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 (<970 pS) and is permeable to large ions including glutamate. These are characteristic of a large, water-filled pore. The cation nonselective, pressuresensitive channel of yeast has a n intermediate conductance of 40 pS, comparable to that of the nicotinic acetylcholine receptor (Hamill and Sakmann, 1981), which is also thought to be a water-filled pore (Dwyer ef al., 1980). With a few exceptions (Deitmer, 1982; Brezden and Gardner, 1986), other known pressure-sensitive channels, such as those in the chick skeletal muscle (Guharay and Sachs, 1984) and the inner ear (Corey and Hudspeth, 1979; Hudspeth, 1983; Ohmori, 1984), are also poorly selective and likely to be wide pores. Whether this similarity has meaning in terms of molecular design or evolutionary history remains to be seen. Data on the primary sequence of ion channels are still sparse. Nevertheless, the sodium channels of vertebrates and insects have been found to be homologous (Salkoff et al., 1986). The acetylcholine receptors of vertebrates and insects are also homologous. These two groups of organisms are at the tips of the two main branches in animal evolution. It is therefore likely that the ancestral sodium channel and acetylcholine receptor appear before the protostome-deuterostome bifurcation. Whether other channel types also have wide taxonomic ranges and deep evolutionary roots remains to be seen. It would also be of interest to see how the primary sequences of the ion channels of Paramecium, yeast, and E . coli compare to those of other organisms. Based on the primary sequences, the structural models of the sodium channel and the acetylcholine receptor employ transmembrane a-helices. Although these models are revised frequently, they often describe the pore as lined with charged sectors of amphipathic a-helices, each contributed by one subunit. Bacterial porins appear to be categorically different. They can be modeled as barrels of folded P-sheets (Benz, 1985). It should be interesting to see the categories into which the ion channels of the microbes described here (Table 1) fall. The state of the art in molecular biology is defined by work done on yeast and E. coli. These two experimental systems have also been used to reveal entire enzymatic cascades in various physiological and cell biological processes through genetic and biochemical analyses. These capabilities should be of great advantages in studying the biophysics and biochemistry of channels native to, or grafted into, these microbes. They

9

1. ION CHANNELS OF UNICELLULAR ORGANISMS

TABLE I PATCH-CLAMP RECORDINGS OF SINGLE CHANNELS

Organism

Type of channel

Known activation factor

IN

LOWERORGANISMS"

Approximate conductance (PS)

< 10

ATP. depol. ?

<430 < I50 40

Pressure." depol.

Ref."

I50 40 70

Ca, depol. Ca, depol. Ca, hyperpol. Ca

Depol. ( > -50 m V ) Pressure,' depol.

Ion selectivity

20 40 <970

"All measurements were done in the presence of 100-300 mM salts. hKey to references: (a) Martinac ef ul. (1986);(b) B. Martinac and Y. Saimi (unpublished); (c) Y. Saimi (unpublished); (d) Gustin et irl. (1986); (e) Gustin el ( I / . (1987); (0 Martinac c>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 <25%) limits the ability to diffuse in heavy atoms, and TABLE I GRAMICIDIN CRYSTALS Components

Space group

Grarnicidin/methanol Gramicidirdethanol Gramicidin/CsCl/methanol Gramicidinldirnyristoylphosphatidyc holine

p2 I p2 12 12 I p2 12I2 I P222,

Unit cell dimensions 15.24 x 26.70 24.8 x 32.4 x 32.12 x 52.10 26.8 x 27.5 x

x 31.71

32.1 x 31.17

32.8

3. GRAMICIDIN, A "SIMPLE" ION CHANNEL

43

also limits the sites available for binding heavy atoms without stereochemical interference. For these reasons, formation of isomorphous derivatives has been difficult, although Koeppe et al. (1978) did succeed in forming gramicidin/CsSCN and gramicidin/KSCN crystals which were isomorphic with each other, although not with ion-free crystals. Using those two crystal forms, they were able to obtain Single Isomorphic Replacement (SIR) phase information. The phase ambiguity from SIR in any region other than the central sections of the map, however, prevents calculation of a Fourier (electron density) map in three dimensions. Patterson analysis, which does not rely on phase information, but only provides information on correlations in the principal planes, indicates that the general shape of the molecule was likely to be a cylinder. More recently, elegant neutron diffraction studies by Koeppe and Schoenborn ( l984), using hydrogen-deuterium exchange to provide SIR phases on the uncomplexed organic solvent form, again indicated at low resolution that the molecule must be cylindrically shaped, but provided no information on the backbone fold or on side-chain positions. To examine in detail the three-dimensional structure of the pore with ions present, we employed an alternative method for phasing the X-ray data-that of single-wavelength anomalous scattering from cesiums incorporated into the crystal (Fig. 4), a method similar to that used for sulfur anomalous phasing of the structure of crambin (Hendrickson and Teeter, 1981). Because in this method the phases are determined from a combination of the Bijvoet differences and the partial structure of the cesium in a single crystal, there is no need to make isomorphous derivatives. The crystals used for these studies have eight cesium sites (four partially occupied) and two independent gramicidin dimers per asymmetric unit. A 1.8 A resolution electron density map (Fig. 5) has been calculated and a molecular model constructed from the data which shows the positions of all (nonhydrogen) backbone and side-chain atoms (Fig. 6) (Wallace and Hendrickson, 1984. 1985; Wallace et ~ 1 . .1988; Wallace and Ravikumar. 1988). Refinement studies currently underway are defining solvent molecule positions at the mouth and in the center of the pore (B. A. Wallace and K. Ravikumar, unpublished observations). The structure of gramicidin in these crystals is a left-handed, antiparallel double helical dimer. It has a p-sheet-type hydrogen bonding pattern with a superhelical twist of 6.4 residues per turn. The ion sites are clearly visible in the center of the pore. They are located 7.2 A from the ends of the pore and separated by 1 I .6 A. The pore diameter (van der Waals to van der Waals) is 4.9 A. The Trp side chains are not stacked normal to the helical axis, but are oriented roughly parallel to the helix axis. The hydrogen bonds of the two chains are staggered relative to each other so that, in three dimensions,

44

B. A. WALLACE

Fm. 4. X-Ray diffraction pattern of the grarnicidinkesium chloride crystals.

the pore opening is a relatively uniform cylinder. We can now compare this to structures that had been previously proposed for the pore: One of the double helical structures (designated the 7 residue per turn model) originally proposed by Veatch ef al. (1974) closely resembles the hydrogen bonding pattern of this structure, although the superhelical pitch is slightly different (and hence the difference in nomenclature). Also, the folding motif suggested by the vibrational spectroscopy studies of Naik and Krimm (1984) is similar to this structure [although in this case, too, the superhelical pitch and therefore the nomenclature (IT,.?) differ somewhat]. The pitch and hand of the structure suggested by the CD studies (Kimball and Wallace, 1982; Wallace, 1986) also corresponds closely to that observed in the crystals. On the other hand, the hydrogen bonding pattern, helix hand. and alignment of chains in the double helical structure proposed by Ar-

3. GRAMICIDIN, A "SIMPLE" ION CHANNEL

45

FIG. 5. Central sections of the three-dimensional electron density map of the gramicidinl cesium complexes, showing views in !he center of the pore (a) along the hetix axis and (b) perpendicular to the helix axis.

46

B. A. WALLACE

FIG.6. Molecular graphics view of the gramicidin pore structure at the current stage of refinement. The cross-hatched lines are the electron density map (data) to which the structure was fit.

seniev et al. (1985a), based on two-dimensional NMR studies of a solution of gramicidin and cesium, are entirely different from that seen in the crystalline gramicidinkesium complex. Finally, the conformation of the ionbound pore derived from model calculations (Koeppe and Kimura, 1984) is of a completely different fold (helical dimer). While the above crystallographic studies have provided us with a detailed view of the molecular structure of gramicidin, it should be noted that spectroscopic and conductance studies also provided critical information for our understanding of this molecule. It is of interest that this structure exhibits a pore size and molecular length compatible with the dimensions proposed for the channel structure based on conductance studies (Hladky and Haydon, 1972; Myers and Haydon, 1972); two cation-binding sites are found per dimer as suggested by conductance measurements (Hladky et al., 1979); the outside of the dimer consists of hydrophobic amino acid side chains which could interact with the hydrophobic lipid fatty acid chains in the membrane; and the pore is lined with the hydrophilic peptide backbone, permitting complex formation with the ions. These features suggest that this form, too, may form a conducting dimer species. Indeed, it may correspond to the minor channel conformation which results in the very long lifetime events. This would be expected of a pore held together

3. GRAMICIDIN, A “SIMPLE” ION CHANNEL

47

by 28 hydrogen bonds, rather than the 6 hydrogen bonds that presumably would hold together the helical dimer. Furthermore, Ovchinnikov and Ivanov ( 1983) have shown that an N-terminal to C-terminal cross-linked dimer is capable of forming active channels, again consistent with a conducting double helix form. Hence, this structure may provide the first view of an ion channel (albeit a minor conformation). To similarly address the question of the conformation of the membranebound form of the molecule, crystals of a complex of gramicidin and either dimyristoyl- or dipalmitoylphosphatidylcholine have been prepared (Wallace, 1983, in press). The molar ratios in the crystals are one gramicidin monomer to two lipid molecules; this was not the input ratio in the crystallization mixture, but the ratio in the ordered complexes formed (Short et al., 1987). Preliminary characterization indicates that the unit cell parameters of these crystal forms are different from any of the crystal forms without lipid (Fig. 7) (Wallace, 1986). This finding suggests that at least the packing, and likely the molecular fold, as well, are different. Raman spectroscopy (Wallace, 1983; Short et al., 1987) indicates that the phospholipids present are highly ordered in the cocrystals. Work is under way to produce isomorphous derivatives. Once both crystal structures have been refined at high resolution, this molecule will be an excellent candidate for dynamics and folding studies. The importance of having a well-determined structure based on experimental results rather than solely on abinitio modeling is evident. Models for the structure which assumed a uniform helical polypeptide backbone can be seen to be wrong in detail now that the structure of the pore is in hand. The backbone irregularities that occur in regions near the ion-binding sites may be the feature that distinguishes specific sites for binding which are highly occupied, as opposed to a rather uniform hole partially occupied along its complete length. Furthermore, the superhelical twist actually found turns out to be different than in any of the models for this form, and results in a rather different stagger of the side chains. In summary, gramicidin represents an excellent system for studying the structural, functional, and folding properties of a transmembrane channel. The vast amount of information on the chemistry and physiology of this molecule, along with the recently available detailed three-dimensional information, means that the structure may be interpreted in terms of functional properties on a molecular level and may form a basis for modeling and design of other ion channels. Finally, it may be useful to note that while gramicidin was initially chosen to be a “simple” model (relative to “more complex” higher-molecular-weight channels). that has not turned out to be the case. Perhaps that is because the very nature of a small molecule is that it is flexible and polymorphic. One hopes that the larger

48

B. A. WALLACE

FIG.7. X-Ray diffraction pattern of the gramicidinldipalmitoylphosphatidylcholine crystals.

molecules may not suffer this fate to quite the same extent because of their increased intramolecular interactions which limit flexibility; therefore, their increase in complexity may not be in direct proportion to their size, and so they may ultimately turn out to be more simple systems for study. ACKNOWLEDGMENTS This work was supported by NSF grants DMB87-96205 and DMBIS-I7866 and. in part, by NIH grant GM27292. During the course of these studies, the author was the recipient of a Camille and Henry Dreyfus Teacher-Scholar Award. REFERENCES Arseniev, A. S . . Barsukov. 1. L.. and Bystrov. V. F. (1985a). FEBS Left. 180, 33-39. Arseniev. A. S.. Barsukov. I. L., Bystrov. V. F.. Lomize, A. L., and Ovchinnikov. Y. A. (1985b). FEBS L e f t . 186. 168-174.

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Bamberg. E.. Apell, H. J.. and Alpes. H. (1977). Proc. N u t / . A m d . Sci. U . S . A . 74, 24022406. Boni. L. T., Connolly. A. J., and Kleinfeld. A. M. (1986). Eiuphvs. J. 49, 122-123. Bradley. R. J . . Urry. D. W.. Okamoto. K., and Rapaka. R. (1978). Scii>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.

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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

54

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 << 1. Since T I , 5 T Z y it, is grossly approximated that W’T; Z which, with 7 ; ’ = kOmmeans that k,, 5 4 x 107/sec.The near extreme narrowing condition also means that T I Cand T,( p may be used in place

lo9.1-

A

.

- -

-

I

/P,~75.4msec

86-

i d

4-

2I

0 lo-

B

9.1-"

I

I

I

I

1

I

L

I

1

,

-

T2"=38.0 msec

86.9'

\~T2'=14.7msec 6-

i 4

4-

2I

0

-

1

I

I

1

A

I

c

lo8.41

1

I

I

L'

T i ' =13.3 msec

8-

*

6.64-

i &

6 \,T2'=8.6msec -

4-

2-

0

1

I

I

I

I

I

I

I

1

1

FIG.18. Spin-echo experiment (Cam-Purcell-Meiboom-Gillpulse sequence) at 30°C for calcium-43 at (A) I M CaCI2in the presence of lysolecithin, (B) at I M CaCI2in the presence

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

81

of TI,and T2f(the values for the ions free in water) in Eqs. (4) and (7) to estimate magnitudes of T, for interaction with the channel when TI > T 2 . In the presence of channels at I M CaCI, and 30"C, the value of TI is 96 msec (see Fig. 17). As shown in Fig. 18B. two transverse relaxation components are just discernible with T2 = 14.7 msec and 7"; = 38 msec and with a mean value for the slope of T2 = 33 msec. This is almost a 300% difference between T i and T2 in the presence of channel, whereas the difference is one-tenth that or 27% in the absence of channels but in the presence of lipid alone. The large line width change can be attributed to interaction with channels. The values in Fig. 18A and B give a ratio, A(R>/R:),of 4.2, which translates by means of Eq. (7) or Fig. 19 into a sec. Using Eq. (A2) and Fig. 19 of the correlation time, T,, of 4.4 x Appendix, this becomes 3.9 x sec. As demonstrated with Cs' and Na', the inverse of T , gives the off-rate constant. Accordingly, at I M CaClzand 30°C, the off-rate constant for calcium ion leaving the binding site in the gramicidin A channel becomes 2.6 x 107/sec. It is also of interest to characterize the calcium ion interaction at a lower concentration. This provides a check on the interpretation that it is a partial single calcium ion occupancy of the gramicidin A channel that is responsible for the observed relaxation. The same relaxation process (the ion entering and leaving the channel) should be dominant at low concentration and at high concentration, that is, within experimental error, the rate constants should be the same. In the presence of lipid alone for 100 mM CaCI, at 3WC, T i , = 54 msec and T,, = 45 msec. This represents a 20% difference between T, and T., i.e., T i , = T Z I .and the near extreme narrowing condition is present. In the presence of 2.9 mM channels with the same amount of lipid at 30°C. T', = 8.6 msec and T'; = 13.3 msec (see Fig. 18C) and the mean value for the slope in Fig. 18C gives a T , of 12 msec. The change in T2 from the absence to the presence of channels is about 40096, and in the presence of channels the difference between T i (50 msec) and T, (12 msec) is also some 40096, compared to only 20% in the absence of channels. Again, the channels can be considered to be the source of the difference in relaxation times. Using Eq. (7) or Fig. 19 and with a A(RL/Ri)ratio of 1.76, the calcium ion correlation time, 7,. calculates to be 1.8 x lO-'sec. giving an off-rate constant of 5.5 x 107/sec.The off-rate constant at 100 mM CaCI, differs only by a factor of about two from that estimated at I M CaCI,. These results demonstrate that, in spite of lysolecithin packaged gramicidin A (3 m M channels) and ( C ) at 0. I M CaCI, also in the presence of lipid plus 3 mM gramicidin channels. In the first curve, only in the presence of the lipid, the transverse relaxation may be calculated very well by a single exponential. In the presence of the channel, however, two transverse relaxation times may be discerned.

82

DAN W. URRY ET AL.

of the calcium ion interaction with the phosphatidylcholine head group of the lipid, it is yet possible to estimate the off-rate constant for calcium ion leaving the channel. IV. APPENDIX: MODIFICATION OF SPIN 312 FORMALISM FOR SPIN 7/2 NUCLEI AND THERMODYNAMIC QUANTITIES FOR THE OFF-RATE CONSTANTS

The enthalpies, A P , and entropies, AS', of activation defined by the thermodynamic form of the Eyring absolute reaction rate equation

kT k' = u-e h

AS'IR

- AH*fAT

were considered briefly above in terms of the interpretation of 7,. In Eq. (Al) k' is the specific rate constant, in our case representing k& and kzfi; K is the transmission coefficient taken as one for the ion transport process; k is the Boltzmann constant (1.38 x erg/deg); h is Planck's constant (6.62 x loT2'erg sec); T is the absolute temperature ("K);and R is the gas constant (1.987 caVmol deg). In order to obtain more meaningful values of A# and AS* for '33Cs,it becomes necessary to consider explicitly the modification of the spin 312 formalism for spin 712 nuclei. At high CsCl concentration (1.5 M ) the plot of In MT versus t in Fig. 1OB is very nearly linear, indicating that the transverse relaxation deviates only to a small extent from being characterized by a single exponential relaxation. This is the near exponential regime for which Halle and Wennerstrom (1981) have carried out a perturbation treatment, giving the modifications of the spin 312 formalism, that is required to be applicable to spin 7/2 nuclei. That treatment is utilized here to obtain a correction to Eq. (7).

where

+

A , = 21, 8J2 B , = 2(12J, + 135,)

A , = 35,

+ 5J, + 2J2

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

3(22) I"

E2

= -@jiT(Jo

-

83

JJ

with

In the terms of Eq. (A2), Eq. (7) becomes simply A,/A I and the additional terms in Eq. (A21 are the perturbation correction. A plot of Eq. (A2), i.e., A(R2/Rl)versus log T ~ is , given in Fig. 19 for IJ3Cs(o = 8.24 x lo7 rad/ sec) and for 43Ca(o = 4.23 x lo7 rad/sec). For each nucleus, Eq. (A2) is the curve on the left and the curve on the right is the plot of Eq. (7). When Eq. (A2) is used for the 30°C data in Fig. 10B as well as similar data at higher temperatures, the open circles of Fig. 1 IB are obtained. Thermodynamic quantities for this corrected curve are AHt = 6.2 kcal/ mol and AS* = -4.2 cal/mol deg, evaluated at 303°K using Eq. ( A l ) . In our nomenclature, these would be and AS:,. These values represent a significant change from those obtained before utilizing the perturbation treatment. For "Na the values at high ion concentration were 5.9 kcall mol and - 5.4 cal/mol deg. Since the barrier for leaving the channel, AH, is greater for Cs', even though Cs' has a greater current, the difference in the entropies of activation would be the basis for Cs' selectivity over Na'. Before this consideration is accepted, however, it should be appreciated that the value for kTff determined by means of Eq. (7) at 30°C (the solid data point at 16.69 in Fig. I IB) is much less than that obtained by means of Eq. (4) (the solid data point at 18.15 in Fig. I I B). It has been shown by the comparison of the calculated and experimental curves in Fig. 9A that, at 3WC, Eq. (4) gives rise to correct values of T, when interpreted as the inverse of the off-rate constant. Previously, comparison with spin 3/2 87Rballowed verification at high ion activities, and the work presented here shows the usefulness of Eq. (4) for "'CS at low and at high ion activities. In Fig. 1 IA at 10 milliactivities, the filled circles utilize Eq. (4) and give rise to values for AH&,andAS& for the ion leaving the singly occupied channel. The use of Eq. (4) is made possible by the discerning of two exponential relaxation processes, as readily seen by the nonlinearity of the data points in Fig. 10A. At 70°C and 10 milliactivities. however, the two components are not separable, yet T, # T2 such that Eq. (7) can be used. But, when Eq. (7) is used to calculate T, for the 70°C data point in Fig. IIA, the calculated value of T, falls clearly below the mean slope that is well-defined at lower temperatures by data using Eq.

w,r

84

DAN W. URRY ET AL.

-- 9

-7

-8

-6

log Tc

FIG.19. Plots of the Rose and Bryant ratio, A(RJR,),as a function of log correlation time, T,, for cesium-I33 at 13. IMHz and calcium-43 at 6.7 MHz resonance frequencies using Eq. (7). Also plotted are the Halle and Wennerstrom perturbation treatment corrections for these spin 7/2 nuclei [see Eq. (A2)] in the nearly exponential relaxation regime which give increasingly shorter correlation times as the ratio increases. The pair of left-hand curves are for cesium-133 and the pair of right-hand curves are for calcium-43, with the curve calculated by the Halle and Wennerstrom treatment being on the left of each pair.

(4). That this is a result of the equations and not a unique process occurring as one goes from 60" to 70°C is demonstrated by the data in Fig. I IS. At high ion activity, 1.5 M (0.77 activity) C,CI, the solid data points using Eq. (7) fall on a mean slope including the 70°C data point. At 3WC, however, it is possible at this high ion activity just to discern two relaxations (see Fig. 10B). When Eq. (4) is used, the data point as plotted in Fig. 1 IS is well above the line defined by the temperature study using Eq. (7). Thus the perspective develops that, with spin 7/2 nuclei, Eq. (7) gives low values. This may be due in part to the different coefficients for the terms in the right-hand side of Eqs. (1) and (3) for the two phenomenological transverse relaxations of spin 7/2 nuclei as opposed to those for spin 312 nuclei, and due to the different way in which those coefficients enter Eqs. (4) and (7). Above, the correction of Eq. (7) using the Halle and Wennerstrom per-

85

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

turbation treatment was applied [Eq. (A211 and found to shift ion correlation times T, to higher values toward but not reaching those of Eq. (4) (see Fig. 1 IS). With respect to understanding the thermodynamics of activation, however, there is a change in slope and different values of AH' and ASf. The question is whether a correction should be applied to data from Eq. (4). It is of interest to note that the same numerical values for the ratios, AWJR,) of Eq. (7) and A(R'JR:) of Eq. (4). give essentially the same value of 7,. To see this, compare the curves in Figs. 19 and 20. Using the correction for the data in Fig. I IA, as was done for Fig. 1 IS, gives the data indicated by open circles and values for AH3 and AS' (in our nomenclature AN, and AS& of 8.8 kcallrnol and 3.6 cal/mol deg. respectively. The effect has been to decrease AH3 and to shift AS*in the negative direction. At this stage, there is confidence in the T, obtained from Eq. (4) at 30°C but uncertainty about the thermodynamics of the activated state for the ion leaving the singly and doubly occupied channel. Yet interesting qualitative statements can be made: An ion leaving the doubly occupied state goes to a state of decreased entropy on passing over the exit barrier; this is the expected entropy change. An ion leaving the singly

20

I5

-N

a \

-

0

-N

a a

5

0

-9

-8

-7

-6

109 TC

FIG.20. Plots of the Bull ratio, A(R!/ Ri).as a function of log correlation time, cesium-I33 and calcium-43 [see Eq. (4)l.

7,. for

86

DAN W. URRY ET AL.

occupied state, however, calculates to go to a state of increased entropy on passing over the exit barrier. One raises the question as to whether this relative difference is relevant to the complicated binding process seen at low ion concentration and relates to an ion-effected change in the state of the gramicidin A channel. The above discussion depends on the relevance of the thermodynamic form of the Eyring rate equation and, in particular, on the relevance of the kT/h term in Eq. (Al) to ion channel transport. This term derives from a gas-phase characterization of the mean velocity that an activated complex moves through a distance S at the top of a potential energy barrier and the unique translational degree of freedom at the top of the barrier for the S distance. At 30°C (303°K) the magnitude of kT/h is 6.29 x lo”, a very large number. If this number is not well-founded, then the value of P’.’*’~ has no relevance as it is a relatively small number. When AS*is 5 , eas”R is about 10’. If kT/h has no valid basis, it could be incorrect by some tens of orders of magnitude. A test of the relevance of AS*derived from Eq. (Al) as applied to ion channel transport was achieved by means of temperature-dependence studies of equilibrium (K:) and rate (&) constants for the sodium ion interaction with the gramicidin A channel. Temperature dependence of K t gave the AH and AS values for ion binding which are well-defined by equilibrium theory. For sodium ion AS: = - 1 1 cal/mol deg, whereas AS:tr = - 5.4 cal/mol deg. This indicates that kTlh is correct to within an order of magnitude for ion channel transport and therefore, that Eq. (All is a useful equation for characterizing ion transport through transmembrane channels when the ion currents are in the lo6 to loMion/ sec range. REFERENCES Apell, H.-J., Bamberg, E., Alpes, H., and Lauger. P. (1977). Formation of ion channels by a negatively charged analog of gramicidin A. J . Mernbr. Biol. 31, 171-188. Bamberg, E . , and Janko, K . (1977). The action of a carbon suboxide dimerized gramicidin A on lipid bilayer membranes. Biochim. Biophys. Acru 465, 486-499. Bamberg, E . , and Lauger, P. (1977). Blocking of the gramicidin channel by divalent cations. J . Membr. Biol. 35, 351-375. Bamberg, E . , Apell, H.-J., and Alpes, H. (1977). Structure of the gramicidin A channel: Discrimination between the T ~and . the ~ p-helix by electrical measurements with lipid bilayer membranes. Proc. Nutl. Acad. Sci. U.S.A. 74, 2402-2406. Bull. T. E. (1972). Nuclear manetic resonance of spin 3/2 nuclei involved in chemical exchange. J . Mugn. Reson. 8,344-353. Bull, T. E., ForsCn, S., and Turner, D. L. (1979). Nuclear magnetic relaxation of spin 5/2 and spin 7/2 nuclei including the effects of chemical exchange. J . Chem. Phys. 70,31063111. Donohue, J . (1953). Hydrogen bond helical configurations of the polypeptide chain. Proc. Nurl. Acud. Sci. U.S.A. 39, 470-478.

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

87

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. In "Physical Chemistry of Transmembrane Ion Motions" ( G . Spach, ed.). pp. 329-348. Elsevier. Amsterdam. Eisenman, G . , and Sandblom, J. P. (1984). Modeling the gramicidin channel. Interpretation of experimental data using rate theory. Biophys. J. 45, 88-90. Forsen. S .. and Lindman, B. (1981). Ion binding in biological systems as studied by NMR spectroscopy. Merhods Biochern. A n d . 27, 289-486. Fossel, E. T., Veatch. W. R.. Ovchinnikov, Y. A.. and Blout, E. R. (1974). A "C nuclear magnetic resonance study of gramicidin A in monomer and dimer forms. Biuchc/ni.s/r.v 13, 5264-5275. Fukushima. E.. and Roeder, S. B. W. (1981). "Experimental Pulse NMR: A Nuts and Bolts Approach." Addison-Wesley, London. England. Glickson. J . D.. Mayers. D. F.. Settine, S. M.. and Urry, D. W. (1972). Spectroscopic studies on the conformation of grarnicidin A'. Proton magnetic resonance assignments. coupling constants, and H-D exchange. Biodwmisfr.y 11, 477-486. Halle, B., and Wennerstrom, H. (1981). Nearly exponential quadrupolar relaxation. A perturbation treatment. J. Mccgn. Reson. 44, 89-100. Henze, R.. Neher. E., Trapane, T. L., and Urry. D. W. (1982). Dielectric relaxation studies of ionic processes in lysolecithin packaged gramicidin channels. J . Mr/nhr. B i d . 64, 233-239. Hinton. J. R., Young. G.. and Millett, F. S. (1982). Thallous ion interaction with gramicidin incorporated in micelles studied by thallium-20s nuclear magnetic resonance. B i o c h e m isrrv 21, 651-654. Hinton, J. F., Koeppe. R. E., 11, Shungu. D.. Whaley, W. L., Paczkowski. J. A., and Millett. F. S. (1986). Equilibrium binding constants for TI* with gramicidins A. B and C in a lysophosphatidylcholine environment determined by '"'TI nuclear magnetic resonance spectroscopy. Biophvs. J. 49, 571-577. Hladky. S. B.. and Haydon, D. A. (1970). Discreteness of conductance change in biomolecular lipid membranes in the presence of certain antibiotics. Nurirre (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. Biochifn. Biophvs. Acrtr 274, 294-312. Hladky, S. B.. and Haydon, D. A. (1984). Ion movements in gramicidin channels. CIII.I.. Top. Memhr. Trunsp. 21, 321-372. Hladky. S. B., Urban, B. W., and Haydon, D. A. (1979). Ion movements in pores formed by gramicidin A. In "Membrane Transport Processes" (C. Stevens. R. Tsien, and W. Chandler. eds.). Vol. 3, pp. 89-103. Raven. New York. James. T. L., and Noggle. J . H. (1969). Sodium-23 nuclear magnetic resonance relaxation studies in ion interaction with soluble RNA. Proc. Niirl. Arcrd. Sci. U.S.A. 62. 644649. Masotti, L.. Spisni. A., and Urry, D. W. (1980). Conformational studies on the gramicidin A transmembrane channel in lipid micelles and liposomes. Cell Biophys. 2, 241-251. Momany. F. A., Carruthers. L. M.. McGuire, R . F.. and Scheraga, H. A. (1974). Intermolecular potentials from crystal data. 111. Determination of empirical potentials and application to the packing configurations and lattice energies in crystals of hydrocarbons. carboxylic acids, amines and amides. J. Phys. Chem. 78, 1595-1620. Momany. F. A., McCuire, R. F., Burgess, A . W.. and Scheraga. H. A. (1975). Energy parameters in polypeptides. VII. Geometric parameters, partial atomic charges, nonbonded interactions, hydrogen bond interactions and intrinsic torsional potentials for the naturally occurring amino acids. J. Phvs. Chem. 79, 2361-2381.

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Mueller, P., and Rudin, D. 0. (1967). Development of K’-Na’ discrimination in experimental biomolecular lipid membranes by macrocyclic antibiotics. Biochem. Biophys. Res. Commiin. 26, 398404. Myers, V. B., and Haydon, D. A. (1972). Ion transfer across lipid membranes in the presence of gramicidin A. 11. The ion selectivity. Biochim. Biophys. Acrci 274, 313-322. Pasquali-Ronchetti, I., Spisni. A.. Casali, E., Masotti, L.. and Urry, D. W. (1983). Gramicidin A induces lysolecithin to form bilayers. Biosci. Rep. 3, 127-133. Prasad, K. U., Alonso-Romanowski, S., Venkatachalam, C. M., Trapane, T. L., and Urry, D. W. (1986). Synthesis, characterization and BLM studies of L . Ala7-gramicidin A. Biochemistry 25, 456-463. Ramachandran, G. N., and Chandrasekharan, R. (1972a). 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.). Vol. 2, pp. 195-215. Gordon & Breach, New York. Ramachandran, G. N . , and Chandrasekharan, R. (1972b). Conformation of peptide chains containing both L- and D-residues: Part I-Helical structures with alternating L- and D-residues with special reference to the LD-ribbon and the LD-helices. Indim Biochwi. Biophys. 9, 1-1 I . Rose, K., and Bryant, R. G. (1978). Electrolyte ion correlation times at protein binding sites. J. Magn. Reson. 31, 4 1 4 7 . Sandblom, J., Eisenman. G., and Hagglund, J . (1983). Multioccupancy models for single filing ionic channels: Theoretical behavior of a four-site channel with three barriers separating the sites. J. Membr. Biol. 71, 61-78. Sarges. R., and Witkop, B. (1965!. Gramicidin A. V. The structure of valine- and isoleucinegramicidin A. J . A m . Chem. Soc. 87, 201 1-2020. Spisni, A., Pasquali-Ronchetti, I., Casali, E., Lindner. L., Cavatorta, P., Masotti. L., and Urry, D. W. (1983). Supramolecular organization of lysophosphatidylcholine-packaged gramicidin A‘. Biochim. Biophys. Acra 732, 58-68. Szabo. G., and Urry, D. W. (1979). N-acetyl gramicidin: Single-channel properties and implications for channel structure. Science 203, 55-57. Urban, B. W., and Hladky, S. B. (1979). Ion transport in the simplest single file pore. Biochim. Biophys. Acta 554, 410429. Urban, B. W.. Hladky, S. B., and Haydon, D. A. (1978). The kinetics of ion movements in the gramicidin channel. Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 2628-2632. Urban, B. W., Hladky. S. B., and Haydon, D. A. (1980). Ion movements in gramicidin pores an example of single-file transport. Biochim. Biophys. Acru 602, 33 1-354. helix. Urry, D. W. (1971). The gramicidin A transmembrane channel: A proposed T,,.,,, Proc. N u t / . Acad. Sci. U.S.A. 68, 672-676. Urry. D. W. (1972a). Protein conformation in biomembranes: Optical rotation and absorption of membrane suspensions. Biochim. Biophys. Acru 265, I 15-168. Urry, D. W. (1972b). A molecular theory of ion conducting channels: A field-dependent transition between conducting and nonconducting conformations. Proc. N u t / . Actid. Sci. U.S.A. 69, 1610-1614. Urry, D. W. (1985a). Chemical basis of ion transport specificity in biological membranes. Top. Curr. Chem. 128, 175-218. Urry, D. W. (1985b). On the molecular structure of the gramicidin transmembrane channel. In “The Enzymes of Biological Membranes” (A. N. Martonosi, ed.). pp. 229-257. Plenum, New York. Urry, D. W. (1987). NMR relaxation studies of alkali metal ion interactions with the gramicidin A transmembrane channel. Bid/. Magn. Reson. 9, 109-131.

4. THE GRAMlClDlN A TRANSMEMBRANE CHANNEL

89

Urry, D. W.. and Ohnishi, T. (1974). Calcium ion binding lo carbonyls of elastin hexapeptide. Bioinorg. Chmn. 3, 305-313. Urry, D. W.. and Trapane, T. L. (1987). Treatment of two phenomenological components in transverse relaxation of rapidly exchanging spin 7/2 cesium-I33 nuclei to calculate correlation times. J . Magn. Resori. 71, 193-200. Urry. D. W., Goodall. M. C.. Glickson, J . D.. and Mayers, D. F. (1971). The gramicidin A transmembrane channel: Characteristics of head-to-head dimerized q,. ,,, helices. P ~ w . N
Urry. D. W., Prasad. K. U.. and Trapane, T. L. (1982a). Location of monovalent cation binding sites in the gramicidin channel. Proc. Ncrtl. Acnd. Sci. U . S . A . 79, 390394. Urry. D. W.. Trapane, T. L.. and Prasad, K. U . (1982b). Molecular structure and ionic mechanisms of an ion-selective transmembrane channel: monovalent vs. divalent cation selectivity. Int. J. Qirclnfron Cliem. Qirun/rrrn Biol. Svmp. 9, 3 1-40. Urry, D. W.. Trapane. T. L.. Walker, J. T., and Prasad, K. U . (1982~).On the relative lipid membrane permeability of Na' and Ca": A physical basis for the messenger role of Ca' ' . J. B i d . Chetn. 257, 6659-6661. Urry. D. W.. Walker. J . T.. and Trapane. T. L. (1982d3. Ion interactions in (I-"C)D . Valx and D . Leu'4 analogs of gramicidin A. the helix sense of the channel and location of ion binding sites. J. Mernhr. Biol. 69, 225-23 I . Urry. D. W., Trapane. T. L.. and Prasad, K. U . (1983). Is the gramicidin A transmembrane channel single-stranded or double-stranded helix? A simple unequivocal determination. Science 221, 1064-1067. Urry, D. W.. Alonso-Romanowski, S., Venkatachalam. C. M.. Trapane, T. L.. Harris. R. D.. and Prasad, K. U. (1984a). Shortened analog of the gramicidin A channel argues for the doubly occupied channel as the dominant conducting state. Biorhim. Biophys. Actu 775, 115-1 19. Urry. D. W.. Alonso-Romanowski. S.. Venkatachalam, C. M., Bradley. R. J.. and Harris, R. D. (1984b). Temperature dependence of single channel currents and the peptide libration mechanism for ion transport through the gramicidin A transmembrane channel. J . Memhr. Biol. 81, 205-217. Urry, D. W., Alonso-Romanowski, S.. Venkatachalam, C. M., Trapane. T. L.. and Prasad. K. U . (1984~).The source of the dispersity of gramicidin A single channel conductances: The L . Leu'-Grdmicidin A analog. Biopliys. J . 46, 259-265. Urry. D. W.. Trapane. T. L.. Brown, R. A.. Venkatachalam. C. M.. and Prasad. K. U . ( 1 9 t h ) . Cesium-I33 NMR longitudinal relaxation study of ion binding to the gramicidin transmembrane channel. J. Magn. Reson. 65, 43-61. Urry, D. W., Trapane. T. L.. Venkatachalam, C. M., and Prasad, K . U . (198Sb). CarbonI3 NMR study of potassium and thallium ion binding to the gramicidin A transmembrane channel. Cfin.J . Chern. 63, 1976-1981. Urry, D. W . , Trapane, T. L., Venkatachatam, C. M., and Prasad, K . U . (1986a). Energy

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profiles for sodium ion passage through the single filing gramicidin transmembrane channel. Int. J. Quantum Chem. Quantum Biol. Symp. I t , 1-13. Urry, D. W., Trapane, T. L., Venkatachalam, C. M., and Prasad, K. U. (1986b). Rubidium87 NMR study of ion interaction with the gramicidin transmembrane channel. J . A m . Cliem. SOC. 108, 1448-1454. Urry. D. W., Trapane, T. L.. Venkatachalam. C. M..and McMichens. R. B. (1989). Interactions at membranous polypeptide sites using NMR: Determining rate and binding constants and site locations. In "Biomembranes. Part R: Transport Theory: Cells and Model Membranes" (S. Fleischer and B. Fleischer. eds.). Methods in Enzymology. Vol. 171. Academic Press, San Diego. California. Veatch, W. R., and Blout, E. R. (1974). The aggregation of gramicidin A in solution. Biochemistp 13, 5257-5264. Veatch, W. R.. Fossel. E. T., and Blout, E. R. (1974). The conformation of gramicidin A. Biochemistp 13, 5249-5256. Venkatachalam, C. M.. and Urry. D. W. (1983). Theoretical conformational analysis of the gramicidin A transmembrane channel. 1. Helix sense and energetics of head-to-head dimerization. J. Compict. Chem. 4, 461-469. Venkatachalam, C. M., and Urry, D. W. (1984). Theoretical analysis of gramicidin A transmembrane channel: 11. Energetics of helical librational states of the channel. J. Comprrt. Chem. 5 , 64-7 I . Venkatachalam. C. M., Alonso-Romanowski. S., Prasad, K. U., and Urry. D. W. (1984). The Leu' gramicidin A analog: Molecular mechanics calculations and analysis of single channel steps related to multiplicity of conducting states. I n t . J. Quuntrrm Chem. Qrrtrnfirm B i d . Svmp. 11, 315-326. Weinstein. S., Wallace, B. A., Blout. E. R., Morrow, J. S . , and Veatch, W. (1979). Conformation of gramicidin A channel in phospholipid vesicles: A "C and ''F nuclear magnetic resonance study. Pror. Nut!. Arud. Sci. U.S.A. 76, 4230-4234. Weinstein, S ., Wallace, B. A., Morrow, J . S., and Veatch, W. R. (1980). Conformation of the gramicidin A transmembrane channel: A ''C nuclear magnetic resonance study of "C-enriched Gramicidin in phosphatidylcholine vesicles. J. Mol. B i d . 143, 1-19.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 33

Chapter 5

Ion Transport through Transmembrane Channels: Ab lnitio Perspectives PETER C . JORDAN Department of Chemistry Brandeis University Waltham, Massachusetts 02254

I.

Introduction

11. Theoretical Approaches Ill. Ab Inirio Methods-General

Considerations IV. Gramicidin A. Introduction B . The Structure of Gramicidin C. Computational Models for Gramicidin D. Quantitative Results V . Applications A. Site-to-Site Transition Rates B. Valence Selectivity VI. Summary References

1.

INTRODUCTION

During the past few years there have been substantial advances toward the goal of establishing the structure of channel proteins. It is reasonable to believe that by combining evidence with inference plausible descriptions of a pore’s molecular architecture will soon be available. With such information at hand, detailed analysis of the interaction of ions and water with those portions of a channel protein which form the water-pore former interface could be carried out. Such investigations would form the basis of a detailed molecular description of channel selectivity. The data presently available (except for gramicidin) do not permit such studies. However, using gramicidin as a guide, we can see what additional information is needed, and what questions theory may soon be able to probe. 91

Copyright 0 1988 by Acddemic Pre\\ Inc All nghts of reproduction in any form rewrved

92

PETER C.JORDAN

In the following sections, some of the different theoretical techniques that have been used to study ion permeation are outlined and the type of problem to which each is best suited is indicated. Then we focus on one general approach, studies at the molecular level, and describe some of the progress that has been made toward understanding the selectivity of gramicidin. It will become clear in the context of this discussion what additional information is needed in order to carry out similar studies on physiologically significant channels.

11.

