, W. A. (1984). Acru Crysrullogr., Secr. A A N , c49. Wallace, B. A.. and Hendrickson. W. A. (1985). Eiopphys. J . 47, 173a. Wallace, B. A.. and Ravikumar. K. (1988). Science. in press. Wallace. B. A.. Veatch. W. R.. and Blout. E. R. (1981). Biochemisfry 20, 5754-5760. Wallace. B. A., Hendrickson. W. A., and Ravikumar. K. (1989). Submitted. Weinstein. S.. Wallace, B. A.. Blout, E. R.. Morrow, .I.S., and Veatch. W. R. (1979). Proc. Nail. Acud. Sci. U.S.A. 76, 4230-4234. Weinstein, S., Wallace, B. A., Morrow, J. S. . and Veatch, W. R. (1980).J . Mol. Eiol. 143, 1-19.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. V O L U M E 33

Chapter 4 Ion Interactions with the Gramicidin A Transmembrane Channel: Cesium-I33 and Calcium-43 NMR Studies DAN W . URRY, NAIJIE JING, TINA L . TRAPANE, CHI-HA0 LUAN, A N D MARSHA WALLER Laboratory of Molecular Biophysics University of Alabama at Birmingham University Station Birmingham, Alabama 35294 I.

Introduction A. Gramicidin A Transmembrane Channel Structure B. Approach to the Determination of the Mechanism of Ion Transport through the Channel C. Background on Ion Interaction Studies D. Cesium-I33 and Calcium-43 Ion Interactions with the Gramicidin A Transmembrane Channel 11. Cesium Ion Transport Studies A. Cesium Ion Single Channel Currents as a Function of Ion Activity t3. Cesium Ion Interactions Using NMR C. Calculation of Single Channel Currents Using NMR-Derived Constants 111. Calcium Ion Interaction Studies A. Ion-Induced Carbonyl Carbon Chemical Shifts B. Calcium-43 N M R Relaxation Studies IV. Appendix: Modification of Spin 3/? Formalism for Spin 7/2 Nuclei and Thermodynamic Quantities for the Off-Rate Constants References

Following an introductory review of the molecular structure and studies of the ionic mechanism of the gramicidin A transmembrane channel, cesium ion conductance data are presented for diphytanoylphosphatidylcholineln-decane membranes at 30°C, 100 mV, and over the ion activity range of 0.06 to 1 .O. Cesium-133 nuclear magnetic resonance ( 13. I MHz) relaxation studies are presented which determine rate constants for ions 51 Copyright (0 IYXX by Academic Pre,,. Inc. All nghtr. of reproduction in any limn rerervcd.

52

DAN W. URRY ET AL.

leaving the singly occupied (0.01 activity CsCI) and doubly occupied (0.77 activity CsCI) channels at 30°C for lysophosphatidylcholine gramicidin A channel lipid bilayer membranes. With these rate constants and previously determined binding constants for single-ion and double-ion occupancy of the channel, it is demonstrated that the single channel currents can be calculated over the activity range of 0.06 to 1.0 to within 15% of the experimental currents. The above efforts demonstrate the capacity to determine meaningful rate constants by means of N M R relaxation studies of spin 7/2 cesium133 and allow these approaches to be considered for spin 7/2 calcium-43. Background N M R data are presented for calcium-43, e.g., CaCI-,concentration dependence and temperature dependence of the longitudinal relaxation time, TI,in D,O, in the presence of phosphatidylcholine lipid and in the presence of phosphatidylcholine lipid plus gramicidin A channels and the transverse relaxation time, T,, for 100 mM and 1 M CaCI, in the presence and absence of channels. These data allow determination of the off-rate constant for calcium ions leaving the channel binding site to be of the order of 5 x 107/sec.With the binding constant demonstrated here to be about l/M for the site at the mouth of the gramicidin A channel and with the experimental off-rate constant, it becomes apparent that the lack of a calcium ion current is due to a high central barrier arising from the large repulsive image force which occurs when a divalent charge is separated from the lipid dielectric constant by no more than a single layer of polypeptide backbone. In the Appendix, it is shown that care is required in interpreting enthalpies and entropies of activation when based on temperature dependence of rate constants determined by means of N M R relaxation studies on spin 7/2 nuclei. It is also noted that the thermodynamic form of the Eyring absolute reaction rate equation is a useful formalism for treating ion channel transport, as demonstrated by previous temperature studies on sodium ion interaction with the gramicidin A transmembrane channel. 1.

INTRODUCTION

A. Gramicidin A Transmembrane Channel Structure

Hladky and Haydon (1970) were the first to observe single channel currents. The molecular system used was gramicidin A, HCO-L-VaI'-Gly'-LA ~ ~ ' - D - L ~ U ~ - L - A ~ ~ ~ - D - V ~ ~ ~ - L - V ~'"-L-Trp' ~ ~ - D I-D- V Leu"~~~-L-T~~' L - T ~ ~ ' ~ - D - L ~ UHCH,CH,OH ' ~ - L - T ~ ~(Sarges ' ~ N and Wit kop, 1965). Shortly afterward, in 1971, the gramicidin A channel structure was proposed (Urry,

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

53

1971; Urry et u I . , 1971). Three elements of the channel structure are the backbone conformation, the helix sense, and the nature of the dimer which forms the channel. The backbone conformation was independently described by Ramachandran and Chandrasekharan ( 1972a,b) and by this laboratory (Urry. 1971; Urry et d.,1971) to be a new class of singlestranded helices originally called L,D-helices or n,,-helices. respectively. The name was changed to P-helices because of the fundamental hydrogenbonding relationships to the parallel and antiparallel P-pleated sheet conformations (Urry, I972b). Having carried out conformational energy calculations using the model, poly(L-Ala-DAla),the studies of Ramachandran and Chandrasekharan (1972a,b) were silent on the issues of helix sense and the nature of the dimeric channel state. Having used conformational analysis of the gramicidin A sequence in combination with the constraints of spanning a lipid bilayer, the original papers (Urry, 1971; Urry et d., 1971) also proposed a left-handed helical sense and described a head-tohead (formyl end to formyl end) hydrogen-bonded dimeric channel. tnterestingly, spectroscopic studies were not used to derive conformation. Spectroscopic studies were used after the fact to assess the reasonableness of the proposed new helical structures under various conditions, and particularly to argue for a new class of helices distinct from the a-helical and other related classical helical conformations (Urry et a / . , 1971, 1972; Glickson et ul., 1972; Donohue, 1953). Subsequently, related doublestranded P-helices were described by Veatch et d. (1974)and Veatch and Blout (1974). Planar bilayer transport studies using derivatives and analogs of gramicidin A. principally the malonyl dimer (Urry et a / . , 1971; Bamberg and Janko, 1977) the 0- and N-pyromellityl derivatives (Apell et al., 1977; Bamberg et al., 1977), and the N-acetyl derivative (Urry, 1972a; Szabo and Urry. 1979) have resulted in the conclusion that the ion-transporting channel structure is formed from single-stranded P-helical monomers hydrogen bonded head-to-head to form the dimeric channel. Spectroscopic studies on suspensions of channels in lipid bilayers have resulted in the same conclusion using NMR methods which localize the amino end deep within the lipid layer and the carboxyl end at the aqueous surface (Weinstein et ul., 1979, 1980). and which determine the ion-induced carbonyl carbon chemical shifts to be incompatible with double-stranded P-helices (Urry et a/., 1983). In the latter case, a left-handed helical sense was concluded for the channel state: i.e., for the head-to-head dimerized singlestranded P'-helices (Urry el ul., 1982d, 1983). The data from planar bilayer transport studies and from spectroscopic studies on lipid bilayer suspensions of channels, which resulted in the conclusion of the original channel structure (Urry et al., 19711, are reviewed elsewhere (Urry, 1985b). The

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DAN W. URRY ET AL.