THEORETICAL APPROACHES

The permeation of ions through channel proteins is influenced by many structural parameters. Among them are the composition of the bathing electrolytes, the structure of the water-membrane interface, the packing of the phospholipid, the folding of the channel protein, and the structure of the pore-water interface; the general features are illustrated in Fig. I . An ideal theory would account for the effect that each of these has on open state conductance. However, given the complexity of the systems of interest, ideality is an unattainable goal. The permeation of a single ion takes less than a microsecond. Various theoretical techniques-continuum electrostatics (for recent review see Jordan, 1986), Brownian dynamics (Lauger and Apell, 1982: Cooper rt al., 1985), conformational analysis (Urry, 197 1 ; Ramachandran and Chan-

Electrolyte ,Ion

Pore 'Constriction \Water

Electrolyte

Rc. I . Topology of the ion-water-pore former-lipid ensemble. (Adapted from Jordan. 1986. with permission.)

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

93

drasekhardn, 1972; Venkatachalem and Urry, 1983, 19841, trh initio methods (for recent reviews see Polymeropoulos and Brickmann. 1985; Jordan, 1987b)-have been used to assess the influence that individual physical and chemical features of the whole ion-aqueous pore-channel proteinmembrane ensemble have on channel conductance. They have vastly different natural time scales ranging from static (continuum electrostatics) to picosecond (molecular dynamics). As a result, each has its own realm of utility. The continuum electrostatic model describes the way that the gross electrical and structural features of the ensemble affect the electric potential within the pore (Parsegian, 1969; Levitt, 1978a; Jordan, 1981, 1982, 1983, 1984a,b). The viewpoint is static. Rapid fluctuation in the electrical potential due to molecular motion can not be treated. Neither can any cooperative influences on the potential due to structural changes caused by ion permeation. This approach is especially valuable for determining the qualitative impact that lipid variation. pore shape perturbation, and changes in solution properties have on the static energy profile that governs channel conductance. As such it is especially useful in helping to correlate electrokinetic data (Levitt, 1978b; Jordan, 1984c, 1986, 1987a). Brownian dynamics treats the ion and the channel water molecules as discrete particles. An equation of motion for the frictionally impeded particles is formulated; the channel protein and the lipid are described in continuum terms and provide a background field influencing the motion of the discrete particles within the pore (Gates et d., 1984; Cooper e f a / . , 1985; Gates and Jakobsson, 1986; Jakobsson and Chiu, 1987). Since Brownian dynamics describe diffusion, which is competitive with permeation, this approach provides a practical way to actually calculate the conductance of a model pore. In this way it is possible to determine, directly, how ionic variation and changes in the background field influence conductance. Conformational analysis has been particularized to treat helical macromolecules (Go and Scheraga, 1973; Go and Okugama, 1976). By establishing the low-energy configurations of a pore former, molecular pictures of the most favorable environment for ion permeation and of those structural rearrangements likely to significantly affect conductance can be determined (Venkatachalem and Urry. 1983, 1984). These calculations discriminate between conformational states separated by only a few kilocalories/mole. Transitions between them occur in nanoseconds. Consequently, this approach can establish what conformational changes may take place during the permeation of a single ion. However, the detailed structural influence of ion-protein and water-protein interactions is neglected.

94

PETER C . JORDAN

Ab initio calculations, which include quantum chemical (Pullman and Etchebest, 1983; Etchebest and Pullman, 1984; Etchebest et ul., 1984; Kim et al., 198% Monte Carlo (Fornili et al., 1984), molecular dynamics (Fischer et al., 1981; Fischer and Brickmann, 1983; Mackay rt al., 1984; Lee and Jordan, 1984; Kappas et al., 1985; Sung and Jordan, 1987, 1988; Skerra and Brickmann, 1987a,b) and correlation function approaches (Vertenstein and Ronis, 1986), can, in principle, provide a complete picture of the most significant interactions within the ion-water-protein system. They describe the system (or somewhat idealized models) in terms of intergroup, interatomic, or electron-nuclear force fields. Depending on how they are used, the focus may be on the most stable state of the system or on dynamical changes occurring as the system evolves with time. They appear ideally suited to establishing the way individual molecular parameters influence channel selectivity. However, each requires large amounts of computer time for even the simplest of calculations. As a result, these studies are usually limited to idealized pores and to picosecond analyses, which require much less computer time than is required for simulating permeation. Because the whole permeation process is influenced by so many different features of the system, it is evident that no one of the theoretical approaches will, by itself, provide a complete interpretive model. Each focusses on the system at a different level of detail and yields different insights. Only by combining the evidence are we likely to develop a convincing picture.

111. Ab Initlo METHODS-GENERAL

CONSIDERATIONS

The basic input needed for a b initio analysis is the channel structure. Unlike the case of enzymes, there are no crystallographic data establishing atomic coordinates of ion pores. Even for grarnicidin, the best characterized system, the atomic structure of the electrically active dimer has only been inferred by means of conformational analysis (Urry et al., 1982a; Koeppe and Kimura, 1984). For such heavily investigated systems as the delayed rectifier potassium channel and the sodium channel, molecular models have been proposed to account for the selectivity patterns (Hille, 1975; Armstrong, 1975). Whether the hypothesized atomic arrangements are actual structural features of the physiological systems is an open question. However, as primary structures of channel proteins become available (Noda et al., 1982, 1983a,b, 1984), conformational analysis may provide a tool for describing the folded structures and identifying selectivity regions (Kosower, 1984;

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

95

Guy, 1984; Guy and Seetharumula, 1986). Structures determined in this way could then be compared with those proposed on the basis of electrophysiological measurements. Theoretical calculations of the energy profile for ion permeation through the selectivity region and for the rate constants of ion passage over the major energy bamers would then provide a way to determine if these models are truly representative of the system of interest. At present such an approach is unrealistic. The structural models are too speculative and the theoretical tools for discriminating among them are too untested. IV. A.

GRAMlClDlN

Introduction

The remainder of this chapter is devoted to considering how ~ r hinitio theory can augment our understanding of ion permeation through the channels formed by gramicidin A and its isomers. The membrane-bound form is ideally valence selective. It mediates the passage of small monovalent cations across lipid bilayer membranes, is blocked by divalent cations, and rejects anions (for recent review see Andersen, 1984). There is a large body of data describing the effect on channel conductance of cation variation, of amino acid variation, of electric field, or of ionic strength. Consequently, gramicidin and its analogs would appear to be perfect candidates to test the reliability and utility of detailed theoretical studies. Ideally, a molecular theory of channel specificity would compute rate constants for ion permeation as functions of all the physical variables accessible experimentally. The required amount of computer time would be astronomical. More realistic approaches would effect comparisons between different ions and focus on how changes in the various accessible variables influence different aspects of the conduction process. The quantity that governs kinetic behavior is the free energy profile along the permeation pathway, illustrated qualitatively in Fig. 2. Its basic features, established from analysis of electrochemical and N M R data, suggest that permeation is essentially a five-step process: diffusion to the mouth of the pore, partial dehydration and association with the pore, translocation, rehydration and dissociation, diffusion away from the mouth. The structure arises from two major influences: ( I ) a slowly varying background potential due to electrostatic interaction between the ion and the dielectrically dissimilar phases (membrane and water); (2) a highly structured portion due to the local interactions among ion, water, and the pore forming protein. Ab inirio approaches only consider the way local interactions contribute

PETER C.JORDAN

96

b

t

C

t

d diffusion

diffusion

translocation

hydration

FIG.2. Schematic representation of (a) the solvation energy profile. (b) the electrostatic energy profile, (c) the total potential energy profile, and (d! the kinetic description of the basic steps in ion permeation. The solvation energy, which is the focus of this chapter, accounts for the short-range interaction between an ion and the polar moieties forming its inner solvation shell. The electrostatic energy incorporates all long-range interactions due to the fact that the lipid, with its low dielectric constant, tends to make the interior of a channel an energetically unfavorable domain. The potential energy profile is the sum of (a) and (b). (Adapted from Jordan, 1984a, with permission.)

to the free energy profile. The simplest calculations approximate it as the internal potential energy profile at 0°K for coupled ion-water motion through the channel (Lee and Jordan, 1984); however, this approach suppresses significant entropic effects, due to the aqueous rearrangements which occur as ions traverse saddle point regions of the full potential energy surface. To calculate a free energy requires computing the potential of mean force along the reaction coordinate for a system maintained at 300' K. The resultant profile can then be used as a basis for further calculation designed to estimate rate constants for ion-water passage across the most significant barriers of the potential energy surface (Fischer and Brickmann, 1983; Vertenstein and Ronis, 1986).

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

97

Rather than focussing on the free energy pathway and using it as a basis for rate constant calculations, translocation (and possibly permeation) times can be computed directly for properly constructed models. The temperature can be increased sufficiently (Fischer et a / . , 1981; Fischer and Brickmann, 1983; Kappas ef al., 1985) or large electric fields can be imposed on the ion (Skerra and Brickmann, 1987a,b); either artifice can substantially accelerate barrier hopping and turn it into a picosecond process. Rate constants calculated in this way can then be extrapolated to lower temperatures or lower fields and used to investigate effects due to ionic and structural variation. All these approaches require knowing the equilibrium structure of the membrane-bound dimer and having a reasonable way to describe the interaction potential between ions, water, and the atoms of the channel former. At present these data can be reasonably estimated but none has been determined definitively.

B. The Structure of Gramicidin

The atomic coordinates for the equilibrium folded structure of the membrane-bound gramicidin dimer are unknown. There are four plausible ways that the monomer, which has the primary structure (Sarges and Witkop, 1965a.b.c) HCO-Val'-~ly'-Ala'-D-Leu4-Ala'-D-Va~~-Val7-D-ValX-Trp'-~L~~'"-T~~"-D-L~U''-T~~''-D-L~~'~-T~~'~-NHCH~C can dimerize. Two are known to exist. The membrane-bound conducting form is a headto-head dimer of helical monomers; the formyl ends of the monomer form the dimer junction (Urry. 1971; Bamberg ef d.,1977; Veatch and Stryer, 1977). The predominant form in organic solvent is an antiparallel doublestranded helix (Veatch et d., 1974; Sychev et d..1980; Wallace, 1983); it is this dimer which crystallizes from organic solvent (Wallace, 1986). The best estimate of the equilibrium structure of the head-to-head dimer is given by conformational analysis. The basic helical backbone, upon which there is general agreement, is illustrated in Fig. 3; the interior is formed by alternately antiparallel peptide linkages. Hydrogen bonding between every sixth unit over most of the length of the channel gives the helix its stability. But for peptides, 1 1 , 13, and 15, in the final turn of the helix, each forms a pair of hydrogen bonds. This structure accounts qualitatively for all major features of the solvation energy profile illustrated in Fig. 2. The channel diameter is -4 A ; thus most of the waters of hydration of an ion must be removed in entering the channel. There is an exceptional domain near the channel entrance where a cation interacts favorably with one of the nonhydrogen-bonded carbonyl groups (#I I) and with the OH of the ethanolamine group (Urry

98

PETER C.JORDAN

FIG.3. Projection of the helical backbone of the head-to-head dimer of gramicidin. The coordinates are those calculated by Koeppe and Kimura (1984). The filled circles are 0 atoms of the CO groups; the small circles are H atoms of the NH group; the circles of the backbone with no ligands are the a-C atoms of the helix. The carbonyl and alcohol carbons are numbered 0 to 16 and 0' to 16' in the right- and left-hand helices, respectively. Numbers I to 15 refer to the corresponding amino acids in the basic pentadecapeptide. Number 0 is the formyl carbon and number 16 is the ethanolamine carbon. The only H atoms illustrated are those of the NH groups. All others, as well as the amino acid side chains are suppressed. The dotted lines indicate hydrogen bonds between COs and NHs. Note that peptides at positions 11, 13, and 15 do not form hydrogen bonds.

et al., 1982b,c); it is a natural binding site for cations. There is a series of roughly equivalent sites in the channel interior at which a cation, while weaking the hydrogen bonds of the helix, can bind to pairs of carbonyl groups; these account for the intermediate minima along the translocation pathway.

C. Computational Models for Gramicidin

Different model potentials provide somewhat different estimates of the most stable conformations in the channel interior. One places the peptide groups nearly parallel to the channel axis (Koeppe and Kimura, 1984); the other suggests that the carbonyl groups tilt -20" toward the axis (Urry et al., 1982a).The equilibrium conformations of the ethanolamine termini and of the amino acid residues can be established with even less certainty. Not only do the individual groups have substantial orientational flexibility, their membrane-bound locations could be significantly influenced by the phospholipids surrounding them. Other uncertainties arise in choosing molecular force fields. While excellent models exist for describing interaction among the atoms of neutral molecules (Jorgensen and Swenson, 1985), the situation is less clear when the focus is on charged species. The difficulty is that ions polarize the functional groups with which they interact most closely, thus significantly

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

99

altering the charge distribution in the groups. The standard molecular force fields do not account for this phenomenon. As a consequence there is no concensus among theorists attempting ah initio descriptions of gramicidin as to the optimum model for describing ion-water-gramicidin interaction. All treatments of the problem have ignored the influence of the phospholipid membrane. While the nonpolar milieu is electrostatically significant, it requires vast amounts of computer time to include these long-range interactions in a microscopic theory. The work that has been carried out severely approximates the dimer-ion-water system. The calculations fall into four major categories: rigid framework models, models which include water; abstract gramicidin-like models, and fully interactive models. Each has its own drawbacks and advantages. The models differ greatly in their essentials. Clementi uses quantum chemistry and evaluates the potential energy profile (at 0" K ) for an ion with associated water molecules within a static gramicidin helix (Kim c ~ t al., 1985). Pullman employs a different set of quantum chemical assumptions to compute the energy profile. She ignores the influence of water, treats the amino acids as if all were glycines and, but for allowing reorientation of the ethanolamine termini at the channel mouths, also freezes the helix (Pullman and Etchebest, 1983; Etchebest and Pullman, 1984; Etchebest et a / . , 1984). Wilson uses classical molecular dynamics to describe a fully interacting thermalized model of gramicidin coupled to an ion and 13 water molecules (Mackay et al., 1984). Brickmann also uses molecular dynamics to focus on kinetic features governed by the helical periodicity of the channel; his model only includes the mobile carbonyl groups which line the helix and describes their thermal interaction with cations and water (Fischer r t al., 1981; Fischer and Brickmann, 1983; Kappas et al., 1985; Skerra and Brickmann, 1987a,b). Our model treats all the amino acid residues as glycines and evaluates the 0" K energy profile for an ion interacting with water and the groups forming the deformable helical backbone of gramicidin (Lee and Jordan, 1984; Sung and Jordan, 1987, 1988). Ronis has a totally different perspective (Vertenstein and Ronis, 1986). He exploits a memory function formalism to derive an expression for the diffusion constant of the ion; this can then be computed from the velocity autocorrelation function of the ion in the fluctuating background potential created by its surroundings. This method has been applied to the study of cation permeation through a water-free, Bnckmannlike helix. Each method uses different force fields to describe the interactions. It is encouraging that, in the regions when the models are comparable, the calculations are in basic agreement with each other and with experiment. In the channel interior, water always forms essentially linear hy-

100

PETER C.JORDAN

drogen-bonded chains. The energy barriers to water-water or water-ion exchange are prohibitive; coupled ion-water motion in the channel must be single file. The theoretical estimates of the number of water molecules that can be accommodated in the channel are consistent with experiment. The calculations indicate that theory can qualitatively account for some aspects of selectivity. Estimates of the relative energy barriers to cation translocation are consistent with trends in the translocation rate constants; Skerra and Brickmann's estimate of cationic mobility is reasonable and Ronis's permeability sequence is qualitatively correct. Cation-binding sites appear near the mouth of the channel. D. Quantitative Results

Quantitative predictions are less encouraging. Quantum chemical calculations exhibit energy profiles with substantially more pronounced wells and barriers than appear to be consistent with either the rate constants of association/dissociation or the translocation rate constant (Etchebest et al., 1984; Kim et al., 1985). Instead of an essentially flat energy profile in the channel interior, translocation bamers in the 10 to 25 kcaVmol range are found. However, these energy calculations do not take into account the influence of the phospholipid membrane. Because an ion polarizes the membrane and because there is an electrical potential between the membrane and water (the membrane is from 200 to 450 m V more positive; Hladky and Haydon, 1973; Pickar and Benz, 1978) the barrier is probably larger.' A resultant translocation barrier of 15 to 30 kcal/mol would be inferred, greater than the 6 5 kcal/mol consistent with experiment. Calculations based upon classical mechanics seem somewhat better. A complete energy profile for the channel interior has been computed for a Cs+(H,O),complex (the ion is sandwiched by two pairs of water molecules). As illustrated in Fig. 4b, there is a succession of energy minima along this pathway (Sung and Jordan, 1987). Here, the total variation in the channel solvation energy, defined as the process M'(H20),,

+ Gram

(H,O),, = (H,O),,

+

M'

. Gram

*

(HzO),, ( I )

is -3.5 kcal/mol. The global energy minimum in the pore interior is near the channel midpoint. With long-range image and dipole effects included, the maximum variation in translocation energy probably rises to -4-6 'In ab inifio calculations no dielectric discontinuities are imposed. If the model is expanded to include lipid, a nonpolarizable domain (vacuum) is replaced by a polarizable one. The extra ion-lipid interactions (which are analogs to the image interaction) thus crffrcrcfions into the channel. However, the influence of the membrane-water potential difference is opposite and still repels ions from the channel.

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

101

I

3 x

-5.

2 -10. 0 L5

-15.

z

!2

2

-20.

8 -15.

-10.

a

-5. ION

0.

5.

15.

10.

POSITION/A

i z

0

-10.

F:

2

8

-15. -15.

b

-10.

-5.

0.

5.

10.

15.

ION POSI"fON/A

Fici. 4. Channel solvation energy profiles for Cs' ( 0 )and CI (*) in a gramicidin-like channel with ( a ) no water molecules and (b) four water molecules present. The position of an ion is measured from the channel midpoint. Those points which are not relative minima denote the lowest energy configuration for an ion constrained in a fixed axial plane. ~

kcaVmol with the peak near the channel midpoint. Similar studies for other cations are needed to determine if these results are general. However, there are some grounds for optimism. Partial calculations have been carried out for the Na+(H,O), complex. AU the minima and one saddle point along the interior pathway have been found. These calculations again indicate an interior global energy minimum near the channel midpoint; however, it is less pronounced than for Cs'(H,O),. If all the saddle point barriers are the same (which is certainly unlikely), the maximum variation in translocation energy (after accounting for the effect of the membrane) would probably be -6-8 kcallmol, larger than that for the Cs+(H,O), complex with the peak again near the channel midpoint. This number is too uncertain to provide a quantitative measure of the di€ference in trans-

PETER

102

C.JORDAN

location energy barriers for Na' and Cs'; it is encouraging that the trend is in the right direction and that the energy differences are not large. While theory does locate ion-binding sites near the channel mouth, there is no agreement as to their location; some calculations find two pronounced exterior sites. In contrast to the available data, which indicate that the position of the binding site is the same regardless of the ion (Anderson et al., 1981; Urry et al., 1982b,c), calculations suggest that there is significant ionic influence on its position. The values range between 8 and 15 A from the channel midpoint. Pullman's quantum chemical study is somewhat encouraging, for Na+- and K'-binding sites are located 10.5 A from the channel midpoint (Etchebest et al., 1984), in agreement with experiment. However, the Cs+-binding site is too close to the midpoint. The same type of calculations that yielded a reasonable translocation energy profile for Cs' is less successful in establishing the location of the cation-binding site (Sung and Jordan, 1986). Here, binding always involves association with the number 1I CO group and the COH of the ethanolamine terminus, but there appears to be some variation in the location of the minimum. For M'(H,O), complexes, the site is found between 11.8 and 12.8 A from the channel midpoint for the alkali cations, a result at variance with the cation independent, 10.5 A location deduced from experiment. Since these results refer to 0" K potential profiles, it is possible that thermal motion wipes out the distinction between cations, a point that is presently under investigation. It is clear that the various ab initio approaches do not always agree quantitatively and are not yet totally reliable. Nonetheless they can be used to develop new insights because they can decouple system parameters inextricably linked in nature.

-

-

V.

APPLICATIONS

A. Site-to-Site Transition Rates

One interesting example, illustrating the interplay of different physical variables, is given by Brickmann's study of cation translocation in a gramicidin-like helix. The model is an infinite sequence of alternately antiparallel carbonyl groups arranged on a helix with the same pitch and radius as gramicidin. As such it can only describe aspects of the translocation process which are not influenced by the dimer junction, by the pore mouth, or by the amino acid side chains. This picture exhibits periodic binding sites which can be identified as intermediate wells along the translocation energy profile (see Fig. 2). Calculations of the site-to-site transition rates,

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

103

which can be identified as transitions between intermediate extrema along the translocation pathway, have been carried out with and without water in the channel and as a function of the ease with which the carbonyl group can reorient itself (Fischer rt al., 1981; Fischer and Brickmann, 1983; Kappas et af.,1985). If we assume that translocation is governed by the rate at which ions can hop between adjacent minima (which is equivalent to assuming that all saddle point barriers along the translocation pathway in gramicidin are roughly the same), the calculation provides a model of the translocation process. When no water is present and the carbonyl group reorients easily, an inverted translocation permeation sequence is found: Li' > 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 <3.5 kcal/mol. The anion binding does not involve association with any particular peptide groups. Rather, it is due to the fact that the head-to-head dimer has a net quadrupole moment that is negative at both mouths and positive at the midpoint; as a result anions can bind in the channel. The most important quantitative features of Fig. 4 are the relative channel solvation energies on a given potential profile and the difference in channel solvation energy between Cs' and CI- as a function of the distance of an ion from the channel midpoint. Because our calculations do not simulate bulk water, the absolute channel solvation energies are substantially too negative. As is apparent from Fig. 4, CI- solvation is much less favorable than Cs' solvation at the mouth of the pore. A series of calculations for Cl-(H20), and Cs+(H20),, complexes (where n = 0, 2, or 4) shows that the channel solvation energy for CI- in the channel mouth should be 12 to 17 kcal/mol greater than that for Cs' in the same domain (Sung and Jordan, 1987). It is just this part of the energy profile which determines the rate of ion entry into a channel. Since bulk hydration has been ignored, this energy difference translates into a substantial energy barrier to CI- entry for the channel-electrolyte system, one that is therefore -12 to 17 kcal/mol greater than that for Cs' entry. On this basis, the rate constant for CI- entry should be to lo-'? times that for Cs' entry. Use of the association rate constants determined for membranes formed from glyceryl monooleate/hexadecane mixtures indicates that CIentry, from 0.1 M salt solution, requires -25 to 10' sec (Urban d ul., 1980). For membranes formed in this way, the time needed for channel dissociation into monomers is -2 sec (Bamberg et ul., 1976; Kolb and Bamberg, 1977); thus the anion would not be able to permeate or block the channel during its lifetime, at least at low to intermediate ionic strength. Not only do the anion and cation potential energy profiles differ, there are significant differences between the low-energy permeation pathways of the two ions. Cs' tries to get as close to the carbonyl groups of the helix as possible (Lee and Jordan, 1984); CI- stays as far away as it can. This is illustrated in Fig. 5 for the region near the channel entrance. In the absence of water Cs+ interacts favorably with the ethanolamine oxygen and binds tightly to the exposed carbonyl group I I , far from the channel

PETER C.JORDAN

106

-4 \

3.5 3.0 2.5 2.0 1.5

1.0 0.5 0.0 6.

0.

10.

12.

14.

ION POSITION/A

3.5 3.0 2.5 0-4 \ 2.0 0

3

1.5 1.0 0.5 0.0 6.

0.

10.

12.

14.

ION POSITION/A FIG.5. Radial distance. p. along the permeation pathway as a function of ion location for Cs' 6-) and CI- (*-) with (a) no water molecules and (b) four water molecules present. The discontinuity in the permeation path of the cation with no water present reflects the fact that it interacts favorably along distinctly different pathways with the three exposed carbonyl groups I I . 13. and 15. When the cation is (13.5 A from the channel midpoint, association with carbonyl group I I alone is favored. Further out. Cs' interacts strongly with carbonyl groups 13 and 15; as a result it moves abruptly toward the channel axis.

107

5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

axis. There is an abrupt change in the most favorable radial location as the Cs' moves out from the channel mouth. Further than - 13.5 from the channel midpoint the cation interacts most strongly with carbonyls 13 and 15; this solvation change accounts for the apparent discontinuity in p, the distance of the ion from the channel axis. With water present, binding is still strong but, as the water molecules also interact with polar groups of the helix, the Cs' site is closer to the axis. There is no longer any sudden change in p as the influence of the surrounding water molecules constrains the low-energy configurations to those in which Cs' binds to carbonyl group 11. There are, however, rapid changes in the geometries of the Cs'(H,O), complex as the cation enters the channel mouth. As the ion translocates through the channel, geometrical constraints force it yet closer to the axis and it remains in a cylindrical shell -0.3 to 0.5 A from the channel axis. In contrast the anion, once inside the channel, never strays more that -0.15 A from the axis at any point along its energy profile; it never associates with any peptide. The same approach can be used to study the antiparallel double helix, the isomer that crystallizes from organic solvent. It is known that this channel preferentially stabilizes anions (Wallace, 1986). Preliminary studies show that the central part of this channel is also more positive than the region near the channel mouths; theory therefore suggests that anions should be bound preferentially by this form of gramicidin, in agreement with observation. This provides some indication that the calculations on anion interaction with the electrically active head-to-head-dimer (for which direct confirmation is unavailable) are reliable.

A

VI.

SUMMARY

There is, at present, uncertainty regarding both the structural and force field parameters needed to describe ionic interaction with channel-forming proteins. No single model calculation has included enough features of the real physical situation, even for a pore former as simple as gramicidin, to provide a definitive theoretical picture of selectivity. However, by comparing results of calculations based on different reasonable models of the ion-water-gramicidin system, significant interpretive insights can be gained. As more structural proposals for describing the selectivity regions of physiologically important channel-forming proteins become available. ah inirio methods can be used to construct permeation free energy profiles and test these models. It should then be possible to study details both of the solvent reorgan-

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PETER C.JORDAN

ization required as an ion enters a channel, loses some of its waters of hydration, and associates with ligands of the pore former and of the less drastic ligand-water rearrangements which occur during ion translocation between binding sites. In this way, we may well be able to probe the delicate balance of interactions that account for channel selectivity. ACKNOWLEDGMENTS This work has been supported by grant GM-28643 from the National Institutes of Health. I wish t o thank Dr. S. S. Sung for his critical commentary and Professor J . A. McCammon for his hospitality. REFERENCES Andersen. 0. S. (1984). Gramicidin channels. Annu. Rev. Plrysiol. 46, 53 1-548. Andersen, 0. S., Barrett. E. W.. and Weiss. L. B. (1981). On the position of the alkali metal cation binding sites in gramicidin channels. Biciphys. J . 33, 63a. Armstrong. C. M. (1975). Ionic pores, gates and gating currents. Q . Rev. Bioipliys. 7, 179210. Bamberg. E., Noda. K.. Gross, E.. and Lauger. P. (1976). Single-channel parameters of gramicidin A. B and C. Biocliiin. Bio)pliys. Acta 419, 223-228. Bamberg. E.. Apell, H.-J., and Alpes. H. (1977). Structure of the gramicidin A channel: Discrimination between the m,..,, and the p helix by electrical measurements with lipid bilayer membranes. Proc. Ncitl. Accrd. Sci. U . S . A . 74, 2402-2406. Cooper. K., Jakobsson. E., and Wolnyes, P. (1985). The theory of ion transport through membrane channels. Progr. Biophys. Mol. Biol. 46, 5 1-96. Dani. J. A.. and Levitt. D. G. (1981). Water transport and ion-water interaction in the gramicidin channel. Biophys. J . 35, 501-508. Etchebest, C., and Pullman. A. (1984). The gramicidin A channel. Role of the ethanolamine end chain on the energy profile for single occupancy by Na'. FEBS Lett. 170, 191195. Etchebest. C.. Ranganathan. S., and Pullman. A. (1984). The gramicidin A channel: comparison of the energy profiles of Na', K ' and Cs'. Influence of the flexibility of the ethanolamine end chain on the profiles. FEBS Left. 173, 301-306. Fischer, W.. and Brickmann. J. (1983). Ion specific diffusion rates through transmembrane protein channels. A molecular dynamics study. Bioplivs. Ckein. 18, 323-337. Fischer. W.. Brickmann. J.. and Lauger, P. (1981). Molecular dynamics study of ion transport in transmembrane protein channels. Biopliys. Clicvn. 13, 105-1 16. Fornili. S. F.. Vercauteren. D. P.. and Clementi. E. (1984). Water structure in the gramicidin A transmembrane channel. dioc;iirn. Bioptiys. Acfrr 771, 151-164. Gates, P., and Jakobsson, E. (1986). Molecular-based calculations of ion flux in gramicidin channels. Biophvs. J . 49, 375a. Gates, P.. Cooper, K.. and Jakobsson, E. (1984). Brownian dynamics simulation of ion movement through channels. Biophys. J . 45, 64a. Go. N . , and Okugama. K. (1976). A method for calculating dihedral angles in helical polymers with given values of unit height and unit rotation. Miic~~ciino/~,i~irlc.s 9, 867-868. Go. N.. and Scheraga. H. A. (1973). Ring closure in chain molecules with C,. I and SL,, symmetry. Moc~~omolecrrlos 6, 273-281. Guy, H. R. (1984). A structural model of the acetylcholine receptor channel based on partition energy and helix packing considerations. Biophys. J . 45, 249-261.

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Guy. H. R.. and Seetharumula. P. (1986). Structural model of the sodium channel based on its sequence. Bioplivs. J. 49. 386a. Heitz. F., Spach. F.. and Trudelle. Y. (1982). Single channels of 9.1 1.13,IS-destryptophylphenalanyl gramicidin A. Biophys. J . 40, 87-89. Heitz. F.. Spach. F.. and Trudelle. Y. (1984). Single channels of various gramicidins. Voltage effects. Biophys. J. 45, 97-99. Hille. B. (1975). Ionic selectivity of Na and K channels of nerve membranes. In "Membranes" ( G . Eisenmann. ed.). Vol. 3. pp. 255-323. Dekker, New York. Hladky. S. B.. and Haydon. D. A. (1973). Membrane conductance and surface potential. Biocliirn. Biopliys. A m 318, 464468. Jakobsson. E.. and Chiu. S.-W. (1987). Stochastic theory of ion movement in channels with single-ion occupancy. Application to sodium permeation of gramicidin channels. Bioplrys. J. 52, 3 3 4 6 . Jordan. P. C. i1981). Energy barriers for the passage of ions through channels. Exact solution of two electrostatic problems. Biopliys. Chern. 13, 203-212. Jordan. P. C. (1982). Electrostatic modeling of ion pores. Energy barriers and electric field profiles. Bictp/iys. J. 39, 157-164. Jordan. P. C. (1983). Electrostatic modeling of ion pores. I I . Effects attributable to the membrane dipole potential. Biopkvs. J. 41, 189-195. Jordan, P. C. (1984a). The effect of pore structure on energy barriers and applied voltage profiles. I. Symmetrical channels. Bioplivs. J. 45, 109 1-1 100. Jordan. P. C. (1984b). The effect of pore structure on energy barriers and applied voltage profiles. I I . Unsymmetrical channels. Biophvs. J. 45, 1101-1 107. . total electrostatic potential in a gramicidin channel. J. Mc,rnhr. Jordan. P. C. ( 1 9 8 4 ~ )The Biol. 78, 91-102. Jordan. P. C. (1986). Ion channel electrostatics and the shapes of channel proteins. In "Ion Channel Reconstitution" (C. Miller. ed.), pp. 37-55. Plenum. New York. Jordan. P. C. (1987a). How pore mouth charge distributions alter the permeability of transmembrane ionic channels. Biopliys. J. 51, 297-31 I . Jordan. P. C. ( 1987b). Microscopic approaches to ion transport through transmembrane channels. The model system gramicidin. J. Phys. Cliern. 91, 6582-6591. Jorgensen. W. L.. and Swenson. C. J. ( 19851. Optimized intermolecular potential functions for amides and peptides. Structure and properties of liquid amides. J . Atn. Clirrri. Soc. 107, 569-578. Kappas. U.. Fischer. W.. Polymeropoulos. E. E.. and Brickmann. J. (1985). Solvent effects in ionic transport through transmembrane protein channels. J . T 1 i ~ t r B . i d . 112, 459464. Kebarle. P. (1977). Ion thermochemistry and solvation from gas phase ion equilibria. A n n / / . Rei,. Phys. Chetn. 28, 445476. Kim, K. S . . Vercauteren. D. P.. Welti. M.. Chin. S. . and Clementi. E. (1985). Interaction of K ' ion with solvated gramicidin A transmembrane channel. Biopliys. J . 47, 327-335. Koeppe. R. E., II, and Kimura. M. (1984). Computer building of P-helical polypeptide models. Biopo/yrner.v 23, 23-38. Kolb. H.-A,. and Bamberg. E. (1977). Influence of membrane thickness and ion concentration on the properties of the gramicidin A channel. Autocorrelation. spectral power density. relaxation and single-channel studies. Biochirn. Bioplivs. Acrtr 464, 127-141. Kosower, E. M. (1984). A molecular model for an acetylcholine binding site. Ion channel and the bilayer helices of the acetycholine receptor assigned using single group rotation theory and electrostatic interactions. Biophys. J. 45, 13-14. Lauger. P.. and Apell. H . J . (1982). Jumping frequencies in membrane channels; comparison

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between stochastic molecular dynamics simulation and rate theory. Biophys. Chern. 16, 209-22 I . Lee, W. K.. and Jordan, P. C. (1984). Molecular dynamics simulation of cation motion in water-filled gramicidinlike pores. Biopphys. J . 46, 805-8 19. Levitt, D. G. (1978a). Electrostatic calculations for an ion channel. I. Energy and potential profiles and interactions between ions. BiupiiYs. J. 22, 209-219. Levitt. D. G. (l978b). Electrostatic calculations for an ion channel. 11 Kinetic behavior of the gramicidin A channel. Biophys. J. 22, 221-248. Mackay, D. H. J., Berens, J., Wilson. K. R.. and Hagler, A. T. (1984). Structure and dynamics of ion transport through gramicidin-A. Biophys. J . 46,229-248. Mazet. J.-L., Andersen. 0. S,, and Koeppe, R. E., 11 (1984). Single channel studies on linear gramicidins with altered amino acid sequences. A comparison of phenylalanine. tryptophane and tyrosine substitutions at positions I and 11. Bicqdrvs. J. 45, 263-274. Morrow, J. S., Veatch. W. R..and Stryer. L. (1979). Transmembrane channel activity of gramicidin A analogues: effects of modification and deletion of the amino-terminal residue. J . Mol. Biol. 132, 733-738. Noda. M., et ul. (1982). Primary structure of &-subunit precursor of Torpedo culifornicu acetylcholine receptor deduced from cDNA sequence. Nutiire (London) 299, 793-797. Noda. M., er a / . (1983a). Primary structures of p- and &subunit precursors of Torpedo culifornicu acetylcholine receptor deduced from cDNA sequence. Nuiure (LondonJ301, 25 1-255. Noda. M.. et ul. (1983b). Structural homology of Torpedo culifornicu acetylcholine receptor subunits. Nuture (London) 302, 528-532. Noda. M., et ul. (1984). Primary structure of Ekctrophorus electricus sodium channel deduced from cDNA sequence. N ~ i t u r e(London) 312, 121-127. Parsegian, V. A. (1969). Energy of an ion crossing a low dielectric membrane: Solution to four relevant electrostatic problems. Nuture (London) 221, 844-846. Pickar. A. D., and Benz. R. (1978). Transport of oppositely charged lipophilic ion probes in lipid bilayers having various structures. J . Mernbr. B i d . 44,353-376. Polymeropoulos, E. E.. and Brickmann. J. (1985). Molecular dynamics of ion transport through transmembrane model channels. Annu. Rev. Biophys. 14, 315-330. Pullman. A.. and Etchebest. C. (1983). The gramicidin A channel: the energy profile for single and double occupancy in a head-to-head p,h,,’-helical dimer backbone. FEBS Leir. 162, 199-202. Ramachandran, G. N.. and Chandrasekharan. R. (1972). Conformation of peptide chains containing both L- and D-residues: I. Helical structures with alternating L- and Dresidues with special reference to the LD-ribbon and the LD-helices. Indiun J . Biochem. Biophys. 9, 1-1 I. Sarges, R., and Witkop. B. (1%5a). Gramicidin A. V. The structure of valine- and isoleucinegramicidin C. J . Am. Cliem. Soc. 87, 201 1-2020. Sarges. R., and Witkop. B. (1965b). Gramicidin VII. The structure of valine- and isoleucinegramicidin B. J . Am. Cliem. Soc. 87, 2027-2030. Sarges, R.. and Witkop. B. (1%5c). Gramicidin VI11. The structure of valine- and isoleucinegramicidin C. Biochemistry 4, 2491-2494. Skerra. A,, and Brickmann, J. (1987a). Structure and dynamics of one-dimensional solutions in biological transmembrane channels. Biophys. J . 51. 969-976. Skerra. A.. and Brickmann, J. (1987b). Simulation of voltage-driven hydrated cation transport through narrow transmembrane channels. Biopliys. J. 51, 977-983. Sung, S.-S., and Jordan. P. C. (1987). Why is gramicidin valence selective
5. ION TRANSPORT THROUGH TRANSMEMBRANE CHANNELS

111

Sung. S.-S.. and Jordan. P. C. (1988). A theoretical study of double ion occupancy in a gramicidin-like channel. J . Pliys. Cliem. 92, 2362-2366. Sychev. S. V.. Nevskaya. N . A.. Jordanov. S.. Shepel. E. N.. Miroshnikov, A. I., Ivanov. V . T.. and Ouchinnikov. Y. A. (1980).The solution conformations of gramicidin A and its analogs. Bioorg. Cliern. 9, 121-151. Urban, B. W.. Hladky. S. B., and Haydon, D. A. (1980). Ion movements in gramicidin pores. An example of single-file transport. Biochitn. Biophys. Acro 602, 33 1-354. Urry. D. W. (1971).The gramicidin A transmembrane channel: A proposed m,, .,,,helix.P m . . Null. Actid. Sci. U.S.A. 68, 672-676. Urry. D. W.. Venkatachalem, C. M.. Prasad. K. U.. Bradley, R . J . , Parenti-Castelli, G.. and Lenaz, C. (19811. Conductance processes of the gramicidin channel. Inr. J . Qiiiinfiiin Clrein. Qiiunlrtm Biol. Svmp. 8, 385-399. Urry. D. W., Trapane, T. L., and Prasad. K. U. (1982a). Molecular structure and ionic mechanisms of an ion-selective transmembrane channel; monovalent vs. divalent cation selectivity. Inl. J . Quunliwn Ckern. Qituntrtm B i d . Svmp. 9, 3140. Urry. D. W.. Prasad, K. U., and Trapane. T. L. (1982b). Location of monovalent cation binding sites in the gramicidin channel. Proc. Null. Acud. Sci. USA 79, 390-394. Urry. D. W.. Walker, J . T., and Trapane. T. L. (1982~).Ion interactions in (I-”C)r)-val” and leu" analogs of gramicidin A, the helix sense of the channel and location of ion binding sites. J . Memhr. Biol. 69, 225-23 I . 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 . Mol. Biol. 113, 89-102. Veatch. W. R.. Fossel. E. T.. and Blout, E. R. (1974). The conformation of gramicidin A. Biocliemistrv 13, 5249-5256. Venkatachalem. C. M..and Urry. D. W. (1983).Theoretical conformational analysis of the gramicidin A transmembrane helix. 1. Helix sense and energetics of head-to-head dimerization. J . Comprtl. Chem. 4, 461-469. Venkatachalem. C. M..and Urry. D. W. (1984).Theoretical analysis of gramicidin A transmembrane channel. II. Energetics of helical librational states of the channel. J . Cornpiir. Cliem. 5, 64-71.

Vertenstein. M..and Ronis. D. (1986). Microscopic theory of membrane transport. 111: Transport in multiple barrier systems. J . Clrem. Plrys. 85, 1628-1649. Wallace. B. A. (1983). Cramicidin A adopts distinctively different conformations in membranes and in organic solvents. Biopolymers 22, 397-402. Wallace, B. A. (1986). The structure of gramicidin A. Biophys. J. 49, 295-306.

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 33

Chapter 6 Rapid Gating Events and Current Fluctuations in Gramicidin A Channels F. J . SIGWORTH AND S. SHENKEL Department of Physiology Yale School of Medicine New Haven, Connecticut W510

1. lntroduction

Methods Properties of Gaps in Channel Currents A. Gaps in GA Channels B. Gaps in N-Acetyl-GA Channels C. Kinetic Significance of the Gaps IV. Open-Channel Noise A. Power Spectra of GA Channel Currents B. Prediction of Spectra from Rate Constants C. A Hypothesis for the Origin of the Open-Channel Noise V . Conclusions References 11.

111.

1.

INTRODUCTION

The ionic conductance produced in lipid membranes by gramicidin A (GA) spontaneously switches on and off according to the kinetics expected for the association and dissociation of two GA molecules. Thus the “gating” of GA channel$ has been explained by a simple dimerization reaction whose rates are sensitive to membrane thickness and surface tension (Elliott ef al., 1983). Upon closer examination, temporal changes in the conductance induced by GA has been seen to be quite complex, involving multiple levels of conductance and more kinetic processes than would be expected for a simple dimerization. Busath and Szabo (1981) investigated ‘mini” conductance channels that appear from even the purest GA preparations (Busath et af., 1987), concluding that the lowered conductance levels can result from conformational changes in one of the monomeric 113

Copyright 0 1988 hy Academic h e \ \ . Inc. All rights of reproduction in any form rewrved

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subunits of the channel. Meanwhile, Ring (1986) has made high-resolution temporal recordings of GA channel currents, finding brief interruptions (gaps) whose kinetic properties depend on voltage and on membrane properties. These gaps last a few milliseconds and occur at rates on the order of 10 sec-’, much faster than the dimerization reaction rates which are of the order of 0.1 sec-’. In the first part of this chapter, we report some observations on gaps in GA channel currents based on high-resolution recordings that were made through the application of patch-clamp technology to black lipid membranes. In our recordings we observe two major classes of gaps. One class has a mean duration of about 0.5 msec and consists of reductions, but not complete interruptions, of the channel current. That some current flows during these “sublevel gaps” allows us to obtain information about the kinetic relationship of these gaps to the main opening-closing (i.e., dimerization) reaction. The other major class of gaps that we observe has a very brief mean duration, about 20 psec (at a membrane potential of 200 mV) which is close to our limit of temporal resolution. Extending Ring’s observation that the frequency of gaps depends on membrane composition, we have made solvent-containing membranes of various thicknesses and find that the kinetics of the 0.5-msec sublevels vary moderately with membrane thickness, but the frequency of occurrence of the 20-psec gaps appears to have a very strong dependence on thickness. Background noise from the recording system prevents one from directly observing changes in channel current on a time scale shorter than -10 psec; however, it is possible to obtain indirect information about rapid interruptions or modulations in channel current by the use of noise analysis techniques. For example, gaps of very short duration can give rise to an “excess” noise that is present when a channel is conducting, but absent when it is not. In the second part of this chapter, we summarize some measurements of open-channel noise in GA channels (Sigworth et al., 1987), and present a tentative interpretation of these results. The noise data can be explained by a theory that postulates the occurrence of gaps of duration 0.5 psec at rates of - 10’ sec-’. In the theory, the frequency of occurrence of these gaps depends on the occupancy of the channel by ions, suggesting that these fluctuations may be closely coupled to the ion transport process. II. METHODS

The experiments reported here were performed with synthetic GA and GA analogs kindly provided by D. Urry. The membranes were formed from glycerol monooleate (GMO; Nu Chek Prep) or diphytanoylphos-

115

6. GRAMlClDlN A CHANNELS

phatidylcholine (Avanti) in hydrocarbon solvents (Aldrich) at lipid concentrations of 20-50 mglml. All recordings were made at room temperature. To obtain low background noise, we recorded the currents from membranes formed at the tips of glass micropipets (tip diameters 3-6 pm). In this technique (originally developed by E. Neher; see Sigworth rt al., 1987)a hemispherical black lipid film is formed at the tip of a I-mm Teflon tube, and lipid from this film is then transferred to a Sylgard-coated, silanetreated micropipet (Fig. 1). The resulting microbilayers typically showed background conductances below 1 pS and allowed us to record GA channels with the same resolution as standard patch-clamp recordings from cell membranes. Gramicidin A or analogs were added from an ethanol stock solution to the bath to a final concentration of -lo-" M. In each case the bath was an unbuffered KCI or CsCl solution. The electrodes were Ag-AgCI wires; in some experiments the bath electrode was connected through an agar salt bridge to control for the possibility of channel block by Ag' ions. Pipet currents were monitored with an EPC-7 patch clamp using its internal 10-kHz Bessel filter, and recorded using one of the two data channels of a modified PCM-501 Digital Audio Processor (Sony) and a VHS video recorder (Bezanilla, 1985). For data analysis the digital data stream obtained from playback of the tape was converted to 16-bit parallel words, read into computer memory through a DMA interface, and written directly to a 16-megabyte disk file. This file would hold 180 sec of continuous data, sampled at the rate of 44.1 kHz that is fixed by the PCM-501.

-

FIG.I . Microbilayer recording system. A spherical "bubble" of lipid is blown by forcing solution out of the Teflon tube (left). After the lipid thins to form a black membrane, the tip of the glass pipet is inserted into the bubble. picking up a membrane. Typically. this membrane is destroyed by a pulse of pressure applied to the pipet. and a new membrane is formed by retracting the pipet and used for recording. The pipet tip diameter is typically 3-6 pm, and is treated by dipping into a solution of dimethyldichlorosilane or by exposure to the vapor. Currents are recorded by a standard patch-clamp amplifier (EPC-7. List Electronics).

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F. J. SIGWORTH AND S. SHENKEL

Power spectra were performed as described (Sigworth et a/., 1987) using masking (Sigworth, 1985) to avoid contamination of the spectra from brief gaps (typical detection limit was 10 psec). Gap durations and amplitudes were analyzed by the half-amplitude threshold method (Colquhoun and Sigworth, 1983) typically using the digitized recording at its full 10 kHz bandwidth. 111.

PROPERTIES OF GAPS IN CHANNEL CURRENTS

Figure 2 shows examples of individual GA channel events recorded in GMO membranes formed with different alkane solvents. Although we did not measure the thickness of the membranes, we expect, as in Hendry et al. (l978), that the various alkanes yielded membranes with thickness increasing as the chain length decreased from hexadecane to decane. The top trace in each panel shows an 80-sec overview of the recording, in which the data points have been averaged to yield an effective time resolution of 100 msec. In the lower panels a portion of the record is shown at 5 kHz bandwidth and at two degrees of expansion to show the considerable amount of “fine structure” in the GA channel currents. In each type of membrane the same kinds of brief gating events were observed; the main effect of membrane composition was on the frequency of occurrence of some events. A. Gaps in GA Channels

The bottom trace in Fig. 2A shows a “channel opening” transition whose time course is indistinguishable from the step response of the recording ’system and is therefore essentially instantaneous. Approximately 40% of all channel opening and closing transitions are not instantaneous, but show a sojourn in an intermediate conductance level, as shown in the expanded trace in Fig. 2B. These “step” sojourns have an average duration of about 0.5 msec and a conductance of some 15% of the full channel conductance. Also visible in Fig. 2B are examples of another kind of gating event, the short 20-psec gaps which are visible here as “needles” that extend only part way to the baseline. The 5 kHz bandwidth shown here corresponds to a filter risetime of 60 psec (Colquhoun and Sigworth, 1983), so that a complete current interruption lasting -30 psec would result in a “needle” extending only halfway to the baseline. When this trace is examined at full (10 kHz) recording bandwidth, the gaps in this trace are seen to last 10-30 psec and appear to represent complete or nearly complete interruptions of the current. Examples of a third class of events are seen later in the trace in Fig.

117

6. GRAMlClDlN A CHANNELS

A

octane

decane

B

i-l

518 msec

.....................

msec

4 mtsc

C

4

tetradecane

D

hexadecane

...................

n

u..

...........

I: 8 sec

..................

...................

2 sec

w FIG.2. Representative recordings from GA channels in symmetrical I M KCI solutions at 200 mV membrane potential, in membranes formed with glycerol monooleate 50 mg/ml in the following solvents: A . octane; B, decane; C, tetradecane: D, hexadecane. The top trace in each panel is an overview of 80 sec of data. with the diamond marking the region shown expanded in the lower two traces. Data were filtered to 5 kHz ( - 3db) bandwidth with a digital Gaussian filter.

2B, as well as in Fig. 2C and D, as longer gaps of 0.3 to 0.8 msec mean duration. Most of these gaps clearly do not reach the baseline. They have a similar amplitude and duration to the intermediate step conductance events (as in Fig. 2B), suggesting that they arise from the same kinetic state. The separation of events into classes of “short gaps” and “long, sublevel

118

F. J. SIGWORTH AND S. SHENKEL

gaps” can be seen in Fig. 3, which plots statistics of gaps that occurred during channel openings to the main conductance level (approximately 9 pA at 200 mV in each case), derived from the same runs as in Fig. 2. For each run an amplitude-duration scatter plot of gaps is shown along with the corresponding semilogarithmic duration histogram. The amplitude values in the scatter plots were calculated in a way to avoid systematic errors from the finite time resolution, but show a scatter that is larger than that expected from background noise and is skewed significantly to positive current levels, representing incomplete channel closures. In the GMO-hexadecane data of Fig. 3D the 0.9-msec “long gap” events are most clearly seen as a more tightly clustered cloud of points in the vicinity of 2 pA in amplitude. In the logarithmically binned histograms shown in the lower panels of Fig. 3, single exponential distributions of dwell times appear as broad peaks (Sigworth and Sine, 19871, and two such components are shown fitted to each set of data. The most dramatic difference among the different membrane compositions is the relative size of the brief and long components of the histogram: in the GMO-decane data of Fig. 3B there were 15 times as many brief gaps as long ones, while in GMO-hexadecane membranes this ratio was only 3. It should be noted that two exponential components do not provide a good fit to the entire spectrum of dwell times that were observed: there appears to be an excess of events at intermediate times between the components as well as an excess of events at long times which we did not attempt to fit because of the limited number of events. Table I summarizes the kinetic parameters obtained from eight experimental runs. In each of these about 180 sec (16 megabytes) of recorded data were analyzed, yielding gap statistics for 10-40 sec of total open time at the main GA conductance level. The scatter in the fitted parameters is considerable, but a clear trend can be seen, in which high rates (A, = 50 sec-’) of short gaps are seen in the thickest (decane) membranes and much lower rates (-3 sec-’) in the thin hexadecane membranes. A similar but smaller difference is seen in the long gap rates A,. The long gap durations appear to lengthen in the thinner membranes. The durations of the short gaps appear to be constant at 19 psec, but since this value is very close to the 15-psec lower limit for event detection in our recordings it is likely that the true gap durations are actually shorter. 6. Gaps in N-Acetyl-GA Channels

An analog of GA in which the N-terminal formyl group is replaced by an acetyl group (Szabo and Urry, 1979) has been shown to yield channel currents with a high frequency of gaps in the 1-msec range (Szabo, 1981).

octane

A

B

decone

20

.

.

. . .. . . <

..

.

-10

-2.0

.nc-

> a, C

w

Dwell t i m e , sec.

Dwell t h e , sec

0188 tiis 147

881 t i i s

36

. h Dwell time. see.

1;-2

Dwell t i m e . s e c

FIG. 3. Scatter plots and dwell-time histograms for channel closing and sublevel events obtained from the runs shown in Fig. 2. Only portions of the recording where a single. full amplitude channel current was present were analyzed. The scatter plots show the mean amplitude of the current during each sojourn below the half-amplitude threshold (-4.5 PA). plotted against the sojourn duration. For events shorter than -50 psec the amplitude could not be reliably estimated and was set to zero. The histograms show the number of durations falling into each of a set of logarithmically spaced bins. I n this type of display an exponential distribution i s represented by a peaked function (Sigworth and Sine. 1987). The visual fits shown were made with two exponential components. one of which was constrained to be 19 psec in each case. ( A ) From a total of 19.6 sec of open time analyzed. 342 events i n a 19-psec component and 51 events in a 0.3-msec component; (B) from 14.3 sec o f open time. 1424 events i n a 19-psec component, and 94 events in a 0.3-msec component; (C) from 13.4 sec of open time, 35 events in a 19-psec component and 1 I events in a 0.9-msec component; (D)from 41.8 sec of open time. 147 events in a 19-psec component and 36 events in a 0.9msec component.

120

F. J. SIGWORTH AND S. SHENKEL

GA GAPPARAMETERS AT 200 mV

TABLE I VARIOUSALKANE MEMBRANE SOLVENIS"

WITH

Alkane

7,

A,

71

XI

Stepsltotal

Octane Decane Tetradecane Hexadecane

0.019 0.019 0.019 0.019

17 ( 1 )

40-98 (3) 2.5-12 (2) 3.4 (2)

0.3 0.3 0.9 0.9

2.6 6.4 0.8-1.5 0.8-1.5

2/10 215 411 416

"Time constants T,, T , (in msec) and rates of occurrence A,. A , are given for the short and long (substate) gaps, respectively, from recordings from GA in I M KCI. The units of the rates are given as events per second of total channel open time. with the number of determinations given in parentheses. The probability of intermediate steps occurring during opening or closing transitions is expressed as "stepsltotal." i.e., the number of subconductance steps detectedlnumber of transitions analyzed between the baseline and the main conductance level. The detection procedure was able to find all steps having dwell times 150 psec at the subconductance level.

Recordings from these channels (Fig. 4) indeed show sublevel gaps that appear very similar to those seen in GA channels, except that their frequency of occurrence is two orders of magnitude higher. Also in the N acetyl-GA currents we observed intermediate steps in 30-40% of the starting and ending transitions of bursts, and these steps had durations and conductances indistinguishable from those of the sublevel gaps. Short gaps are not visible in the distributions shown in Fig. 4B and D, but this may be due to the lower time resolution of the recordings and the possibility that the short gap distribution is lost in the abundance of longer gaps. C. Kinetic Significance of the Gaps

Because the long gaps are not complete "closures" of the channel but represent transitions to a sublevel of conductance, it should be possible to determine the kinetic relationship between this sublevel state and the fully closed channel state, assumed here to arise from the dissociation of the GA dimer, which lasts on the order of seconds or longer. Indeed, transitions from fully open to a sublevel and then to the fully closed state are observed in a significant fraction (20-60%) of all closing transitions, and sublevel steps are similarly seen in the same fraction of opening transitions. Because the conductance and lifetime of these sublevel sojourns are similar to the sublevel gaps, we assume that they represent the same kinetic state. In the various membranes we tested, the sublevel gaps accounted for only 0.1 to I% of the total open time of the channel. Therefore it is sur-

121

6. GRAMlClDlN A CHANNELS

Decone

A

4 0 1

...................................

B

..........................

,456 ms

msec 4

Dwell time. sec

Hexodecone

C

4ot

.................................................................

I: 1

8 sec

11

1

D

...

$$.?*:*+~-,.: .,

a 0.0

E

d -I

u

-201,

,

,

, 4 4 1 ins

Dwell tlnie, sec

FIG.4. Gaps in N-acetyl-gramicidin A currents. ( A ) Representative events obtained in a GMO-decane membrane with I M KCI, 100 mV applied potential. ( B ) Gap statistics displayed as in Fig. 3. Note the clustering of gap amplitude values around a nonzero value of about 0.6 PA. The gap dwell-time distribution is well fitted by a single exponential component (from 0.78 sec total open time, 514 events. 0.46 msec). (C) Events obtained in GMO-hexadecane membranes under the same conditions. Note the longer burst duration. ( D ) Gap statistics. Again. the distribution is well fitted by a single exponential (0.46 sec total open time. 176 events. 0.44 msec).

122

F. J. SIGWORTH AND S.SHENKEL

prising that 2040% of the opening and closing transitions involved sojourns in the sublevel state. The implication is that there is a strong coupling between the dissociation of the GA dimer and the appearance of the sublevel, such that a dimer is 60-200 times more likely to dissociate from its sublevel state than from the main conductance state. Thus this state is likely to represent an intermediate in the dimerization process. The N-acetyl-GA channels showed sublevel gaps of similar duration, but at frequencies two orders of magnitude higher than seen in GA channels. Structurally, N-acetyl-GA channels have an additional methyl group at the N-terminus of each chain, which might alter the energetics of the head-to-head junction. The difference in kinetics implies that the N-acetylGA channels have a less stable main conductance state, having a lifetime two orders of magnitude shorter than in GA. The short gaps are too brief and their conductance is too close to zero to allow their detection in association with opening and closing events. However, there is circumstantial evidence for associating these events with the dimerization process as well. Their rate of occurrence has a strong dependence on membrane composition, changing by more than a factor of 10 between decane-GMO and hexadecane-GMO membranes. This change was larger than the change in channel open time (taken here to be the lifetime of a GA dimer), which we estimate to have changed by at most a factor of 3 under the same conditions. Thus the mechanism underlying these gaps may be very sensitive to some of the properties which affect the dimerization process, such as membrane thickness and surface tension or surface deformation forces (Elliott et al., 1983; Huang, 1986). IV. OPEN-CHANNEL NOISE

Because the flux of ions through an open channel is so large-on the order of lo7 per second-any internal motion in the channel protein that influences ion transport rates can result in a relatively large change in ion flux, yielding a measurable signal. From this point of view, then, the fluctuations in current through single channels provide a unique way to study the internal motions of proteins. If there are motions that are so rapid to be on the time scale of the ion transport process itself, even these can give rise to measurable current fluctuations because they can cause the transport of ions to show a coarse graininess due to temporal correlations in ion transit events. Exploiting the sensitivity of the patch-clamp technique, which allows even the small “shot noise” from uncorrelated ion transport events to be observed, we have measured the open-channel current noise in acetylcholine receptor (AChR) channels (Sigworth, 1985,

6. GRAMlClDlN A CHANNELS

123

1986), and in the channels formed by GA and analogs (Sigworth et ul., 1987). The AChR currents yielded noise spectra with a low-frequency excess that probably arises from confonnational fluctuations in the channel protein on a time scale of -0.5 msec. In contrast, the noise in GA channels, as well as in channels formed by four GA analogs, showed a flat spectral density in the frequency range up to 20 kHz. This sort of spectrum is to be expected for underlying fluctuations that have a time scale shorter than 10 ksec. A.

Power Spectra of GA Channel Currents

Figure 5 shows spectra obtained from the Ala7-GA analog. We favor working with this analog over GA (which normally has an L- Val residue at position 7) because it has less dispersion in single-channel conductance values (Prasad et al., 1986). Background noise spectra were obtained from portions of the recording with no channels open, and spectra were also obtained from portions having the current from one open channel (Fig. 5B). The difference of these is the spectral contribution of the open-channel noise, shown in Fig. 5C, for currents in symmetrical 200 mM CsCI. Although the spectra are featureless in that they are flat, the magnitude of the spectral density is interesting: in each case it is larger than that expected for simple shot noise, and it varies with Cs concentration. The spectral density at 1 M CsCl (Fig. 5D)is lower than that at 200 mM, and is nearly equal to the spectral density at 50 mM; at these concentrations the channel currents are 9.3 and 4.1 PA, as compared to 8.9 pA at I M. The fluctuations are therefore relatively large at the lower Cs concentrations.

B. Prediction of Spectra from Rate Constants We first wondered if the size of the spectral densities could be explained by standard theories for the kinetics of ion permeation in GA channels, such as the two-site model of Finkelstein and Andersen (1981) or the foursite model of Eisenman and Sandblom (1983). We applied the theory of Frehland (1978) to these two models to predict the expected power spectra. Frehland’s theory starts with the description of the ion transport process as a set of states, each representing a particular configuration of ions in the channel, and the rate constants that connect these states. Figure 6 shows the kinetic scheme of the two-site model, which has four states representing empty, singly occupied, and doubly occupied channels [a fifth state is also shown at the bottom of the figure; we added it to explain the observed noise spectrum (see below)]. The power spectrum of fluc-

124

F. J. SIGWORTH AND S.SHENKEL

A1n7-GA. 200 mV.

0. 2Y C.' 2 kHz

ll0pA"

A

50 m s

B

m 1 I

m I

0.

I shm.1 1

0p.n

2

I

I

3

4

Log froquoncy.

ci

1-0

0lli.r.n.a..

m

1

I

I

2

3

4

Log froquoncy.

GI

-I

C

2

3

Hz

C

4

i

Log frequency, Hz

Hz

FIG.5 . Open-channel noise in Ala7-GA channels at 200 mV. (A) An increase in noise is visible in a recording in 0.2 M CsCI, filtered at 2 kHz a s the number of channels open increases from zero up to three. Only short portions of the recording are shown; typical channel open dwell times are several seconds. ( B ) Average power spectra obtained with no channels open (average of 1256 spectra) and with one channel open to the main current level of 9.3 pA at 200 m M CsCl (average of 1296 spectra). (C) Different spectrum from the data of B. giving the open-channel noise contribution. The solid line shows the average spectral density of 7.2 x lo-"' A'/Hz (averaged over the frequency range 300 Hz-3 kHz) and the dashed line shows the expected shot noise density of 3.2 x lo-"' A'IHz for this current. (D) Different spectrum obtained with I M CsCI. The average spectral density was 3.5 x lo-"' A'/Hz. while the expected shot noise for the 8.9 pA current was 3.0 x lo-'" A'/Hz.

tuations in the current is obtained as the Fourier transform of an autocovariance function R(T)which is given by R(T)

=

c.

YP" Y K P

CII".Ko(4

P K P

where y P vand yuprepresent the charge movements that occur in the state transitions v + p and p + K, respectively. The function C F U , J ~is) the joint probability density of the occurrence of these two transitions sep-

125

6. GRAMlClDlN A CHANNELS

n

I0 U

g . 90 .V

* 9o/v

n

,Io,

'00'

U

FIG.6. The kinetic scheme and rate constants of Finkelstein and Andersen (1981) for GA ion transport. to which an additional state (*I i s added to account for the observed openchannel noise. The rate constants are given in inverse microseconds; the factor C indicates molar Cs' concentration. V indicates a Boltzmann factor of voltage. Each state represents a different configuration of ions in the channel, where 0 indicates an empty binding site. and I represents a binding site occupied by an ion.

arated by the interval T. Thus the autocovariance function reflects the temporal correlations in the transport of ions, with those transport steps that involve large charge movements being weighted most heavily. If, for example, there is a positive correlation in the movement of ions through the channel, such that several ions tend to move in rapid succession giving bursts of current, this will result in a large autocovariance and therefore a large spectral density. In the other extreme case of independent, unidirectional hopping of ions with charge 4 at rate A across a single barrier, the autocovariance reduces to R(T) = hq'6(7)

The corresponding spectral density is independent of the frequencyfand is given by Ssho,f'j) = 2iq, where i = A4 is the mean single-channel current. This is the classical shot noise formula (Schottky, 1918). Figure 7A compares the behavior of the four-state model with the observed concentration dependence of the single-channel current i and the normalized spectral density S/S,,,,,. Unlike the experimental data points, the predicted spectral densities lie very close to the shot noise level, as

126

F. J. SIGWORTH AND S. SHENKEL

CONCENTRATION, M

FIG.7. Predictions of the kinetic scheme of Fig. 6 for open-channel noise. (A) Predictions from the Finkelstein and Andersen model (without the blocking state) at 200 mV, for the cs concentration dependence (a), the relative power spectral density s/s,h,, where Sshol is the expected classical shot noise spectral density; (b) the single-channel current i; and (c) the probability of channel occupancy by 0, 1, or 2 ions. The points are experimental values from Ala’-GA channels. (B) Predictions of the model of Fig. 6 including the added blocking state, with the same data points superimposed. (C) Corresponding predictions and data at a membrane potential of 100 mV.

a result of relatively weak temporal correlations between the transport of ions. Actually, negative correlations are predicted in the concentration range between 0.1 and 10 M Cs’, causing the spectral density to lie below the shot noise level. This is to be expected in the cases where one ion must “wait” for another ion to leave the channel before it can be transported. Spectral densities that are similarly close to the shot noise level are predicted by the 16-state model of Eisenman and Sandblom. C. A Hypothesis for the Origin of the Open-Channel Noise

Almost any imaginable variation or blocking of the channel conductance can produce the flat frequency dependence that we observed in the noise spectra, provided that the time scale of the fluctuations is short enough so that frequency-dependent features of the spectrum are outside of our 20-kHz range of measurement. The magnitude of the spectral density and its concentration dependence can, however, be used to constrain simple theories for the origin of the noise. Because the spectral density was relatively large at the lower Cs’ concentrations, it seemed reasonable to us that there could be brief interruptions in the channel current that depended on the ion occupancy of the channel. A very simple model of this kind was made by adding to the four-state scheme (Fig. 6) an “occluded” state into which a channel can go when it is unoccupied by ions. Such a model

127

6. GRAMlClDlN A CHANNELS

could explain the relative absence of excess noise at high ion concentrations, since then the probability of an unoccupied channel is low. In comparing the predictions of low-frequency spectral density from the model with the data points available, we found that two rate constants associated with the new state were well constrained by the concentration dependence of the spectral density (Fig. 7B and C). The values obtained, occlusion rate p = 0.6 x lo6 sec-' and return rate a = 2 x lo6 sec-', predict that at limiting low concentrations the channel would show 0.5-psec gaps occurring every 2 psec. At 50 mM C s + , the lowest concentration tested, the channel is unoccupied by ions only about half the time, and the gaps would occur at roughly 3-psec intervals. This kinetic scheme is perhaps the simplest of many different possibilities for the origin of the noise. To test the hypothesis it would be very desirable to have an independent estimate of the time scale of the fluctuations. One approach would be to measure the power spectrum up to about 300 kHz directly. The theory predicts a feature in the spectrum in this frequency range (Fig. 8) which corresponds to the 0.5-psec gaps that were postulated. Another approach would be to analyze the third and higher moments of

4 states

+

blocking

c e

d

c a L

4

I

5

I

6 7 Log frpqwncy. Hr

I

e

r.01 M

FIG.8. Predicted frequency dependence of the spectral density from the model of Fig. 6 at different Cs' concentrations. Spectral densities are normalized to S,,., (shown as dashed lines) and are plotted on a linear scale. The low frequency asymptotes (shown here down to lo4 Hz) are seen to be flat, but a prominent Lorentzian component is predicted in the vicinity of 300 kHz, especially at Cs + concentrations near 100 mM.

128

F. J. SIGWORTH AND S. SHENKEL

the amplitude distribution of the current fluctuations: interruptions in the channel current, being asymmetrical fluctuations, should cause the distribution to be skewed in the direction of reduced channel current. A preliminary analysis of the third moment of the amplitude distribution in the data shown in Fig. 5A and B shows it to be consistent with current interruptions in the -1-psec range.

V. CONCLUSIONS

From previous studies it has been known that GA channels display rapid conformational changes. On the one hand, Ring (1986) measured brief gaps in the channel current on the millisecond time scale, and at the other extreme, molecular dynamics calculations (Fischer and Brickmann, 1983; Mackay et al., 1984) show that internal motions of the channel protein contribute strongly to the rapid transport of ions through the channel. Our work is an effort to extend Ring’s study of the “fine structure” of GA channel kinetics to shorter time scales using higher-resolution recording techniques and the indirect methods of noise analysis. We find evidence for fluctuations in channel currents occurring on at least three distinct time scales, and each of these kinds of fluctuations have intriguing properties that may allow their structural basis to be elucidated. The “long” (-0.5 msec) sublevel gaps that we observe are very closely coupled to the process by which a GA dimer is formed and the channel conductance is switched on. These gaps are most likely reporting an intermediate state in the formation and dissociation of the channel dimer, since the first opening and final closing of a channel often involves a transition through this sublevel state. On the other hand, the “brief’ (-20 psec) gaps are too short to allow such a coupling with channel formation to be detected. They do, however, show a large dependence on membrane properties such as the thickness, as does the lifetime of the channel dimer (Elliott et al., 1983). We infer the existence of a third class of gaps on the basis of open-channel noise measurements. The current noise shows a flat spectrum and suggests a substantial fluctuation in the channel current on a time scale of a few microseconds or less. From the concentration dependence of the spectral density we present a simple hypothesis in which the gaps would have a mean duration of 0.5 psec and would represent the occasional switching of the empty channel into an “occluded” conformation that would not accept ions. This hypothesis is consistent with the idea that bound ions might stabilize the channel in its conducting conformation.

129

6 . GRAMlClDlN A CHANNELS

ACKNOWLEDGMENTS We are grateful to D. Urry and K. U . Prasad for the gramicidin A and analogs used in this study, and to E. Neher who established the microbilayer technique. This work has been supported by grant NS-21501 from the National Institutes of Health. REFERENCES Bezanilla. F. (1985). A high capacity data recording device based on a digital audio processor and a video cassette recorder. Biopliv.~.J . 47, 437-441. Busath. D.. and Szabo. G. ( 1981). Gramicidin forms multi-state rectifying channels. Ntrirrrc~ (Londcin) 294, 371-373. Busath. 0..Andersen. 0 . S.. and Koeppe, R. E. (1987). On the conductance heterogeneity in membrane channels formed by gramicidin A. A cooperative study. Bioplrys. J . 51, 79-88. Colquhoun. D.. and Sigworth. F. J. (1983). Fitting and statistical analysis of single-channel records. I n “Single Channel Recording” ( B . Sakmann and E . Neher. eds.). pp. 191263. Plenum. New York. Eisenman. G.. and Sandblom. J . (1983). Energy barriers in ionic channels: data for gramicidin A interpreted using a single-file (3B4S”) model having 3 barriers separating 4 sites. 111 “Physical Chemistry of Transmembrane Ion Motions” ( G . Sprach. ed.), pp. 329-348. Elsevier, Amsterdam. Elliott. J . R.. Needham, D.. Dilger. J . P.. and Haydon. D. A. (1983). The effects of bilayer thickness and tension on gramicidin single-channel lifetime. Biochim. Biophys. Acilr 735, 95-103. Finkelstein. A,. and Andersen. 0.S. (1981). The gramicidin A channel: a review of its permeability characteristics with special reference to the single-file aspect of transport. J . Mrinhr. Biol. 59, 155-171. Fischer. W.. and Brickmann. J . (1983). Ion-specific diffusion rates through transmembrane protein channels. A molecular dynamics study. Biophys. Chi~n7.18. 323-337. Frehland. E. ( 1978).Current noise around steady states in discrete transport systems. Bio~pliy.~. Chein. 8. 255-265. 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. B i o i h i m . B i o p k y s . Acitr 513, 106-116. Huang, H. W. (1986). Deformation free energy of bilayer membrane and its effect on gramicidin channel lifetime. Biophvs. J . 50, 1061-1070. Mackay. 0. H., Berens. P. H.. and Wilson, K. R. (1984). Structure and dynamics of ion transport through gramicidin A. Biophys. J . 46, 229-248. Prasad, K. U.. Alonso-Romanowski. S.. Venkatachalam. C. M.. Trapane. T. L.. and Urry. 0 . W. (1986). Synthesis. characterization and BLM studies of L.Ala’-Gramicidin A. Biocliriiiistry 25, 456-463. Ring. A. ( 1986). Brief closures of gramicidin A channels in lipid bilayer membranes. & J l ’ / i h 7 . B i o p h y s . Acicr 856, 64-53, Schottky. W. (1918). Ann. Phvs. (Leipzig) 57, 541-567. Sigworth. F. J . (198s).Open channel noise. 1. Noise in acetylcholine receptor currents suggest> conformational fluctuations. Biophys. J . 47. 709-720. Sigworth. F. J . (1986). Open channel noise. 11. A test for coupling between current fluctuations and conformational transitions in the acetylcholine receptor. Biopliys. J . 49, I04 I - 1046.

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Sigworth, F. J.. and Sine, S. M. (1987). Data transformations for improved display and fitting of single-channel kinetics. Biophys. J . 52, 1047-1054. Sigworth, F. J., Urry, D. W., and Prasad. K. U. (1987). Open channel noise. 111. Highresolution recording show rapid current fluctuations in gramicidin A and four chemical analogs. Biophys. J. 52, 1055-1064. Szabo. G. (1981). Internal gating of ion conductance in a gramicidin channel. B i o p h y s . J. 33, 64a. Szabo. G . , and Urry, D. W. (1979). N-acetyl-gramicidin: single-channel properties and implications for channel structure. Science 203, 55-57.

Part II

Nicotinic Acetylcholine Receptors

This Page Intentionally Left Blank

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. V O L U M E 33

Chapter 7 Function of Mammalian Nicotinic Acetylcholine Receptors: Agonist Concentration Dependence of Single Channel Current Kinetics STEVEN M . SINE Department of Physiology Yale University School of Medicine New Haven. Connecticut 06510 AND

JOE HENRY STEINBACH Departments of Anesthesiology and Anatomy and Neurobiology Washington University School of Medicine St. Louis. Missouri 63110

1. Introduction 11. Methods

I l l . Results and Discussion A. General Features of Ach-Induced Single Channel Currents in BC3H-I Cells B. Analysis of Open-Duration Histograms at Different Ach Concentrations C. Analysis of Closed-Duration Histograms at Different Ach Concentrations D. Concentration Dependence of Closed-Duration Histograms E. Limiting Low Ach Concentrations F. Saturating Ach Concentrations G. Closed Duration Histograms at Intermediate Ach Concentrations H. Concentration Dependence of the Effective Channel Opening Rate 1. Summary of the Closed-Duration Analysis J . Functional Implications of the Scheme I Transition Rates References

133

Copyright 0 1988 by Acadernlc Prehr. Inc. All righls 01 reproduction in any form re\erved.

134

STEVEN M. SINE AND JOE HENRY STEINBACH

1.

INTRODUCTION

This chapter summarizes our studies of the activation of acetylcholine receptors (AchRs) of BC3H-1 mammalian clonal muscle cells (Sine and Steinbach, 1986b, 1987). Receptor activation is described in the context of a linear four-state scheme, and transition rates are estimated for each reaction step in this scheme. Attention is turned to a description of activation by acetylcholine (Ach) and to the dependence of single channel current kinetics on Ach concentration. The present analysis recasts our original dwell time histograms using a logarithmic time axis as described by Sigworth and Sine (1987). The linear to logarithmic transformation compresses a single dwell time distribution, allowing it to be displayed on a single panel. Histograms from several experiments, then, may be compared more readily, such as closed time histograms at different agonist concentrations. The BC3H-1 clonal cell line is a homogeneous population of nonfusing muscle cells isolated from an intracranial tumor in C3H mice (Schubert et a l . , 1974). Following differentiation, BC3H-1 cells express surface AchRs which distribute at a uniform density of about 100 p M - ' . The receptor is a 9 s monomer consisting of the four subunits typical of end plate receptors (Boulter and Patrick, 1977).BC3H-I AchRs also bind a-bungarotoxin, classical nicotinic agonists and antagonists, and local anesthetics (Sine and Taylor, 1979, 1981, 1982). Finally, the BC3H-1 AchR subunits exhibit strong sequence homology to subunits of the Torpedo and the skeletal muscle AchR (Noda et af., 1983; Boulter et ul., 1985). The AchR of BC3H-1 cells is well characterized, and thus well suited for future studies of structure-function relationships. II. METHODS

Standard patch-clamp techniques were used to record currents through single AchR channels (Sine and Steinbach, 1986a,b, 1987). The following recording conditions were used throughout: the cell-attached patch configuration, a temperature of 11"C, an absolute membrane potential of - 70 or - 100 mV (cell interior negative), high potassium normal divalent cation saline in the external and pipet solutions, and a recording bandwidth of 7000-8000 Hz. Logarithmic dwell time histograms were constructed as described by Sigworth and Sine (1987). The histograms were fitted to the sum of several exponential components using the method of maximum likelihood (Colquhoun and Sigworth, 1983).

7. ACTIVATION OF ACETYLCHOLINE RECEPTORS

111. A.

135

RESULTS AND DISCUSSION

General Features of Ach-Induced Single Channel Currents in BCSH-1 Cells

Single channel currents exhibit distinct patterns of activity at low and at high Ach concentrations (Sine and Steinbach, 1986a, 1987).At low concentrations (100 nM Ach), two types of currents are observed: solitary brief-duration openings, and bursts of long-duration openings interrupted by brief-duration closed periods. Both brief openings and bursts of openings appear as random independent events, and typically occur at a frequency of about one per second. In contrast, at high concentrations (above 20 pM Ach) currents appear as long trains of channel openings, termed clusters, which are separated by intercluster closed periods lasting tens of seconds. Clusters, in turn, consist of several groups of more closely spaced openings, with the closures between groups lasting several hundred milliseconds. Solitary openings are also seen at high concentrations; these belong primarily to the brief-duration opening population. In the present kinetic analysis, attention is turned to bursts of long-duration openings at low Ach concentrations, and to groups of long-duration openings at high concentrations.

B. Analysis of Open-Duration Histograms at Different Ach Concentrations Figure 1 exhibits open-duration histograms at increasing Ach concentrations. At each concentration, two separate classes of openings are apparent: a major long-duration class with a mean duration of I5 to 25 msec, and a minor short-duration class with a mean of 100 to 200 psec. Shortduration openings, in general, occur as isolated openings at low agonist concentrations, but as both isolated and grouped openings at high concentrations. Long-duration openings occur both isolated and in bursts at low concentrations, and isolated and in groups at high concentrations. Long-duration openings elicit 99.9% of the current flow induced by Ach; they therefore represent the major active receptor state. The ratio of the number of short- to long-duration openings decreases only slightly over the 10,000-fold concentration range examined. This nearconstant ratio suggests that both short- and long-duration openings arise from doubly occupied receptors, and that few if any, short-duration openings result from singly occupied receptors. We also find that short- and long-duration openings become temporally associated at high agonist con-

136 117

I

STEVEN M. SINE AND JOE HENRY STEINBACH ,

,

,

,

,

,

,

I

,

I

,-

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I

A

36

49

m

rn \

25

3 16

c

B

8 4

4 0

0 -5 10

-4 10

-3 10

-2 10

-1

10

10

0

10

1

10

2

10

3

-5 lo

Time (sec)

-5

-4

-3

1o

-2 1o

-1 1o

n

I

I

14

,

,

,

-4

1o

-3

-2

-1

1o

Time (sec)

loo

lol

lo2

lo3

Time (sec)

Time (sec) ,

I

,

,

14 n

I h n

81

81

m

m

\

\

3 36

2 36

c

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t5

9 0

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-5

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10

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-3

10

"

-2

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-1

10

I

10

0

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10

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10

2

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Time (sec)

Time (sec)

62

32

36

191

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m

1 mMAch

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; 81

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c

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1

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10

0

lo1

lo2

103

137

7. ACTfVATlON OF ACETYLCHOLINE RECEPTORS

TABLE I

F i n t o PAHAMEPEKS FOH OPEN

AND CLOSED

T I M EHISTOGRAMS“

Acetylcholine concentrations

Open times Total openings “I,

n,, “1 I

(’?

I1 2

Closed times Total closings c4l 1111 “I

It I C?

II “3

n, “I

n, “ 5

If.

100 nM

20 p M

1.138 36.8 0.687 579 0.091 9.029 0.222

4.921 52.4 0.726 10.504 0.274

1,870 1.14 0.622 756 0.059 14,283 0.319

-

-

60 pM

2.120 65.4

I mM

4.738 70.9

0.906

om

6.398 0.094

5.631 0.1 17

-

-

-

-

-

-

12.131 0.757 0.0089 8.84 0.036 64.0 0.079 474 0.082 3,984 0. I30 30. I22 0.663

5.Y5 I 0.268 0.0066 2.61 0.0166 26.4 0.033 378 0. I66 3.466 0.121 3 1.989 0.657

35.286 0.185 0.0027 3.56 0.0055 39. I 0.0 I 3 433 0.03x 4.369 0.063 37.176 0.879

“Lower case c denotes the decay rate in reciprocal seconds. and n. the fraction of the total events in the specified component. The total number of events is the total area under the fitted curve.

centrations, indicating that the same doubly occupied receptor can produce both types of openings (Sine and Steinbach, 1984). Papkey and Oswald (19861, however, observe a change in the ratio of short- to long-duration openings at Ach concentrations below 100 nM, suggesting that brief-duration openings represent singly occupied receptors at very low Ach concentrations. Brief-duration openings, then, may result from single agonist

FIG.I . Open- (right column) and closed-duration (left column) histograms are displayed on a logarithmic time axis as described by Sigworth and Sine ( 1987). The overall f i t is the

upper continuous curve. and the individual components are the dashed curves. Solid curves highlight the closed-duration components associated with receptor activation steps in Scheme I . The fitted decay rates and fractional areas are given in Table I.