1971 channel structure is shown in Fig. 1 by means of space filling models. The currently most detailed representation of the channel structure in Fig. 1, obtained using the potential energy functions of the Scheraga group (Momany et al., 1974, 1975), is given in Fig. 2 as stereo plots (Venkatachalam and Urry, 1983). The channel comprises two single-stranded, left-handed, head-to-head dimerized p-helices. The helical parameters are n = - 3.1 dipeptideshrn and h = 1.53 A translation along the helix axis per dipeptide; the head-to-head docking geometries are given in terms of the intermonomer distance, d = 1.8 with an N . . . 0 distance of 3.2 A,and in terms of a rotation on the helix axis, 8, of one monomer with respect to the other (Venkatachalam and Urry, 1983). The channel length is about 26 A and the channel diameter is just under 4 A. The in vucuo minimum energy conformation has a channel radius of 1.5 A defined as the radial distance from the helix axis to the van der Waals radius of the L-residue carbonyl oxygens (Venkatachalam and Urry, 1984). It is important to appreciate that gramicidin A can exist in solution in

A

FIG. I . Space tilling model of the molecular structure of the gramicidin A transmembrane channel. (A) Channel view showing the approximately 4 bi channel diameter with the Trp", Trp'', and Trp" carbonyls directed outward into the solution. (B) Side view showing two gramicidin molecules dimerized head-to-head with the hydrogens of the formyl blocking groups of the amino termini at the center. (Adapted with permission from Urry e / d.,1975.)

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

55

FIG.2. Stereo plots of the minimum energy conformation of the gramicidin A channel showing the dimerized left-handed @-helices in side view (A) and along the helix axis (B). Plots are given for either distance, wall-eye viewing (two right-hand figures) or for close, cross-eye viewing (two left-hand figures). (Adapted with permission from Venkatachalam and Urry, 1983.)

many conformations (Fossel rt af., 1974; Urry er d.,1975) and can also associate with lipid in different conformations (Masotti r t ul., 1980).Thus the problem of obtaining the channel structure by X-ray diffraction techniques is substantial. When the channel structure is determined by diffraction methods, it is our view that its primary addition to our present knowledge will be to refine average helical (n and h ) and docking ( d and 0) parameters and to detail local deformations from the mean helical parameters due to the variable side chains. Another important contribution

56

DAN W. URRY ET AL.

of a diffraction structure would be to provide information on side-chain distributions, as these are considered to be quite variable and to result in different single channel current magnitudes. The present state of diffraction studies on gramicidin A is given in Chapter 3 of this volume.

B. Approach to the Determination of the Mechanism of Ion Transport through the Channel This laboratory uses a four-component approach in elucidating the mechanism of ion transport through the gramicidin channel. One thrust is the usual aspect of experimentally measuring ion type, ion concentration, applied potential, and temperature dependences of single channel currents (or conductances) by means of electrical measurements on planar lipid bilayers containing channels, that is, the approach due originally to Mueller and Rudin (1%7). The second thrust is to examine channels in suspensions of lipid bilayers comprising a related lipid for the purposes of locating ion binding sites and of determining binding and rate constants related to elemental steps contributing to the single channel current (Urry, 1985a). A third component is to use the information of binding site location and of binding and rate constants to calculate the single channel currents as a function of ion type, concentration, etc. Finally, a fourth component is to prepare and similarly characterize analogs of the channel for the purpose of testing and further developing the emerging ion transport mechanisms (Urry et al., 1984a,b; Prasad et al., 1986). In this chapter the first three components are utilized. C. Background on ion interaction Studies 1. OBTAINING THE LIPIDBILAYERCHANNEL STATE

The system which this laboratory has found to be useful in characterizing ion channel interactions has been the heat incorporation of gramicidin A and its analogs with L-a-lysolecithin (lyso-PC). In determining that this system results in the channel state, perturbation of lipid mobility was demonstrated and there was, on heating, a simultaneous development of a unique circular dichroism pattern, with development of strong ion interactions as demonstrated by a large sodium ion resonance chemical shift and a large change (decrease) in longitudinal relaxation time. It is necessary with each heat incorporation to verify that the channel state has been achieved by using the circular dichroism and 13Nachemical shift criteria. Because of the latter, our incorporations generally have 0.5 mM NaCl present. As shown in Fig. 3, heat incorporation of lyso-PC with gramicidin

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

57

FIG. 3. Electron micrograph of gramicidin A heat incorporated into L-a-lysolecithin ( 1 : 15 molar ratio) for 20 hr at 70°C. then negatively stained with I% uranyl acetate. pH

4.5. Vesicle-like structures are observed along with many folded structures showing single bilayer thickness. (Adapted with permission from Spisni el a / . , 1983.)

A results in a vesicle-like state and, when the concentration of lipid and gramicidin are high, a multilamellar state can be obtained that has periodicities typical of a lipid bilayer state (Pasquali-Ronchetti er al., 1983; Spisni er al., 1983). The particular advantage of the lyso-PC-gramicidin channel system is that the lipid bilayer state does not result in well-sealed vesicles but rather can be considered to be lipid bilayer sheets containing

58

DAN W. URRY ET AL.

channels without the problem of intravesicular and extravesicular ion pools. When determining binding constants and rate constants by means of relaxation methods, it is an advantage to have a single equilibrated pool of ions. For the several articles from this laboratory that demonstrate the above, the reader is referred to two reviews from which additional original references may be obtained (Urry, 1985a.b). 2. ION-INDUCED CARBONYL CARBONCHEMICAL SHIFTS

Location of Two Binding Sites. Using a series of synthetic gramicidin A molecules in each of which a single carbonyl was enriched with 90% carbon-13, it has been possible to determine the ion-induced carbonyl carbon chemical shifts for individual carbonyl carbon resonances along the sequence (Urry et al., 1982a,d, 1983). A plot of the ion-induced carbonyl carbon chemical shifts as a function of the channel structure shown in Figs. I and 2 is given in Fig. 4. The ion-binding sites are seen to be very well localized and to be related by the twofold symmetry of the channel structure. A plot of ion-induced carbonyl carbon chemical shift as a function of log (ion activity) (see Fig. 5 ) shows two binding processes, a tight

FIG.4. (A) Wire model of the channel structure along with (B) a plot of the ion-induced carbonyl carbon chemical shift as a function of position along the helix axis for 100 mM Na' and 83 m M TI + , locating the position of the binding sites for these ions in the channel. (Adapted with permission from Urry ei a / . , 1982d, 1983.)

59

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

I

1

I

I I l l l l

I

10

I

log

[ TIAc]

I

I

I I I I I I

100 (mM)

FIG.5 . Thallous ion-induced carbonyl carbon chemical shift for I-"C-enriched Trp" ( 0 ) and Trp" ( 0 )residues as a function of thallium acetate concentration. The dashed curve represents a tit to the data for two interactive ion-binding sites in the channel, and the solid curve represents the best tit assuming only one site. (Adapted with permission from Urry et d.,198Sb.)

binding process and a weak binding process. It is significant to recognize that, for example, at the concentration where the tight binding process is being completed, the system is one of half-occupancy of all binding sites. This is apparent from the carbonyl carbon chemical shift data. The ion pool is in fast exchange with all of the carbonyl carbons of residues in the binding site. As the first (tight) binding process proceeds, all of the carbonyl carbon resonances of a single enriched residue, e.g., all Trp" carbonyl carbon resonances, are being shifted as the first ion enters the channel. There is no pool of inaccessible or less accessible Trp" carbonyls. This is apparent by examining Fig. 6A and B (Urry ct al., 1982a). These are data for 100 mM NaCl (Fig. 6A; near the maximum for single sodium ion occupancy of the channel) and for 83 mM thallium acetate (between single and double thallium ion occupancy; Fig. 6B). Figure 6C is a schematic representation of a resonance at high field (on the right-hand side) that becomes shifted to lower field upon interaction with rapidly exchanging ions. If only one-half of the sites were accessible, one-half of the high field resonance would remain unshifted and only the other half of the intensity would shift downfield. as shown in Fig. 6D. The result would

60

DAN W. URRY ET AL.

C

#

"

175

'

'

l

171

l

PPm

l

I67

4

b

163

"

I75

'

'

171

'

' ' I67 ppm

~

163

FIG.6. Carbon-I3 NMR spectra at 25 MHz of the carbonyl region of lysolecithin packaged carbon-I3 enriched Trp" gramicidin A at 70°C in the presence of I 0 0 mM NaCl (A) and 83 mM thallium acetate (B). In each case, two spectra are overlayed, one with only 0.5 mM NaCl (no ion binding) and the second in the presence of the ion showing the downfield shift of the entire carbonyl carbon resonance. The sharp resonance at -174 ppm is from the lipid carbonyl which shows no interaction upon addition of ion. (Adapted from Uny er a/., 1982a.) (C) Schematic representation of a resonance at high field (right-hand side) which shifts entirely to lower field upon interaction with rapidly exchanging ions. (D).Schematic representation (using resonances of C ) of the result of accessibility of only one-half of carbonyl carbons with the pool of ions resulting in only partial shifting of the resonance to lower field. The summed curve would be the experimental result. This would be the case if only one-half of the sites were accessible.

be the summed curve of Fig. 6D. Clearly, this is not representative of Fig. 6A and B. When properly incorporated and verified, it is incorrect to state that one-half of the sites are inaccessible (Hinton et al., 1986). Because of the slow rotational motion at 30°C of the vesicle-like structures of Fig. 3, the data on ion binding using carbonyl carbon chemical shift were obtained at 70°C which is the temperature used to heat-incorporate gramicidin A as the channel state in lipid bilayers. Another point to note in Fig. 5 is that the dashed curve (which is a calculated curve using binding constants of 5000lM and 70lM for the tight and weak sites,

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

61

respectively) does not fit the data well at the low concentration range. This is indicative of a more complex ion interaction process at low ion concentrations. Analogous problems have been noted when attempting at low ion concentrations to fit sodium ion chemical shifts and rubidium ion-induced carbonyl chemical shifts (Urry er al., 1986b);the binding constant derived from ion chemical shift appears to be tighter as one moves to lower concentration and/or there appears to be a more complex process, possibly a cooperative binding process in the early stages of single ion occupancy. To date, the analysis of the binding constants has ignored this complexity at low ion concentration. The binding and rate constant values reviewed elsewhere and reported below using NMR relaxation methods relate to data above about 20 mM ion concentration. As will be seen below, these data can be used to calculate single channel currents and can be considered to have described the ion transport mechanism of the gramicidin A transmembrane channel in the concentration range above 20 mM. Complete concurrence with the conductance data and completion of the detailed mechanism will require an unraveling of the complexity at low ion concentration. 3. ION NMR STUDIES

Using thallium-205 ion chemical shift studies, Hinton and co-workers (1982, 1986)have estimated a binding constant for thallium ion. In a desire also to obtain rate constants and because of the difficulty of using ion chemical shift To identify the weak binding process (because the ion pool is so large and there is such a small effect of the weak binding process on the ion pool chemical shift at high ion concentrations), this laboratory has utilized the NMR relaxation methods. This takes advantage of the efficient quadrupolar nuclear relaxation mechanism present for the alkali metal nuclei (7Li, '3Na, "K, 87Rb,and '33Cs).Utilizing the quadrupolar nuclear relaxation mechanism available to alkali metal ion interaction studies is particularly appropriate to ion transport through channels. This is because evaluation of the ion correlation time, T,, is facilitated when 1.2 < ( 1 + w ~ T 7 ~ )2000, where w = 2 m , and u, is the nuclear magnetic resonance observation frequency. For ion interactions with the gramicidin A transmembrane channel, it can be shown that 7,' = ken, i.e., the reciprocal of the ion correlation time is the ion off-rate constant. Accordingly, a workable range of off-rate constants can be from 0 . 0 3 ~ to 20. For the most accessible NMR spectrometers, e.g., with a 23.5 kilogauss (kG) electromagnet, the values of w for the alkali metal ions are 'Li, 2.44 x lO%ec; "Na, 1.66 x 108/sec;39K, 2.93 x 107/sec;X7Rb,2.05 x 10H/sec; and '"Cs, 8.23 x 107/sec.With an off-rate constant approximating the

62

DAN W. URRY ET AL.

current for ion flow through a channel, the situation can be favorable for ion currents in the 10' to 10' ion/sec range. As two recent reviews have been written on alkali metal ion interactions with the gramicidin A transmembrane channel using NMR, one for readers of Methods in Enzymology (Urry et al., 1988) and a second for readers of Bulletin of Magnetic Resonance (Urry, 19871, these data will not be reviewed here. The special issue of spin 7/2 cesium-I33 will be considered, however, as it relates to the 'j3Cs data reported here and to the extension of the approach to spin 712 calcium-43. Analyses for spin 3/2 nuclei, which include the alkali metal ion nuclei of 7Li, "Na, 39K, and X7Rb,have been carried out principally by Bull, Forsen, and colleagues (Bull, 1972; Bull et al., 1979; Forsen and Lindman, 1981) for obtaining ion correlation times. Because Rb' and Cs' exhibit similar gramicidin A single channel currents at high ion activities and because they have been shown to exhibit similar weak binding constants (3.9/M for Rb' and 4.2/M for Cs'; see Urry ez d.,1985a, 1986b), this requires that they have similar off-rate constants at high ion activities. Phenomenologically, the "'Cs resonance exhibits a narrow and a broad component when interacting with the gramicidin A transmembrane channel, just as does H7Rb.When the spin 7/2 '"Cs data are analyzed using the spin 3/2 formalism, Cs' at ISM CsCl in previous studies was calculated to have an off-rate constant (-6 x 107/sec)which is 80% that obtained for Rb' (Urry and Trapane, 1987). The result is that the calculated single channel currents approximated at high ion activities differ by only about 20%, with that for Rb' being greater. The result is consistent with the experimental single channel currents obtained for phosphatidylcholine membranes. Thus, in this example, the study of alkali metal ion interactions with the gramicidin A channel has been helpful in extending the interpretation and usefulness of NMR relaxation data on spin 7/2 nuclei.