138

STEVEN M. SINE AND JOE HENRY STEINBACH

occupancy at low agonist concentrations, but arise primarily from double occupancy above 100 nM Ach. A third class of openings is apparent at 100 nM Ach, with an intermediate mean duration of about 1 msec, and a relative area of about 5%. Intermediate openings are seen in about 60% of recordings obtained at potentials between -70 and - 190 mV, but are resolved most often at potentials more negative than - 130 mV. Although clearly apparent in open-duration histograms at low Ach concentrations, intermediate openings are seldom resolved at high agonist concentrations. They are, however, resolved in histograms of solitary openings even at high agonist concentrations (Sine and Steinbach, 1987). In contrast to short-duration openings, intermediate openings are never temporally associated with long-duration openings. C. Analysis of Closed-Duration Histograms at Different Ach Concentrations

The results presented below are interpreted in the context of a linear four-state activation Scheme ( I ) :

2A

+R

k,

k:

P

k-,

k-2

a

s AR s A2R e AzR*

(1)

In Scheme 1, two agonists, A, combine with the receptor, R, with association rate constants, k, and k2, and dissociate with rate constants, k _ , and k P 2 .The open channel state, A2R*, arises from the fully occupied receptor, A2R, with the opening rate constant, p, and dissipates with the closing rate constant, a. Colquhoun and Hawkes (1981) describe the Scheme 1 predictions for the probability density function of closed durations as a function of agonist concentration. The probability density function, p(t), is the sum of three exponentials:

+ Q,lle-"' + Q2I2e-l2'

p ( l ) = Q,,lue-'"'

The areas, Q , and decay rates, I, describe the three time-dependent components, and are defined by the agonist concentration and the rate constants in Scheme 1 (i.e., k,, k2,k - I , k P z ,and p; see Colquhoun and Hawkes, 1981). Although in theory, Scheme 1 predicts three closed-duration components, in practice, the choice of rate constants and agonist concentration makes one or more components disappear. The masking of components arises either because the decay rates exceed the bandwidth capabilities of the recording system (about 8000 Hz), or because the relative area of a component is too small. For the rate constants consistent with the data

7. ACTIVATION OF ACETYLCHOLINE RECEPTORS

139

presented below, the following closed-duration components are expected to be resolved experimentally. At limiting low agonist concentrations, one brief-duration component is predicted with the decay rate (p + k - ? ) . At intermediate concentrations, two brief-duration components are forecast, one with the decay rate k - ,, and the other with the rate p’, the effective channel opening rate. The concentration dependence of the p’ decay rate may also be fitted to obtain estimates of the two association rates, k , and k,. At saturating concentrations, only one component is expected, with the decay rate p. Thus by examining closed-duration distributions from low to high agonist concentrations, the entire set of Scheme 1 rate constants may be estimated.

D. Concentration Dependence of Closed-Duration Histograms Figure 1 also exhibits closed-duration histograms at increasing Ach concentrations. The histograms are described by the sum of several exponential components; three components for 0.1 p M Ach, and six components for 20 p M to I mM Ach. Even first inspection, therefore, reveals more closed-duration components than are predicted by Scheme 1. However, Scheme l may represent the core description of the activation process, accounting for one or more of the observed components. As described below, the solid curves highlight components likely to be associated with receptor activation steps in Scheme 1. The remaining dashed components represent additional activatable states as well as two desensitized states at concentrations between 20 p M and I mM. The following analysis points out both the Scheme 1 and the additional activatable components for each histogram, starting with the 0.1 p M histogram (a limiting low agonist concentration), then the 1 mM histogram (a saturating agonist concentration), and finally the 20 and 60 p M histograms, representing intermediate agonist concentrations. E. Limiting Low Ach Concentrations

At 0.1 p M Ach, three closed-duration components are observed. Although, at limiting low agonist concentrations, Scheme l predicts up to three exponential components, only two components are expected to be observed experimentally. One of these is the major long-duration component, representing closed periods between independent bursts of openings. Its time constant is not readily interpreted in terms of the Scheme I rate constants, because it depends on the number of receptors in the membrane patch. The two brief-duration components represent closures within bursts of openings. The more robust brief component has a time

140

STEVEN M. SINE AND JOE HENRY STEINBACH

constant of about 70 psec and occurs at a rate of 50 to 60 per second of open time. Both its time constant and its occurrence rate are the same for a variety of agonists: acetylcholine, carbamylcholine. suberyldicholine, and the weak agonist, dimethyl-d-tubocurarine (Sine and Steinbach, 1986a,b). The agonist invariant properties of the 70-psec closures suggest they represent a closed state intrinsic to the open channel per se. The remaining intraburst component, shown by the solid curve, has a time constant of intermediate duration, about 1 msec, and occurs at a rate of 7 per second of open time. Both its time constant and its occurrence rate differ among agonists (Sine and Steinbach, 1986a), suggesting that intermediate closed periods arise through dwells in the A2R state followed by reopening to the A,R* state. At limiting low agonist concentrations, Scheme I predicts that dwells in the A2Rclosed state decay with the rate, p + k - , , and that they occur at the frequency, P/k-,, per burst of openings. Using these relationships, analysis of intermediate-duration intraburst closures yields estimates of p between 320 and 490 sec-', and of k - , between 860 and 1230 secc'. The channel closing rate, a, is estimated as the total open time within bursts divided by the sum of the number of closures in the intermediateplus the long-duration components. Thus the low concentration a estimate is 23.5 f 6.5 sec-l at a voltage of - 100 mV, or about 35 sec-' at -70 mV. F. Saturating Ach Concentrations

At saturating agonist concentrations, Scheme I predicts only one closedduration component: it arises from dwells in the A,R closed state. In the presence of 1 mM Ach, the closed-duration histogram is described as the sum of six well-resolved exponentials, with time constants spaced about a decade apart. The two long-duration components represent the distribution of intercluster and intergroup closed periods, and the component of shortest duration, the agonist-independent closures seen at limiting low agonist concentrations. Thus three intragroup closures remain as candidates for dwells representing the A2R closed state. Two quantities are considered in order to identify the AIR dwells. First, at the saturating concentration of 1 mM, the component reflecting A2R dwells should exhibit a decay rate matching the low concentration estimate of p. Second, the occurrence frequency of these activation-related closures should approach a, the channel closing rate, which is also estimated at low agonist concentration. Of the three remaining intragroup closures, the c3 component shown by the solid curve best fits the two criteria for dwells in A,R. The decay

141

7. ACTIVATION OF ACETYLCHOLINE RECEPTORS

rate of the c, component is close to the low concentration estimate of p: for three experiments the decay rate is 465 f 30 sec-l (mean fSD). Closures of the c , component occur at a rate of 3 I .5 9.6 per second of open time, close to the low concentration a estimate of 35 sec-'. Closures of the L',component, then, are associated with dwells arising from the A,R to A2R* transition. The two remaining intragroup closures, the c, and c2components, exhibit kinetic properties distinct from the A2R to A,R* transition predicted by the low concentration intermediate-duration closures. The c4 closures show a decay rate of 3700 sec- I , and an occurrence rate of 37.2 k 9. I per second of open time. Although the c, occurrence rate is close to the low concentration a estimate, the e, decay rate is 10-fold greater than the p predicted from the low concentration intermediate closures. The c, closures show a decay rate of 40 sec-', and an occurrence rate of 13 per second of open time; neither of these properties is consistent with the low concentration estimates of p and a.Because they occur at a saturating agonist concentration, and because their properties differ from those predicted for the A,R dwells, the c, and c2 closures are proposed to represent additional doubly occupied closed states branching from Scheme I .

*

G. Closed-Duration Histograms at Intermediate Ach Concentrations Depending on the choice of Scheme 1 rate constants, at least two closedduration components may be observed at intermediate agonist concentrations (Sine and Steinbach, 1987). In particular, if the product of agonist concentration and the second agonist association rate, Ak,. is comparable to the first agonist dissociation rate, k _ ,, two equal-sized components are forecast. The decay rate of one component gives k - ,, if the agonist concentration is kept relatively low, and the decay rate of the other component gives p', the effective channel opening rate. At 20 p M Ach, an intermediate agonist concentration, the closed-duration histogram is described as the sum of six exponentials. As described for the I mM histogram, the two slowest components represent intergroup and intercluster closures, and the fastest component represents agonistindependent closures seen at low concentrations. Of the three remaining intragroup components, the c2 and c, components are interpreted as arising from receptor activation steps of Scheme I ; they are shown as continuous curves in the 20 p M histogram. Thus the c2 component reflects periods in which the channel closes, loses and rebinds two agonist molecules, and reopens: its decay rate equals p', the effective channel opening rate at 20 p M Ach. The c j decay rate, then, gives an upper limit estimate of the

142

STEVEN M. SINE AND JOE HENRY STEINBACH

first agonist dissociation rate, k - , , of 560 sec-I. As predicted for activationrelated closures, the combined c2 and c3 occurrence rate, 36.6 sec-’, closely matches a,the channel closing rate observed at low agonist concentration. At 60 p M Ach, the closed-duration histogram is again described as the sum of six exponentials. In contrast to the 20 p M histogram, the components of the 60 p M histogram appear similar to the those of the 1 mM histogram, suggesting that 60 p M approaches a saturating agonist concentration. The c3 decay rate, however, is consistently lower than the I mM c3 decay rate; at 60 p M the mean c3 decay rate is 342 2 55.4 sec-I, compared to 443 15 sec-’ at 1 mM. At 60 p M , then, the c3 decay rate equals p‘, close to the intrinsic channel opening rate, p. At 60 p M , the c3 occurrence rate is 33.7 6.6 sec-I, again approaching the low concentration a estimate. The c3 component, then, appears to arise from receptor activation processes described by Scheme 1; it is shown as the solid curve in the 60 p M histogram.

*

*

H. Concentration Dependence of the Effective Channel Opening Rate

Figure 2 shows the concentration dependence of the effective channel opening rate, p’ (c, decay rate at 20 p M , and c3 decay rate at 60 p M and above). The effective channel opening rate increases at intermediate concentrations and shows saturation at high concentrations. The curve shown represents the empirical Hill equation with a fitted dissociation constant of 40 pM and an assigned Hill coefficient of 2.0. Thus p’ rises steeply with agonist concentration, suggesting that the dissociation constants differ for the first and second agonist-binding steps. The rate constants for agonist association were estimated by fitting the concentration dependence of p’ to the relationships given by Colquhoun and Hawkes (1981). In particular, one of the three predicted closed-duration components is associated with the p’ decay rate; it depends on agonist concentration and the set of five rate constants in Scheme 1. Three of these five rate constants have already been assigned: the limiting low concentration measurements estimate k - z , both the low and the saturating concentration measurements yield p, and the 20 pM measurements give k - , . Thus p, k-,, and k - , were fixed, and the two association rates, k , and k2, were varied to fit the predicted decay rate to the measured p‘ at each agonist concentration. The fitting analysis reveals k , and k, estimates of 1 x lo7 and 1 x 10’ M-lsec-I; dividing these by the dissociation rate constants gives dissociation constant estimates of 10 p M and 50 pM,respectively. Agonist binding, then, shows positive cooperativity. Since the

143

7. ACTIVATION OF ACETYLCHOLINE RECEPTORS 1 -

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.

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.

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-7

-5

~

-4

-3

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LOG (Acetylcholine) FIG.2. Concentration dependence of the effective channel opening rate. The effective channel opening rate. p', i s expressed relative to the maximum channel opening rate, Pm.,,. and the ratio i s plotted against Ach concentration. The data are fitted to the nonlinear Hill equation: p'/Pma. = I/[ I ( K / L ) " ]K. i s the empirical dissociation constant. L the Ach concentration, and n the Hill coefficient. The curve shown i s the Hill equation with an assumed Hill coefficient of 2.0 and a fitted dissociation constant of 41 (LM.

+

,

dissociation rates, k - and k - 4 2 , are equal, the cooperativity arises through different agonist association rates, k , and k,. 1.

Summary of the Closed-Duration Analysis

At saturating Ach concentrations, a major closed-duration component, designated c3, is associated with the A,R to A,R* transition of Scheme 1. This association is supported by the observations that the c3decay rate approaches p estimated at low Ach concentrations, and that the c3 occurrence frequency is close to a, also estimated at low concentrations. The remaining three high concentration components are less likely candidates for the channel opening transition because they diverge from the low concentration estimates of p and a. At intermediate agonist concentrations, two closed-duration components are associated with receptor activation processes. The decay rate of the slower component, labeled p', increases over a narrow range of Ach concentration, saturating at about 130 p M Ach. The steep concentration dependence is consistent with positive cooperativity in agonist binding, with dissociation constants of 50 and 10 p M . The second activation component is clearly resolved only at 20 pM Ach where its decay rate yields the dissociation rate constant, k - , and its area equals that of the p' component. Both its decay rate and its

,

144

STEVEN M. SINE AND JOE HENRY STEINBACH

relatively large area are consistent with positive cooperativity in agonist binding. Because the two agonist dissociation rates, k - , and k - 2 / 2 , are essentially equal, the different agonist association rates are the primary source of binding cooperativity. J. Functional Implications of the Scheme 1 Transition Rates

Qualitatively, the Ach receptor of BC3H- 1 cells functions appropriately for receptors at the neuromuscular junction. The channel opening rate, p, is 10-fold greater than the channel closing rate, a. Thus doubly occupied receptors have a high probability of being open, and they open within milliseconds. The agonist dissociation rate is relatively high, so bound Ach is released shortly after a transient increase of the Ach concentration. The apparent positive cooperativity in agonist binding increases the fraction of doubly occupied receptors over a narrow range of Ach concentrations. Receptor activation, then, is sensitive to small changes of the Ach concentration. Although BC3H- 1 receptors function qualitatively as expected for Ach receptors at the adult mammalian neuromuscular junction, they exhibit significant quantitative differences. Essentially, BC3H-1 receptors appear slower than adult junctional receptors. Both the burst duration and the estimated l/a are larger in BC3H-1 cells than the time constant for miniature end plate current decays at the adult mouse end plate; a,therefore, must be larger for adult end plate receptors (Linder et al., 1984). Similarly, miniature end plate currents rise much faster than would be predicted by our estimated sum, (p + k - 2 ) (Linder et al., 1984). Studying receptors at frog neuromuscular junctions, Colquhoun and Sakmann (1985) estimated p. a, and k - 2 . and found values roughly 10-fold greater than ours. For snake junctional receptors, however, Leibowitz and Dionne (1984) estimate p and k - 2 comparable to ours. Overall, the functional properties of BC3H1 receptors are closer to those observed for neonatal muscle (Sakmann and Brenner, 1978; Steele and Steinbach, 1986). ACKNOWLEDGMENT We thank Fred Sigworth for sharing his idea of the logarithmic and square root histogram transformations. REFERENCES Boulter, J., and Patrick, J . (1977). Purification of an acetylcholine receptor from a nonfusing muscle cell line. Biochemistry 16, 4900-4908. Boulter, J . , Luyten, W.. Evans, K.. Mason, P., Ballivet, M., Goldman, D., Stengelin. S., Martin, G . , Heinemann, S. , and Patrick, J . (1985). Isolation of a clone coding for the a-subunit of a mouse acetylcholine receptor. J . Neurosci. 5, 2545-2552.

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Colquhoun. D., and Hawkes. A. G. (1981). On the stochastic properties of single ion channels. Proc.. R . Soc. London. Ser. B 211, 205-235. Colquhoun, D., and Sakmann, B. (1985). Fast events in single channel currents activated by acetylcholine and its analogs at the frog muscle endplate. J. Plrysiol. fLonclonl 369, 501-557. Colquhoun, D.. and Sigworth. F. J. (1983). Fitting and statistical analysis of single channel records. In “Single Channel Recording” lB. Sakmann and E. Neher, eds.). pp. 191264. Plenum, New York. Leibowitz, M. D., and Dionne. V. E. (1984). Single channel acetylcholine receptor kinetics. Biophys. 1.45, 153-164. Linder, T. M., Pennefather, P., and Quastel, D. M. J . (1984). The timecourse of miniature endplate currents and its modification by receptor blockade and ethanol. J . Gen. Plivsiol. 83, 435-468. Noda. M., Takahashi. T., Tanabe. M..Toyosato, S. , Kikyotani. Y.. Furutani, T.. Hirose, H.. Takashima. S.. Inayama, T., Miyata, and Numa, S. (1983). Structural homology of Torpedo californicu acetylcholine receptor subunits. Nutitre (London) 302, 528-532. Papkey, R. L.. and Oswald, R. E. (1986). Effects of allosteric ligands on the gating of single channel currents in BC3H-I cells. In “The Nicotinic Acetylcholine Receptor: Structure and Function” (A. Maelicke, ed.), pp. 243-257. Springer-Verlag. Berlin and New York. Sakmann, B., and Brenner. H. (1978). Change in synaptic channel gating during neuromuscular development. Nature (London) 276, 401402. Schubert, D., Harris. A. J.. Devine. C. E., and Heinemann, S. (1974). Characterization of a unique muscle cell line. J. CeII B i d . 61, 398-413. Sigworth. F. J., and Sine. S. M. (1987). Fitting and display of single channel dwell time histograms. Bioplrys. J . 52, 1047-1054. Sine, S. M.. and Steinbach, J . H. (1984). Activation of a nicotinic acetylcholine receptor. B i ~ p h y .J. ~ . 45, 175-185. Sine, S. M.. and Steinbach. J. H. (198ha). Acetylcholine receptor activation by a site selective ligand: Nature of brief open and closed states in BC3H-I cells. J . Pltysio/. fLondon) 370, 357-379. Sine. S . M.. and Steinbach, J . H. (l986b). Activation of acetylcholine receptors on cloniil mammalian BC3H- I cells by low concentrations of agonist. J . Physio/. (London) 373, 129- 162. Sine. S. M., and Steinbach, J. H. (1987). Activation of acetylcholine receptors on clonal mammalian BC3H- I cells by high concentrations of agonist. J. P l i y ~ i ~fLontlon) l. (in press). Sine. S. M . . and Taylor. P. ( 1979). Functional consequences of agonist mediated state transitions in the cholinergic receptor: Studies in cultured muscle cells. J. B i d . Clwrrr. 254. 33 15-3375. Sine. S. M.. and Taylor. P. (1981). Relationship between reversible antagonist occupancy and the functional capacity of the acetylcholine receptor. J. Biol. Clrc,ur. 256. 66926699. Sine. S. M.. and Taylor. P. (1982). Local anesthetics and histrionicotoxin itre iillosteric inhibitors of the acetylcholine receptor. J . B i d . Clw/n. 257, X106-XI 14. Steele. J . A,. and Steinbach. J . H. (1986). Single channel studies reveal three clasceh of acetylcholine-activated channels in mouse skeletal muscle. Bioplrys. J. 49, 36la.

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CURRENT TOPICS IN MEMBKANES AND TRANSPORT. VOLUME 33

Chapter 8 Regulation of the Nicotinic Acetylcholine Receptor Channel by Protein Phosphorylation RICHARD L. HUGANIR Laboratory of Molecular and Cellular Neuroscience The Rockefeller University New York, New York 10021

1. Protein Phosphorylation 11. The Nicotinic Acetylcholine Receptor

HI.

Protein Phosphorylation of the Nicotinic Acetylcholine Receptor Biochemical Characterization of the Phosphorylation of the Nicotinic Acetylcholine Receptor B. Physiological Significance of Phosphorylation of the Nicotinic Acetylcholine Receptor Conclusions References

A.

IV .

1.

PROTEIN PHOSPHORYLATION

Protein phosphorylation has been widely recognized as one of the major regulatory mechanisms in the control of cellular metabolism (Cohen, 1982). Protein phosphorylation has been shown to regulate such diverse functions as glycogen metabolism, lipid metabolism, muscle contraction, and neurotransmitter synthesis (Cohen, 1982; Nairn et al., 1985; Browning et al., 1985; Huganir, 1986a,b). It is likely that almost all cellular pathways are regulated to some extent by protein phosphorylation. Recent studies have provided evidence that protein phosphorylation plays a major role in the regulation of ion channel function (Nairn ef al., 1985; Browning et al., 1985; Huganir, 1986a,b). Protein phosphorylation systems consist of three primary components: a protein kinase, a substrate protein, and a protein phosphatase (see Fig. 1) (Cohen, 1982; Nairn et al., 1985; Browning et a l . , 1985; Huganir, 147 Copyripht $ 1 , 1988 hy Acadcmic Prew. Inc. All rights of reproduclion in m y form r e w v e d .

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RICHARD L. HUGANIR

ADP

Protein K i n a s e

~

Protein P ho sp ha t a s.e

Phosphoprotein

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Pi

FIG.I . Phosphorylation-dephosphorylation cycle of substrate proteins.

1986a,b). Protein kinases are enzymes that catalyze the covalent transfer of the terminal phosphate of ATP to specific substrate proteins. The addition of the highly charged phosphate molecule alters the structure of the phosphorylated protein and thereby regulates its function. The phosphorylated protein then directly or indirectly modulates the physiological properties of the cell (Cohen, 1982; Browning et al., 1985; Nairn et af., 1985; Huganir, 1986a,b). This process can be reversed by protein phosphatases which remove the phosphate from the substrate protein and return the substrate protein and the cell to their basal states (Ingebritsen and Cohen, 1983). The activity of many protein kinases is regulated by the level of intracellular second messengers such as CAMP, cGMP, calcium, and diacylglycerol (Cohen, 1982; Browning et a f . , 1985; Nairn et al., 1985; Huganir, 1986a,b). The protein kinases regulated by second messengers can be divided into four major classes: CAMP-dependent protein kinases, cGMPdependent protein kinases, calciudcalmodulin-dependent protein kinases, and calciudphospholipid-dependent protein kinase (protein kinase C) (see Table I) (Cohen, 1982; Browning et af., 1985; Nairn et a f . , 1985; Huganir, 1986a,b). All of these protein kinases exclusively phosphorylate serine and/or threonine residues of their respective substrate proteins. In addition, a new class of protein kinase has recently been described which exclusively phosphorylates tyrosine residues of their substrate proteins (Sefton and Hunter, 1984; Hunter and Cooper, 1985). The tyrosine-specific protein kinases were initially discovered because they were the products of genes

TABLE 1 DIFFERENT CLASSESOF PROTEIN KINASES CAMP-dependent protein kinase cGMP-dependent protein kinase Calcium/calmodulin-dependent protein kinases Calcium/phospholipid-dependent protein kinase Tyrosine-specific protein kinases

8. NICOTINIC ACETYLCHOLINE RECEPTOR

149

responsible for transforming cells by oncogenic retroviruses (Sefton and Hunter, 1984; Hunter and Cooper, 1985). Most of these viral tyrosinespecific protein kinases have been shown to have normal cellular homologs which are very similar in structure to the viral proteins (Sefton and Hunter, 1984; Hunter and Cooper, 1985). Recent studies have shown that normal cellular homologs of the tyrosine-specific protein kinases may be important in neuronal function and ion channel modulation (Barnekow et ul., 1982; Cotton and Brugge, 1983; Sorge et ul., 1984; Levy ef al., 1984; Huganir et a / . , 1984). II. THE NICOTINIC ACETYLCHOLINE RECEPTOR

The nicotinic acetylcholine receptor provides an ideal model system for the study of the regulation of ion channel function by protein phosphorylation. The nicotinic acetylcholine receptor is a neurotransmitter-dependent ion channel that mediates the depolarization of the postsynaptic membrane of nicotinic cholinergic synapses (Changeux, I98 I; Changeux et al., 1984). Acetylcholine released from the presynaptic nerve terminal binds to the nicotinic acetylcholine receptor in the postsynaptic membrane. This causes the rapid opening of an ion channel that is permeable to sodium, potassium, and calcium (Changeux, 1981; Changeux et u/., 1984). The resulting depolarization of the membrane produces an action potential in the postsynaptic cell. The relative ease of electrophysiological studies at the neuromuscular junction as well as the abundance of the nicotinic acetylcholine receptor in the electric organs of electric fish have made the acetylcholine receptor the most completely characterized neurotransmitter receptor and ion channel in biology today (Changeux, 1981; Changeux ef al., 1984). It has served as an excellent model system for the study of the structure, function, and regulation of membrane receptors and ion channels. Postsynaptic membranes highly enriched in the nicotinic acetylcholine receptor can be isolated from the electric organs of Electrophorirs dwtricus and Torpedo and from mammalian skeletal muscle (Changeux, 1981; Changeux et nl., 1984). The nicotinic receptor can be solubilized from these postsynaptic membrane preparations and has been purified to homogeneity (Changeux, 1981; Changeux et d.,1984; Reynolds and Karlin, 1978; Huganir and Racker, 1982). The purified receptor is an M , 255.000 pentameric complex which consists of four types of subunits, a ( M , 40.000), p ( M , SO,OOO), y ( M , 60.000), and 6 ( M , 65,000) in the stoichiometry of a,pyG (Reynolds and Karlin. 1978). The purified receptor is biologically functional when reconstituted into phospholipid vesicles and displays all

150

RICHARD L. HUGANIR

the known biological properties of the nicotinic acetylcholine receptor in the native membrane (Huganir and Racker, 1982; Tank et al., 1983). Although the four subunits are distinct and are encoded by different genes, they are highly homologous in amino acid sequence and structure (Noda et al., 1982, 1983a,b; Claudio et al., 1983; Devillers-Thiery et al., 1983). Each subunit spans the membrane and it has been proposed that the five subunits are arranged in a pentameric rosette around a central ion channel (see Fig. 2). In addition, by analyzing the amino acid sequence of each subunit for hydrophobic and hydrophilic regions, models have been proposed for the transmembrane structure of each subunit (Claudio et al., 1983; Devillers-Thiery et ul., 1983; Finer-Moore and Stroud, 1984). In these models, each subunit has a large N-terminal region that is extracellular and four hydrophobic transmembrane segments (M 1-M4) (see Fig. 3). A fifth transmembrane segment has been proposed to form an amphipathic a-helix (MS) (Finer-Moore and Stroud, 1984). It is thought that the hydrophobic half of this a-helix faces the membrane while the hydrophilic half lines the pore of the ion channel wall. Each subunit would thus contribute one amphipathic a-helix to form the ion channel (FinerMoore and Stroud, 1984). 111. A.

PROTEIN PHOSPHORYLATION OF THE NICOTINIC ACETYLCHOLINE RECEPTOR

Biochemical Characterization of Phosphorylation of the Nicotinic Acetylchollne Receptor

Gordon et al. (1977a) and Teichberg and Changeux (1977) first demonstrated that postsynaptic membranes enriched in the nicotinic acetylcholine receptor contained protein kinase activity. The protein kinase was subsequently shown to phosphorylate the nicotinic acetylcholine receptor (Gordon et al., 1977b; Teichberg et al., 1977). Initial studies reported that the y- and &subunits were phosphorylated (Teichberg et al., 1977; Saitoh and Changeux, 1981) and indirect evidence suggested that the a- and psubunits were also phosphorylated (Teichberg et al., 1977; Smilowitz er al., 1981). These early studies were unable to demonstrate the regulation of this protein phosphorylation by CAMP, cGMP, calcium, or calcium/ calmodulin (Gordon et al., 1977a; Saitoh and Changeux, 1981; Davis et al., 1982). Later studies reported that the phosphorylation of the receptor was regulated by calcium and calmodulin (Smilowitz et al., 1981). Calcium and calmodulin, however, do not appear to regulate the phosphorylation of the receptor but regulate the phosphorylation of proteins in the post-

8. NICOTINIC ACETYLCHOLINE RECEPTOR

151

FIG.2. Schematic model of the structure of the nicotinic acetylcholine receptor. (A) Arrangement of the five subunits around the central pit as viewed from above the plane of the membrane. (B) Cross section of the receptor in the plane of the membrane.

FIG.3. Schematic model of the transmembrane topography of each subunit of the acetylcholine receptor. P indicates the area of each subunit which is proposed to be phosphorylated by the various protein kinases.

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RICHARD L. HUGANIR

synaptic membranes which comigrate with the receptor subunits on SDSpolyacrylamide gels (Huganir and Greengard, 1983). Recent studies have demonstrated that the isolated postsynaptic membranes enriched in the nicotinic acetylcholine receptor contain at least four different protein kinases, CAMP-dependent protein kinase (Huganir and Greengard, 1983), calciumlcalmodulin-dependentprotein kinase (Huganir and Greengard, 1983), protein kinase C (Huganir et al., 1983), and a tyrosine-specific protein kinase (Huganir et al., 1984). Three of the endogenous protein kinases phosphorylate the nicotinic acetylcholine receptor in the isolated postsynaptic membranes. The CAMP-dependent protein kinase phosphorylates the y- and 8-subunits (Huganir and Greengard, 1983), protein kinase C phosphorylates the 8- and a-subunits (Huganir et al., 1983), and the tyrosine-specific protein kinase phosphorylates the p-, y-, and 8-subunits (Huganir et al., 1984) (see Fig. 4). In addition, studies using purified CAMP-dependent protein kinase, protein kinase C, tyrosinespecific protein kinases, and purified nicotinic acetylcholine receptor have demonstrated that these kinases phosphorylate the purified receptor with the same subunit specificity as the endogenous protein kinases in the postsynaptic membrane (Huganir et al., 1983, 1984; Huganir and Greengard, 1983). Since the cDNA for all four subunits has been cloned (Noda et al., 1982, 1983a.b; Claudio et al., 1983; Devillers-Thiery et al., 1983), the amino acid sequences of all four subunits has been examined for possible phosphorylation sites for the three protein kinases (Huganir et af., 1984). Locations for all seven phosphorylation sites have been proposed, taking into account ( 1 ) the specificity of the three protein kinases for the subunits of the receptor, (2) peptide maps of the subunits phosphorylated by the three protein kinases using proteases and cyanogen bromide (CNBr). and (3) the known primary amino acid sequence preferences of CAMP-dependent protein kinase, protein kinase C, and tyrosine-specific protein kinases (see Fig. 5 ) . The two serine residues proposed as the phosphorylation sites on the y- and &subunits for the CAMP-dependent protein kinase are preceded by three (y-subunit) and two @-subunit)arginine residues, characteristic of other known substrates for CAMP-dependent protein kinase (Kemp et d., 1977). The two serine residues that are proposed to be phosphorylated by protein kinase C on the a- and 8-subunits are surrounded by lysine and arginine residues, characteristic of other known substrates for protein kinase C (Nishizuka, 1980; Hunter et al., 1984).The three tyrosine residues which are proposed to be the phosphorylation sites on the p-, y-, and &subunits for the tyrosine-specific protein kinase are preceded by acidic amino acids such as glutamic acid or aspartic acid residues, characteristic of other known substrates for tyrosine-specific protein kinases (Patschinsky et al., 1982; Hunter, 1982; Pike at al., 1982).

8. NICOTINIC ACETYLCHOLINE RECEPTOR

153

FIG.4. Subunit specificity of the three different protein kinases which phosphorylate the nicotinic acetylcholine receptor. Polyacrylamide-gel electrophoresis of acetylcholine receptor purified after phosphorylation by cyclic AMP-dependent protein kinase, CAMP K ; protein kinase C, PKC; tyrosine-specific protein kinase, Tyr K .

Recent studies using synthetic peptides containing the sequences of the proposed phosphorylation sites on the &subunit have supported the proposed location of the CAMP-dependent phosphorylation sites (Souroujon et al., 1986; Safran et al., 1986). Peptides corresponding to residues 354367, 364-374, and 373-387 of the &subunit were synthesized and antibodies to each of these peptides were made. It was found that peptide 354-367

154

3 58

RICHARD L. HUGANIR

P

P

P

- - R -S E B - S E R - V ~ L - G L Y - T Y R - I L E - S E R - L Y S - A L l r - E L N

378

Y-SUBUNIT

8-SUBUlyLT 340

P

360

SER-PRO-ASP-SER-LYS-PRO-THR-IL-ILE-SER-ARG-A~-~-~P-GW-~-PHE-ILE-ARG-LYS-PRO U-SUBUNn

P 334 LYS-ILE-PHE-ILE-ASP-THR-ILE-PRO-ASN-VAL-~~T-PHE-PHE-SER-THR-PIET-~ 314

FIG.5 . Proposed locations of the phosphorylated amino acid residues on the a-,p-, y. and &subunits of the nicotinic acetylcholine receptor. The kinases and their proposed phosphorylation sites are tyrosine-specific protein kinase @-subunit, Tyr-355; y-subunit, Tyr364; &subunit. Tyr-372). CAMP-dependent protein kinase (y-subunit, Ser 353; &subunit, Ser 361). and protein kinase C (a-subunit. Ser-333; &subunit, Ser-377).

served as a substrate for purified CAMP-dependent protein kinase while the other two peptides did not. In addition, the antibodies to peptide 354367 recognize the y- and &-subunitsby immunoblot techniques and also inhibited the phosphorylation of the y- and 3-subunits by CAMP-dependent protein kinase (Souroujon et al., 1986). The antibody to peptide 354-367 reacted well with nonphosphorylated receptor but reacted poorly with the phosphorylated receptor (Safran et al., 1986). These results strongly suggest that the CAMP-dependent phosphorylation site on 3 is located between residues 354 and 367 and that the site on the y-subunit is located on the homologous site between residues 346 and 369. The proposed phosphorylation sites for the CAMP-dependent protein kinase on the y- and 3-subunits have been recently demonstrated by protein sequencing techniques to be correct (Yee and Huganir, 1988). The purified nicotinic acetylcholine receptor was phosphorylated with purified catalytic subunit of CAMP-dependent protein kinase to a high stoichiometry. The y- and 8-subunits were then isolated by preparative SDS-polyacrylamide gel electrophoresis and chemically cleaved with CNBr. The CNBr-digested subunits were subjected to reversed-phase high-performance liquid chromatography (HPLC) using a Vydac C-18 column to isolate the phosphorylated peptides. These "P-labeled peptides were then digested with the protease trypsin. Phosphopeptides from the tryptic digest were separated by reversed-phase HPLC on a Vydac C-18 column. The phosphopeptides

8. NICOTINIC ACETYLCHOLINE RECEPTOR

155

were then pooled and sequenced on a gas phase sequencer. The sequences obtained for the tryptic peptides containing the CAMP-dependent protein phosphorylation sites were identical to those previously proposed (Huganir et al., 1984). The amino acid sequence immediately surrounding the phosphorylation site on the a-subunit of Torpedo californica is conserved in the amino acid sequences of human and calf a-subunit (Noda et al., 1983c), while both phosphorylation sites on the y-subunit of Torpedo are conserved in the sequences of the chicken y-subunit (Nef et al., 1984), and all three phosphorylation sites on the &-subunitare conserved in chicken (Nef et al., 1984) and mouse 8-subunit sequences (LaPolla et al., 1984). The conservation of the phosphorylation sites in different species suggests that phosphorylation is an important regulatory mechanism in receptor function. All of the proposed phosphorylation sites are located on a common region of each of the subunits with the three phosphorylation sites on the 8-subunit being within 20 amino acids of each other (see Fig. 5 ) . This suggests that phosphorylation of the acetylcholine receptor by these three protein kinases may be involved in regulating a common property of the receptor. The phosphorylation sites are located on the major intracellular loop between M3 and M5 in the theoretical models (Finer-Moore and Stroud, 1984) of the structure of the receptor subunits (see Fig. 3). Phosphorylation of these areas of the subunits may regulate the interaction of the subunits with cytoskeletal elements and affect the localization of the receptor in the membrane. Alternatively, phosphorylation of these areas, which are adjacent to the membrane-spanning region (MS)(see Fig. 3) that has been proposed to form the ion channel (Finer-Moore and Stroud, 1984), might regulate the ion channel properties of the receptor.

B. Physiological Significance of Phosphorylation of the Nicotinic Acetylcholine Receptor The physiological significance of the phosphorylation of the nicotinic acetylcholine receptor had been unclear for years. Phosphorylation-dephosphorylation is not necessary for the opening and closing of the ion channel since purified receptor preparations are active in the absence of ATP (Huganir and Racker, 1982; Tank el al., 1983) and have no detectable protein kinase activity (Huganir and Greengard, 1983). It has been proposed that phosphorylation of the receptor could modulate the ion channel properties of the receptor such as the mean channel open time, the conductance of the channel, and the cation selectivity or the rate of desensitization (Huganir and Greengard, 1983). Alternatively, it has also been proposed that phosphorylation could regulate other properties of the re-

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RICHARD L. HUGANIR

ceptor such as the localization and stabilization of the receptor at the synapse (Changeux, 1981; Changeux et al., 1984). Recently, the functional effects of phosphorylation of the nicotinic acetylcholine receptor by CAMP-dependent protein kinase have been directly examined (Huganir et al., 1986). The ion transport properties of the purified and reconstituted acetylcholine receptor were investigated before and after phosphorylation. It was found that phosphorylation of the nicotinic acetylcholine receptor on the y- and 8-subunits by CAMP-dependent protein kinase increases the rate of the rapid desensitization of the receptor, a process by which the receptor is inactivated in the presence of acetylcholine (Huganir et al., 1986). The nicotinic acetylcholine receptor in Torpedo californica postsynaptic membrane preparations was phosphorylated using purified catalytic subunit of CAMP-dependent protein kinase in the presence of ATP. Under the standard conditions used, the y- and b-subunits were each phosphorylated to a final stoichiometry of 0 . 6 0 . 7 mol phosphate per mole of subunit. Following the phosphorylation reaction, the membranes were solubilized with 1% sodium cholate and the nicotinic acetylcholine receptor purified by affinity chromatography (Huganir and Racker, 1982). Control nonphosphorylated receptor was prepared under identical conditions except that the catalytic subunit of CAMP-dependent protein kinase or ATP or both were omitted from the reaction mixture. (The three types of control preparations gave virtually identical results.) The purified receptor preparations were then reconstituted into phospholipid vesicles by cholate dialysis, and quench-flow and stop-flow rapid kinetic techniques were used to analyze the properties of the acetylcholinedependent ion transport of the nonphosphorylated and phosphorylated receptor preparations (Huganir et al., 1986). Using these reconstitution techniques, the initial rates of acetylcholinedependent ion transport by the nonphosphorylated and phosphorylated acetylcholine receptor were determined over a wide range of acetylcholine concentrations. The rates of ion transport of the nonphosphorylated and phosphorylated receptor had the same dependence on acetylcholine concentration. This indicated that the initial rate of ion transport and the dissociation constant of acetylcholine for the sites that activate the receptor was unchanged by phosphorylation (Huganir et al., 1986). In contrast, when the rates of the rapid desensitization of the nonphosphorylated and phosphorylated receptor were measured directly, a striking difference was observed (Fig. 6). The rapid desensitization was measured by a quench-flow technique by preincubation of reconstituted vesicles with acetylcholine for various periods of time before the determination of the rate of ion transport (Huganir et al., 1986). The data in Fig. 6 show the percent of ion transport activity remaining after prein-

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>-

t

L

5 10 0

1

2

3

4

PREINCUBATION TIME [SEC)

FIG.6 . Desensitization o f the nonphosphorylated (0) and phosphorylated (W) nicotinic acetylcholine receptor. The reconstituted vesicles were preincubated with 10 p M acetylcholine for the times indicated and then the ion transport activity was measured for 12 msec with 50 p M acetylcholine using "'Kb' (32). The data were fitted to the following equation using a nonlinear least-squares program: = ( J . , ) ! , l b t ~" I . where (J,,)/ is the ion transport rate coefficient after preincubation o f the receptor with acetylcholine for the period o f time (7) given on the abscissa of the graph. ( J s ) , ,, i s the ion transport rate coefficient prior to desensitization. and Q is the desensitization rate Coefficient. The "activity remaining" given Average data obtained with the three nonon the ordinate i s [ ( J J l = (Jn)/, ,)I x 100. 0. phosphorylated preparations (a = 0. I S 0.02 sec I): average data obtained with two preparations o f receptors phosphorylated to a stoichionietry o f 0.6 mol phosphate per mole y- and &subunits ( a = I . I 2 0. I sec - I ) . ~

..

cubation of the nonphosphorylated and phosphorylated receptor preparations with 10 pM acetylcholine for the indicated times. The ion transport activity of both receptor preparations decreased as the preincubation time was increased and was described by a first-order rate law. The rate of desensitization of the nonphosphorylated receptor was similar to the rate previously described for the rapid desensitization in Torpedo (Hess er d., 1982). The rate of desensitization of the phosphorylated receptor was 78 times faster than the rate of desensitization of the nonphosphorylated receptor (Huganir et al., 1986). Recent studies on the effect of agents that raise intracellular levels of cAMP on the function of the nicotinic acetylcholine receptor in rat muscle complement the in vitro studies on the regulation of desensitization of the nicotinic acetylcholine receptor by CAMP-dependent protein kinase (Middleton et ul., 1986a; Albuquerque er al., 1986). These studies have demonstrated that forskolin, a potent activator of adenylate cyclase and cAMP phosphodiesterase inhibitor promote a striking increase in the rate of de-

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sensitization of the nicotinic acetylcholine receptor in intact rat soleus muscle (Middleton et al., 1986a; Albuquerque er af., 1986). The desensitization of the nicotinic receptor was measured by studying the amplitude of the membrane depolarization evoked by consecutive pulses of iontophoretically applied acetylcholine. Brief acetylcholine pulses (0.2-2 msec) delivered at 7-8 Hz evoked constant responses at untreated end plates (Middleton et al., 1986a; Albuquerque et af., 1986). In contrast, after treatment of the muscle with 1.&100 p M forskolin, the amplitude of the response to consecutive pulses of acetylcholine was decreased by as much as 80% within 1 sec (Middleton et al., 1986a; Albuquerque et al., 1986). The sensitivity of the muscle to acetylcholine recovered completely within 1 sec after the pulses were stopped and fell again when the pulses were resumed (Middleton et al., 1986a; Albuquerque ef af., 1986). Similarly, when longer iontophoretic pulses ( I see) were applied to muscle, a relatively constant response was evoked at control end plates, but after forskolin treatment the depolarization decreased by 50% within 200 msec (Middleton et af., 1986a). These results indicate that forskolin increases the rate of desensitization of the nicotinic acetylcholine receptor in rat soleus muscle. It was also shown that inhibitors of CAMP-phosphodiesterase had effects similar but smaller to that of forskolin and that these inhibitors potentiated the effects of forskolin (Middleton et al., 1986a). Forskolin analogs that do not activate adenylate cyclase had no effect on desensitization (Albuquerque et af., 1986). It appears therefore that the effect of these agents on the desensitization rate of the receptor is mediated by increased levels of intracellular CAMP. To determine whether the increased rate of desensitization of the nicotinic acetylcholine receptor observed in rat muscle after forskolin treatment is due to phosphorylation of the receptor by CAMP-dependent protein kinase, the phosphorylation of the nicotinic receptor in rat rnyotubes has recently been studied (Anthony er af., 1986). Cultured primary rat myotubes were incubated with "P-orthophosphate for several hours and then exposed to forskolin in the presence and absence of phosphodiesterase inhibitors. The myotubes were then solubilized with nonionic detergents in the presence of phosphatase inhibitors and protease inhibitors and the nicotinic receptor purified by affinity chromatography and analyzed for phosphorylation (Anthony et al., 1986). Two subunits with similar molecular weights to those of the y- and b-subunits of Torpedo nicotinic acetylcholine receptor were observed to be basally phosphorylated (Anthony ef al., 1986). After the addition of forskolin or forskolin and a phosphodiesterase inhibitor, the phosphorylation of the apparent y- and b-subunits increased up to I0-fold. This effect of forskolin on the phosphorylation state of the receptor was dose-dependent with a half-maximal effect at

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-10 F M , which is in agreement with the dose-response curves for activation of adenylate cyclase by forskolin in many cell types (Seamon et al., 1981). The effect of forskolin on the phosphorylation of the nicotinic acetylcholine receptor was rapid, reaching a maximum within 5-10 min after treatment of the muscles with 10 p M forskolin (Anthony et al., 1986). The effect of forskolin on the rate of desensitization of the nicotinic acetylcholine receptor is also seen in cultured rat myotubes (Middleton et al., 1986b). Therefore, the observed physiological effects of forskolin treatment could be correlated with the phosphorylation state of the receptor in the cultured myotubes. It was found that the observed increase in the desensitization rate after forskolin treatment showed a very similar dose-response curve to the increase in the phosphorylation of the receptor subunits. In addition, the time course of the development of the increase in desensitization rate after forskolin treatment is identical to the increase in the phosphorylation of the receptor subunits (Middleton et al., 1986b; Anthony et al., 1986). These in vivo results, together with the in vitro results described earlier, provide strong evidence that phosphorylation of the nicotinic acetylcholine receptor by CAMP-dependent protein kinase increases the desensitization rate of the receptor. In contrast, the role of phosphorylation of the receptor by protein kinase C and the tyrosine-specific protein kinase is not clear. It has recently been reported that agents which activate protein kinase C (i.e., phorbol esters) increase the rate of desensitization of the nicotinic receptor in cultured myotubes (Eusebi et al., 1985). This suggests that protein kinase C phosphorylation of the receptor also increases the desensitization of the receptor. This is not surprising in light of the proposal (Huganir et a/., 1984) that the phosphorylation sites for CAMP-dependent protein kinase and protein kinase C on the &subunit are located within 20 amino acids of each other. Moreover, since the tyrosine phosphorylation sites are so close to the serine phosphorylation sites it is tempting to speculate that tyrosine phosphorylation regulates the desensitization of the receptor as well. IV.