D. Cesium-133 and Calcium-43 Ion Interactions with the Gramicidin A Transmembrane Channel Included in this chapter is the plot of the single channel current versus ion activity data for gramicidin A in diphytanoylphosphatidylcholinelndecane membranes at 30°C and 100 mV applied potential. Then the determination of cesium ion correlation times as a function of temperature and their interpretation in terms of channel off-rate constants, taken together with previously determined binding constants, are used to calculate the single channel currents as a function of ion concentration for comparison with the experimental single channel current data over the ion activity range of 0.06 to 1.0.

4. THE GRAMlClOlN A TRANSMEMBRANE CHANNEL

63

As cesium-133 and calcium-43 have related NMR properties (both being spin 7/2 nuclei and having lower electric quadrupole moments than sodium23. which is assisted by larger Sternheimer antishielding factors) and as both exhibit rapid exchange with the channel, it becomes possible to estimate the off-rate constant for "'Ca leaving the gramicidin A channel binding sites. An objective is for the results presented here to bring the quadrupolar relaxation methodology for ion interaction with channels to potential application to Ca channels. II. CESIUM ION TRANSPORT STUDIES

A.

Cesium Ion Single Channel Currents as a Function of Ion Activity

Single channel currents vary with the lipid used to form the lipid bilayer. As lysophosphatidylcholineis used to package gramicidin channels in lipid bilayers for the ion interaction studies, a phosphatidylcholine lipid bilayer membrane is used to determine the single channel currents. Black lipid membranes were formed on a 0.43-mm diameter aperture separating two Teflon chambers, each filled with 7 ml of CsCl solution at various activities (0.06, 0. I , 0.3, 0.6, 0.8, 1 .O). The lipid solutions used to form the membrane consisted of 2% (wt/vol) diphytanoyl lecithin (DPhL) in n-decane. Picomolar concentrations of synthetic HPLC-purified "C-Val'-gramicidin A were added to the bath from methanolic stock solution. Data were acquired only after membrane stability was achieved, usually about 30 min. The temperature of the cell was maintained at 30°C to within ? 0.3"C by means of a Peltier Cell. The electrical measurements were made with Ag-AgCI electrodes, and the applied voltage clamp was 100 mV. The cell and the voltage source are placed in a refrigerator, which acts as a Faraday cage as well as to aid in stabilizing the temperature. The entire apparatus sits on a vibration-free air-isolated Micro-g table. The block diagram of the set-up used for observing, recording, and analyzing single channel events is shown in Fig. 7. The channel signals coming from the cell are amplified using a Burr-Brown OPAl1 IAM operational amplifier as a current to voltage converter, filtered by means of the KrohnHite Model 3342 (2 x 20 dB at 100 Hz) filter, recorded on a video casette recorder (VCR) utilizing the pulse code modulator PCM-I (Medical Electronics) for digitization, and analyzed using a Tektronix 4054A computer graphics terminal. The details of the data collection and analyses of the single channel current are described elsewhere (Urry et al., 1984b; Venkatachalam et al., 1984).

64

DAN W. URRY ET AL. OPAIIIAM

-I7Amplifier

I - - - - - - -

L

-

cell

-1-

shield

Krohn- hite 3342 filter

PCM-I

ICO

4907 File Manager

-

Tektronix 5111A oscilloscope

et

Tektronix 4054 A Computer

-

4662 Digital Plotter

-

FIG. 7. Block diagram of the experimental set-up utilized for observing, recording, and analyzing single channel events for the gramicidin A channel in planar black lipid membranes.

The histograms of gramicidin A single channel frequency of occurrence versus conductance (see Fig. 8) were obtained from the digital plotter after analyzing the data by computer. Although the distribution of conductance steps in the histogram for gramicidin A is broad, most events are concentrated in a 4-6 pS range. Therefore, a cut-off number relative to the maximal frequency of occurrence has been selected such that a most probable distribution of gramicidin A conductance can be stated and used to obtain a mean conductance. A cut-off of one-fifth maximal frequency of occurrence was chosen which generally gave a most probable mean conductance for more than 50% of the total events. A plot of the cesium ion currents (mean conductance x 0.1 Vh.6 x lo-’” C) at different

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

65 number of wents=1121 mean of most probable conductance peak40.6BpS

0.06A I

1

-

number of wents=1656

I

mean of mort probable conductonce peak = 18.25pS 0.1 A

uctance peak =43.48pS

mean of mast probable

mean of most probable conductance peak 158.68pS

conductance peak'54.92pS

5PS

FIG. 8 . Single channel conductance histograms for gramicidin A in diphytanoylphosphatidylcholineln-decane membranes at 30°C and 100 mV applied potential at cesium ion activities of 0.06, 0.1, 0.3, 0.6, 0.8. and 1.0. The total number of events giving rise to each histogram is indicated as well as the mean of the most probable conductance peak as defined in the text.

ion activities (0.06, 0.1, 0.3, 0.6, 0.8. and 1.0) is given in Fig. 9A using the conditions noted above. These experimental currents will be compared to the calculated currents using the NMR-derived binding and rate constants.

66

DAN W. URRY ET AL.

A k

I .06 x 10'/uc

k27.60~ IOT/src

$ =60/M K: * 4 /M

ji

\

Y)

.Q F

PX .-

log [activity CS+] FIG. 9. (A) Calculated single channel currents over a range of ion activities for cesium ion in the gramicidin channel, using NMR-derived binding and rate constants, with the location of ion-binding sites as indicated in Fig. 4, and with the introduction of voltage dependence using Eyring rate theory and assuming a linear potential gradient across the channel. Two curves are given with rate constants across the central barrier (kch)of 5 x 107/sec (based on dielectric relaxation data for TI') and of I x IO"/sec to show the effect of changing k,,. The experimentally measured most probable single channel conductances from the data in Fig. 8 are plotted as open circles (dashed curve). (B) Calculated probabilities of mole fractions of the channel states: x,, is the unoccupied, x , is the singly occupied, and xd is the doubly occupied channel using the NMR-derived binding constants of 6O/M and 4 / M . (Adapted with permission from Urry and Trapane, 1987.)

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

67

B. Cesium Ion Interactions Using NMR 1. ESTIMATION OF BINDING CONSTANTS When cesium nuclei are placed in a magnetic field, their nuclei tend to align and precess, giving a net macroscopic magnetic moment, M,,, in the direction, z, of the magnetic field. By means of a short intense radio frequency pulse directed along an axis perpendicular to the magnetic field direction, the macroscopic magnetic moment can be inverted to -M,,. The rate at which the inverted macroscopic moment returns to the original orientation for quadrupolar nuclei, i.e., nuclei with spins greater than I / 2, depends on fluctuating electric field gradients in the immediate environment (lattice) of the nuclei. For spin 312 nuclei the macroscopic magnetic moment along the z-axis, M y , at any time t is written (Bull, 1972) MAt)

=

MdO.2 exp( - t/T',) + 0.8 exp( - t/T;')]

(1)

where T , and T'; are the longitudinal relaxation times for the two nuclear relaxations of spin 312 nuclei. As is evidenced by the coefficient of 0.8, it is apparent that the magnitude of M,is dominated by a single term. This has also been shown to be the case for spin 512 and spin 7/2 nuclei (Bull et a / . , 1979). Accordingly for spin 7/2 Cesium-133, it is usually sufficient to consider the expression for the relaxation of an inverted macroscopic moment to be M;(T)= Mo[I - 2 exp( -7/Tl)]

A plot of In[M,, - M,(T)]versus time, T , with T being the interval between the 180" and 90" pulses in the inversion recovery pulse sequence (180"7-90"), yields a straight line with the slope of - T ; I. With the relaxation rate in the absence of channels, R,, = l/Tlf, and the relaxation rate in the presence of channels as R , = l/T,, a plot of the inverse of the excess relaxation rate ( R , - R J ' as a function of ion activity for a single binding process yields a straight line (James and Noggle, 1969). The negative x-axis intercept of the extrapolated straight line gives the reciprocal of the binding constant. For a more complex binding process, the plot is not linear. For two binding processes, a tight process and a weak process as exhibited by cesium ion, the extrapolated intercept at limiting low ion activity gives an apparent tight binding constant, and the extrapolated intercept for the limiting high ion activity range gives the apparent weak binding constant. These values for cesium ion interaction with the gramicidin A transmembrane channel have been determined as = 4/M (Urry et ul., 1985a). Ph= 60/M and

68

DAN W. URRY ET AL.

2. ESTIMATION OF RATECONSTANTS FOR CS' LEAVING THE CHANNEL a . Determination of Transverse Relaxation Times. With quadrupolar nuclei, it is often possible to observe resonance lines which are composed of two Lorentzian components, a broad component and a narrow component, When field homogeneity is not a problem, the two components can be resolved and their line widths at half intensity, u,,,, define transverse ) - ' T'i = ( ~ F Y ' ; / relaxation times for the two components: T', = ( ~ F V ' ~ / ~and ,)-,. For cesium-133 in water, the line width can be very narrow such that field inhomogeneity can be a concern. In general the spin-echo method can be useful. In this method, instead of inverting the macroscopic magnetic moment with a 180" pulse as in the above method for determining TI, a 90" pulse is used and the relaxation of magnetization in the plane perpendicular to the magnetic field direction, i.e., in the transverse direction, is monitored as a function of time, t (Fukushima and Roeder, 1981). For spin 3/2 nuclei the expression for the relaxation of the transverse magnetization, M T , is written (Bull, 1972) MT(t) =

MT(0)[0.6exp( - t/T'J

+ 0.4 exp( - t/T$)J

(3)

Using values from a spin echo experiment, a plot of In M T versus f can be used to evaluate T,and T$. Particularly as T,approaches T';, the estimates of these quantities can more effectively be achieved by the curve stripping of logarithmic plots of two exponential relaxations as in Fig. 10 than by curve resolution into two Lorentzian curves and determining line widths at half intensity for the resolved curves. At 30°C in the presence of 3 mM channels, the spin-echo data obtained for 1 I mM CsCl are given in Fig. 10A and for I .5 M CsCl in Fig. 10B. In Fig. 1OA two relaxation processes are clearly observed; curve stripping = 0.26 msec and T'; = 12.8 msec. It may also be yields values of noted that, at this low concentration, both broad and narrow components are readily observed in the resonance line, as has also been reported at 210 mM CsCl (Urry et al., 1985a). At 1.5 M CsCl, two components are not as readily apparent: only a small deviation is seen in Fig. IOB from a single exponential relaxation. By fitting the curve for times greater than 0.07 sec to obtain Z": = 215 msec and by taking the difference values in MT below 0.07 sec, a new curve is obtained at short times with a T', of 98 msec. From Eq. (3) the ratio of the t = 0 intercepts for spin 3/2 nuclei should be 0.6/0.4 = 1.5, whereas the ratios in Fig. 10 vary from greater than 3 to less than 1. This demonstrates that, even though spin 7/2 '"Cs nuclei exhibit two phenomenological components in their transverse relaxation, the relative magnitudes are not as given in Eq. (3) and are not constant with ion concentration.

69

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

0

I

0

2

4

time, msec

6

8

I

I

I

I

0.I

0.2

0.3

0.4

10

1

0.5

time,sec

FIG. 10. Spin-echo experiment (Carr-Purcell-Meiboom-Gill pulse sequence) for cesium133 in the presence of 3 mM gramicidin channels at 30°C. In A. the CsCl concentration is I I m M (10 milliactivity) where two transverse relaxations are clearly seen for the ion interacting with the tight site in the channel. In B, the CsCl concentration is much higher (1.5 M or 0.77 activity) and the tiansverse relaxation is only slightly nonexponential for ions interacting with the weak site or doubly occupied channel.

6. Determinution of Ion Correlution Time from Transverse Reluxution Times. What is normally obtained from the T'? and T'i values is the ion correlation time, 7,. It has been previously shown for the spin 3/2 alkali metal ions interacting with the gramicidin A transmembrane channel, most explicitly for '3Na interaction, that 7 ; ' = &, where klff is the off-rate constant for an ion leaving the doubly occupied channel. Significantly, it has also been shown for spin 312 "Rb and spin 7/2 '33Csthat their weak

70

DAN W. URRY ET AL.

binding constants are similar, i.e., K t = 4/M (Urry et al., 1985a, 1986b). As Rb' and Cs' exhibit very similar single channel currents at high ion concentrations, this requires that these ions have similar off-rate constants at high ion activities.' When the following equation (Bull, 1972)

for spin 3/2 nuclei is applied to the data for "Rb and '33Cs,similar values (within 20%) are indeed obtained for T , and k:fi ( = 7 , ' ) at high ion concentrations. This provides the practical demonstration that Eq. (4) can be used to determine T , for '3'Cs at high ion concentrations. [Plots of Eq. (4) for '13Cs and 4'Ca are given in the Appendix as Fig. 20.1 With the transverse relaxation times in the absence of channel but presence of the lipid at 30°C taken as 8.4 sec at low and high ion concentrations, the data in Fig. 10 give ion correlation times of 9.4 x lopRsec at 11 m M CsCl and 1.3 x lo-* sec at 1.5 M CsCI. In the following section, these values of 7, will be considered in terms of their relevance to the off-rate constants. c . Interpretation of Ion Correlation Times as Ofl-Rate Constants. The sources of the fluctuating - electric field gradient that could give rise to the experimental T~ are considered For interaction with gramicidin channels in lipid bilayer membranes

_1 -- -1 7,

7,

+ - 1+ - + ITvib

Tcb

I

(5)

Tb

where T~is the reorientation correlation time for the channel binding site in the magnetic field, T~~~is the vibrational correlation time for the ligands coordinating the ion at the binding site, T,b is the time for an ion to jump from one binding site to the other inside the channel, i.e., the intrachannel ion translocation time to pass over the central barrier, and 7 b is the ion 'At high ion concentrations, the current, i , through a single channel can be given to a good approximation by

, = - lK;:+ K'" [exp(lVFE/2dR7) - exp( ;: '

- I,rFE/2dRT]

With a common weak binding constant (G= 4/M) and binding sites for Rb' and Cs'. ,( = ,b = 3 A as the distance from binding site to barrier, z the charge on the ion of one, F the Faraday (23 kcal/m$e-volt), E the applied potential (0.1 volt), 2d the total distance across the channel (30 A), R the gas constant (1.987 cal/deg), T the absolute temperature (303°K) and kZrr = T ~ . ' then , i simply becomes 0.62 T,-' for both Rb' and Cs' (Urry 1'1 d., 1985a. 1986b; Urry and Trapane, 1987).