CONCLUSIONS

Protein phosphorylation is one of the major regulatory mechanisms in the control of cellular metabolism and plays an important role in the regulation of ion channel function. The nicotinic acetylcholine receptor has been an excellent model system to study in detail the regulation of ion channels by protein phosphorylation. The nicotinic acetylcholine receptor is a multisubunit (a2py8) neurotransmitter-dependent ion channel which

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has been shown to be phosphorylated on seven different sites by three different protein kinases in postsynaptic membranes isolated from Torpedo californica. CAMP-dependent protein kinase phosphorylates the y- and 6-subunits, protein kinase C phosphorylates the 6- and a-subunits, while a tyrosine-specific protein kinase phosphorylates the p-, y-, and 6-subunits of the receptor. Phosphorylation of the y- and &subunits of the purified nicotinic receptor by purified CAMP-dependent protein kinase in vitro dramatically increases the rate of desensitization of the receptor. The nicotinic acetylcholine receptor in rat primary muscle cell cultures is basally phosphorylated and this phosphorylation is regulated by the intracellular concentration of CAMP. Stimulation of the phosphorylation of the receptor in response to increases in intracellular CAMPlevels is accompanied by an increase in the rate of desensitization of the muscle receptor. Phosphorylation of the receptor by protein kinase C also appears to regulate the rate of desensitization since treatment of muscle cells with agents which activate protein kinase C increases the rate of desensitization of the receptor. It is likely that phosphorylation of the nicotinic receptor by the tyrosine-specific protein kinase will also regulate the desensitization rate of the receptor. These results unequivocally demonstrate that protein phosphorylation of an ion channel regulates its function and suggest that phosphorylation of neurotransmitter-dependent ion channels in general may be a major mechanism in the regulation of synaptic transmission. REFERENCES Albuquerque, E. X..Deshpande, S. S., Aracava, Y.,Alkondon, M.. and Daly. J. W. (1986). A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor: A study with forskolin and its analogs. FEES Lett. 199, 113120.

Anthony, D. T.. Rubin, L. L., Miles, K., and Huganir, R. L. (1986). Forskolin regulates phosphorylation of the nicotinic acetylcholine receptor in rat primary muscle cell cultures. Abstr. Annu. Meet. Soc. Neurosci. 16th. p. 148. Barnekow, A., Schartl, B., Anders, F., and Bauer, A. (1982). Identification of a fish protein associated with a kinase activity and related to the rous sarcoma virus transforming protein. Cancer Res. 42, 2429-2433. Browning, M. D., Huganir, R. L., and Greengard, P. (1985). Protein phosphorylation and neuronal function. J . Neurochern. 45, 11-23, Changeux. J.-P. (1981). The acetylcholine receptor. An allosteric membrane protein. Harvey Lect. 75, 85-255. Changeux, J.-P., Devillers-Thiery, A., and Chemouilli, P. (1984). Acetylcholine receptor: an allosteric protein. Science 225, 1335-1345. Claudio, T., Ballivert, M., Patrick, J., and Heinemann, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo californicu acetylcholine receptor y subunit. Proc. Nur/.Acud. Sci. U . S . A . 80, 1111-1115. Cohen, P. (1982). The role of protein phosphorylation in neural and hormonal control of cellular activity. Nufure (London) 296, 613-620.

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Cotton. P. C.. and Brugge. J . S. (1983). Neural tissues express high levels o f f the cellular src gene product pp60’~”‘. Mol. Cell. B i d . 3, 1157-1 162. Davis, C. G.. Gordon, A. S., and Diamond, I. (1982). Specificity and localization of the acetylcholine receptor kinase. Proc. Null. Acud. Sci. U . S . A . 79, 3666-3670. Devillers-Thiery, A., Giraudat, J., Bentaboulet, M.. and Changeux, J.-P. (1983). Complete mRNA coding sequence of the acetylcholine binding a-subunit of Torpedo murmorura acetylcholine receptor: A model for the transmembrane organization of polypeptide chain. Proc. Nutl. Acad. Sci. U . S . A . 80, 2067-2071. Eusebi. F.. Molinaro, M., and Zani, B. M. (1985). Agents that activate protein kinase C reduce acetylcholine sensitivity in cultured myotubes. J . Cell B i d . 100, 1339-1342. Finer-Moore, J., and Stroud, R. M. (1984). Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. Nurl. A u d . Sci. U . S . A . 81, 155159.

Gordon, A. S.. Davis, C. G., and Diamond, I. (1977a). Phosphorylation of membrane proteins at a cholinergic synapse. Proc. Null. Acud. Sci. U . S . A . 74, 263-267. Gordon, A. S., Davis, C. G., Milfay, D.. and Diamond, I. (1977b). Phosphorylation of acetylcholine receptor by endogenous membrane protein kinase in receptor enriched membranes of Torpedo culifornica. Nutitre (London) 267, 539-542. Hess. G. P.. Pasquale. E. B., Walker, J . W., and McNamee, M. G. (1982). Comparison of acetylcholine receptor-controlled cation flux in membrane vesicles from Torpedo i d ifornicu and Electrophoriis electricits: Chemical kinetic measurements in the millisecond region. Proc. N u / / . Acud. Sci. U . S . A . 79, 963-967. Huganir, R. L. (1986a). Biochemical mechanisms in the modulation of the ion channel function. In “Neuromodulation” (L. Kaczmarek and 1. Levitan. eds.). Raven. New York. In press. Huganir. R. L. (1986b). Phosphorylation of purified ion channel proteins. I n “Neuromodulation” (L. Kaczmarek and I. Levitan. eds.). Raven. New York. In press. Huganir, R. L., and Greengard, P. (1983). CAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc. Null. Acud. Sci. U . S . A . 80, 1130-1134. Huganir. R. L., and Racker, E. (1982). Properties of proteoliposomes reconstituted with acetylcholine receptor from Torpedo colifornicu. J . B i d . Chem. 257, 9372-9378. Huganir, R. L., Albert, K. A.. and Greengard. P. (1983). Phosphorylation of the nicotinic acetylcholine receptor by Ca”lphospho1ipid-dependent protein kinase, and comparison with its phosphorylation by CAMP-dependent protein kinase. Soc. Neurosci. Ahstr. 9, 578. Huganir, R. L., Miles, K., and Greengard. P. (1984). Phosphorylation of the nicotinic acetylcholine receptor by an endogenous tyrosine-specific protein kinase. Proc. N d . Acud. Sci. U . S . A . 81,6963-6972. Huganir, R. L.. Delcour, A. H.. Greengard, P., and Hess, G. P. (1986). Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Ntitrrre (London) 321, 774-776. Hunter, T. (1982). Synthetic peptide substrates for a tyrosine protein kinase. J. B i d . Chem. 257, 48434848. Hunter. T., and Cooper, J. A. (1985). Protein-tyrosine kinases. Annu. Rev. Biochem. 54, 897-930. Hunter, T., Ling. N.. and Cooper, J . A. (1984). Protein kinase C phosphorylation of the EGF receptor at a threonine residue close to the cytoplasmic face of the plasma membrane. Nurure (London) 311, 450483. Ingebritsen, T. S.. and Cohen. P. (1983). Protein phosphatases: Properties and role in cellular regulation. Science 221, 33 1-338.

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Kemp. B. E., Graves, D. J., Benjamin, E., and Krebs, E. G. (1977). Role of multiple basic residues in determining the substrate specificity of cyclic AMP-dependent protein kinase. J. Biol. Chem. 252, 48884894. LaPolla, R. J., Mayne, K. M., and Davidson, N. (1984). Isolation and characterization of cDNA clone for the complete protein coding region of the 6 subunit of the mouse acetylcholine receptor. Proc. Natl. Acad. Sci. U.S.A. 81, 7970-7974. Levy, B. T., Sorge, L. K., Meymandi, A., and Maness, P. F. (1984). pp60'-'" kinase is in chick and human embryonic tissues. Dev. Biol. 104, 9-17. Middleton, P., Jaramillo, F., and Schuetze, S. M. (1986a). Forskolin increases the rate of acetylcholine receptor desensitization at rat soleus endplates. Proc. Natl. Acad. Sci. U . S . A . 83, 4967-4971. Middleton, P., Rubin, L. L., and Schuetze, S. M. (1986b). Forskolin increases the rate of acetylcholine receptor desensitization on rat myotubes in vitro. Abstr. Annu. Meet. SOC.Neurosci. 16th, p. 148. Nairn, A. C., Hemmings, H. C., Jr., and Greengard, P. (1985). Protein kinases in the brain. Annu. Rev. Biochem. 54, 931-976. Nef, P., Mauron, A., Stalder, R., Alliod, C., and Ballivet, M. (1984). Structure, linkage, and sequence of the two genes encoding the 6 and y subunits of the nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. U . S . A . 81, 7975-7979. Nishizuka. Y.(1980). Three multifunctional protein kinase systems in transmembrane control. Mol. Biol. Biochem. Biophys. 32. Noda, M.. Takahashi, H.. Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T.. Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982). Primary structure ofu-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nuture (London) 299,793-797. Noda, M., Takahashi, H., Tanabe. T., Toyosato, M., Kikyotani, S., Furutani, Y.. Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983a). Structural homology of Torpedo californica acetylcholine receptor subunits. Nature (London) 302, 528-532. Noda, M., Takahashi, H., Hirose, T.. Asai, M., Takashima, H., Inayama, S.. Miyate. T., and Numa, S. (1983b). Primary structures of p- and y-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature (London) 301, 251-255. Noda, M., Furutani, Y ., Takahashi, H., Toyosato, M., Tanabe, T., Shimizu. S., Kikyotani, S., Kayano, T., Hirose, T., Inayama, S., and Numa, S. (1983~).Cloning and sequence analysis of calf cDNA and human genomic DNA encoding a-subunit precursor of muscle acetylcholine receptor. Nature (London) 305, 818-823. Patschinsky. T., Hunter, T., Esch, F. S., Cooper, J. A., and Sefton, B. M. (1982).Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc. Natl. Acad. Sci. U . S . A . 19, 973-977. Pike, L. J., Gallis, B., Casnellie, J. E., Bornstein, P., and Krebs, E. G. (1982). Proc. Natl. Acad. Sci. U . S . A . 19, 1443-1447. Reynolds, J. A., and Karlin, A. (1978). Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry 11, 2035. Safran, A., Neumann, D., and Fuchs, S. (1986). Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies. EMBO J . 5, 3175-3178. Saitoh, T., and Changeux. J.-P. (1981).Change in the state of phosphorylation of acetylcholine receptor during maturation of the electromotor synapse in Torpedo mrrrmorutct electric organ. Proc. Nut/. Acud. Sci. U.S.A. 18, 44304434. Seamon, K. B., Padgett, W.. and Daly, J. W. (1981).Forskolin: Unique diterpene activator

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of adenylate cyclase in membranes and in intact cells. Proc. Nurl. Acud. Sci. U . S . A . 78, 3363-3367. Sefton. B. M., and Hunter, T. (1984). Tyrosine protein kinases. In Adv. Cvclic Nucleotide Protein Phosphorylation Res. 18, 195-226. Smilowitz, H.. Hadjian, R. A.. Dwyer, J . , and Feinstein, M. B. (1981). Regulation of acetylcholine receptor phosphorylation by calcium and calmodulin. Proc. Null. Acad. Sci. U . S . A . 78, 47084712. Sorge, J . P., Sorge. L. K.. and Maness, P. F. (1984). pp60'~"" is expressed in human fetal and adult brain. Am. J. Purhol. 119, 151-157. Souroujon. M. C., Neumann. D.. Pizzighella, S., Fridkin, M., and Fuchs. S. ( 1986). Mapping of the CAMP-dependent phosphorylation sites on the acetylcholine receptor. EMBO J . 5, 543-546. Tank. D. W., Huganir, R. L., Greengard, P.. and Webb, W. W. (1983). Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor. Proc. Natl. Acud. Sci. U.S.A. 80, 5129-5133. Teichberg, V. I . , and Changeux. J.-P. (1977). Evidence for protein phosphorylation and dephosphorylation in membrane fragments isolated from the electric organs of Electrophorus elecrricus. FEBS Lett. 74, 76. Teichberg, V. I . , Sobel, A., and Changeux. J.-P. (1977). I n virro phosphorylation of the acetylcholine receptor. Narure (London) 267, 540-542. Yee, G. H.. and Huganir. R. L. (1987). Determination of the sites of CAMP-dependent phosphorylation on the nicotinic acetylcholine receptor. J. B i d . Chern. 262, 1674816753.

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CURRENT TOPICS IN MEMBRANES A N D TRANSPORT, VOLUME 33

Chapter 9 Synthetic Peptides in the Study of the Nicotinic Acetylcholine Receptor EDWARD HAWROT, KIMBERLY L . COLSON, THOMAS L . LENTZ,* AND PAUL T. WILSON* Departments of Pharmacology and *Cell Biology Yale University School of Medicine New Haven, Connecticut 06510

1. Overview 11. Synthetic Peptides in the Application of lmmunochemical Tests of AChR Structure A. The Use of Synthetic Peptides to Generate Antibodies to Predetermined Domains B. Domain-directed Antibodies in the Topological Mapping of the AChR 111. Functional Activities Associated with Synthetic Peptides A. A Synthetic Peptide Corresponding to Sequences within the a-Subunit of the AChR Binds a-Bungarotoxin B. Receptor Binding Activities of Synthetic Peptides Derived from Curaremimetic Neurotoxin Sequences and from Rabies Virus Glycoprotein Sequences C. Other Methodologies for Detecting Functional Activity IV. Determination of the Solution Conformation of Small Synthetic Peptides Relevant to the Ligand-Binding Site of the AChR References

1.

OVERVIEW

As the technologies for DNA sequencing and the synthesis of small polypeptides have advanced in the last few years, a wide range of exciting, new approaches to questions involving protein structure and function have been introduced which could not have been foreseen even a few years ago. With the advent and refinement of recombinant DNA techniques, the traditional role of protein purification as a means of obtaining sequence and structural information has been diverted to one of obtaining enough 165 Copyright 1 4 IYXX by Academic h e \ \ . Inc. All right\ of reproduction in any form reherved.

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sequence information to allow production of synthetic oligonucleotides that can be used as probes of appropriate cDNA libraries. Alternatively, protein purification is used to generate sufficient protein that specific antibodies can be prepared and used to screen expression libraries for the gene of interest. Application of these strategies has yielded a large number of primary amino acid sequences corresponding either to known or hypothetical proteins. Current areas of interest center on defining the function of putative protein sequences, predicting the tertiary structure of the encoded protein sequences, assigning functionality to particular structural domains, and in the case of membrane proteins, predicting the transmembrane disposition of the polypeptide chain. A major goal in protein chemistry is to understand the rules governing how primary amino acid sequences specify the folding of peptide chains; this involves the integration of information provided by X-ray crystallography, NMR assignments of interproton distances (which can contribute to restraints on solution conformation), and the calculated molecular dynamic simulations of protein structure (Clore et al., 1985). In all of these areas, important contributions will be made through the study of synthetic peptides corresponding to protein regions of particular structural interest. Ideally, we would like to be able to use primary amino acid sequence information to assign specific functional domains such as ligand-binding sites or iop channels, to particular sequences. Based upon analysis of the primary amino acid sequence of the nicotinic acetylcholine receptor (AChR) subunits, Finer-Moore and Stroud (1984) and Guy (1984) propose a model involving amphipathic helices in the formation of the AChR ion channel. Testing such predictions necessitates the application of various new experimental strategies including site-directed mutagenesis (Mishina et al., 1985), production of chimeric fusion proteins in expression systems (Barkas et al., 1987), and the use of domain-directed antibodies (see, e.g., Ratnam et al., 1986a,b). In the latter approach, synthetic peptides are proving to be very instrumental in facilitating the interpretation of experimental results. This chapter focuses on the information obtained through the use of synthetic peptides corresponding to the primary amino acid sequence of the four subunits in the nicotinic AChR. The discussion includes how such synthetic peptides have facilitated the identification of the bungarotoxin (BGTX)-binding site on the a-subunit, how peptides have been used to identify specificities of preexisting monoclonal antibodies (MAbs), and how some peptides have facilitated the production of MAbs with domaindirected specificity. Similar approaches involving synthetic peptides may prove useful in the study of other membrane proteins.

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II. SYNTHETIC PEPTIDES IN THE APPLICATION OF IMMUNOCHEMICAL TESTS OF AChR STRUCTURE A. The Use of Synthetic Peptides to Generate Antibodies to Predetermined Domains

Much of the impetus for the use of synthetic peptides to generate sitespecific antibodies arose following a number of articles demonstrating that small (10-30 amino acid) peptides were efficient in eliciting antisera that cross-react with the native protein from which the sequence was obtained (Shinnick et af., 1983; Sutcliffe et al., 1983; Niman et al., 1983). Furthermore, it appeared that certain amino acid sequences, which were normally not immunogenic when the entire native protein was used as immunogen, could however, elicit an immune response when the appropriate short peptides were used as immunogens. It now appears that almost any part of the protein surface may be antigenic under the appropriate conditions despite the fact that antisera raised against native protein are restricted to a limited number of sites (epitopes) (Benjamin et al., 1984). In general, in order to elicit a strong immune response, short peptides need to be coupled to a carrier protein such as bovine serum albumin prior to being used as an immunogen. Both antisera and MAbs can be raised in this way, although carrier coupling may not be necessary for some longer peptides (>23 residues) (Benjamin et al., 1984). 6. Domain-Directed Antibodies in the Topological Mapping of the AChR

I . MEMBRANE-SIDEDNESS OF T H E N SUBUNITS

AND

C-TERMINI OF THE AChR

Since the four subunits (a,p, -y, 6) of the AChR are highly homologous, it is probable that the transmembrane orientation of the four subunits will be very similar. Several models have been proposed to describe the topology of the peptide chain within the membrane and with respect to the external (ectodomain) and internal (cytoplasmic) faces of the membrane (Claudio et ul., 1983; Noda et ul., 1983; Guy, 1984; Criado et d., 1985a,b; Finer-Moore and Stroud, 1984). One strategy of experimental verification of the models focuses on the orientation of the N- and C-termini of the polypeptide chain. There is some direct indication that the N-terminus of the &-chainlies in the ectodomain or external face of the plasma membrane. The evidence for this comes from analysis of the protease sensitivity of

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&-subunit synthesized in a cell-free system utilizing dog pancreas microsomal membranes (Anderson et al., 1982). The orientation of microsomal membrane vesicles is the reverse of that for the intact plasma membrane, with the cytoplasmic face of the microsomal membrane being susceptible to added proteases. It was shown that the N-terminus of the membrane-integrated 8-subunit is resistant to cleavage by externally added protease and remains in association with a 44kDa membrane-associated fragment. Furthermore, partial sequence analysis demonstrates that a 21-residue leader sequence is removed from the &-subunit during the process of membrane integration (Anderson et ul., 1982), as would be expected if the leader sequence were removed by a signal peptidase located on the lumenal face of the vesicles. Due to the inaccessibility of the N-terminus to protease digestion in the microsomal system and the cleavage of the leader sequence, it was concluded that the N-terminus must lie on the external face of the plasma membrane. Attempts have been made to directly demonstrate the proposed localization using antibodies directed to the N-terminus. Neumann et ul. (1984) prepared antisera to the N-terminal20 amino acids from the a-subunit of Torpedo AChR whereas Ratnam and Lindstrom (1984) prepared antisera to the N-terminal 10 amino acids from each of the four subunits of Torpedo AChR. Although antisera to an N-terminal 20-amino acid peptide reacted well with SDS-denatured AChR, the reactivity with AChR after solubilization with nonionic detergents was greatly reduced. Moreover, the antisera did not react appreciably with AChR-enriched Torpedo electric organ membranes (Neumann et al., 1984). These findings suggested that the Nterminus of the membrane-bound AChR is either not exposed or is in such a conformation that it cannot be recognized by the antisera. Even when solubilized with a nondenaturing detergent, the N-terminus is apparently not as accessible as it is in the denatured form of the AChR. Ratnam and Lindstrom (1984) arrived at the same conclusion with their antisera. They showed that the N-terminal 10-amino acid peptide from the @-subunitdid produce antisera which could react with denatured P-subunit. These antisera did not, however, react with membrane-bound AChR, nor with lithium diiodosalicylate (LIS) permeabilized vesicles, suggesting that the N-terminus of the @-subunit is either buried or sterically constrained in the native receptor. Therefore, there is as yet no direct evidence that the Nterminus of any of the subunits lies in the ectodomain of the AChR. Despite the lack of direct evidence, it is generally assumed that the Nterminus is on the external side of the plasma membrane based on the cell-free synthesis studies. Localization of the C-terminus region would provide information as to how many crossings of the membrane would

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be required for the proper transmembrane disposition of the N- and Ctermini. If the C-terminus were also in the ectodomain, then an even number of membrane crossings would be required (e.g., model A in Fig. I ) . On the other hand, if the C-terminus is on the cytoplasmic face then an odd number of membrane crossings would be necessary, disproving the original models (Noda el a / . , 1983; Claudio et n l . , 1983) (see models BD. Fig. I ) . Ratnam and Lindstrom (1984) prepared antisera to the C-terminal 10 amino acid peptides for each of the four subunits by conjugating the peptides to keyhole limpet hemocyanin using bisdiazatized benzidine. The peptide conjugates were immunogenic and the resulting antisera reacted with the corresponding denatured subunits, with solubilized receptor, and with Torpedo membrane vesicles that had been permeabilized with LIS. The antisera did not react, however, with intact membrane vesicles, suggesting that the C-termini of all four subunits are on the cytoplasmic face of the plasma membrane. The findings of Young ef t i / . (1985) who prepared antisera to a C-terminal 16-aminoacid peptide corresponding to the Torpedo 6-subunit support this conclusion. Antisera reacted with Torpc~domembranes only after detergent permeabilization. I n addition, on the basis of electron microscopy with colloidal gold-conjugated second antibodies, it was possible to obtain ultrastructural evidence that the Cterminus of the 6-subunit is on the cytoplasmic face of the plasma membrane. Antisera raised against a peptide containing residues 350-358 of the p-subunit reacted in the same way, suggesting that this sequence is also on the cytoplasmic surface and that there must be either no or an even number of transmembrane segments between residues 358 and the C-terminus. These observations supported the model depicting five transmembrane segments, with one of the segments, M5 (see model B, Fig. I ) , being an amphipathic a-helix that could form the lining of the ion channel (Guy, 1984; Finer-Moore and Stroud. 1984). The conclusion that the C-termini of the subunits are on the cytoplasmic surface was also confirmed using preexisting subunit-specific antisera and anti-p-subunit MAbs. The presumed epitopes for these antibodies were delineated by examination of their cross-reactivity with a panel of synthetic peptides corresponding to the C-termini of the four subunits (Lindstrom ef a / . , 1984). Three MAbs reacted with the p C-terminal peptide. and three polyclonal antisera reacted with the p, y, and 6 C-terminal peptides. respectively. No antisera were found which reacted specifically with the a C-terminus. All three antisera and the three anti-p MAbs (#163, 170, and 172) reacted with AChR-rich membranes only after the membranes were permeabilized, providing further evidence that the C-terminus is on the cytoplasmic side of the plasma membrane.

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FIG. I . Four models are presented that depict the possible transmembrane orientation of the a polypeptide chain in the Torpedo AChR. Model A is an adaptation of the essential features of the model as proposed by Claudio el (I/. (1983) and Noda i>f a / . ( 1983) which postulated four transmembrane a-helices (i.e.. MI, M2. M3, M4). Model B includes the fifth transmembranous amphipathic a-helix. M5, suggested by Guy ( 1984) and Finer-Moore and Stroud (1984), placing the C-terminus on the cytoplasmic face of the membrane. Model C. as described by Ratnam ef ul. (1986b) also proposed five transmembrane crossings of the polypeptide chain but substituted regions M6 and M7 for M4 and M5 based on immunochemical evidence. Model D suggests that an orientation with only three transmembrane crossings involving the original MI, M2. and M3 helices can account for almost all of the observed experimental findings. The only data not accounted for in this model are the suggested cytoplasmic localization of MAbs directed to sequences 152-159 of a (Criado ('I d., 1985a).

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

2.

171

IDENTIFICATION A N D MAPPINGOF PROTEOLYTIC FRAGMENTS OF AChR SUBUNITS U S I N G ANTI-PEPTIDE ANIIBODIES

Since many antibodies prepared against peptides corresponding to AChR sequences react with the denatured form of the AChR subunits, these antibodies can serve as useful reagents in identifying the origin of proteolytic fragments derived from AChR subunits. The procedure for accomplishing this identification rests on the ability to immobilize resolved fragments by protein blotting. The immobilized fragments can then be specifically visualized by the binding of appropriate anti-peptide antibodies. For example, Barkas et al. (1984) prepared antisera to two peptides, PI, which corresponds to residues 151-169 of a. and P3. which corresponds to the C-terminus of a, residues 426-437. After trypsinization of Torprdo membranes, a 35-kDa fragment and a 9- to 10-kDa fragment were obtained. The 35-kDa fragment labeled with the anti-PI antisera, whereas the smaller fragment labeled with the anti-P3 antisera on protein blots. These findings suggested that the smaller fragment contained the C-terminus and the larger fragment contained the sequences 151-169. Similarly, anti-peptide antibodies were used to identify proteolytic fragments of the a-subunit derived from V8 protease digestion (Neumann et d.,1985). Antisera to peptide 1-20 and to peptide 126-143 of a were used in immunoblotting experiments to show that a 26-kDa fragment labeled with both antisera, and therefore must contain the N-terminus and extend as far as amino acids 126-143. On the other hand, a 17-kDa proteolytic fragment labeled only with the peptide 126-143 antisera. An IS-kDa fragment that bound a BGTX probe (see below) was not labeled with either antisera and conversely, the 26- and the 17-kDa fragments did not bind BGTX. It was concluded that the toxin-binding site must reside on the C-terminal side of the first potential V8 protease cleavage site following residues 126-143, namely aspartate-152. Similar strategies can be used to map any other sequence specific sites that can be probed by immunoblotting. These would include putative glycosylation sites (that could be probed by binding of the appropriate labeled lectins). sequences recognized by MAbs that had been generated against the intact protein, and sequences that can bind any other ligands or cofactors. Ratnam et d.(l986a) used MAb immunoblotting to localize the antigenic determinants on the primary amino acid sequence of AChR subunits with respect to the C-terminus. The C-terminal-derived proteolytic fragments, identified by reaction with anti-C-terminal peptide antisera, were tested for MAb binding, using a library of previously prepared MAbs. The size of the cross-reacting fragments set approximate limits to the sequences specifying the MAb-binding sites. In this way, it was possible to

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deduce the approximate location of the residues recognized by various MAbs. In those cases where a particular MAb produces marked functional effects upon binding to the target protein sequence, such analyses would, in general, be very important in identifying the sequences responsible for those particular functional domains. 3. ANTI-PEPTIDE ANTIBODIES IN T H E LOCALIZATION OF THE MAIN

IMMUNOGENIC REGION ON

THE

SUBUNIT OF T H E AChR

The general strategy described above has also been applied to localize the sequences specifying the so-called main-immunogenic region (MIR) on the a-subunit. The MIR is made up of a set of conformationally dependent epitopes that bind the majority of anti-receptor antibodies found in sera from patients with myasthenia gravis (Tzartos et a / . , 1982). When animals are immunized with nondenatured, detergent-solubilized Torpedo AChR, the majority of the antibodies produced bind to the MIR as defined above. Experimentally, anti-MIR antibodies are most readily assayed by competition with an established anti-MIR MAb (Tzartos and Lindstrom, 1980). It was initially proposed that the highly hydrophilic region centering around residues 161-166 on the a-subunit might represent an important antigenic site such as the MIR (Noda et al., 1983). Juillerat et ul. (1984) prepared a peptide of sequence 151-169 and showed that antisera to nondenatured AChR did not recognize this peptide and that three MAbs with specificity for the MIR did not bind to this peptide. In addition, antisera to peptide (a151-169) bound to denatured AChR but not to the nondenatured AChR, suggesting that residues 15 1-169 are normally inaccessible to antibodies. Similar conclusions were reached by Lindstrom et LII. (1984) using peptides corresponding to residues 159-169 and 152-167 of the asubunit. Of six MAbs which reacted both with the MIR and with denatured a-subunit, none bound to peptide (1x159-169). Furthermore, a MAb directed against peptide (a152-167) bound to intact AChR in a solid-phase enzyme-linked immunosorbent assay (ELISA) but the binding could not be competed by other MAbs with known specificity for the MIR. Furthermore, this MAb failed to bind to native membrane-bound AChR (Criado e t a / . , 1985a). It was concluded that the sequence 152-167 is not part of the MIR and probably is not contiguous with the MIR on the surface of the AChR. By analyzing papain-derived proteolytic fragments of the a-subunit with anti-MIR MAbs and with antisera to peptide P1 (a151-169), Barkas e t a / . (1986) showed that the sequences making up the MIR must be located on the N-terminal side of all or part of the region containing residues 151169. It was concluded that a papain cleavage site must lie between as-

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173

paragine-141, the apparent site of N-linked gycosylation (Noda et ul., 1983). and the region defined by peptide PI. In a separate study, Ratnam er ul. (1986a) utilized a similar approach to localize the binding site for MAb 210, a MAb against the MIR. They concluded that the MIR must lie between residues 46 and 127 on the a-subunit, consistent with the findings of Barkas et ul. (1986). Recent findings with fusion proteins expressed in Escherichiu coli (Barkas et ul., 1987) suggest that residues 6-85 are sufficient to form a binding site for six MAbs directed against the MIR. However, not all MAbs to the MIR reacted with this fusion protein, consistent with the notion that several epitopes may contribute to the MIR. Three of the six MAbs also reacted with a fusion protein containing residues 37-216. Thus at least one epitope of the MIR may lie between residues 37 and 85, the overlap region defined by the two fusion proteins. Combining these results with the conclusions of Ratnam et al. (1986a) suggests that the sequence between residues 46 and 85 may provide one epitope for the MIR. Synthetic peptides corresponding to 35% of the entire primary sequence of the a-subunit were used by Ratnam et al. (1986a) to map the overall antigenic structure of this subunit. Antisera raised against denatured asubunit or against whole AChR were tested for immunoprecipitation of radioactively labeled synthetic peptides. It appears that 73% of the total antibody titer in antisera to the denatured a-subunit is directed against less than 20% of the primary sequence, most of these sequences being near the C-terminal region of the a-subunit. A similar distribution of binding specificities was observed when antisera to nondenatured AChR were tested against synthetic peptides. The binding activities observed do not account for the total titer since many antibodies in such an antiserum are directed toward conformationally sensitive domains which cannot be detected by immunoblotting or by immunoprecipitation of denatured subunits and short peptides. Based on the inability to identify many anti-peptide specificities in an analysis of anti-AChR antisera, Ratnam et LII. (1986a) suggest that much of the primary amino acid sequence of the a-subunit is immunologically silent. Given the bias of the assays used in this study, however, it is possible that many of the “silent” antigenic determinants are conformationally dependent. Souroujon et ul. (1986) mapped in detail the binding regions of seven MAbs to the a-subunit by imrnunoblotting as previously described. Five of the seven MAbs bound to a I4-kDa, V8 protease-derived fragment that shared recognition with an antiserum to a peptide corresponding to residues 351-368 of the a-subunit. Thus, the 14-kDa fragment was derived from the C-terminal portion of the a-subunit and the majority of the tested MAbs (5 of 7) reacted with the C-terminal region. Both studies are in general

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agreement with the work of Froehner (l98l), who showed that when denatured AChR or subunits were used as immunogens, the majority of the antibodies did not bind to domains accessible on the membrane-bound AChR, but rather bound to the cytoplasmic domain of the AChR. Now that there is considerable evidence that the C-termini of the subunits are oriented within the cytoplasm, these earlier observations can readily be accounted for. 4. ANTI-PEPTIDE ANTIBODIES A S PROBES OF T H E LIGAND-BINDING SITEON THE AChR

With native AChR as an immunogen, a number of MAbs have been prepared that bind to the ligand-binding site of the AChR. This conclusion is based on the finding that these MAbs can compete with cholinergic ligands in binding studies (see, e.g., Mochly-Rosen and Fuchs, 1981). Although these MAbs would appear to be excellent candidates for use in an immunoblot analysis to deduce the location of the ligand-binding site, this has not been the case. To date, MAbs that bind to the cholinergic ligand-binding site react very poorly, if at all, with the denatured a-subunit. Based upon inspection of the primary amino acid sequence of the asubunit, it had initially been proposed that the region between cysteines 128 and 142 might be involved in the formation of the ligand-binding site (Noda et al., 1983). To test this hypothesis, Plumer et al. (1984) prepared antisera and MAbs to a peptide corresponding to amino acids 127-132 of the a-subunit. The antibodies to this peptide (127-132) were specific for the a-subunit and cross-reacted with native, denatured, or membranebound AChR. High concentrations of various cholinergic ligands including a-cobratoxin failed to interfere with the binding of these anti-peptide (127132) MAbs, suggesting that residues 127-132 were not part of the cholinergic binding site. In another study, Lennon e? al. (1985) reported that a peptide with sequence 125-147 of a could induce experimental autoimmune myasthenia gravis in rats and that this region was accessible on the surface of membrane-bound AChR. Others, however, have not observed muscle weakness following immunization with peptides corresponding to the same region. Furthermore, antibodies against the 125-147 peptide did not interfere with BGTX binding to the AChR, in agreement with the studies of Plumer ef al. (1984). Criado et al. (1986) prepared a synthetic peptide of a residues 127-143 that was cyclized by the disulfide bond between cysteines 128 and 142. Several MAbs prepared against this peptide recognized purified AChR in a solid-phase ELISA, but reacted considerably less with membrane-bound AChR, suggesting that this sequence (128-142) is not fully exposed on the

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175

extracellular surface of the AChR. High concentrations of the cholinergic ligands, carbamylcholine, benzoquinonium, tubocurarine, and a-BGTX, produced very little interference with binding of the MAbs. Conversely, addition of excess MAb had little if any effect on the binding of labeled BGTX to AChR. Based on the lack of cross-interference between MAbs and cholinergic ligands, it was concluded that this region (residues 127143) was not a part of the acetylcholine-binding site. When existing antisera to the a-subunit were tested for immunoprecipitation of the synthetic peptide (127-143). only I% of the antibodies cross-reacted with the peptide and none of 34 MAbs against the a-subunit bound this peptide. Thus, the cyclized peptide (127-143) does not appear to be a major antigenic determinant for immune recognition. Although Lennon e f al. (1985) had suggested that the region of 125-147 is a major antigenic site, their conclusion is not consistent with the findings of Criado et al. (1986) or with Souroujon et ul. (1986).

5. FURTHER REFINEMENT A N D DEFINITION ORIENTATION OF THE AChR SUBUNITS

OF THE

TRANSMEMBKANE

In the course of mapping the antigenic determinants along the primary amino acid sequence of the P-subunit, Ratnam et trl. (1986b) obtained evidence that the sequence 429-441 was on the cytoplasmic side of the plasma membrane. According to the model proposed by Stroud, however (see Fig. IB), sequence 429-441 should be just to the N-terminal side of M4. If the C-terminal is indeed cytoplasmic and M4 is a transmembrane region, then it follows that the sequence 429-441 should be extracellular, linking M4 and M5. The evidence placing sequence 429-441 to the intracellular domain was based on findings using MAb 125, an antibody raised against Torpedo AChR which was roughly mapped to the region 429-441 on the basis of cross-reaction with C-terminal-containing proteolytic fragments of the p-subunit . Electron microscopy with colloidal gold-labeled second antibodies was used to demonstrate that MAb 125 bound to the intracellular side of the Torpedo membranes, placing this sequence on the intracellular side. Since the observed transmembrane binding of MAb 125 was not consistent with the localization of M4 as a transmembrane segment (assuming the C-terminus is intracellular), further studies were performed using the analogous region on the a-subunit. Five peptides were synthesized covering the region between residues 330 and 408 of the a-subunit (Ratnam et al., 1986b). When screened with an existing library of MAbs to the AChR, MAbs to four of the five peptides were identified and used for additional studies. Antisera were prepared against the synthetic peptide

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EDWARD HAWROT ET AL.

(389-408) in order to provide a probe for the region not covered by any of the existing MAbs. Immunochemical studies as well as electron microscopy suggested that residues 330-408 were intracellular. Since M5, the amphipathic helix, was predicted to include residues 364-390, these results contradicted the predictions of the amphipathic-based model (see Fig. IB) and argue that the hydrophobic helix M4 and the amphipathic helix M5 are not transmembranous segments. These two regions may, however, be membrane associated to some degree. The immunochemical evidence that MI is a transmembrane segment comes from the finding that a MAb (#255) that binds to an (Y peptide with sequence of 235-242 localizes to the intracellular side of Torpedo membranes, as determined by the use of intact or permeabilized vesicles (Criado et al., 1985b). All current models (Fig. I , A-D) predict that the region 235-242 acts as a cytoplasmic linker between M I and M2. Since MAb 255 binds to the intracellular side and the N-terminus is presumed to be external, there must be at least one or an odd number of transmembrane segments between the N-terminus and residues 235-242 of a. If the putative M2 and M3 helices are also transmembrane regions, then residues 262277 should be on the external face of the plasma membrane. This has not yet been tested directly. The finding by Criado et ul. (1985a) that the (Y sequence 152-159 may also lie on the cytoplasmic side of Torpedo membrane vesicles contradicts the earlier models (Fig. IA and B). Their localization is based on the binding characteristics of two MAbs [#236 and 237 (similar to #236 but of lower affinity)] to intact or permeabilized Torpedo vesicles. A synthetic peptide ((~152-167)is recognized by MAb 236 which does not, however, bind to another, shorter, overlapping peptide (a159-169). From this it was concluded that the specificity of MAb 236 must be to residues 152-159. This MAb (#236) did not bind to intact, right-side-out, Torpedo membrane vesicles but did bind to permeabilized vesicles, arguing that the sequence 152-159 must be on the cytoplasmic face. In order to accommodate this observation with the presumed external location of the N-linked glycosylation site (ASN-141) and of the two cysteines at positions 192 and 193 (since they can be affinity alkylated in intact membranes; see Kao et nl., 1984), there must be another pair of transmembrane segments between residues 141 and 192. Ratnam et uf. (1986b) proposed two new transmembrane segments, M6, roughly from 143 to I5 I , and M7. an amphipathic helix extending approximately from residues 160 to 191 (Fig. 1 0 . Since the M6 region is too short to form a helix, this model requires some other secondary structure. Further, this model places the region containing residues 161-166, previously hypothesized to be the MIR because of its hydrophilic nature, in the midst of a transmembrane segment (M7). Since

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177

an electron microscopic localization of MAb 236 has not been carried out, a direct demonstration that M6 and M7 are transmembrane regions is based only on a single line of evidence provided by the binding characteristics of the two MAbs. An alternative model which is consistent with most all of the immunochemical studies described above is shown in Fig. ID. Here we show the transmembrane distribution of a subunit with only three transmembrane crossings, M I , M2, and M3. Except for the incompatibility of this model with the results reported by Criado cr d.(1985a) for the cytoplasmic localization of residues 152-159 (those results considered weak for the reasons discussed above), the model appears to fit all other data. Unlike previous models, model D does not propose any amphipathic helices to be involved in channel formation since ( 1 ) charged residues may not be necessary for the formation of a water-filled channel, (2) the polar nature of the polypeptide backbone may be sufficient for the formation of a waterfilled pore, and (3) a channel lined with charges would provide a large number of intermediate binding sites that might slow down the movement of cations through the channel, which is in contrast to the high flux rates observed with the nicotinic AChR channel. Recent studies have indicated that some noncompetitive antagonists which interact with the open channel can be cross-linked to residues contained within the MI region (Karlin, personal communication) suggesting that it is M I and not an amphipathic helix that may be involved in channel formation. Three-dimensional electron image analyses of tubular crystals of the AChR suggest that the subunit cross-section in the area of the bilayer is of the order of 500 A2 (Brisson and Unwin, 1985). which is consistent with five transmembrane crosses. If the hydrophobic helical domain of M4 were associated with the membrane bilayer, however, perhaps lying parallel to the membrane, then the observed cross-sectional area would also be consistent with model D with its three transmembranous helices. 111.