71

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

occupancy time in the binding site which is the inverse of the off-rate constant. All of these times would give rise to electric field gradient fluctuations for the ion interaction with the channel. As the reorientation correlation time for the membranes shown in Fig. 3 would be of the order of IO-'sec, T,-' is at least an order of magnitude too small to contribute significantly to the measured value of 7,. In evaluating T"~,, the temperature dependence of T, is considered as plotted in Fig. 11A and B. At low concentrations of CsCI, two transverse relaxation components could be resolved over the 30" to 60°C temperature range. This allows use of Eq. (4) to determine 7,. When In T, is plotted as a function of T - ' (OK)as in Fig. I IA, a reasonably straight line is obtained. Using Eyring rate theory [see Eq. ( A l ) in the Appendix], AH' is I'

0

I

.f

;'

4 I

I

I 3.4

I

1

3.2

1

I

1

3.0 I/ThtlxlO-'

FIG.1 I . Temperature dependence of the off-rates (k$, and k!&) from the singly and doubly occupied gramicidin channels. (A) At I 1 mM CsCl two transverse relaxations could be experimentally determined over the temperature range, allowing the use of the ratio A(RiIR:) of Eq. (4) as derived by Bull (1972) to calculate the correlation time, T , , for the ion in the channel which is taken as the inverse of the off-rate from the tight site k$, (see discussion and Appendix). (B) At I .5 M CsCl the transverse relaxation is nearly exponential over the same temperature range, making necessary the use of the ratio A(R,/R,)of Eq. (7) as expressed by Rose and Bryant (1978) to calculate the faster off-rate from the weak site kZR. In each part. a second curve is plotted ( 0 ) ;these are the off-rates calculated by applying the perturbation treatment of Halle and Wennerstrom (1981) to the spin 312 formalism needed to correct for the phenomenological transverse relaxation observed for spin 7/2 nuclei (see Appendix).

I 2.8

72

DAN W. URRY ET AL.

10.8 kcal/mol and, evaluating at 303"K, AS' is 9.4 cal/mol deg. Based on a harmonic oscillator partition function to estimate entropy, as in Eq. (6) and Fig. 12,

in order for a vibrational process to have an entropy of 9.4 cal/mol deg, the frequency would be about lO"/sec and, consequently, the correlation time would be about 1.6 X lo-" sec (i.e., ?vib = 1/2nvi). Such a value for 7,ib could not be responsible for an experimental correlation time of about 90 nsec. Also, an activation energy of 10.8 kcal/mol is obviously not relevant to a vibrational process where 1 kcal/mol is a large quantity. At high concentration above 30°C, it is not possible to resolve the two transverse components, as the relaxation times are too nearly the same. In this situation, a mean transverse relaxation time can be used in com-

e

109 ui

FIG.12. Frequency dependence of entropy, S,, based on a harmonic oscillator partition function as in Eq. (6). From the curve in Fig. I I A , the entropy evaluated at 30°C would be sec, 9.4 cal/mol deg, giving ui of -lO"/sec and, consequently, a correlation time of -10a value which is significantly out of the range of the experimentally observed value of about 90 nsec. Therefore, the correlation times giving rise to the experimentally determined values cannot be due to vibrational processes.

73

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

bination with a mean longitudinal relaxation time to estimate expression (Rose and Bryant, 1978)

T,

by the

A plot of Eq. (7) is given in the Appendix in Fig. 19. The result is the plot of filled circles in Fig. I I B which, when utilizing Eyring rate theory, give AH*of 7.7 kcal/mol and AS* of 3.0 cal/mol deg. The values are again not consistent with the characterization of a vibrational process. (These enthalpies and entropies of activation will be considered in more detail in the Appendix.) When considering sodium-23, it was argued that the experimental T , could not be due to T,h because the same value was obtained for gramicidin A and the malonyl gramicidin A, which give different currents primarily due to a difference in the rate over the central barrier (Urry et al., 1986a). Also for sodium-23 at low ion concentration where single ion occupancy occurs and the intrachannel ion translocation would have its greatest effect, the interaction with the channel is such that the broad component is too broad to observe, which indicates an off-rate constant of IO'/sec or less (Urry. 1987). In the case of 'z3Cs,different values are obtained for T , at high and at low CsCl concentrations such that Tch does not provide the dominant fluctuations of the electric field gradient as it clearly did not for '3Na. Accordingly, we neglect this possible contribution to 7,. There is a curious element of the '33Csresonance that appears and disappears as the ion titration proceeds. This is the occurrence of a third, low intensity. relatively narrow component that is observed near the T values at which M , = 0 for the other two components. The appearance of this third component could be the result of the presence of the four possible transitions for spin 7/2 nuclei (Urry et al., 1985a). Curiously, its appearance coincides with the concentration range for maximal single ion occupancy. Considering this peak in terms of Eq. (7) gives a T , of the order of 5 x lo-' sec. One possibility not entirely ruled out is that this third component could be due to intrachannel ions just sufficiently isolated from the ion pool, due to differences in correlation time and chemical shift during a phase of the titration, to be separately observed. This seems unlikely, however, because at near-maximal single ion occupancy (0.125 activity in Fig. 9B) the line widths of the intense narrow and broad components can be determined near the null (Urry et ul., 1985a), and, by Eq. (4). the ion correlation time is essentially the same, 4 x IO-'/sec. Having explicitly considered and discounted the first three terms on the right-hand side of Eq. ( 3 ,the interpretation of T , becomes that of ion

DAN W. URRY ET AL.

74

occupancy times, T ~ .Since the reciprocal of the occupancy time is the off-rate constant the result is

_1 --_1 -Tc

k,,

Th

C. Calculation of Single Channel Currents Using NMR-Derived Constants

From the binding constants for Cs' of p h = 60/M and G = 4/M (Urry et al., 1985a), the mole fractions of unoccupied, X,, of singly occupied, X , , and of doubly occupied, X,, channels are plotted in Fig. 9B. With this information, with the rate constants for ions leaving the singly occupied channel (k& = 1.06 x 107/sec)and for ions leaving the doubly occupied channel (k:ff = 7.6 x 107/sec),with the location of ion-binding sites as indicated in Fig. 4, with the rate over the central barrier taken as kch = 5 x 107/sec,with assumption of a linear potential gradient across the channel, and with the introduction of voltage dependence using Eyring rate theory, it is possible to calculate the single channel currents over a substantial range of ion activity. The calculation is as previously presented in detail (Urry et a / . , 1980). The calculated curve using kch = 5 x lo7/ sec, which was based on dielectric relaxation data on TI' (R.Henze and D. W. Urry, unpublished observations; Henze et al., 1982), is included with the experimental data in Fig. 9A as the solid curve. Included for comparison as the dotted curve is the calculation with k c h = I x 10X/sec. The two values of kchshow the effect of this rate constant on the location of the maximum in the current versus activity curve. In this calculation of single channel currents not a single parameter is used: all of the binding constants and rate constants and binding site locations are based on physical measurements independent of the electrical measurement of currents. While there is greater detail to be worked out, particularly at low ion concentrations, the comparison of the calculated curves strongly indicates the basic correctness of the ionic mechanism. The basic mechanism is close to the two-site three-barrier model considered much earlier by Hladky, Haydon, and Urban (Hladky et al., 1979; Urban and Hladky, 1979; Urban et al., 1978, 1980; Hladky and Haydon, 1984). Further consideration of the low concentration data, however, could possibly result in the identification of an outer complex as the initial interaction at low concentration, i.e., an additional pair of sites, which would then provide experimental evidence for a more complex mechanism analogous to the four-site mathematical model of Eisenman and Sandblom (Eisenman and Sandblom, 1983, 1984; Sandblom et al., 1983), though a single channel would not have simultaneously four occupiable sites.

75

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

111.

CALCIUM ION INTERACTION STUDIES

The divalent calcium cation is not transported by the gramicidin A transmembrane channel (Hladky and Haydon, 1972; Myers and Haydon, 1972), but calcium ion does competitively decrease the magnitude of single channel currents of transported ions (Bamberg et al., 1977). An NMR characterization of Ca" interaction with the channel provides both detailed information on the mechanism of competitive inhibition without transport and the basis for selectivity against divalent cations.

A.

lon-Induced Carbonyl Carbon Chemical Shifts

As demonstrated by the calcium ion-induced carbonyl carbon chemical shifts in Fig. 13, there is a calcium ion-binding site at the mouth of the gramicidin A channel; it utilizes the carbonyls of residues 15, 14, 13. and 1 1 (Urry et al., 1982~).In comparison to that of sodium ion, the binding site for calcium ion is pressed outward toward solution by about I A. such that the chemical shift for the well-resolved and relatively sharp resonance of residue 15 is almost as large as that of the broader resonance of residue 13. Accordingly, a titration can be effectively carried out using [ I-'3C]Trp''-gramicidin A. The data are plotted in Fig. 14, and a fit to this data provides an estimate for the binding constant at 70°C of 0.7/M with a chemical shift of I .2 ppm for the state where half of the sites are occupied (i.e., one calcium ion per channel). The binding constant is reasonable

r

X

1

X

M

12 I

15

1

8

4

1 1 1 1 1 1 1 1 1 1 1

II

7

3

12'

8'

4'

1 1 ' 1 1 1 1 1 1 1 1 1 1 1 1 1 1

NF'

NF

3

7

I I

1

1

15

C a r b o n y l Oxygen P o s i t i o n FIG. 13. Calcium ion-induced carbonyl carbon chemical shifts for the I-"C-enriched Lresidues of Trp'. Trp". Trp". and Trp" of the gramicidin A channel in the presence of I M CaCI, (as in Fig. 4). Shown a s the open circles are the shifts for the same residues in the presence of 0. I M NaCI. The chemical shift for the D - L ~ u "carbonyl is also given in the presence of Ca" ( + ) and Na' ( x 1. (Reproduced with permission from Urry 1'1 u / . , 1982c.)

76

DAN W. URRY ET AL.

[ Ca CI,]

(molar)

FIG. 14. Carbon-I3 NMR chemical shift as a function of calcium ion concentration for the Trpl carbonyl carbon of the gramicidin channel. The solid curve is the best fit to the ,data, assuming a single binding site for the divalent calcium ion at the mouth of the channel.

and compares favorably with the conducting channel, as Bamberg and Lauger (1977) had estimated a value of IIM from the effects of calcium ion inhibition of monovalent cation single channel currents. The chemical shift is reasonable for half occupancy of sites, as the total ion-induced carbonyl carbon chemical shift of just greater than 2 ppm has been demonstrated for similar binding of calcium ion to peptide carbonyls (Urry and Ohnishi, 1974). As shown in Fig. 15, these data again demonstrate the accessibility of all sites at the carboxyl (ethanolamine) end of the molecule. In particular, with 1.2 M CaCI,, it is apparent in Fig. I5 (as above in Fig. 6 for [I-'3C]Trp" carbonyls) that all I-'3C-enriched Trp" carbonyl carbons are being shifted as the calcium ion exchanges rapidly with all of the binding sites. When properly incorporated with lysolecithin to form lipid bilayer sheets containing channels, all sites are accessible. It is necessary to verify by circular dichroism that the channel state has been achieved, and by the .'"a chemical shift at 0.5 mM NaCl that the expected number of channel sites are accessible. The lack of a divalent ion-induced carbonyl chemical shift by [ l-"C]Val'-gramicidin A (Urry et al., 1983) demonstrates that the amino (formyl) end of the molecule is inaccessible to divalent ions, consistent with the head (formyl) end being buried deeply within the lipid bilayer on head-to-head association to form the conducting dimer.

77

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

,

175

173

171 ppm

w,

169

167

FIG.15. Carbon-I3 N M R spectra at 25 MHz of the carbonyl region of lysolecithin packaged I-"C-enriched Trp" gramicidin A at 70°C. Two spectra are overlaid, one in the presence of 0.5 rnM NaCl ( I ) , which gives the resonance of the carbonyl carbon without ion interaction, and one in the presence of 1.2 M CaCI, (2) which shows the downfield shift of the resonance upon interaction with the divalent cation. The resonance at -174 pprn is due to the lipid carbonyl. which does not show any shift upon addition of ion.

B. Calcium43 NMR Relaxation Studies The only calcium nucleus amenable to NMR observation is calcium43. with a natural abundance of 0.13%. This requires the purchase of an enriched source; 80% 4'CaC0, is obtainable from Oak Ridge National Laboratories, Tennessee. Addition of HCI provides the calcium chloride salt. For a 23.5 kG magnet the observation frequency is 6.7 MHz. I. LONGITUDINAL RELAXATIONTIMESTUDIES Before utilizing calcium-43 NMR for characterizing interactions with channels in lipids, it is necessary to obtain a set of background data. There is a temperature dependence of the longitudinal relaxation time, as shown in Fig. 16A for 0.05 and 1 A4 CaClz in DzO. The dependence of T , on

70

DAN W. URRY ET AL.

2.0

1.6 I

-80

,

I

/

-i

I

1.2

,*

,

,'8'

la1 0

0.8 I

20

,

I

,

40 Temperalure

I

.

'

0.I2

'

I 0.4

[CoCl,]

'

I 0.6

'

1

0.8

'

(Molar1

60 PC)

FIG.16. (A) Temperature dependence of calcium-43 longitudinal relaxation time, TI, in 'HZOat 0.05 and 1 .O M CaCL (B) Concentration dependence of calcium-43 TI at 30°C.