FUNCTIONAL ACTIVITIES ASSOCIATED WITH SYNTHETIC PEPTIDES

A. A Synthetic Peptide Corresponding to Sequences within the aSubunit of the AChR Binds a-Bungarotoxin A number of laboratories have shown that the isolated a-subunit of the AChR binds a-BGTX albeit with reduced affinity as compared to native AChR (Haggerty and Froehner, 1981). Furthermore, proteolytic fragments of the a-subunit that bind BGTX can be generated (Tzartos and Changeux,

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1983; Wilson el al., 1985; Neumann et al., 1985). Analysis of the proteolytic fragments on the basis of size, protease specificity, and the presence of ASN-141 (e.g., susceptibility to digestion by endoglycosidase H) led to the assignment of the BGTX-binding site to a 90-amino acid sequence (residues 153-243) within the a-subunit (Wilson et al., 1985). Figure 2 shows an autoradiogram in which labeled a-BGTX or concanavalin A (Con

FIG.2. "'I-Labeled BGTX and "'I-Labeled Con A binding to protein transfers of proteolytic fragments of the subunit. Isolated subunit (20 pg) was incubated without protease (lanes A and B). or with chymotrypsin (lanes C and D). or papain (lanes E and F). After I hr at 23°C. proteolysis was stopped by the addition of protease inhibitors. Each sample was then split into two, and endoglycosidase H was added to one-half of each protease digestion. Following 24 hr at 37°C. the aliquots were placed in sample buffer and were loaded onto the lanes of a 15% SDS-polyacrylamide gel, electrophoresed. transferred. overlayed with "'I-labeled BGTX. and autoradiographed. Lanes A, C, and E. no endoglycosidase H; lanes B. D. and F. endoglycosidase H treated; lanes G and H. proteolytic digestion of isolated subunit using VX protease. Lane G, "'I-labeled BGTX binding; lane H, "'I-labeled Con A binding. The positions of the a subunit and of the 28-kDa papain-derived and I9-kDa VX protease-derived fragments are indicated. The arrows indicate the position of the IS-kDa papain fragment without (lane E) or after (lane F) endo H treatment (I2-kDa).(From Wilson 1'1 l d . ,

1985.)

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

179

A) was bound to immobilized proteolytic fragments of the a-subunit. The presence of asparagine- 141 within a particular fragment was assessed by binding of labeled Con A, a lectin which binds to the mannose components of the N-linked carbohydrate, or by sensitivity to endoglycosidase H treatment, as evidenced by an increased electrophoretic mobility. A BGTX-binding fragment that is unaffected by endogl ycosidase H cannot contain asparagine-141, the sole site of N-linked glycosylation on the asubunit. As indicated in Fig. 2, both chymotrypsin and papain produce distinctive 27- to 28-kDa fragments that are resistant to endoglycosidase H and thus cannot contain asparagine-I41 (Fig. 2, lanes C, D, E, F). These results and the fact that the largest possible BGTX-binding fragment that would not contain asparagine-141 would have to be 140 amino acids long with a maximal M , of approximately 16 kDa, considerably less than the 27- to 28-kDa fragments seen in Fig. 2, indicate that the BGTX-binding site cannot lie between the N-terminus of a and asparagine-141 and must reside to the C-terminal side of asparagine-141. Upon papain digestion of the a subunit, a 15-kDa BGTX-binding fragment is generated which is reduced to 12 kDa after endoglycosidase H treatment (Fig. 2, lanes E and F). This fragment must therefore contain asparagine-141 and thus the BGTX-binding site must lie within 100 residues (100 x I I5 Da = 11.5 kDa) of asparagine-141. The BGTX-binding site can thus be restricted to the region between residues 141 and 241. Treatment with V8 protease produces an 19- to 20-kDa BGTX-binding fragment that does not bind labeled Con A (Fig. 2, lanes G and H). Since V8 protease cleaves to the carboxyl side aspartate or glutamate residues, the N-terminal limit of the BGTX-binding site must be the first aspartate or glutamate past asparagine-141, which is aspartate-152. Therefore the BGTX-binding site must be contained within the 88 residues between 153 to 241. Using specific anti-peptide antibodies, Neumann ei al. ( 1985) also concluded that the BGTX-binding site must lie to the carboxyl side of aspartate-152. Analysis of two fragments produced by V8 protease cleavage of the asubunit (18 and 20 kDa) has led to some confusion in the literature as to the relationship of the BGTX-binding site to these fragments. Some workers have suggested that both the 18- and 20-kDa fragments have N-termini beginning at residue 46, and that the two fragments differ in their extent of glycosylation and may be derived from structurally heterogeneous asubunits (Ratnam et d.,1986~).Pedersen ei d.(1986) have shown, however, that the 20-kDa fragment begins at residue 173, not valine-46, and that, on occasion, an incompletely cleaved form of the I8-kDa fragment which does begin at valine-46 comigrates with the 20-kDa fragment. Thus, heterogeneity within the 20-kDa region may explain some of the experimentally inconsistent results that have appeared in the literature. Pedersen et a / . (1986) have shown that the 20-kDa fragment contained the reactive

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EDWARD HAWROT ET AL.

sites for three different affinity labels: the allosteric antagonist, meproadifen; the competitive antagonist, tubocurarine; and the affinity alkylating agent, 4-(N-maleimido)-a-benzyltrimethylammonium iodide (MBTA). These observations are consistent with the identification of cysteine- 192 and cysteine-193 as the residues labeled with MBTA (Kao et al., 1984). Figure 3 summarizes the information provided by the analysis of proteolytic fragments that bind BGTX and shows the overlap region identified as the putative BGTX-binding site on the basis of the experiments described above. Two independent lines of evidence, BGTX binding to proteolytic fragments and MBTA labeling of cysteine-192 and cysteine-193, have focused attention on the region surrounding cysteine- 192 and cysteine-193 as being important in BGTX binding. To test this hypothesis, a 32-amino acid peptide (32-mer) was synthesized which corresponded to residues 173 to 204 of the a-subunit. When tested for BGTX binding, this synthetic peptide bound BGTX with the same apparent affinity (50-100 nM) as the immobilized a-subunit or as other protease-generated fragments (Wilson et al., 1985). Furthermore, BGTX binding to the 32-mer could be competed with tubocurarine with an IC,,, of approximately 0.1 mM or with a-cobratoxin at an IC,, of 0.4 p M (Fig. 4), whereas much higher concentrations of the muscarinic antagonist, atropine, were required, demonstrating apparent nicotinic specificity for BGTX binding. Agonists, however, were essentially ineffective in competing for BGTX binding to the 32-mer when compared with NaCl or choline chloride controls, suggesting that the structure of the agonist-binding domain may not be readily reformed in the synthetic peptide. Since all of the antagonists are relatively

1

$er----------------------------t-NHp-teni nal d m a i n

141 152 173 192 193 204 24 1 Asn---Asp---Ser---Cys-Cys----Hi~-------Gl"-----------------------t COOH-tenninal domain -1P-kOa papain f r a g m e n t d -19-kDa V8 fragment+BGTX binding domain+ t32-mer+

+

431 GlY

>

FIG.3. Mapping of the BGTX-binding site onto the primary AA sequence of the subunit. The subunit was arbitrarily divided at asparagine-141 into two domains: a 16-kDa (140 AA) NH,-terminal domain and a 34-kDa (296 A A ) COOH-terminal domain. The positions of fragments used to map the BGTX-binding site are indicated. The maximum region of the COOHterminal domain that the 12-kDa. endo H-sensitive fragment generated by digestion with papain could occupy is determined by assuming that its N,H-terminus is asparagine-141. The region occupied by the 19-kDa VX protease-derived fragment is determined by assuming its NH,-terminus is glycine-153, the first residue past the first V8 cleavage site in the COOHterminal domain. The BGTX-binding domain is taken as the region common to these two fragments. The position within this domain of the 32-mer is indicated. (From Wilson d., 1985.)

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181

110

-10

-8

-6

-4

-2

0

k a n d l (109 M)

FIG.4. Binding of "'I-labeled BGTX to Torprdii 32-mer and competition with antagonists. Wells of microtiter plates were coated with 5 p,g 32-mer, quenched, and incubated with I nM "'I-labeled BGTX. The effects of the following agents on toxin binding were tested: abungarotoxin (BGTX), a-cobratoxin (CTX), d-tubocurarine (dTC). and NaCI. After washing, bound radioactivity was removed by SDS/NaOH and counted in a gamma counter.

large molecules in comparison to the cholinergic agonists, it is likely that the structural rigidity of the antagonists contributes significantly to the binding energy upon complex formation. The agonists, being much more flexible, may not be capable of inducing the appropriate conformational change in the synthetic peptide. Alternatively, additional residues that are outside the region 173-204 may be involved in the configuration of the agonist-binding subsite. Neumann ef ul. (1986a) have prepared a IZamino acid synthetic peptide, corresponding to residues 185 to 196, that binds BGTX in a manner that can be competed with 10 mM tubocurarine, although the apparent affinity for BGTX was not reported. Similarly, Mulac-Jericevic and Atassi ( 1986) found that the synthetic peptide corresponding to residues 182-198 of the a-subunit also bound BGTX. Table 1 provides a compilation of the known sequences in this region from a variety of Torpedo a-subunit related proteins. The muscle-like as from Torpedo, human, mouse, calf, and chicken are highly homologous in this region but are quite different from the corresponding region of the

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TABLE I COMPARISON OF THE SEQUENCES HOMOLOGOUS TO THE Torpedo 32-MEH" Torpedo a Chicken a a Human Calf a Mouse a Rat nerve "a" T~rppd~ p

I73 I80 I90 200 SGEWVMKDYRGWKHWVYYTCCPDTPYLDITYH SGEWVMKDYRG WKH WVY Y ACCPDTPY LDlTY H SGEWVIKESRG WKHSVTY SCCPDTPY LDlTY H SGEWVJKESRGWKHWVFYACCPSTPYLDITYH

SGEWVIKEARGWKHWVFYSCCPTTPYLDITYH SGEWAllKAPGY KHEIKYNCOEE-IYQDITYS NGQWSIEH-KPSRK-NWRSDDP-SYEDVTFY

"Sequences extracted from data provided in Boulter e f a / . (1985. 1986).

Torpedo p-subunit, which overall, is known to be homologous to the asubunit. It should also be noted that 8 of the 32 residues in the Torpedo sequence in this region are aromatic amino acids and that 6 of these are conserved in the human sequence. We take this as suggesting that aromatic interactions may be important in BGTX binding. Residues 173-176 are always SGEW in the muscle-like as, residue 180 is always an acidic amino acid, residue 182 is an arginine, residues 184186 are always WKH, residue 188 is valine, tyrosine is invariably at position 190, residues 192-194 are always CCP, and residues 197-204 are always PYLDITYH. The rat nerve a-like sequence derived from a rat pheochromocytoma cDNA library is much less homologous in this region, displaying 17 residue differences out of 32 as compared to the 6-residue differences between Torpedo and human, mouse, or calf a.The decreased homology between Torpedo a and the rat nerve a may be related to the fact that the a-subunit of the neuronal form of the nicotinic AChR may not bind BGTX, as a large body of evidence now suggests (Boulter e f al., 1986). It is now of considerable interest to determine which of the invariant residues among the muscle-like sequences contribute to the formation of the BGTX-binding site and whether an intact disulfide between cysteine192 and cysteine-193 is required for BGTX binding. Neumann et al. (l986b) have prepared the 12-amino acid synthetic peptide corresponding to residues 185-196 of the human a-subunit which differs in 3 of the 12 residues from the corresponding Torpedo sequence. They found that the humanderived peptide did not bind BGTX whereas the Torpedo-derived peptide bound BGTX with an apparent affinity of 3.5 x lo-' M. The apparent reduction in affinity of the Torpedo 12-mer (185-196) in comparison to the Torpedo 32-mer (173-204; K , = lo-' M ) may be due to the smaller size of the peptide or the fact that the smaller peptide was covalently

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

183

linked to Sepharose beads during the binding assay. A direct comparison of the affinities of the 32-mer and the 12-mer has not been reported. The result with the human-derived peptide ( 185-196) is surprising since human AChR binds BGTX. The human 12-mer has a serine replacing the tryptophan at position 187 which is conserved in all the other species, suggesting that this tryptophan may play an important role in the binding activity within this peptide region. In fact, direct chemical modification of the tryptophan in the Torpedo-derived peptide decreased BGTX binding (Neumann et al., 1986b). Besides the difference at residue 187, however, the human sequence also contains a threonine at position 189 where all other species have an aromatic residue. Both residues may play an important role in this region. Since human AChR, despite the absence of tryptophan-I87 and tyrosine 189, does bind BGTX. the region 185-196 may be only one of several regions involved in specifying the BGTXbinding site. We have recently tested a 32-mer corresponding to the human (Y sequence of residues 173-204 and measured an apparent affinity constant of 6 kA4 for BGTX. The reduction in affinity of the human 32-mer in comparison to the Torpedo 32-mer thus parallels the results obtained with the shorter human peptide (185-196). The 32-mer appears to contain a major determinant or determinants important for BGTX binding, but may not contain all of the residues that interact with BGTX in binding to native AChR. Even within the 32-mer, a number of residues dispersed along the entire sequence may participate in the recognition of BGTX. We have synthesized a number of overlapping small peptides within the 173-204 region and are presently assessing their ability to bind BGTX. Preliminary studies suggest that several different small peptides bind BGTX but with reduced affinity when compared to the 32-mer (E. Hawrot et al., unpublished observations). Reduction and carboxymethylation of the cysteines in the Torpedo 12-mer (185-196) have been found to result in a loss of BGTX-binding activity (Neumann r t d., 1986b),suggesting that the disulfide bridge between cysteine-192 and cysteine-193 is important for BGTX binding to the peptide. In preliminary studies, we have obtained similar results with the 32-mer. When the treated peptide was analyzed for amino acid composition, however, it appeared that the tryptophan at position 187 had also been modified (E. Hawrot r t al., unpublished observations). Thus, the disulfide requirement for BGTX binding still remains unsettled. Further evidence that the region containing residues 173-204 is functionally important comes from the observation that mice immunized with the 32-mer developed clinical and electromyographic signs of experimental autoimmune myasthenia gravis (EAMG) and exhibited significant T cell responses (Brooks et al., 1986). These findings demonstrate that the entire

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EDWARD HAWROT ET AL.

AChR molecule is not required for the induction of autoimmune disease and that small synthetic peptides may provide useful tools for the study of autoimmune disorders such as myasthenia gravis. Immunosuppression of the response to such small disease-inducing synthetic peptides may provide one approach to the therapy of autoimmune disorders (Brooks et ul., 1986).

B. Receptor Binding Activities of Synthetic Peptides Derived from Curaremimetic Neurotoxin Sequences and from Rabies Virus Glycoprotein Sequences The snake venom-derived curaremimetic neurotoxins form a large family of structurally related, intermediate-sized (7-8 kDa) polypeptides that exhibit high affinity for the AChR and act as competitive antagonists of acetylcholine binding to the AChR. Functional blockade of the AChR results from the antagonistic binding of these neurotoxins. A comparison of the areas of maximal homology between the neurotoxins suggests that the region corresponding to the end of the so-called “toxic” loop 2 may be very important in the binding of the neurotoxins to the AChR and may mimic the structure of the acetylcholine molecule. In line with this suggestion, Juillerat e f ul. (1982) have prepared a 33-amino acid synthetic peptide corresponding to residues 16-48 of toxic loop 2 from the major neurotoxin in the venom of Nuju nuju philippinensis. This synthetic peptide bound to the Torpedo AChR with an apparent affinity of 2.2 x lo-’ M. The affinity of the peptide for the AChR was reduced in comparison to the native neurotoxin, but was higher than that of tubocurarine. Synthetic peptides corresponding to neurotoxin sequences may thus provide an experimental avenue for defining which specific toxin residues are involved in receptor recognition at the ligand-binding site. There is some evidence that direct binding of rabies virus to the nicotinic AChR may be mediated through the viral glycoprotein and that this interaction may contribute to the observed neurotropism of this virus. A comparison of the amino acid sequence of the rabies virus glycoprotein with the neurotoxin sequences indicates that a segment of the rabies virus glycoprotein shares homology with the entire sequence of the long curaremimetic neurotoxins (Lentz et ul., 1984). The greatest identity between the two sequences occurs in toxic loop 2-the region of the neurotoxin molecule believed to be important for neurotoxicity. If this region of the virus glycoprotein is responsible for viral interaction with the AChR, then it may be possible to show that synthetic peptides derived from these sequences may also interact with the AChR. To test such an interaction,

185

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

one can monitor the competitive interference of the viral-derived synthetic peptides as well as the corresponding neurotoxin peptides in the binding of labeled BGTX to purified Torpedo AChR. The sequence of a BGTXderived 10-amino acid peptide and two rabies virus glycoprotein-derived peptides (a 10-mer, which is identical to the BGTX-derived 10-mer in 6 of the 10 residues, and an overlapping 13-mer) used to test this hypothesis are shown in Fig. 5 . When tested for inhibition of labeled BGTX binding to Torpedo AChR in a solid-phase assay system, all three synthetic peptides inhibited BGTX binding in the millimolar range (Fig. 6). Although none of the synthetic peptides were as potent as tubocurarine. their potency was comparable to other small cholinergic ligands when tested in the same assay system. In fact, the rabies virus glycoprotein-derived 13mer was significantly more potent than either carbamylcholine or nicotine in competing with labeled BGTX for binding to the Torpedo AChR. Our findings are consistent with the view that the viral glycoprotein contains sequences which define domains that can bind to the binding site of nicotinic AChR. Furthermore, these experiments indicate that small synthetic peptides corresponding to well-defined domains of larger macromolecules can interact with receptor-binding sites and will be useful for defining the molecular determinants of binding sites. Analysis of analogous synthetic peptides with single amino acid substitutions should provide additional information on the relative importance of each residue within the peptide sequence. This approach with small modified synthetic peptides can therefore supplement information obtained from studies utilizing site-directed mutagenesis of the larger macromolecule.

28

. .. .. 1

. 4 0 I I

I

.

.

.

.

.

.

a

.

.

.

T-W-C-D-G-F-C-S-S-R-GK-R (BGR l()tlER) D-A-F-C-S-S-RG-K-V RABIES GLYCOPROTEIN (CVS l [ k u ~ ) D I-F-T-N-S-R-GK-R RABIES GLYCDPROTEIN (CVS 1%~) T-P-C-D-I-F-T-N-S-RG-K-R 187 199

u -

RIXIN

B

FIG.5 . Alignment of a portion of long snake venom neurotoxin sequences (residues 2840) with a segment of the rabies virus (CVS strain) glycoprotein sequence (residues 18719). The toxin residues are located at the end of loop 2 of the neurotoxins. The glycoprotein sequence occurs prior to a site for N-linked glycosylation at position 204 of the CVS strain. (*), Arginine-37 which may represent the counterpart of the quaternary ammonium of acetylcholine; (:), identity between 0.hannali and CVS; (.), conservative substitution.

186

EDWARD HAWROT ET AL. 110

I

3

t

v

i X

c W

m I

H

N

-7

1

FIG.6. Binding-inhibition assay. The abilities of d-tubocurarine (d-TC); nicotine (Nic); carbachol (Carb); rabies virus glycoprotein, CVS strain; 10-residue peptide, residues 190199 (CVS 10-mer) and 13-residue peptide, residues 198-199 (CVS 13-mer); and an a-BGTX 10-residue peptide, residues 3 1-40 (BTX 10-mer) to inhibit binding of "'I-labeled a-BGTX were compared. Wells of microtiter plates were coated with 0.15 pg AChR and incubated for 10 min with 5 nM labeled toxin and increasing concentrations of ligand. After washing, bound radioactivity was removed and measured. A line is drawn at 50% "'I-labeled a-BGTX binding. Values represent mean of three replicates.

C. Other Methodologies for Detecting Functional Activity Soon after the development of solid-phase peptide synthesis, Merrifield and co-workers (Gutte et al., 1972) demonstrated that a tetradecapeptide corresponding to the C-terminus of ribonuclease A (residues I I 1 to 124) noncovalently reactivated an inactive form of ribonuclease A that ended at residue 118. Up to 98% of the activity was regained, suggesting that histidine-I 18 on the synthetic peptide could contribute to the functionally active site of the enzyme. Furthermore, the dissociation constant ( K , ) of the complex was 2 x lo-' M and decreased to 7 x lo-' M in the presence of substrate. Although the tetradecapeptide was in itself inactive, it could, with high efficiency and affinity, reconstitute and participate in the active site of this enzyme through a noncovalent interaction. These and other

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187

early studies suggested that synthetic peptides could adopt biologically relevant conformations and subserve functional activities such as binding of enzymatic substrates. The observation that small synthetic peptides are efficient immunogens in generating antibodies that cross-react with the corresponding sequences in the native protein suggests that small peptides may, on a significant time scale, reside in conformations in solution which mimic those secondary structures found in the native protein. This hypothesis has important ramifications for various models of protein folding during biosynthesis. Small peptides such as the S-peptide, the 20-amino acid N-terminal peptide of ribonuclease A, show partial a-helix formation in aqueous solutions (Kim and Baldwin, 1984). In native ribonuclease A, residues 313 are known to exist in an a-helix. A nonapeptide that acts as an immunodominant synthetic immunogen for an influenza virus hemagglutinin (Dyson et al., 1985) was analyzed by high-field nuclear magnetic resonance (NMR) spectroscopic techniques and shown to exhibit a conformational preference in water for a type-I1 reverse turn of the polypeptide backbone. Similarly, studies with a synthetic octapeptide from an ATPase indicated that this peptide may function as an ion-binding site (Gangola and Shamoo, 1986). In another case, a synthetic pentapeptide from fibronectin acted as a competitive, reversible inhibitor of cellular adhesion (Humphries ct al., 1986). Thus, a number of examples now exist that indicate small peptides can exhibit conformations in solution that may form biologically relevant functional domains such as ligand-binding sites. IV. DETERMINATION OF THE SOLUTION CONFORMATION OF SMALL SYNTHETIC PEPTIDES RELEVANT TO THE LIGANDBINDING SITE OF THE AChR

The molecular details of how acetylcholine binding leads to transient opening of the channel within the AChR complex is a question of fundamental and wide-ranging interest in neurobiology. Ideally, one would like to be able to perform X-ray crystallographic analysis of the AChR in its various states (e.g., closed, ligand-bound closed, ligand-bound activated, desensitized) in order to correlate structural changes with functional states. The formation of crystals suitable for X-ray analysis is problematic with membrane proteins and thus other approaches are required. One such powerful approach is to focus, using appropriate synthetic peptides, on small functionally relevant domains of the AChR, such as the ligand-binding site, and to describe the interaction of these small AChR domains with

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EDWARD HAWROT ET AL.

the ligand of interest. A variety of physicochemical techniques are now available to facilitate the description of the molecular interactions between small peptides in solution. A first step in this direction, however, is to use these techniques to determine the native structure, if any, of the synthetic peptide domains in solution. Circular dichroism provides one means of determining secondary structure and monitoring structural transitions (Clore et al., 1985). Although circular dichroism provides valuable information as to the overall structure of the peptide, it does not provide detailed information on the environment of specific residues within the peptide. Two-dimensional NMR spectroscopy, on the other hand, can provide detailed structural information at the single proton level. This information can be used to define the secondary structure within the polypeptide and can also be used to provide distance constraints to the three-dimensional configuration of the polypeptide's individual residues (Have1 and Wuthrich, 1985; Williamson et d . , 1985). With the aim of ultimately describing the molecular interaction of BGTX with the residues involved in the ligand-binding site on the AChR, we have begun to characterize the solution structure of short synthetic peptides (an 11-mer, cx residues 186196, and a 12-mer, a residues 185-196) that bind BGTX in a solid-phase assay (Neumann et al., 1986; E. Hawrot et al., unpublished observations). As a first step toward analyzing the structure of these peptides, we have performed one- and two-dimensional NMR spectroscopy and have identified the residues responsible for a number of the resonance peaks in the NMR spectrum. Figure 7 shows a connectivity map obtained by two-dimensional correlated spectroscopy (COSY) as well as the corresponding one-dimensional spectrum in the aromatic region with the assignment of peaks to individual protons in the I I-mer. COSY is applied to homonuclear and heteronuclear systems for the determination of scalar (i.e., through-bond couplings) and is used for the assignment of spectral resonances. The diagonal peaks correspond to the one-dimensional spectrum whereas the off-diagonal peaks (cross peaks) indicate the scalar couplings between protons. These off-diagonal peaks, designating the scalar coupling, which are often buried within the complexity of the one-dimensional spectrum, are due to through-bond connectivities between hydrogen atoms that are, in this spectrum under the conditions used, separated by no more than three chemical bonds in the covalent primary structure of the peptide. Longer range couplings can be enhanced by selecting slightly modified measurement conditions, Thus, COSY is a two-dimensional technique that greatly simplifies the assignment of complex one-dimensional N M R spectra. The 'H resonances in the aromatic region of the I 1-mer were completely assigned with this technique,

189

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

Y ,C2,6

Y ,C3,5

wc y

H C2

0 r\

a@

Q

E a 0

m

7,O

8.0 PPM

FIG.7. The ' H COSY spectrum of the aromatic protons of an I I-mer (residues 186-196) specifying a portion of the a-BGTX-binding site on the a-subunit of the Torpedo acetylcholine receptor. The spectrum was acquired using a W " ( 4 , ) - -'K)"(+,)-Acquire pulse sequence in D,O at pH 3.7. The initial / I delay was 3 x c,-'sec. A 351 x 1024 data matrix wiis = 0". 180". YO". 270"). we used a 16-step sequence acquired. For phase cycling = 0". with the phases of both pulses incremented by W" for each 4-step sequence and with quadrature detection.

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EDWARD HAWROT ET AL.

as shown in the Fig. 7. This information should permit us to identify those residues involved in any structural changes that occur upon interaction with BGTX. Using another powerful NMR technique, that of measuring amide (peptide NH) proton-deuterium exchange rates, we have found that the 12mer (185-196) contains significant secondary structure in solution. It is well known that, in native proteins, the rate of exchange of amide protons with the deuterium in solution is greatly reduced compared to the rate of exchange obtained with the unfolded protein (Loftus et al., 1986). This is thought to be due to solvent exclusion produced by steric constraints resulting from peptide folding as well as involvement of the amide protons in H-bonding. Both a-helices and p-sheet structures involve H-bonding through amide protons, and thus protein fragments containing such structures will exhibit differential amide-proton exchange rates as measured by NMR techniques (Loftus et al., 1986). One commonly used procedure to assess amide-proton exchange is to monitor the temperature dependence of amide-proton exchange. Increased temperature will increase the proton-deuterium exchange rate and indicate which amide-protons are readily exchanged. In contrast, if other amide-protons resist exchange under elevated temperature conditions, then this can be taken as an indication of a structural constraint within the peptide. Figure 8 is such an experiment examining the temperature dependence of amide-proton exchange within the 12-mer in solution. As the amide-proton exchanges with the deuterium in solution, the resonance due to that proton disappears from the NMR spectrum. The protons resonating at 6.87 ppm rapidly exchange with the solvent at T = 343"K, as indicated by the disappearance of the signal into the baseline. At this temperature, however, the protons resonating at 8.15 ppm are only slowly exchanging with the solvent. These findings, demonstrating a differential exchange rate between different amide-protons within the 12-mer, are consistent with the hypothesis that this peptide can maintain a secondary structure. The importance of these structural constraints in the interaction of the peptide with BGTX will require additional NMR studies of the peptide and of the peptide-BGTX complex. It is hoped that, by focusing on such well delimited peptidepeptide interactions, we will be able to obtain insights into the structural parameters that are critical for the proper functioning of the ligand-binding site on the AChR. ACKNOWLEDGMENTS We wish to thank Dr. Ian Armitage for valuable discussions regarding application of NMR technologies to synthetic peptides and Dr. Susan B. Edelstein for a critical review of this manuscript. We thank Dr. Kenneth Williams of the Protein Chemistry Facility at Yale for

191

9. SYNTHETIC PEPTIDES IN THE STUDY OF AChR

T=343K

T=333K

T=323K

8.0

P PM

7.0

FIG.8. The downfield region of the 'H spectrum of the 12-mer (185-196) in 85% HJY 15% D,O at pH 3.7 as a function of temperature. Presaturation was performed to reduce the H,O resonance intensity. Several exchangable protons resonate at 6.87 ppm. The spectra were referenced to the upfield methyl resonance of the valine residue.

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the synthesis of several peptides used in this study. The original research described here was supported by NIH GM32629, NSF Grant BNS 85-06404, and the Muscular Dystrophy Association. E. H. is an Established Investigator of the American Heart Association. REFERENCES Anderson, D. J.. Walter. P., and Blobel, G . (1982). Signal recognition protein is required for the integration of acetylcholine receptor 6 subunit, a transmembrane glycoprotein, into the endoplasmic reticulum membrane. J. Cell Biol. 93, 501-506. Barkas. T., Juillerat, M., Kistler, J., Schwendimann, B., and Moody, J. (1984). Antibodies to synthetic peptides as probes of acetylcholine receptor structure. Eur. J. Bioclwm. 143, 309-314. Barkas. T., Gabriel, J.-M.. Juillerat, M.. Kokla, A., and Tzartos. S. J. (1986). Localisation of the main immunogenic region of the nicotinic acetylcholine receptor. FEBS Lett. 196, 237-241. Barkas, T., Mauron, A., Roth, B., Alliod, C., Tzartos, S. J., and Ballivet, M. ( 1987). Fusion proteins delineate the binding sites of the nicotinic acetylcholine receptor for antibodies to the main immunogenic region and for a-bungarotoxin. Science 235, 77-80. Benjamin, D. C., Berzofsky, J. A., East, I. J., Gurd, F. R. N., Hannum, C., Leach, S. J., Margoliash, E., Michael, J. G., Miller, A., Prager, E. M., Reichlin, M., Sercarz. E. E.. Smith-Gill. S. J.. Todd, P. E., and Wilson, A. C. (1984). The antigenic structure of proteins: A reappraisal. Annu. Rev. Immunol. 2, 67-101. Boulter, J.. Luyten, W.. Evans, K., Mason, P., Ballivet, M., Goldman, D., Stengelin. S., Martin. G . . Heinemann, S . , and Patrick, J. (1985). Isolation of a clone coding for the a-subunit of a mouse acetylcholine receptor. J . Neurosci. 5, 2545-2552. Boulter, J . . Evans, K., Goldman, D., Martin, G., Treco, D., Heinemann, S.. and Patrick, J. (1986). Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor subunit. Nature (London) 319, 368-374. Brisson, A., and Unwin, P. N. T. (1985). Quaternary structure of the acetylcholine receptor. Nuture (London) 315,474-477. Brooks, E. B.. Hawrot, E.. Kantor. F. S . . Lentz. T. L.. and Pachner, R. (1986). Induction of experimental myasthenia in mice by immunization with a synthetic peptide of the acetylcholine receptor. Annu. Meet. Soc. Neirro.sci.. 16th Abstr. 338.2. Claudio, T.. Ballivet, M., Patrick, J., and Heinemann, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo culifornica acetylcholine receptor gamma-subunit. Proc. Null. Acad. Sci. U.S.A. 80, I 1 11-1 115. Clore. G. M., Gronenborn, A. M., Brunger, A. T., and Karplus, M. (1985). Solution conformation of a heptadecapeptide comprising the DNA binding helix F of the cyclic AMP receptor protein of Escherichia coli. Combined use of 'H nuclear magnetic resonance and restrained molecular dynamics. J . Mol. B i d . 186, 435-455. Criado, M., Hochschwender, S., Sarin, V., Fox, J. L., and Lindstrom, J. (1985a). Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits. Proc. Natl. Acud. Sci. U.S.A. 82, 2004-2008. Criado. M.. Sarin. V.. Fox, J. L., and Lindstrom. J. (1985b). Structural localization of the sequence ~1235-242 of the nicotinic acetylcholine receptor. Biochem. Biophys. Res. Comrnun. 128, 864-87 I . Criado, M., Sarin. V.. Fox, J. L.. and Lindstrom, J. (1986). Evidence that the acetylcholine binding site is not formed by the sequence a127-143 of the acetylcholine receptor. Biochemistiy 25, 2839-2846. Dyson. H. J.. Cross, K. J., Houghten, R. A., Wilson, I. A,, Wright, P. E.. and Lerner. R.

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A. ( 1985). The immunodominant site of a synthetic immunogen has a conformational preference in water for a type-ll reverse turn. Natrtre (LondonJ 318, 480-483. Finer-Moore, J.. and Stroud, R. M. (1984). Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. N o t / . Acrid. Sci. U . S . A . 81, 155159.

Froehner, S. C. (1981). Identification of exposed and buried determinants of the membrane20, 4905-4915. bound acetylcholine receptor from Torpedo ccrliforniccr. Bioc~l~emistry Gangola. P.. and Shamoo, A. E. (1986). Synthesis and characterization of a peptide segment of (Ca" + Mg")-ATPase. J . Biol. Chem. 261, 8601-8603. Gutte. 9.. Lin, M. C.. Caldi, D. G., and Merrifield, R. B. (1972). Reactivation of des( 119, 120-. or 121-124) ribonuclease A by mixture with synthetic COOH-termical peptides of varying lengths. J. Biol. Chem. 247, 4763-4767. Guy, H . R. ( 1984). A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations. Biopliys. J. 45, 249-261. Haggerty. J. G., and Froehner. S. C. (1981). Restoration of "'I-a-bungarotoxin binding activity to the Q subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate. J. Biol. Chem. 256, 8294-8297. Havel. T. F.. and Wiithrich. K. (1985). An evaluation of the combined use of nuclear magnetic resonance and distance geometry for the determination of protein conformations in solution. J. Mol. Biol. 182, 281-294. Humphries, M. J.. Olden. K., and Yamada, K. M. (1986). A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science 233, 467-470. Juillerat, M. A.. Schwendimann. B.. Hauert. J.. Fulpius, B. W., and Bargetzi. J . P. (1982). Specific binding to isolated acetylcholine receptor of a synthetic peptide duplicating the sequence of the presumed active center of a lethal toxin from snake venom. J . Biol. Cliein. 257, 290 1-2907. Juillerat. M. A.. Barkas. T.. and Tzartos, S . J . (1984). Antigenic sites of the nicotinic acetylcholine receptor cannot be predicted from the hydrophilicity profile. FEBS Lett. 168, 143- 148. Kao, P. N . . Dwork, A. J., Kaldany, R.-R. J.. Silver, M. L., Wideman, J . , Stein. S., and Karlin, A. (1984). Identification of the Q subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site. J . Biol. Cliein. 259, 1166211665.

Kim, P. S., and Baldwin, R. L. (1984). A helix stop signal in the isolated S-peptide of ribonuclease A. Narrrre (London) 307, 329-334. Lennon. V. A,. McCormick, D. J.. Lambert. E. H.. Griesmann. G. E.. and Atassi. M. Z. (1985). Region of peptide 125-147 of acetylcholine receptor a subunit is exposed at neuromuscular junction and induces experimental autoimmune myasthenia gravis. Tcell immunity, and modulating autoantibodies. Proc. Ncrtl. Acctd. Sci. U . S . A . 82,88058809. Lentz. T. L.. Wilson, P. T., Hawrot, E., and Speicher, D. W. (1984).Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins. Science 226, 847-848. Lindstrom. J.. Criado, M.. Hochschwender. S., Fox, J. L., and Sarin. V. (1984). Immunochemical tests of acetylcholine receptor subunit models. Nutrtre (London) 31 I , 573575. Loftus, D., Gbenle. G. 0.. Kim. P. S.. and Baldwin. R. L. (1986). Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates. Biochemistry 25, 1428-1436. Mishina, M., Tobimatsu, T., Imoto, K.. Tanaka, K.4, Fujita. Y.. Fukuda. K.. Kurasaki.

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M.. Takahashi, H.. Morimoto, Y., Hirose, T.. Inayama. S., Takahashi, T.. Kuno. M., and Numa, S. (1985). Location of functional regions of acetylcholine receptor a-subunit by site-directed mutagenesis. Nurure (London) 313, 364-369. Mochly-Rosen, D., and Fuchs, S. (19811. Monoclonal anti-acetylcholine-receptor antibodies directed against the cholinergic binding site. Eiochemisfry 20, 5920-5924. Mulac-Jericevic, B.. and Atassi, M. Z. (1986). Segment 182-198 of Torpedo culifornicu acetylcholine receptor contains a second toxin-binding region and binds anti-receptor antibodies. FEES Lett. 199, 68-74. Neumann, D . . Fridkin, M., and Fuchs, S. (1984). Anti-acetylcholine receptor response achieved by immunization with a synthetic peptide from the receptor sequence. Biochem. Eiophys. Res. Comrnun. 121,673-679. Neumann. D.. Gershoni. J . M.. Fridkin, M.. and Fuchs. S. (1985). Antibodies to synthetic peptides as probes for the binding site on the a subunit of the acetylcholine receptor. Proc. Natl. Acud. Sci. U.S.A. 82, 3490-3493. Neumann. D.. Barchan, D., Safran. A., Gershoni. J. M., and Fuchs, S. (1986a). Mapping of the a-bungarotoxin binding site within the Q subunit of the acetylcholine receptor. Proc. Nut/. Acad. Sci. U.S.A. 83, 3008-301 I . Neumann, D., Barchan. D.. Fridkin, M.. and Fuchs, S. (1986b). Analysis of ligand binding to the synthetic dodecapeptide 185-196 of the acetylcholine receptor a-subunit. Proc. Nntl. Acad. Sci. U.S.A. 83, 9250-9253. Niman. H. L.. Houghten, R. A., Walker, L. E., Reisfeld, R. A,, Wilson, I. A.. Hogle. J. M., and Lerner, R. A. (1983).Generation of protein-reactive antibodies by short peptides is an event of high frequency: lmplications for the structural basis of immune recognition. Proc. Nutl. Acud. Sci. U.S.A. 80, 49494953. Noda. M., Takahashi, H., Tanabe, T.,Toyosato, M.. Kikyotani, S., Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S . (1983). Structural homology of Torpedo culifornica acetylcholine receptor subunits. Nuture (London) 302,528-532. Pedersen, S . E., Dreyer, V. B., and Cohen, J. B. (1986). Location of ligand-binding sites on the nicotinic acetylcholine receptor a-subunit. J . Eiol. Chem. 261, 13735-13743. Plumer. R., Fels. G . . and Maelicke, A. (1984). Antibodies against preselected peptides to map functional sites on the acetylcholine receptor. FEES Left. 178, 204-208. Ratnam, M., and Lindstrom, J. (1984). Structural features of the nicotinic acetylcholine receptor revealed by antibodies to synthetic peptides. Biochem. Eiophys. Res. Comrnun. 122, 1225-1233. Ratnam. M., Sargent, P. B., Sarin, V., Fox, J. L., Nguyen, D. L.. Rivier, J., Criado. M., and Lindstrom, J. (1986a). Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochemistry 25, 2621-2632. Ratnam, M.. Nguyen. D. L., Rivier, J., Sargent, P. B., and Lindstrom, J. (1986b). Transmembrane topography of nicotinic acetylcholine receptor: Immunochemical tests contradict theoretical predictions based on hydrophobicity profiles. Eiochernistry 25,26332643. Ratnam, M., Gullick. W.. Spiess, J., Wan, K., Criado, M., and Lindstrom, J . (1986~). Structural heterogeneity of the a subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding. Biochemistry 25, 4268-4275. Shinnick, T. M., Sutcliff. J. G . , Green, N., and Lerner, R. A. (1983). Synthetic peptide irnmunogens as vaccines. Annu. Rev. Microbiol. 37, 425-446. Souroujon, M. C . . Neumann, D., Pizzighella, S., Safran, A., and Fuchs, S. (1986). Localization of a highly immunogenic region on the acetylcholine receptor a-subunit. Biochem. Eiophys. Res. Comrnun. 135, 82-89.

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Sutcliffe. J . G.. Shinnick. T. M., Green, N.. and Lerner. R. A. (1983). Antibodies that react with predetermined sites on proteins. Science 219, 660-666. Tzartos. S. J., and Changeux. J . P. (1983). High afinity binding of a-bungarotoxin to the purified a-subunit and to its 27,000-Dalton proteolytic peptide from Torpedo inurmor(rili acetylcholine receptor. Requirement for sodium dodecyl sulfate. EMBU J . 2, 38 1-387. Tzartos. S. J.. and Lindstrom, J. M. (1980).Monoclonal antibodies used to probe acetylcholine receptor structure: Localization of the main immunogenic region and detection of similarities between subunits. Proc. Nail. Acud. Sci. U . S . A . 77, 755-759. Tzartos, S. J . , Seybold. M. E., and Lindstrom. J . M. (1982). Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc. Nail. Acud. Sci. U.S.A. 79, 188-192. Williamson. M. P.. Havel, T. F.. and Wuthrich. K. (1985). Solution conformation of proteinase inhibitor 1lA from bull seminal plasma by 'H nuclear magnetic resonance and distance geometry. J. Mot. B i d . 182, 295-315. Wilson, P. T.. Lentz. T. L.. and Hawrot, E. (1985). Determination of the primary amino acid sequence specifying the a-bungarotoxin binding site on the a-subunit of the acetylcholine receptor for Torpedo californic,ti. Proc. Nail. Accrd. Sci. U.S.A. 82, 87908794. Young. E. F., Ralson, E.. Blake, J . , Ramachandran, J . , Hall, Z. W . , and Stroud, R. M. ( 1985). Topological mapping of acetylcholine receptor: Evidence for a model with five transmembrane segments and a cytoplasmic COOH-terminal peptide. Proc. Nut/. Actrd. Sci. U . S . A . 82, 62-30.

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CURRL(NT TOPICS I N MEMBRANES AND TRANSPORT. VOLUME 33

Chapter 10 Expression of Acetylcholine Receptor Subunits in Saccharomyces cerevisiae (Yeast) MELODY T. SWEET,* JON LINDSTROM,** NORIHISA FUJITA,*' KATHRIN JANSEN,* CHURL K . MIN,* TONI CLAUDIO,' NATHAN NELSON,' THOMAS D . FOX,' AND GEORGE P . HESS* *Sections of Biochemistry, Molecular and Cell Biology, and of 'Genetics and Development Division of Biological Sciences Cornell Universio Ithaca. New York 14853 **The Salk Institute of Biological Studies San Diego, California 92138 BDepartment of Physiology Yale University School of Medicine New Haven, Connecticut 06510 AND 'Department of Biochemistry Roche Institute of Molecular Biology Nutley, New Jersey 07011

Many of the proteins that control the transmission of signals between cells of the nervous system occur in very low concentrations. New approaches are needed to produce these proteins in quantities that are large enough for their structure and function to be studied. Here we will describe one such new approach, the expression of one of these proteins, the ace'Present address: MRC Molecular Neurobiology Unit, UniverPity of Cambridge Medical School, Cambridge EB2 2QH. England.

197 Copyright 11 19XX by Academic h e r , . Inc All right\ ot reproduction in any form resxved

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tylcholine receptor, in yeast. The best known acetylcholine receptor is the one found in the electric organ of certain eels and fish such as Torpedo cnlifornic-a, where it occurs in large amounts. The protein consists of five polypeptide chains (a2py8).of molecular weights ranging from 40 kDa for the a-subunit to 65 kDa for the &subunit (Weill et [ I / . , 1974; Reynolds and Karlin, 1978). and the a-subunit carries the binding site for acetylcholine (Weill et d . , 1974: Reynolds and Karlin, 1978). The receptor from Electrophorrrs olecfricrrs, Torpedo species, and mammalian muscle cells have similar antigenic properties (Claudio and Raftery, 1977; Patrick and Stallcup, 1979; Lindstrom et d.,1980; Tzartos et N I . , 1986). Newly developed experimental approaches, using the techniques of structural analysis, kinetics, and molecular biology, have made possible some dramatic advances in the study of the acetylcholine receptor from the electric organ of Torpedo; some of these results will be described in other chapters in this volume. The receptor is by far the best known of the approximately 20 membrane proteins that have been identified as being important in signal transmission, simply because it exists in such large quantities in electric organs. In addition to the work described elsewhere in this volume, a number of other studies require large amounts of proteins, for example structure determination. The result of a three-dimensional image analysis, by Brisson and Unwin (1985), of tubular crystals of the Torpedo receptor is shown in Fig. I . This is the view of the receptor when one looks directly on to the plane of the membrane. The protein protrudes from the bilayer in a funnel-shaped structure, and the five subunits surround the centrally located transmembrane channel. The funnel is tapered to an 8 transmembrane pore, which can regulate the flow of inorganic ions through the membrane. Another type of investigation that requires relatively large amounts of protein is the chemical kinetics approach. The chemical reactions mediated by the acetylcholine receptor, which, in the absence of much additional information, is taken as a model for other receptors, are illustrated in Fig. 2. Two molecules of acetylcholine bind to one molecule of the acetylcholine receptor protein (Cash and Hess, 1980), and the transmembrane receptor channel opens transiently. The resulting exchange of inorganic ions through the channel gives rise to an electrical signal, which is a prerequisite for signal transmission to occur. The elementary steps in channel opening can be conveniently examined by the single-channel current recording technique of Neher and Sakmann (1976). The method is very versatile and enables one to study receptors directly on cell surfaces. A number of other processes determine, and can modulate, the formation of transmembrane channels and the transmission of signals: the associ-

A

10. EXPRESSION OF ACh RECEPTOR IN YEAST

199

FIG.I . Three-dimensional map of receptor molecules in the crystal lattice as they would appear viewed from the synaptic cleft; successive sheets are of sections parallel to the membrane plane separated by spaces corresponding to 5 A. The center-to-center separation between receptors in the horizontal direction corresponds to 90 A. (Reprinted with permission from Brisson and Unwin, 1985.)

ation and dissociation of ligands from sites that control channel opening, the actual opening of the channel, and the inhibition of the receptor by acetylcholine itself (Takeyasu et a / . , 1983). The inhibitory site is characterized by a dissociation constant that is dependent on the transmembrane voltage of the cell membrane (Takeyasu rt d., 1983, 1986; Shiono pr a / . , 1984). A second inhibitory site, to which many compounds of pharmacological interest bind (Neher and Steinbach, 1978; Oswald p t d..1983;

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A I

r

I n W

a

Channel activation

a (0

3

Desensitized inactive receptor form

I

t

-

sa

Channel closing

3

g

Channel opening

sor n W (0 I

t

Open-channel receptor form allowing transmembrane movement of cations (M+) FIG.2. Regulatory mechanisms of the acetylcholine receptor. The open circles represent the sites where competitive agonists and antagonists bind to the receptor. The triangle represents the voltage-dependent. inhibitory hinding site.

Cox et al., 1984; Heidmann and Changeux, 19841, is not shown in Fig. 2. This second inhibitory site is characterized by a dissociation constant distinctive of the particular inhibitor (Neher and Steinbach, 1978; Adams, 1981; Oswald et al., 1983; Heidmann and Changeux. 1984). All the constants pertinent to the processes that determine the fraction of receptors that form transmembrane channels could easily be determined,

10. EXPRESSION OF ACh RECEPTOR IN YEAST

20 1

except for one problem. The receptor undergoes two changes in conformation: a slow inactivation (desensitization), which occurs in the second time region and was first described by Katz and Thesleff in 1957, and a fast desensitization, which occurs in the millisecond time region and was first discovered in 1978 by Stan Lipkowitz and Gary Struve (Hess et a / . , 1978). Receptor desensitization is accompanied by changes in the binding constants of compounds that affect channel opening (Katz and Thesleff, 1957; Hess et a / . , 1978, 1979). Therefore, the binding processes responsible for signal transmission must be measured in the millisecond time region before the receptor desensitizes. Derek Cash and Hitoshi Aoshima (Hess et ul., 1979; Cash e t a / . , 1980; Cash and Hess, 1980; Aoshima et al., 1981) developed fast reaction techniques suitable for measuring the properties of the receptor in the millisecond time region, that is before it undergoes the rapid desensitization and changes its ligand-binding properties (Aoshima et ul.. 1981). Like the structural studies, chemical kinetic measurements, which include labeling of specific binding sites and other covalent modifications of the receptor, require relatively large quantities of receptor protein. The application of molecular genetic techniques in neurobiology made investigations of the interrelationships between the five receptor subunits and their roles in signal transmission possible. Complementary DNAs for the subunits of the Torpedo acetylcholine receptor were first cloned successfully, the a-,p-, and &-subunitsby Numa's group at Kyoto University (Noda et uf., 1982, 1983a) and the y-subunit by a group at the Salk Institute (Ballivet et ul., 1982). The primary structure of each of the four polypeptides has been determined from the nucleotide sequences of the cDNAs (Noda et u/., 1982, 1983a; Claudio r t a/., 1983; Devillers-Thiery or d.. 1983). Clones coding for the acetylcholine receptor subunits from a mouse muscle cell line (BC,HI; LaPolla rt d.,1984; Boulter el d., 1985), a rat chromaffin cell line (PC12; Boulter "t a/., 1986), chicken embryo cells (Nef et u / . , 19841, calf muscle (Noda et a / . , 1983b; Takai e l d..1985), and human cells (Noda et al.. 1983b) have also been isolated and characterized. These cDNAs can be used in an expression system to instruct cells to make the protein encoded by the cDNA. The importance of efficient expression systems lies in the ability to produce large quantities of the many different receptors that do not occur in abundance naturally, so that their structure and function can be investigated. The question being addressed in this laboratory, therefore, is: How can large quantities of these proteins be produced so that they can be studied '? Eric Barnard and his colleagues have shown that small amounts of receptor protein can be made by microinjecting mRNA for the Torpedo acetylcholine receptor into frog oocytes (Barnard et ul., 1982; Sumikawa

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al., 1981). Although the quantities of protein produced are much smaller than those needed to study the structure and function of the acetylcholine receptor (which so far can only be done with the receptor from the electric organ of certain fish), a great deal of information has been obtained (Mishina et al., 1984, 1985, 1986; Sakmann et al., 1985). In Chapter I 1 the expression of receptors in cells grown in culture is discussed. The approach we are taking involves another type of eukaryotic cell, namely the yeast Saccharomyces cerevisiae. The difference in the expression of a foreign protein in cultured cells and in the oocyte system is that it is possible to integrate cDNA into the genome of replicating cells whereas mRNA must be microinjected into individual oocytes. We have chosen yeast rather than other cells because it is possible to grow it in large quantities, and because its genetics are relatively well understood. In the experiments with yeast, two important questions needed to be answered first: (1) Can a completely foreign protein, a neuronal receptor protein, be expressed in yeast? These proteins are not expressed in Escherichia coli. ( 2 ) Does yeast have a mechanism for inserting this protein into its plasma membrane? In the first experiments with yeast (Fujita et al., 1986a); the structural gene for the a-subunit of the T. californica acetylcholine receptor, cloned by Toni Claudio at Yale, was inserted into a yeast expression vector, obtained from Professor Ben Hall of the University of Washington in Seattle (McKnight and McConaughey, 1983). The plasmid (Fig. 3a) contains the yeast alcohol dehydrogenase gene promoter, the yeast cytochrome c gene terminator, and a region of the yeast 2 p plasmid that allows the plasmid to be replicated in yeast. The plasmid also contains the TRPl gene as a positive marker, so that a successful transformation of a trpl mutant yeast strain can be assessed by screening for colonies that no longer et

FIG.3. (a) Construction of the pYTca1 and pYTc8l plasmids containing cDNA encoding for the a-and &subunits of the T. ci&fi>micuacetylcholine receptor. respectively. (b) Immunoblot detection of the a- and &-subunits of the T. cir/ifomicicacetylcholine receptor in yeast. Yeast cells were transformed with the plasmid construct pYTcal or pYTcSI. Control yeast were transformed with the plasmid pMAC561, which does not contain cDNA encoding an acetylcholine receptor subunit. The cell walls of the yeast were digested with zymolyase and the resultant spheroplasts were disrupted by vortexing with glass beads. Plasma membrane fractions were isolated by differential centrifugation. The proteins from the plasma membrane fraction were separated by electrophoresis on a 10% SDS-polyacrylamide gel. The proteins were transferred to nitrocellulose paper, incubated with monoclonal antibodies # I42 and #I29 (lanes 2-5) against the a-and the &subunit. respectively (Gullick and Lindstrom. 1983; Criado e / d.,1985). or antibody #I29 alone (lane I). and subsequently treated with sheep anti-rat IgG and '"1-labeled protein A (Nelson, 1983). The resultant autoradiograms of the membrane fractions are shown. Lane 5 contains plasma membrane from the T. c~r/ifiwnic,tr electroplax (-10 pg protein).

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MELODY T. SWEET ET AL.

require tryptophan in the culture medium. A similar approach was adopted with the other subunits, and to date the b- and y-subunits have also been expressed successfully in separate yeast clones (Fujita ef al., 1986b, and unpublished results). Expression of the receptor subunits was detected by immune blotting extracts from transformed yeast cells. Yeast membrane proteins were separated by gel electrophoresis and blotted on to a nitrocellulose filter. The receptor subunits were detected by using rat monoclonal antibodies specific for the individual subunits. Anti-rat antibody (IgG) and ["'I]-labeled protein A were then used to detect the monoclonal antibody. Shown on the right hand side of Fig. 3b are the a- and b-subunits obtained from the T . californica electric organ, and made visible by reaction with specific monoclonal antibodies for these two subunits. In the two central lanes (3 and 4) are proteins from yeast cells transformed with a plasmid that does not contain cDNA encoding a receptor subunit; neither the a- nor the 8subunit is detected in these cells. In the two lanes on the left (lanes I and 2) the a- and &subunit of the Torpedo receptor isolated from the plasma membrane of transformed yeast can be seen. Only yeast cells that contained a plasmid carrying the structural gene for a subunit contained a polypeptide that was immunoreactive. Moreover, the labeled proteins had the same mobility as the corresponding subunits from native Torpedo membranes. It appears, therefore, that yeast can synthesize membranebound receptor polypeptides, and that the polypeptides have the expected molecular weight, primary structure, and antigenic specificity. The fact that the mobility of the subunits from Torpedo membranes and from yeast is the same suggests that, like the native receptor subunits, posttranslational modification of the subunits has occurred in yeast. Numa and co-workers (Noda et ul., 1982) have shown that the a-subunit of the Torpedo acetylcholine receptor contains a single potential N-glycosidation site, an asparagine residue #141. When this residue was converted to aspartic acid, the a-subunit migrated faster than the wild type on an SDSpolyacrylamide gel (Mishina ef al., 1985). The experiments illustrated in Fig. 4 further demonstrate that yeast can not only express a foreign protein but can also insert it into its plasma membrane. Yeast cells that were transformed with pYTcal were first treated with zymolyase to prepare spheroplasts. cells in which the plasma membrane is exposed. The spheroplasts were then fixed with formaldehyde and treated with various monoclonal antibodies, which in turn were decorated with a fluorescent second antibody. On the left are phase contrast pictures of spheroplasts and on the right are immunofluorescence pictures of the same cells. The top row shows yeast cells that were treated with the fluorescent second antibody but not with a monoclonal antibody against

10. EXPRESSION OF ACh RECEPTOR IN YEAST

205

FIG.4. lmmunofluorescence detection of the a-subunit of the acetylcholine receptor expressed in yeast. Yeast transformed with pYTcal (a plasmid containing cDNA encoding the a-subunit) were converted to spheroplasts and fixed in 3% formaldehyde. The fixed spheroplasts were either incubated first with monoclonal antibody # 142 (Gullick and Lindstrom. 1983) and then with fluorescein-conjugated rabbit anti-rat IgG (c and d) or were incubated with only fluorescein-conjugated anti-rat IgG (a and b). I n a and c are the yeast containing the a-subunit as seen under phase contrast. and in b and d are the same a yeast cells when illuminated by a mercury lamp using a 474-nm barrier filter (conditions optimizing fluorescence emission). Spheroplasts are magnified INM times.

the Torpedo a-subunit. Spheroplasts in the bottom row were treated with both the monoclonal and the fluorescent antibodies. The top right side shows that yeast that were not treated with the monoclonal antibody against the a-subunit do not demonstrate fluorescence. This is the control. The lower right side shows that the a-subunit monoclonal antibody reacts with the transformed yeast cell membrane and can be visualized with the second antibody. Figure 5 contains quantitative fluorescence measurements made on Professor Watt Webb's digitized video-image microscopy system at Cornell (Gross and Webb, 1987). The round spots are yeast cells and below them are histograms of the fluorescence intensity associated with the cell. Figure

MELODY T. SWEET ET AL.

10. EXPRESSION OF ACh RECEPTOR IN YEAST

207

5b shows cells that have been transformed with pYTc8l and that expressed the 8-subunit and inserted it into their plasma membranes. Figure 5a is a parallel control experiment: cells that were transformed with a plasmid that did not contain the &subunit cDNA and, therefore, gave the background fluorescence level. Additional experiments were done in order to determine whether the antibodies could penetrate the yeast cell membrane, to make certain that the receptor subunits detected are in the membrane and not in the cytoplasm of the cell. These results are shown in Fig. 6. An antibody that is specific for porin, an outer mitochondria1 membrane protein (Riezman ef al., 1983). was used. The fluorescence of yeast cells treated with antibodies against porin is shown in Fig. 6a. The intensity was about the same as that obtained with cells treated only with the fluorescent second antibody (not shown). Figure 6b shows an example of yeast in which the plasma membrane was made permeable with Triton X-100.The antibody was able to penetrate the yeast membrane and a 100% increase in the fluorescence signal was observed. The experiments with antiporin show that, unless the yeast cell membrane was permeabilized, it was not penetrated by the antibodies. This demonstrates that in the experiments described above the a- and &subunit have been inserted into the yeast membrane and react with antibodies on the cell surface. One could then ask the following questions: ( I ) What is the orientation of the a-subunit in the plasma membrane when it is expressed by itself in yeast? (2) What are the ligand-binding properties of the a-subunit in the yeast membrane? It is known that in the intact receptor the a-subunit contains binding sites for both acetylcholine, as shown by Karlin (Weill et al., 1974),and inhibitors such as the snake toxin, a-bungarotoxin, as shown by Wilson at Yale University (Wilson ef a/., 198% by Fuchs at the Weizman Institute (Neumann et al., 1986), and by Lindstrom at the Salk Institute (Criado et al.. 1986). If the a-subunit in yeast also contains these binding sites it should be possible, using site-specific mutation of specific nucleotide sequences, to determine the contribution individual amino acid residues make FIG.5. Detection of the cloned &subunit of the acetylcholine receptor in the yeast plasma membrane by irnmunofluorescence. Spheroplasts were produced from yeast cells transformed with pYTc8l or pMAC56I (control), fixed with 3% formaldehyde. and incubated with monoclonal antibody #I28 (Sargent ef d.,1984). lrnrnunocomplexes were visualized by subsequent addition of fluorescein isothiocyanate-labeled rabbit anti-rat IgG. Digitized video-image microscopy was used to measure the intensity of yeast cell fluorescence as shown in the histograms. The horizontal lines through the individual cells indicate where the yeast cells were scanned for fluorescence intensity. Cells are magnified 700 times. (a) Yeast cells transformed with pMACS61 (control): (b) yeast cells transformed with pYTc8I.

208

MELODY T. SWEET ET AL.

10. EXPRESSION OF ACh RECEPTOR IN YEAST

209

to the ligand-binding process. Intact yeast containing the a-subunit do indeed bind a-bungarotoxin at a specific site on the a-subunit (Fig. 7). The binding is saturable (Fig. 7a) and Fig. 7b shows that the dissociation constant for this binding site, K D , has a value of 0.2 p M . The binding of a-bungarotoxin is 100-fold weaker than to the native a-subunit in the pentameric structure; however, isolated a-subunits from Torpc.do yield a comparable binding constant (Haggert and Froehner, 1981: Tzartos and Changuex, 1983). This, with the appropriate control experiments. indicates that the binding site is on the outside of the yeast membrane. The site is also located on the outside of the cell membrane in the native pentameric acetylcholine receptor (Ratnam et al., 1986a). a-Bungarotoxin is the classic inhibitory ligand of the nicotinic acetylcholine receptor (Lee, 1972). Using radiolabeled toxin as an indicator, it was possible to demonstrate that other compounds that bind to the receptor sites, and cause the receptor channel to open or inhibit channel formation, also bind to the a-subunit when it is inserted into the yeast membrane (Fig. 7c). The nicotinic receptor antagonists a-bungarotoxin (aBTX), a Nrlju toxin (aNajaTx), and d-tubocurarine, and the receptor agonist suberyldicholine were able to displace a-bungarotoxin from its binding site on the a-subunit. Less potent agonists, acetylcholine and carbamoylcholine. and sodium chloride, at concentrations less than 1 mM, were not able to displace the bound a-bungarotoxin. The dissociation constants for a-yeast are much higher than those for the native acetylcholine receptor, indicating that when the a-subunit of the receptor is expressed in the yeast membrane, it retains its ability to bind a-bungarotoxin and compounds that control channel opening, but to much less an extent than when the asubunit is part of the native pentameric Torpedo acetylcholine receptor. The yeast expression system is, therefore, suitable for studying the structure and function of the a-subunit. and perhaps the other subunits, in yeast. From the use of monoclonal antibodies, particularly by Jon Lindstrom and his group (Sargent et af., 1984; Criado et al., 1985; Ratnam r t d., 1986a.b). a considerable amount is known about the orientation of the asubunit in the membrane when this subunit is part of the pentameric re-

FIG.6. Detection of penetration of the plasma membrane by antibodies. Yeast cells were transformed. converted into spheroplasts, and fixed with formaldehyde. Fixed spheroplasts were incubated with an antibody against porin, a major protein of the outer mitochondria1 membrane, in (a) the absence and (b) the presence of I% Triton X-100.Fluorescein-isothiocyanate-conjugated goat anti-rabbit IgC was used to visualize the bound antiporin. The intensity of yeast cell fluorescence, indicated in the histograms, was measured by digitized video-image microscopy. Cells are magnified 700 times.

a

a-Bungarotoxin : a-subunit Complex (nM)

C

I

10. EXPRESSION OF ACh RECEPTOR IN YEAST

21 1

ceptor (Fig. 8) (Ratnam et a/., 1986b).The peptide chain crosses the cell membrane, indicated by the lipid bilayer, five times. Four of the segments crossing the membrane are believed to be in helical conformation, indicated by the spirals, and one section of the chain crosses the membrane in a different conformation. The amino-terminal region of the chain faces the outside and is indicated by an N ; the carbohydrates are indicated by the bush-like structure. The C-terminal portion of the chain is inside the cell, and is represented by residue 437. The thickened areas are regions on the chain for which specific monoclonal antibodies have been prepared in Lindstrom's laboratory at the Salk Institute. Using a variety of these monoclonal antibodies, and in collaboration with Pat Donaldson in Webb's laboratory at Cornell University, both similarities and differences were found in the orientation of the a-subunit in.the pentameric Torpedo receptor and as it is expressed in yeast. The main immunogenic region (Tzartos et a/., 1981) (contained within amino acids 46 to 127) of the a-subunit in yeast, and the acetylcholineand toxin-binding sites are all outside the membrane as they are in the native receptor in the Torpedo membrane (Ratnam et af.. 1986a,b). The difference lies in a later region (amino acid residues 339 to 396) of the achain. When the a-subunit is part of the pentameric native receptor in the Torpedo electric organ, this portion of the chain is on the cytoplasmic face of the membrane. When the a-subunit is expressed by itself in yeast it is on the outside. What happens when all the subunits are expressed together in yeast is one of the interesting questions still to be answered. The a-subunit constitutes approximately I% of the total protein in the yeast membrane (Fujita rt id., 1986a). Its density is approximately the same in the plasma membrane of yeast as it is in the receptor-rich plasma membrane of the electric organ of E. e/rc.tric.us, with which this laboratory

FIG.7. u-Bungarotoxin binding to the u-subunit produced in yeast. (a)and (b) Spheroplasts were prepared and suspended in 1.25 osmolar buffer containing 250 mM sodium chloride. 5 mM potassium chloride. 0. I mM EDTA. 10 mM MOPS. pH 7.4. and a balance of Sorbitol. The spheroplasts ( - I mg wet weight) were incubated for 2 hr with '"I-labeled a-bungarotoxin in the presence (nonspecific binding) or absence (total binding) of an excess of 40 pM unlabeled u-bungarotoxin. These reaction mixtures were then filtered through I .2 pm diameter pore size Millipore membrane filters and washed with buffer. Specific binding was calculated as the difference between the total and nonspecifically bound toxin (Weiland et d.,1978). About 4 5 9 of the a-bungarotoxin binding was specific. ( a ) Saturation curve of specific '"Ilabeled u-bungarotoxin binding. ( b ) Scatchard plot of specific toxin binding. The K , , is 0. I pM. ( c ) The membranes prepared from spheroplasts disrupted by mixing with glass bead5 and isolated by differential centrifugation (0.I mg/ml) were incubated for 2 hr with 60 nM ['"I-labeled] u-bungarotoxin ((r-BTX) in the presence of varying concentrations of competing ligands. The reaction mixtures were filtered and specific binding was calculated as described previously (Haggert and Froehner. 1981: Tzartos and Changeux. 1983).

MELODY T. SWEET ET AL.

212

............. ...... ..... ..... :-...: iM3: ...... ....*..:: .* :."'

2771

......

43

15 .-,

.*." *.

8

''298

......................

F~G. 8. Model of the transmembrane orientation of the polypeptide chain in a-subunits. Thick line segments indicate sequences for which there is evidence of transmembrane orientation. The shrub-like symbol indicates the glycosylation site. (Reprinted with permission from Ratnam e t a / . , 1986b.3

made most of our rapid kinetic experiments (for reviews see Hess et al., 1983; Udgaonkar and Hess, 1986, 1987). The other subunits, although there is a great deal of homology between the amino acid sequences, are expressed to much less an extent. The @-subunitis transcribed but expression has not been detected so far. The sequences that are important for expression and insertion of proteins into the plasma membrane are quite difierent

213

10. EXPRESSION OF ACh RECEPTOR IN YEAST

in the a- and p-subunits (Noda servations):

ef id.,

1982; T. Claudio, unpublished ob-

Signul Peptides

acDNA: ATG ATT CTG TGC ACT TAT TGG CAT GTA -__GGG TTG GTG CTA CTG TTA TTT Met Ile Leu Cys Ser Tyr Trp His Val Gly Leu Val Leu Leu Leu Phe --TCG TGT TGT GGT CTG GTA CTA GGU . . . Ser Cys Cys Gly Leu Val Leu Gly . . .

PcDNA: ATG GAG AAC GTG AGG AGG ATG GCG CTG --GGT CTG GTG GTG ATG ATG GCG Met Glu Asn Val Arg Arg Met Ala Leu -Gly Leu __ - Val Val Met Met Ala ASP Val

CTGGCCCTCAGCGGCGTGGGGGCG . . . Leu Ala Leu Ser Gly Val Gly Ala . . . ~

Lecider Sequences PyTca: -

. . . AAAGCTGAAGAATG. __ . . PyTcp: -

. . . GAATTCCGCGATG.. . These differences provide a unique opportunity to determine which sequences of the subunit are important for expression and for membrane insertion. It is possible to replace the leader sequence of the p-subunit with the leader sequence of the a-subunit, and the signal peptide of the P-subunit with the signal peptide of the &-subunit; it is also possible to use the same codons that specify specific amino acids in the a-subunit in the p-subunit. I n summary, it has been shown that 1 . Yeast has the necessary apparatus to express membrane-bound receptor proteins and, more remarkably, to insert such foreign proteins into the plasma membrane. 2. The a-subunit synthesized by yeast contains a ligand-binding site inserted in the correct orientation for binding in the yeast membrane. 3. The relationship between the structure and function of the a-subunit can now be investigated using the yeast expression system.

There exists in yeast an excellent opportunity to investigate the assembly of subunits into a functional protein. Two approaches can be used: (i) one can isolate the subunits and then study their assembly in artificial membranes; (ii) one can introduce the cDNA corresponding to all the subunits

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of the receptor into the genome of yeast and also study assembly in the plasma membrane. In general, the mechanism by which proteins are processed from the site of synthesis t o the point of insertion into plasma membranes is not yet well understood, and yeast cells, which express the a-subunit of the acetylcholine receptor subunits in quantity, appear ideal for such studies because the protein is inserted into the membrane. The cells can easily be grown in large quantities and the methods for separating the plasma membrane and the various organelles of the yeast cells are well worked out (Nelson, 1983). It is possible then to start investigating further questions important for understanding the expression of proteins in yeast: (1) Which cDNA sequences are important for expression and for insertion? (2) Which cDNA sequences and which properties of the yeast cells are important in the assembly of a multisubunit protein into a functional receptor? At the beginning of this chapter, we presented some of the reasons why we chose to investigate yeast as an expression system for neuronal receptors, namely to use yeast to amplify the amounts of receptor proteins that normally exist in only small quantities in cells so that their structure and function can be studied. Because yeast can easily be grown in large quantities, it has obvious advantages over oocytes, an expression system in which each cell has to be injected to obtain relatively little protein. Therefore, with the development of the technology to produce large enough quantities of membrane proteins in yeast to investigate their structure and function, yeast cells can become a good source for the approximately 20 neuronal receptors that exist in only small quantities in cells and about which relatively little is known. ACKNOWLEDGMENTS Supported by a grant from the Cornell Biotechnology Program, which i s supported by the New York State Science and Technology Foundation and a consortium of industries. We thank Ben Hall for the yeast expression vector pMAC561. M. T. S. was supported by a Muscular Dystrophy Association Postdoctoral Fellowship, K. J . by a Feodor Lynen Postdoctoral Fellowship awarded by the Alexander von Humboldt Foundation, and C. K. M. by a scholarship from the Korean Ministry of Education. REFERENCES Adams, P. R. (1981). Acetylcholine receptor kinetics. J . Membr. Eiol. 58, 161-174. Aoshima, H., Cash, D. J., and Hess, G. P. (1981). The mechanism of the inactivation (desensitization) of the acetylcholine receptor. Investigations by fast reaction techniques with membrane vesicles. Biochemistry 20, 3467-3474. Ballivet. M.. Patrick, J., Lee, J., and Heinemann, S. (1982). Molecular cloning of cDNA coding for the y-subunit of Torpedo acetylcholine receptor. Proc. Nurl. Acud. Sci. U.S.A. 79, 4466-4470.

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Barnard, E.. Miledi, R.. and Sumikawa, K. (1982). Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proc. Roy. Soc. London, Ser. B 215, 241-246. Boulter, J., Luyten, W.. Evans, K.. Mason, P.. Ballivet, M., Goldman. D.. Stengelin, S . . Martin, G . . Heinemann, S., and Patrick, J. (1985). Isolation of a clone coding for the a-subunit of a mouse acetylcholine receptor. J . Neurosci. 5, 2545-2552. Boulter, J.. Evans, K., Goldman. D.. Martin. G.. Treco, D.. Heinemann. S.. and Patrick J . (1986). Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor a-subunit. Nutrrre (London) 319, 368-374. Brisson, A., and Unwin, P. N. T. (1985). Quaternary structure of the acetylcholine receptor. Nulitre (London) 315, 474477. Cash, D. J., and Hess, G. P. (1980). Molecular mechanism of acetylcholine-receptor-controlled ion translocation across cell membranes. Proc. Null. Acud. Sci. U . S . A . 77, 842-846. Cash, D. J.. Aoshima. H., and Hess, G. P. (1980). Acetylcholine-induced cation translocation across cell membrane and inactivation of the acetylcholine receptor: Chemical kinetic measurements in the msec time region. Proc. Null. Acud. Sci. U . S . A . 78, 3318-3322. Claudio. T.. and Raftery, M. A. (1977). Immunological comparison of acetylcholine receptors and their subunits from species of electric ray. Arcli. Biochem. Biopliys. 181, 484-489. Claudio, T.. Ballivet. M., Patrick, J.. and Heinemann, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo culifornicri acetylcholine receptor y-subunit. Proc. N U / / .Arcid. Sci. U . S . A . 80, 1111-1115. Cox, R. N.. Kaldany, R.-R., Brandt, P. W.. Ferren, B., Hudson, R. A.. and Karlin. A. ( 1984). A continuous-flow. rapid-mixing photolabeling technique applied to the acetylcholine receptor. Anal. Biocliem. 136, 476-486. Criado, M . , Hochschwender, S.. Sarin, V., Fox, J. L.. and Lindstrom, J. (1985). Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits. Proc. Null. Acud. Sci. U . S . A . 82, 2004-2008. Criado, M.. Sarin. V., Fox, J. L., and Lindstrom, J. (1986). Evidence that the acetylcholine binding site is not formed by the sequence 1x127-143 of the acetylcholine receptor. Biocliemis/~y25, 2839-2846. Devillers-Thiery. Giraudat, J., Bentaboulet, M., and Changeux. J.-P. ( 1983). Complete mRNA coding sequence of the acetylcholine binding a-subunit of Torpedo rnurmorutu acetylcholine receptor: A model for the transmembrane organization of the polypeptide chain. Proc. Null. Acud. Sci. U . S . A . 80, 2067-2071. Fujita. N.. Nelson, N., Fox. T. D.. Claudio. T.. Lindstrom, J., Riezman, H., and Hess. G. P. ( 1986a). Biosynthesis of the Torpedo rdifornicu acetylcholine receptor a-subunit in yeast. Science 231, 1284-1287. Fujita. N.. Sweet, M. T.. Fox, T. D., Nelson, N . . Claudio. T.. Lindstrom, J.. and Hess. G. P. (1986b). Expression of cDNAs for acetylcholine receptor subunits in the yeast cell plasma membrane. Bioclwm. Soc. S y m p . 52, 41-56. Gross, D . . and Webb. W. W. (1987). Cell surface clustering and mobility of the liganded LDL receptor measured by digital video fluorescence microscopy. In "Spectroscopic Membrane Probes" ( L . M. Loew. ed.). CRC Press. Miami, Florida. In press. Gullick, W. J., and Lindstrom, J. M. (1983). Mapping the binding of monoclonal antibodies to the acetylcholine receptor from Torpedo cu/iJi,rnicu. Biochemisrrv 22, 3312-3320. Haggert. J. A.. and Froehner, S. C. (1981). Restoration of '"l-a-bungarotoxin binding activity to the a-subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate. J. B i d . Chem. 256, 8294-8297. Heidmann. T., and Changeux. J.-P. (1984). Time-resolved photolabeling by the noncom-

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petitive blocker chlorpromazine of the acetylcholine receptor in its transiently open and closed ion channel conformations. Proc. Nutl. Acud. Sci. U . S . A . 81, 1897-1901. Hess, G. P., Lipkowitz, S., and Struve. G. E. (1978). Acetylcholine receptor-mediated ion flux in electroplax membrane microsacs (vesicles): Change in mechanism produced by asymmetrical distribution of sodium and potassium ions. Proc. Nut/. Acud. Sci. U . S . A . 75, 1703-1707. Hess, G. P.. Cash, D. J., and Aoshima, H. (1979). Acetylcholine receptor-controlled ion fluxes in membrane vesicles investigated by fast reaction techniques. Nuture (London) 282, 329-33 1. Hess, G. P., Cash, D. J., and Aoshima. H. (1983). Acetylcholine-receptor-controlled ion translocation. Chemical kinetic investigations of the mechanism. Annu. Rev. Biophgs. Bioeng. 12, 443-473. Katz, B., and Thesleff. S. (1957). A study of the desensitization produced by acetylcholine at the motor endplate. J . Physiol. (London) 138, 63-80. LaPolla, R. J.. Mayne, K. M., and Davidson, N. (1984). Isolation and characterization of a cDNA clone for the complete protein coding region of the 6-subunit of the mouse acetylcholine receptor. Proc. Nutl. Acud. Sci. U.S.A. 81, 7970-7974. Lee, C. Y. (1972). Chemistry and pharmacology of polypeptide toxins in snake venoms. Annu. Rev. Phurmucol. 12, 265-286. Lindstrom. J., Cooper, J., and Tzartos, S. (1980). Acetylcholine receptors from Torpedo and Elecrrophorus have similar subunit structures. Biochemistry 19, 1454-1458. McKnight, G., and McConaughey. B. (1983). Selection of functional cDNAs by complementation in yeast. Proc. Nutl. Acud. Sci. U . S . A . 80, 4 4 1 2 4 1 6 . Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto. Y ., Noda, M., Yamamoto, T.. Terao. M., Lindstrom. J., Takahashi, T., Kuno, M., and Numa, S. (1984). Expression of functional acetylcholine receptor from cloned cDNAs. Nuture (London) 307, 604408. Mishina, M.. Tobimatsu. T.. Imoto, K., Tanaka, K., Fujita, Y., Kukuda, K.. Kurasaki. M., Takahashi, H., Morimoto, Y., Hirose, T., Inayama, S., Takahashi, T.. Kuno. M.. and Numa, S. (1985). Location of functional regions of acetylcholine receptor a-subunit by site-directed mutagenesis. Nuture (London) 313, 364-369. Mishina, M., Takai. T., Imoto, K., Noda, M., Takahashi. T.. Numa, S., Methfessel. C., and Sakmann, 9. (1986). Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nuture (London) 321, 406-41 I . Nef, P., Mauron. A., Stalder, R.,Alloid. C., and Ballivet, M. (1984). Structure, linkage, and sequence of the two genes encoding the 6- and y-subunits of the nicotinic acetylcholine receptor. Proc. Nutl. Acud. Sci. U . S . A . 81, 7975-7979. Neher. E., and Sakmann. 9. (1976). Single-channel currents recorded from membrane of denervated frog muscle fibers. Nuture (London) 260, 779-802. Neher, E., and Steinbach, J. H. (1978). Local anesthetics transiently block currents through single acetylcholine-receptor channels. J. Physiol. (London) 277, 153-176. Nelson, N. (1983). Structure and synthesis of chloroplast ATPase. Methods Enzymol. 97, 510-523. Neumann. D., Barchan. D., Safran, A., Gershoni, J. M., and Fuchs, S. (1986). Mapping of the a-bungarotoxin binding site within the a-subunit of the acetylcholine receptor. Proc. Nut/. Acud. Sci. U.S.A. 83, 3008-3011. Noda, M., Takahashi. H.. Tanabe, T., Toyosata, M.. Furutani, Y., Hirose. T.. Asai, M., Inayama, S.. Miyata, T.. and Numa, S. (1982). Primary structure ofa-subunit precursor of Torpedo culifornicu acetylcholine receptor deduced from cDNA sequence. Nutrrre (London) 299, 793-797.