CaCI, concentration in D 2 0 is given in Fig. 16B. As the lipid used in our present studies on the gramicidin A channel in lipid bilayers is lysophosphatidylcholine, the CaCI, concentration dependence of T, in the presence of lysolecithin is given at 30" and 70°C in Fig. 17. Also included in Fig. 17 are the plots of 43Ca T, as a function of CaCI, concentration in the presence of 2.84 mM channels at 30°C and 2.59 mM channels at 70°C. What is apparent on comparison of Figs. 16B and 17 is a substantial interaction of calcium ion with the lipid. The values for T , in the absence of lipid (i.e., in D,O) are in the 1 to 2 sec range, but on addition of lysolecithin the values for TI drop to the 50-80 msec range at 30°C and to the 100-220 msec range at 70°C. Even though 43Cais a spin 7/2 nucleus, longitudinal relaxation studies on this nucleus can, in general, be used to estimate binding constants because a single component dominates the longitudinal relaxation (Bull et al., 1979). At 30"C, however, TI is smaller in the presence of lysolecithin alone than with lysolecithin plus channels. Therefore, it is not possible to use the data to estimate channel binding constants as has been so effectively utilized for the alkali metal ions (Urry et al., 1988; Urry, 1987). A crude estimate of the binding constant at 70°C using the low concentration data and neglecting the lipid interaction gives a value of about 4 / M , which differs by a factor of five from that obtained at the same temperature using the carbonyl carbon chemical shift data. An excess longitudinal relaxation rate plot for interaction with lysolecithin alone gives a similar apparent binding constant, indicating that the value measured in the presence of channels is primarily due to interaction with the lipid.

I

79

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

I

200

- 150 u

0,

t-

--*------*--.-----.

9/ /

-

I

/

0

100

_,---e---

lysolecithin + channels 30°C -_ _-* - -- - --- -

/

I I

0

lysoleciihin + chonnelr 70°C

0’

/Y’

I /

0 *)

.m----

/

/ /

/

: b

/

i /

*’/

/ /

/

;

i

50 -

d/

,,&I ,

- - - c --- -------o

_c-

_ _ _ _ _ - --

-0

Ax

-0

30’

lysoleciihin

/ ; ’ //

//

;y

I

I

I

I

[COCI,]

I

1

1

I

I

(Molar)

FIG.17. Experimental data for calcium-43 T , in the presence of lysolecithin micelles and in the presence of lysolecithin packaged gramicidin channels at 30” and 70°C as a function of CaCl, concentration. I n all samples. the lipid concentration was 0. I M and. in the gramicidin A-containing samples. the channel concentration was 3 mM.

2. TRANSVERSE RELAXATION TIMESTUDIES RATE CONSTANTS FROM THE CHANNEL

AND

ESTIMATION OF OFF-

In spite of the limitations of T Idata, transverse relaxation time, T,, data (alone and in combination with T , data) can be quite useful in providing estimates for off-rate constants. Using the spin-echo method, i.e., a CarrPurcell-Meiboom-Gill (CPMG) pulse sequence (see Fig. I8A), the transverse relaxation time due to lipid, T,,, is found to be 75 msec for 1 M CaCl, and 30°C. This is quite close to the value of the longitudinal relaxation time due to lipid, T I , = 80 msec, at 1 M CaCI, as shown in Fig. 17. When T I = T,, this is called the extreme narrowing condition, which means that w2?f Na' > K' > Rb'. As carbonyl reorientation becomes more difficult (still without water in the channel), the selectivity sequence changes: the lighter atoms find translocation more difficult and the order is K' > Rb' > Na' > Li'. Only when water is included in the calculation can the experimental sequence be reproduced: Rb' > K' > Na'. The significant feature of these studies is that they demonstrate that the translocation process may be highly cooperative. The small ions bind more strongly to the carbonyl groups; however, it is only because the channel is fairly rigid that they can not permeate readily. In terms of the helical backbone illustrated in Fig. 3, the interior minima are formed by cation association with pairs of carbonyl groups: 9 and 14, 7 and 12, 5 and 10, etc. An easily deformable channel would appear to permit sufficient motion of the lighter ion-carbonyl complexes to allow more rapid transfer of cations from one binding site to the next. If carbonyl groups 9 and 14 could reorient easily, they could librate enough to carry the ion close to groups 7 and 12 and thus facilitate hopping. In the rigid channel a cation apparently must separate from one of its carbonyls before it can approach the next pair; thus the binding affinity, which is greatest for the small ions, is more important. Furthermore, translocation over intermediate barriers appears not only to involve a balance between binding and deformability; aqueous friction also seems to play a role in affecting the relative permeability of the ions. Recent work, by imposing an external electric field, has simulated ionic motion across the internal barriers (Skerra and Brickmann, I987b). The data provided can yield an estimate of the translocational ability for Na' . The value deduced from these simulations, -5 x lo-" m' sec-', is quite consistent with experimental results (Dani and Levitt, 1981). As informative as these studies are, one can not be completely certain that they actually describe rate-limiting features of motion through the channel interior. Site-to-site transitions have a major influence on translocation only if the intermediate free energy barriers are comparable to or larger than the central barrier to translocation. This barrier is sensitive

104

PETER C.JORDAN

to a number of features missing from Brickmann's study: the presence of the dimer junction, the influence of the pore mouth, the amino acid side chains, and the long-range electrostatic interaction between ion, membrane, and water. As already mentioned, energy profile calculations on Cs'(H,O), complexes in the channel suggest that the translocation barrier is due as much to the long-range polarization interaction as it is to the ion-water-gramicidin potential energy. Preliminary studies of the Na'(H,O), complex suggest similar conclusions. If this is also true for more realistically solvated ions, the general shape of the translocation free energy profile would be more important than its detailed structure in determining the rate of translocation. These questions can only be resolved by further calculations using a model of gramicidin that has both a junction and the mouth, and attempts to incorporate side chain effects, which are known to influence translocation (Bamberg et ul., 1976; Morrow et ul., 1979; Heitz et ul., 1982, 1984; Mazet et al., 1984). Reliable estimates of both the global and the intermediate free energy barriers for more fully hydrated ions are needed, as both functions of ionic variation and of conformational flexibility, to clearly distinguish between these options. B. Valence Selectivity

Theory can also probe questions not readily accessible to experiment. An example is the valence selectivity exhibited by gramicidin. Here the significant region in the free energy profile is the region near the entrance to the channel. Although the channel is large enough to permit anion entry, there is no anionic contribution to conductance (except perhaps at high salt concentration). Why do the anion and cation free energy profiles differ so greatly? One view suggests that an anion could not bind in the channel (Urry et ul., 1981; Venkatachalem and Urry, 1984). Energy profile studies of the interacting ion-water-dimer system suggest this should not be the case (Sung and Jordan, 1987). The channel solvation energy, Eq. (I), is illustrated in Fig. 4 as a function of the location of the ion in the pore. The figure contrasts potential energy profiles for Cs' and CI- in two cases, with no water present and with four water molecules associated with the ions. These ions are chosen because they have almost the same size and polarizability and their ability to solvate water in the gas phase is nearly identical (Kebarle, 1977); they only differ in charge. There are a number of significant features. The overall shapes of the profiles 'differ substantially. For Cs' there is a pronounced binding site near the pore mouth; the channel solvation energy in the interior of the pore varies relatively little. The energy barriers to hopping between the intermediate binding sites in the region from -9 to 9 A are

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

105

all S2.5 kcal/mol. In contrast to Cs'. CI- solvation is relatively unfavorable in the channel mouth. However, there is a deep potential minimum near the channel midpoint; in the absence of water there is only a single binding site. The existence of other anion binding sites reflects the fact that there is a series of favorable locations for water molecules in the channel which maximizes their capacity to interact with the peptide backbone. Here too, there are only fairly small barriers to hopping between intermediate sites; they are all