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Noda. M.. Takahashi. H.. Tanabe. T., Toyosato, M.. Kikyotani. S.. Hirose. T.. Asai. M.. Takashina, H.. Inayama. S., Miyata. T.. and Numa. S. (1983a). Primary structures of p- and &-subunitprecursors of Torpedo colijbrnicu acetylcholine receptor deduced from cDNA sequences. Nu/iire (London) 301, 251-255. Noda, M.. Furutani. Y..Takahashi, H.. Toyosato. M.. Tanabe, T.. Shimizu. S . . Kikyotani, S., Kayano. T., Hirose. T., Inayama. S.. and Numa. S. ( 1983b).Cloning and sequence analysis of calf cDNA and human genomic DNA encoding a-subunit precursor of muscle acetylcholine receptor. Notitre (London) 305, 818-823. Oswald, R. E., Heidmann, T.. and Changeux, J.-P. (1983). Multiple affinity states for noncompetitive blockers revealed by I 'Hjphencyclidine binding to acetylcholine receptorrich membrane fragments from Torpedo murmoru/tr. Biochernisiry 22, 3 128-3 136. Patrick, J., and Stallcup. W. B. (1979). Immunological distinction between acetylcholine receptor and the a-bungarotoxin binding component on sympathetic neurons. Proc. N a i l . Acud. Sci. U . S . A . 74, 46894692. Ratnam. M., Sargent. P.. Sarin. V., Fox, J. L., Le Nguyen, D., Rivier, J.. Criado. M.. and Lindstrom. J. (1986a). Location of antigenic determinants of primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping. Biochernisrrv 25, 26212632. Ratnam. M.. Le Nguyen. D., Rivier, J., Sargent. P.. and Lindstrom, J. (1986b). Transmembrane topography of nicotinic acetylcholine receptor: lmmunochemical tests contradict theoretical predictions based on hydrophobicity profiles. Biochemisii? 25, 262 I 2632. Reynolds, J.. and Karlin, A. (1978). Molecular weight in detergent solution of acetylcholine receptor from Torpedo culifornicri. Biocliemi.s/r?, 17, 2035-2038. Riezman. H.. Hay, R., Gasser, S., Daum, G.. Schneider. G., Witte, C., and Schatz. G. (1983). The outer membrane of yeast mitochondria: isolation of outside-out sealed vesicles. E M B O J . 2, 1105-1 I I I . Sakmann. B.. Methfessel. C., Mishina. M., Takahashi, T.. Takai, T.. Kurasaki. M., Fukuda. K., and Numa, S. (1985).Role of acetylcholine receptor subunits in gating of the channel. Nature (London) 318, 538-543. Sargent, P. B.. Hedges. B. E.. Tsavaler, L., Clemmons, L.. Tzartos, S., and Lindstrom. J. M. (1984). Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies. J. CeII Biol. 98, 609-618. Shiono. S., Takeyasu. K.. Udgaonkar, J . B.. Delcour. A. H., Fujita, N.. and Hess. G . P. (1984). Regulatory properties of acetylcholine receptor: Evidence for two different inhibitory sites. one for acetylcholine and the other for a noncompetitive inhibitor of receptor function (procaine). Bioc~hemisiry23, 6889-6893. Sumikawa. K., Houghton. M.. Emtage, J. S.. Richards. €3. M.. and Barnard, E. A. (1981). Active multi-subunit acetylcholine receptor assembled by translation of heterologous mRNA in Xenoprts oocytes. Nurure (London) 292, 862-864. Takai, T.. Noda, M.. Mishina, M.. Shimizu, S.. Furutani, Y.,Kayano, T., Ikeda, T., Kubo, T., Takahashi. H.. Takahashi. T.. Kuno, M., and Numa. S. (1985). Cloning, sequencing and expression of cDNA for a novel subunit of acetylcholine receptor from calf muscle. Nuitire (London) 315, 761-764. Takeyasu, K.. Udgaonkar. J. B., and Hess. G. P. (1983). Acetylcholine receptor: Evidence for a voltage-dependent regulatory site for acetylcholine chemical kinetic measurements in membrane vesicles using a voltage clamp. Biochemisrry 22, 5973-5978. Takeyasu. K.. Shiono, S.. Udgaonkar. J. B.. Fujita. N.. and Hess, G. P. (1986). Acetylcholine

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receptor: Characterization of the voltage-dependent regulatory (inhibitory) site for acetylcholine in membrane vesicles from Torpedo culifornicu electroplax. Biochemistry 25, 177Q-1776. Tzartos, S . , and Changeux, J.-P. (1983). High afinity binding of a-bungarotoxin to the purified a-subunit and to its 27,000-dalton proteolytic peptide from Torpedo mtwnorutu acetylcholine receptor. Requirement for sodium dodecyl sulfate. EMBO J. 2, 38 1-387. Tzartos, S. J . , Rand, D. E.. Einarson. B. L.. and Lindstrom. J . M. (1981). Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies. J. Biol. Cliem. 256, 8635-8645. Tzartos. S., Langeberg, J . , Hochschwinder, S.. Swanson, L., and Lindstrom. J . (1986). Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: Species, subunit and region specificity. J . Nrirroitnmrmol. 10, 235253. Udgaonkar. J. B.. and Hess, G. P. (1986). Acetylcholine receptor kinetics: Chemical kinetics. J. Memhr.. Biol. 93, 93-109. Udgaonkar. J . B.. and Hess. G. P. (1987). Chemical kinetic measurements of transmembrane processes using rapid reaction techniques: Acetylcholine receptor. Annrr. Rev. Bioplipllvs. Bioplivs. Cliem. 16, 507-534. Weiland. G.. Frisman. D., and Taylor. P. (1978). Affinity labeling of the subunits of the membrane associated cholinergic receptor. Mol. Phurrnucol. 15, 213-226. Weill. C. L., McNamee, M., and Karlin, A. (1974). Afinity-labeling of purified acetylcholine receptor from Torpedo idifornictr. Biochem. Bicipliys. Res. Coinmiin. 61, 997-1003. Wilson. P. T., Lentz, T. L., and Hawrot, E. (1985). Determination of the primary amino acid sequence specifying the a-bungarotoxin binding site on the a-subunit of the acetylcholine receptor from Torpedo culifornicu. Proc. Nutl. Actid. Sci. U.S.A. 82, 87908794.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 33

Chapter 11

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

I. Introduction 11. Materials and Methods

A. Construction of cDNA Libraries B. Screening the Libraries for AChR Subunit Clones C. Preparation of mRNA Transcripts D. Preparation of Oocytes E. Microinjection F. ["'I]-BuTX Binding G. ACh-Induced "Na Uptake H . ACh-Induced Single Channel Currents I. DNA-Mediated Gene Transfer J . Viral Infection K . Labeling and Imrnunoprecipitations 111. Results A. Identifying Full-Length Clones B. Expression of Functional Cell Surface AChRs in Oocytes C. Cotransformation D. Protein Expression of Transfected DNA E. Viral Infection F. Expression of Torpedo a Protein in Fibroblasts and Muscle Cells G. Optimized Labeling and lmmunoprecipitation Conditions H. Partial Characterization of Torpedo a-Subunits in Fibroblasts and Muscle Cells IV. Discussion References 219 Copyright (0 1988 by Academic Press. Inc. All rights of reproductiun in any form reserved.

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1.

INTRODUCTION

The acetylcholine receptor (AChR) is an oligomeric, intrinsic membrane glycoprotein consisting of four different subunits (a,p, y, 6) with the stoichiometry a$yG. The molecular masses of the subunits are approximately 40,000,50,000,60,000, and 65,000 Da, respectively. The AChR is the best characterized ionic channel protein in vertebrates and, although a tremendous amount of information has been amassed concerning its structure, functional properties, role in various diseases, developmental regulation, and interaction with other proteins, much still remains to be elucidated (for reviews see Popot and Changeux, 1984; Dolly and Barnard, 1984: Anholt et al., 1984; McCarthy et ul., 1986; Karlin et a l . , 1986; Claudio, 1986). The complete primary amino acid sequence of each subunit was first deduced from clones isolated from Torpedo californica electric organ cDNA libraries (Noda et al., 1982, 1983a; Claudio et ul., 1983). Many predictions were then made concerning post-translational modifications of the receptor, the topology of folding of the polypeptide chains and the location of important functional domains such as the ligand binding site, the ion channel, and epitopes possibly involved in the autoimmune disease myasthenia gravis. The clones have been used effectively to isolate AChR subunit cDNAs and genes from other tissue sources in which the AChR is not a prevalent component: human (Noda et d . , 1983b), bovine (Noda et al., 1983b),chicken (Nef et ul., 19841, the mouse muscle cell line BC,H- I (LaPolla et al., 1984), the phaeochromocytoma cell line PC12 (Boulter e t al., 19861, Drosophilu melanogaster (Hermans-Borgmeyer e? d . , 1986), and Xenopus faevis (Baldwin et al., 1988; D. Hartman, W. N . Green, and T. Claudio, unpublished observations). Studies have also begun to correlate functional properties with structural domains. Several methods have been used: replacement of entire subunits with homologous subunits from different species, formation of chimeric subunits (one subunit composed of sequences derived from two different species), and specific point mutations or deletions within a subunit. The first approach was used in order to determine if different properties of the AChR could be ascribed to individual subunits. Some combinations of subunits have proved to be very interesting. When bovine &subunit mRNA was mixed with Torpedo a, p, and y mRNAs for example, the receptor formed had the same channel conductance as an all-bovine receptor. Using site-directed mutagenesis techniques, a chimeric S-subunit was made in which 87 base pairs (bp) of the Torpedo &subunit were replaced with the corresponding bovine sequences. lmoto et al. (1986) made an AChR that was composed of this 6 chimera subunit plus Torpedo a-,p-, and y-subunits, and showed that a channel conductance similar to an all-bovine receptor was obtained.

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In order to undertake such structure-function studies, one needs a system in which to express the altered genes or cDNAs. Until very recently (Claudio et u l . , 1987). expression of functional AChRs has only been achieved transiently in Xenopus oocytes. When normal or altered cDNAs are used as templates to make SP6 mRNA transcripts in vitro and the RNAs are microinjected into oocytes, transient expression of acetylcholine receptors has been observed (Mishina et ul., 1985; White et ul., 1985; Claudio, 1987). Another expression system currently being investigated is transient or stable expression of AChRs in yeast (Fujita et ul., 1986; see Chapter 10, this volume). Our laboratory is establishing yet another system, the stable expression of AChRs in mammalian tissue culture cell lines (Claudio, 1987; Claudio et al., 1987). A stable expression system has several advantages over a transient system. ( 1 ) New cell lines can be derived from a single transduced cell. This means that each cell is identical to every other cell and the cell line need only be characterized once. (2) Large quantities of identical cells are easily obtained, thus many types of biochemical studies can be performed on these cells that would be difficult or impractical to perform on individually microinjected oocytes. (3) The lines are stable and expression is continuous, thereby eliminating the need to reestablish expression every time an experiment will be performed. Also relevant to this point is t h e observation that oocytes tend to be seasonal; it is often difficult or impossible to obtain expression in the oocyte system during the summer months. (4) Several cell biological processes and protein-protein interactions can only be studied in this system, and certain electrophysiological techniques (such as single channel recording using a patch clamp) are more easily performed on tissue culture cells than on oocytes. Some of the properties of the receptor that our laboratory is interested in investigating can best be studied in a system where AChR is continuously expressed and expressed in large quantities. For example, a fibroblast cell line that stably expresses AChR can be used to study nerve-AChR interactions by coculturing this line with nerve cells. Such a system would allow us to determine the specific effect of the nerve on the AChR without the interference of other muscle-specific proteins. Alternatively, one may wish to investigate the specific interaction between AChRs and muscle-specific proteins. This could be achieved by stably expressing a foreign AChR (such as the Torpedo AChR) in a cultured muscle cell line where one can specifically manipulate the foreign AChR. Before attempting stable expression of Torpedo AChRs in mammalian tissue culture cells, however, we had to accomplish the following tasks: (1) make a T. californicu electric organ cDNA library, (2) isolate the four full-length AChR subunit cDNA clones, and (3) show that these cDNAs did indeed encode polypeptides that could form a functional AChR. The

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results of these three studies are described in this chapter. In order to establish the stable expression system, two problems typical of a protein such as the AChR must be overcome. First, because the AChR is composed of four different subunits, it is necessary to integrate four different cDNAs into the chromosomes of the same cell. Second, because the AChR is a multisubunit complex with several functions, it might be necessary to express the receptor in a specialized environment, such as a muscle cell, in order to obtain correct subunit assembly and to regenerate all of its functions. We have addressed these problems by using two different methods for introducing AChR cDNAs into the genomes of tissue culture cells: DNA-mediated gene transfer and viral infection using retroviral recombinants. The results of these studies are also described in this chapter in addition to a partial characterization of the expressed proteins. II. MATERIALS AND METHODS A.

Construction of cDNA Libraries

Polyadenylated mRNA was obtained from T. californica electric organ using the method of Chirgwin et al. (1979). Tissue frozen at -75°C was pulverized in a mortar and pestel on dry ice, homogenized in guanidinium thiocyanate solution using a Polytron homogenizer, spun at 8000 rpm for 10 min at S'C, and the supernatant collected. The pH was adjusted to 5.3 with acetic acid, 0.75 volume of ethanol was added, the solution was placed at - 20°C overnight then spun at 6000 rpm for 10 min at - 10°C. The pellet was homogenized in guanidinum hydrochloride solution, 0.5 volume of ethanol was added, the solution was placed at - 20°C for 3 hr, then it was spun at 6000 rpm for 10 min at - 10°C. The pellet was again dissolved in guanidinum hydrochloride solution and ethanol precipitated as just described, then washed in 70% ethanol and dissolved in water. Particulates were removed by spinning at 10,000 rpm for 10 rnin. The RNA was precipitated with 0.1 volume sodium acetate and 2.5 volumes of ethanol. The final pellet was dissolved in water. The polyadenylated fraction of mRNA was obtained by passing the RNA two times over an oligo(dT)-cellulose column. The first strand was synthesized using avian myeloblastosis virus reverse transcriptase and oligo(dT),,.-,, (Collaborative Research) primed mRNA in the presence of 40 kg/ml actinomycin D and human placental RNase inhibitor (RNasin from Sigma). The size of the first strand synthesized ranged from -500 to -7000 bp with the majority at -2000 bp. The second strand was synthesized with Escherichea coli DNA polymerase I in the presence of RNase H (P-L Biochemical) (Okayama and Berg,

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1982). The double-stranded cDNA was treated for 1 hr at 37°C with EcoRI methylase [in 100 mM Tris, pH 7.5, 10 mM ethylenediaminetetraacetic acid (EDTA). 6 p M S-adenosylmethionine] and the ends were made flush by filling in with the Klenow fragment of DNA polymerase I. EcoRI linkers (NEB # 1004) were phosphorylated using polynucleotide kinase, they were ligated to the termini of the cDNA, and then digested with EcoRl restriction enzyme. The cDNA was next inserted into a unique EcoRI site in the phage cloning vector hgtlO (Huynh et al., 1983, the vector was packaged into phage, and the phage were grown in the bacterial strain, C,,jk-mk-hfl. A Agt I I library was also constructed by ligating the doublestranded cDNA into the EcoRl site of this vector and growing the phage in the bacterial strain, LE392 (Young and Davis, 1983). With the hgt 10 library, the transformation efficiency was 2 x lo7 plaque forming units (pfu)/pg of double-stranded cDNA and I .5 x lo-' different recombinants were amplified to a titer of 5 x 10' pfu/ml. With the hgtl 1 library, the transformation efficiency was 2.6 x 10" pfu/pg double-stranded cDNA, and the library contained 6.7 x lo5 recombinants which were amplified to a titer of 8 x 10' pfu/ml(6.2% of which contains inserts yielding a titer of 5 x lo8 pfu/ml with inserts). B. Screening the Libraries for AChR Subunit Clones

Probes used for identifying AChR subunit clones in the AgtlO library were a nick-translated (Rigby et al., 1977) BglI to EcoRl fragment from y (Claudio et al., 1983), nick-translated partial p and 6 clones (Hershey et al., 1983), and a "P end-labeled synthetic IColigonucleotide sequence corresponding to the 5' untranslated region of a. Screening of duplicate filters was carried out according to standard procedures (Maniatis et al., 1982). Hybridization, using nick-translated probes, was carried out in 10 x Denhardt's and 4 x SET ( I x SET is 0. I5 M NaCI, 0.04 M Tris, pH 7.8, 20 mM EDTA) at 68°C. The washes were done at 68°C with a final wash in 1 x SET. Screening the library with the oligonucleotide probe was carried out in 5 x Denhardt's, 6 x SET at 25°C with washes at 30°C and 37°C in 6 x SET. C. Preparation of mRNA Transcripts

SP6 mRNA transcripts were made from full-length clones as described (White et al., 1985). The a and p cDNAs were cloned into the plasmid pSP62-PL and the y and 6 cDNAs were cloned into the plasmid pSP64PL (Melton et al., 1984). Plasmids containing a, p, y, or 6 inserts were

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linearized with the restriction enzymes EcoRI, Xmnl, Pstl. and AutII, respectively. Linearized DNAs were purified on 1% agarose gels. One hundred-microliter transcription reactions were incubated at 40°C for 70 min and contained 25 pg/ml linearized DNA; SP6 polymerase at 160 units/ ml; RNasin at 1000 units/ml; 0.5 mM each of ATP, CTP, and UTP; [a-"PICTP at 50 pCi/ml (Amersham); 0.1 mM GTP; 0.5 mM diguanosine triphosphate; in 40 mM Tris (ph 7.5). 10 mM NaCI, 10 rnM dithiothreitol, 6 mM MgCL, 4 mM spermidine. DNA templates were removed by incubating with RNase-free DNase at 20 pg/ml for 10 min at 37°C. The reaction mix was phenol extracted and unincorporated nucleotides were removed by column centrifugation (Penefsky, 1977) in 10 mM sodium phosphate (pH 7.0). RNA was recovered by precipitation in 2.5 M sodium acetate and 2.5 volumes of ethanol. The pSP64-PL vectors consistently gave 5-10 times the yield of RNA as the pSP62-PL vectors. Labeled transcripts were sized on glyoxal gels but unlabeled RNA transcripts were used routinely for oocyte injections. Labeled transcript, however, did not appear deleterious to the oocyte and gave the same levels of expression as unlabeled transcripts when injected within -2 weeks of being labeled. D. Preparation of Oocytes

Oocytes were obtained from Xenopus laevis females (Nasco in Fort Atkinson, WI) and prepared for microinjection using methods adapted from Methfessel et al. (1986) and White et al. (1985). A portion of ovary was removed from the frog, sectioned into I cm' pieces, and washed with four 50-ml aliquots of calcium-free Ringer's [82.5 mM NaCI, 2 mM KCI, 1 mM MgCl,, 5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), adjusted to pH 7.6 with NaOH]. Washed oocytes were taken up in 15 ml of calcium-free Ringer's containing 2 mg/ml collagenase (Sigma Type lA), and incubated at 19" to 21°C until individual oocytes dislodged from their follicle cell covering (4 to 8 hr). After decanting the collagenase solution, oocytes were washed with four 50-ml aliquots of Barth's saline supplemented with 2.5 mM sodium pyruvate and 10 units/ml of penicillinstreptomycin (Gibco), then stored overnight in supplemented Barth's saline. Healthy oocytes (stage 5 to 6) were then selected for microinjection. E. Microinjection

RNA at a molar ratio of 1 : 1 : 1 : I was dissolved in distilled water at 0.125 mg/ml and taken up into an injection pipet having a blunt tip 20 to 30 pm in diameter. After penetrating the vegetal hemisphere, 50-111 aliquots of RNA were delivered to each oocyte. After 3 to 5 days at 19" to 21°C

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225

injected oocytes were examined for AChR expression by measuring the binding of 'Z'I-labeled a-bungarotoxin ([ '2SI]a-BuTX),ACh-induced "Na uptake, and ACh-induced single channel currents. F. ['251]cu-BuTXBinding

The number of cell-surface AChRs capable of binding a-BuTX was determined by incubating batches of five oocytes for 90 min at 21°C in 100 pI of Barth's saline containing 1 nM ['251]a-BuTX.Oocytes were then washed with four 8-ml aliquots of Barth's saline, and counted individually in a gamma counter. G. ACh-Induced =Na Uptake

Batches of five oocytes were incubated at 0°C in 100 pI of calcium-free Ringer solution supplemented with I pCi of "Na and 5 pM ACh. After a 15-min uptake period, oocytes were washed with four 8-ml aliquots of ice-cold Barth's saline (the first of which contained 0.5 mM d-tubocurarine chloride) and then transferred to individual gamma-counting tubes containing 0.5 ml of Ringer's solution. H. ACh-Induced Single Channel Currents

Single channel currents were recorded from small patches of oocyte membrane using standard patch-clamp techniques (Hamill ef al., 1981; Methfessel ef a / . , 1986). Oocytes stripped of their vitelline membrane were incubated in standard Ringer solution ( 1 15 mM NaCI, 2.5 mM KCI, 1 .O mM CaCI,, 1.0 mM MgCI2, 10 mM HEPES, ph 7.2) at 12"C, and approached with a patch pipet typically containing I CI.MACh, 20 mM KCI, 80 mM KF, 1 mM MgCI2, 10 mM HEPES, pH 7.2. After establishing a giga-seal, currents were recorded from cell-attached patches, or from cellfree outside-out patches exposed to bath-applied ACh. 1.

DNA-Mediated Gene Transfer

The a-,p-, and 8-subunit clones from the AgtlO library were subcloned into the EcoRI site of the vector pSS-2 (generously provided by S. Silverstein) (Fig. la). This vector is basically a pBR322 derivative, PAT 153. in which the 412 bp Hind111 to BarnHI fragment was replaced by the 340 bp Hind111 to PvuII fragment from SV40. The y clone (extending from the A h 1 site at -21 bp to the PvuII site in the 3' untranslated region) (Claudio et ul., 1983) was subcloned into the Hind111 site of pSS-2. Murine

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pss-2

- 4.3 kb a

FIG. I . Mammalian cell expression vectors. The arrow indicates the direction of transcription; the wedges indicate the sites of insertion for the AChR cDNA clones; amp' indicates ampicillin resistance; tet' indicates tetracycline resistance; H3 indicates Hindlll; R indicates EcoRI; B indicates BarnHI. The vector pSS-2 is shown in (a); the vector pVCOS is shown in (b); and the vector pDOP is shown in (c). More details can be found in Section 11, Materials and Methods.

fibroblast cells deficient in thymidine kinase and adenine phosphoribosyltransferase (Ltk-aprt -) were obtained from R. Axel and maintained in Dulbecco's modified Eagle's medium (DME) containing 10% calf serum and 50 kg/ml diaminopurine. The rat fusing muscle cell line, L6 (Yaffe, 1968), was obtained from D. Schubert and maintained in DME plus 10% calf serum, The mouse nonfusing muscle cell line, BC3H-l (Schubert et ul., 1974), was obtained from P. Taylor and maintained in DME plus 10% fetal calf serum. Ltk-aprt- cells were transfected with the four subunit cDNAs in the vector pSS-2 along with the uprt gene using the calcium phosphate precipitation procedure of Graham and van der Eb (1973) as

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227

modified by Wigler et al. (1977). Cells were put into selective azaserine, adenine medium and after 12 days, 11 colonies expressing the aprt+ phenotype were isolated and grown into individual stable cell lines. The a cDNA was also engineered into the expression vector pVCOS (generously provided by S. Goff) (Fig. lb). The vector contains about 3 kb of Moloney murine leukemia virus (M-MuLV)DNA including the two long terminal repeats (LTR) engineered into the cosmid pHC79 (Hohn and Collins, 1980) at the EcoRI site. The gag, pol, and env genes of the virus were deleted from the PstI site at the 5' end of the gag gene to the HpaI site at the 3' end of the pol gene. An EcoRI cloning site was introduced between the LTRs with a polylinker. A cDNA can be inserted at this site such that the 5' LTR provides a promoter for the cDNA and the 3' LTR provides signals for polyadenylation. J. Viral Infection

The murine retroviral expression vector pDOP (Fig. lc) was constructed by R. C. Mulligan. In this vector, the gag, pol, and env genes of murine sarcoma virus have been deleted and a cDNA can be introduced at a unique BarnHI site between the LTRs. Downstream from the cDNA insertion site are a pBR322 origin of replication, an SV40 early promoter, and the bacterial neomycin phosphotransferase gene. The neomycin gene provides a dominant selectable marker. DNA sequences were derived from the transposon Tn5 which encode G418 (Geneticin, Gibco) resistance in mammalian cells (Davies and Jimenez, 1980) and kanamycin resistance in E. coli (Jorgensen et al., 1979). Also included in this vector are 3.8 kb of polyoma DNA including the origin of replication and about 3 kb of pBR322 DNA. The vector encodes two independent transcription units: one extending from the 5' LTR to the 3' LTR, and the other extending from the SV40 promoter to the 3' LTR. This vector thus permits the simultaneous expression of the inserted protein coding sequences (driven from the viral LTR) and the dominant selectable marker (driven from the internal SV40 promoter). Murine NIH3T3 fibroblast cells and (p2 cells (Mann et al., 1983) were obtained from R. C. Mulligan and maintained in DME and 10% calf serum. The 9 2 cell line can package a replicationdefective retroviral recombinant and thus produce stocks of helper-free defective retroviruses. The line was derived from an NIH3T3 cell that was infected with a mutant M-MuLV (Mann et al., 1983). The Torpedo a cDNA clone used was from the XgtlO library. The insert was isolated after digestion with EcoRI, BarnHI linkers were ligated to the termini, and the insert was cloned into the BarnHI site of the pDOP vector. Ten micrograms of pDOP-a plasmid DNA was transfected onto 2 x lo6 92

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cells using the procedure of Graham and van der Eb (1973) as modified by Parker and Stark (1979). Eighteen hours after the glycerol shock, culture medium was collected and filtered through a 0.45 pm Gelman acrodisc. One milliliter of supernatant was added to lo6 NIH3T3 and L6 cells in 10-cm plates and incubated for 2.5 hr at 37°C in the presence of 8 pg/ml Polybrene (Aldrich). DME plus 10% calf serum was added, the cultures were incubated for 2-3 days, and then split 1 : 20 into DME containing 10% calf serum and 0.6 mg/ml G418 (Gibco). K. Labeling and lmmunoprecipitations

Confluent 10-cm dishes of cells were washed twice with phosphatebuffered saline then incubated for 15 min at 37°C with media lacking the specific amino acid(s) used to radiolabel proteins. Dishes were washed once with phosphate-buffered saline, 2 ml of the above media containing 200-400 pCi of the radiolabeled amino acid were added, and the cells were incubated for 20 min at 37°C. In the cold room, plates of cells were washed twice with 4°C phosphate-buffered saline and lysed with 700 pI of lysis buffer (150 mM NaCI, 5 mM EDTA. 50 mM Tris, pH 7.4, 0.02% NaN,, 0.5% Nonidet P-40, 1 mg/ml hemoglobin) containing fresh 2 mM phenylmethylsulfonyl fluoride and 2 mM N-ethylmaleimide. Cells were then scraped into 1.5-ml Eppendorf tubes, vortexed a few times over a 10-min period at 4"C, and spun for 10 min at 4°C in an Eppendorf centrifuge. Supernatants were collected, SDS was added to I%, and the samples were boiled for 3 min. After the samples cooled, Nonidet P-40 was added to 5% followed by 50 pI of protein A Sepharose (diluted two times in lysis buffer). The samples were rocked for 15 min at 4"C, spun for about 30 sec, antisera were added to the supernatants, and the samples were rocked overnight at 4°C. Fifty microliters of 2 x diluted protein A Sepharose was added, the samples were rocked for a minimum of 3 hr at 4"C, spun for about I min, the supernatants were removed, the resins were washed twice in lysis buffer containing 0.5 M NaCI, then washed three times in lysis buffer. After the final spin, supernatants were removed, 35 pI of 2 x gel loading buffer (4% SDS, 20% glycerol, 0.125 M Tris, pH 6.8, 0.01% bromphenol blue) containing fresh 10 mM dithiothreitol was added to pellets, the samples were boiled for 3 min, the resins were removed by centrifugation, and the supernatants were loaded onto 10% discontinuous SDS gels (Laemmli, 1979). The gels were fixed for 30 min in 25% methanol, 10% acetic acid, soaked in Amplify (Amersham) for 30 min, dried on a gel dryer, then put on film at -70°C with an intensifying screen. In the initial experiments, proteins were labeled with 400 pCi of

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11. ESTABLISHING A STABLE EXPRESSION SYSTEM

["Slmethionine (Amersham). In later experiments, proteins were labeled with 200 FCi each of [3H]leucine and [3H]isoleucine (Amersham). 111.

RESULTS

A. Identifying Full-Length Clones The Torpedo electric organ hgt 10 library was screened with the four subunit-specific cDNA probes and approximately 0.25% of the plaques was positive for each subunit. Of the positive plaques, 10 were chosen from each of the different subunit screens and analyzed further for the presence of full-length clones. Phage DNA was prepared from purified plaques, the DNA was digested with the restriction enzyme EcoRI, and the digests were run on 1% agarose gels. The inserts were isolated from gels and the DNA was mapped using various restriction enzymes. By comparing our mapping results with published sequence data, we were able to determine that we had isolated full-length a,y, and 6 clones. We sequenced (by the method of Maxam and Gilbert, 1980) the 5' ends of two of the p clones and determined that one of the two clones was full length. The clones have the lengths shown in the following tabulation: Clone a

P Y 8

5' Untranslated -80 2 -10 -40

bp

bp bp bp

3' Untranslated -300 -200 -500 -200

bp bp bp bp

Length of clone

- 1760 bp - I720 hp -2030 bp - 1800 bp

B. Expression of Functional Cell Surface AChRs in Oocytes Although the clones appeared to be full length by restriction endonuclease mapping and partial DNA sequence analysis, it was still necessary to determine that they could encode subunits that would form a fully functional AChR complex. This was done by making SP6 mRNA transcripts in vitro (using the clones as templates), then injecting the RNA into Xrnopus oocytes and assaying for the presence of functional AChR. The results of the toxin-binding experiments and the **Na uptake experiments are shown in Table I. In Table I A , three of the four microinjected oocytes

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TONI CLAUD10 ET AL. TABLE I CELL SURFACE CI-BuTX-BINDING SITES A N D "Na UPTAKE MEASUREMENTS

Oocyte

Injected

Oocyte

Noninjected

A. ["'I]a-BuTX bound (cpmY

90 70 80 80

I 2 3 4

1530 1840

9 10

II

3030 2500 2280

I3 14 15

12

190

16

100

I320

B. "Na uptake (cpm)" 480 300 40 80

"Assays were performed in standard Xenopus Ringer's solution (Hamill P / d.,1981) in the presence of I nM [l"l]u-BuTX. "Oocytes were tested for the ability to take up "Na (Sine and Taylor, 1979) after a 15min exposure to 5 (IM ACh.

bound approximately 2.5 fmol of ['"I]a-BuTX. Since each AChR molecule binds two toxin molecules, one can calculate that approximately 7.5 x 10' molecules of toxin-binding AChR were expressed on the surface of each oocyte, giving a receptor density of approximately 240 molecules per pm'. This result demonstrates that the individual subunits were made, assembled into AChR complexes, and inserted into the plasma membrane. Next, we tested if these cell surface receptors could bind their natural ligand, ACh, and form functional ligand-gated channels. In Table IB, three of the four injected oocytes responded to ACh and took up "Na. The responding oocytes took up approximately 10 times the level of 22Naas noninjected oocytes, clearly demonstrating that the injected oocytes express functional AChRs. Single channel studies ideally require a receptor density of approximately 10-100 AChRs per pm'. The results of the toxinbinding experiments showed that we had a surface density of -240 molecules per pm' and the results of the "Na uptake experiments demonstrated that functional receptors were expressed. We therefore expected that we would be able to detect single channels in oocytes. Indeed, single channel recordings were obtained using either a cell-attached patch (Fig. 2A) or an outside-out patch (Fig. 2B). Evidence that these were in fact AChR channels included the following: ( I ) no channel activity was observed in the absence of ACh in either the cell-attached or the outsideout configuration, (2) channels appeared upon the addition of ACh to the outside-out patch, and (3) channel activity desensitized completely at

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r

c B

r

FIG.2. Single channel recordings. Single channel activity was recorded in standard Xcwoprrs Ringer's solution from oocytes expressing AChRs as determined by "Na flux studies.

Records were obtained from a cell-attached patch ( A ) at 10°C and - 70 mV. and from an outside-out patch (B) at 10°C and -60 mV. I n A. the ACh concentration in the pipet was I pM: in B. ACh was added to a final concentration of 100 pM. The horizontal scale bar is 10 msec, and the vertical scale bar i s 5 PA. The record shown in B was taken during the course of ACh mixing. At equilibrium. channel activity disappeared as a result of desensitization.

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100 p M ACh and could not be restored by washing out the ACh. The conclusion from this set of experiments was that the clones encoded proper AChR subunit polypeptides. These same clones might therefore be expressed properly when introduced into mammalian cells.

C. Cotransformation

In order to establish a tissue culture cell line that continuously expresses T. californica AChRs on its cell surface, it is first necessary to incorporate all four AChR cDNAs into the chromosomes of the same cell. It is also desirable to incorporate similar copy numbers of each cDNA because the stoichiometry of the subunits in the receptor complex is 2 : 1 : 1 : 1. In order to determine whether DNA-mediated cotransformation would be a suitable method for establishing such a system, we had to determine how efficiently five different genes or cDNAs would be introduced into the same cell and what the copy number would be for each of these DNAs. The answers to these questions would determine the suitability of using DNA-mediated gene transfer as a method for establishing stable expression of AChRs in cultured cells. The subunit cDNAs were subcloned into the vector pSS-2 and transfected onto Ltk-aprt- (murine fibroblast) cells along with the uprt gene; the cells were put into selective media, and 11 individual colonies expressing the aprtf phenotype were isolated and grown into stable cell lines. DNA was isolated from each cell line, digested with the restriction enzyme EcoRI, run on gels, transferred to nitrocellulose (according to the procedure of Southern, 19751, and hybridized to four separate nicktranslated probes for a, p. y, and 6. The results of the Southern blots are shown in Fig. 3. Cell line 1 incorporated only the uprt gene. Cell line 7 incorporated the aprt gene, the p cDNA, and the 6 cDNA. The other nine lines incorporated the aprt gene and all four AChR subunit cDNAs. Thus, 80% of the cells that incorporated the uprt gene into their chromosomes also incorporated all four receptor subunit cDNAs. In Fig. 3, the lanes containing 100 ng of pure plasmid (Fig. 3, lanes P) would represent approximately 10 copies of the cDNA per cell. By comparing the intensity of each cDNA hybridization band on the Southern blot to the plasmid bands (P), one can see that the amount of each cDNA incorporated varied from approximately I to 10-20 copies per cell. However, five of these lines had approximately equal copy numbers of each cDNA. These results demonstrate that DNA-mediated gene transfer is a method that can be used for easily introducing the four AChR subunit cDNAs into the same cell, and that this procedure introduces multiple genes with approximately equal copy numbers at a relatively high efficiency (Claudio. 1987).

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233

FIG.3. DNA blots of I I stable cell lines cotransfected with mixtures of Torpedo u, 8. y. and 6 cDNAs plus an tip,? gene. L. untransformed Ltk-aprt cellular DNA; P, plasmid DNA containing the cDNA from which the individual probes were made: 1-1 I . individual transformed cell lines: 2.0. 2.3. and 4.4,molecular weight markers (in kilobases) of A DNA digested with HirrdllI restriction enzyme. Blots were hybridized t o nick-translated a. f3, y. and 6 probes (market alpha. beta, gamma. or delta on the respective blots).

D. Protein Expression of Transfected DNA Having shown that these cDNAs were readily incorporated into Ltk-aprt cells, we wished to determine whether Torpedo subunits could be expressed in fibroblasts and if the correct posttranslational modifications would occur. We decided to put one of the subunit cDNAs into an expression vector, introduce it stably into an Ltk-aprt- cell, and assay for the correct expression of just this one subunit before attempting the expression of all of the subunits. The a cDNA was therefore engineered into the expression vector pVCOS (generously provided by S. Goff). Ltk-aprt cells were transfected with pVCOS-cw and aprt as described above for the pSS-2 vectors and, after I 1 days in selection, four colonies were isolated and grown into stable cell lines. Confluent dishes of cells were labeled with [3'S]rnethionine, solubilized, irnmunoprecipitated with anti-cw antisera (polyclonal subunit-specific antisera were prepared in rabbits as previously ~

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described, Claudio and Raftery, 1977), run on discontinuous SDS gels, and autoradiographed (Claudio, 1987). The results are shown in Fig. 4. a-Subunit was seen only in those lanes containing anti-a antibody immunoprecipitations of cells transfected with pVCOS-a (Fig. 4, lanes 1 A, 2A, 3A). There was no a-protein seen in the untransformed Ltk-aprtcells that were immunoprecipitated with anti-a antisera (Fig. 4, lane LA) and no a-protein seen in transformed cell lines that were immunoprecipitated with preimmune serum (Fig. 4, lanes lP, 2P, 3P). These results demonstrate that Torpedo a-subunit can be expressed in mouse fibroblast cells (Ltk-aprt-) and that the expressed subunit contains at least some of the same antigenic determinants as native Torpedo a-subunits. Because

FIG.4. lmmunoprecipitation of Torpedo u protein stably expressed in Ltk-aprt- cells. Three different ["S]methionine-labeled. immunoprecipitated, stable Ltk-aprt cell lines cotransformed with the Torpedo a cDNA containing plasmid (pVCOS-u) and aprt were run on 10% SDS-polyacryamide gels and autoradiographed. Each lane represents approximately 7.5 x IOh cells labeled with I mCi of ["Slmethionine. L, untransformed Ltk-aprt- cells; 1-3 are the three transformed cell lines; P, preimmune serum: A, anti-u antiserum. The positions of unlabeled T. culifornicu electric organ AChR subunits are marked (a,p. y. 6). The arrows indicate the positions of Torpedo u-subunits in the immunoprecipitation. ~

11. ESTABLISHING A STABLE EXPRESSION SYSTEM

235

the a-subunits synthesized in Ltk-aprt - cells comigrated precisely with native Torpedo a-subunits, it suggested that the subunits were being made and processed correctly in this foreign environment. Further encouragement that expression of functional AChRs would be achieved in fibroblasts was derived from the observation that a number of different genes and cDNAs have been successfully expressed in mammalian tissue culture cells (reviewed in Gluzman, 1982; Temin, 1986). In addition, integral membrane glycoproteins have been expressed on foreign cell surfaces in a configuration that displays antigenic and biological activity (Gething and Sambrook, 1981) as well as enzymatic activity (Davis et al., 1983). Assembly of a multimeric complex has also been achieved: HLA-DR a and p polypeptide chains have been expressed in mouse fibroblast cells, assembled as dimers, and inserted into the plasma membrane (Rabourdin-Combe and Mach, 1983; Korman et al., 1987). Because of the successful expression of other proteins in systems similar to ours and the encouraging results we had obtained thus far with Torpedo ci expression, we were very hopeful that AChR subunits would assemble into functional receptor complexes in fibroblasts. It was possible, however, that certain properties would not be properly reconstituted in fibroblasts because other muscle-specific proteins were required. One method for overcoming possible inadequacies in the fibroblast system would be to express Torpedo AChRs in muscle cells. The muscle system would also be a method for investigating muscle-specific proteinprotein interactions. For example, nerve-induced AChR clustering on cell surfaces might not occur in fibroblasts but would occur in muscle cells. This would be a very interesting result in itself. If the clustering process required one or perhaps a few proteins in addition to nerve and AChR, then we could add those genes (for example, the 43,000-Da protein; Neubig et al., 1979) to our fibroblast-AChR cell lines and look for clustering in these new cell lines. If clustering is a much more complex process than this, and we are forced to use muscle cells in order to study this process, then we need a method of introducing Torpedo cDNAs into muscle cells. Two muscle cell lines (L6 and BC,H-I) were tested for the ability to take up DNA by the calcium phosphate precipitation method. Cells were transfected with a dominant selectable marker, the neomycin resistance gene, and put into (3418 selective media (see Section 111, E). Whereas the transformation protocol routinely gave efficiencies of about 40 colonies per lo6 cells per nanogram of aprt in Ltk-aprt- cells, the efficiency was only about 0.5 colonies per 10' cells per nanogram of the neo' gene in L6 cells and less than 0.005 colonies per 10' cells per nanogram of the neo' gene in BC,H-I cells. We therefore sought a more efficient method for introducing material into these muscle cell lines.

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TONI CLAUD10 ET AL.

E. Viral Infection

A very efficient method for introducing DNA or RNA into cells is by viral infection. Most of the fibroblast and muscle cell lines in which we wish to express Torpedo AChRs are of murine or rat origin. We therefore decided to try murine retroviral recombinants and viral infection (Mann et al., 1983) as a method of introducing our cDNAs into fibroblast and muscle cell lines. We initially used the murine retroviral expression vector pDOP (generously provided by R. C. Mulligan) in which we inserted our Torpedo CY cDNA downstream from the M-MuLV LTR. This DNA was transfected onto 9 2 cells, and RNA was made and packaged into replication-defective viral particles. Infectious virus particles containing the packaged recombinant were collected ((p2 supernatants) and used to infect NIH3T3 and L6 cells. Once the cells were confluent, they were split into G418-selective media (the pDOP vector also contains the neo' gene driven from the internal SV40 promoter), and individual colonies were isolated. Six colonies of pDOP-a-3T3 cells, eight colonies of pDOP-a-L6 cells, and two colonies of pDOP-L6 cells were isolated using cloning cylinders and grown into new cell lines. F. Expression of Torpedo a-Protein in Fibroblasts and Muscle Cells

3/4-confluent dishes of fibroblast and muscle cells were labeled with ['H]leucine and [3H]isoleucine (Fig. 5B) or ["S]methionine (Fig. 5A), solubilized, immunoprecipitated with anti-a antisera, run on discontinuous SDS gels, and autoradiographed. The a-subunit was seen only in those lanes containing immunoprecipitations with anti-a antisera of cells infected with pDOP-a. There was no a-protein seen in the noninfected 3T3 cells and no a-protein seen in those cells infected with the pDOP vector alone that lacked an a cDNA insert. The L6 cells were harvested at a stage when cells were subconfluent and not fused, and therefore not expressing endogenous AChR. The results obtained in 3T3 cells with the retrovirus a cDNA recombinant were in complete agreement with those obtained in Ltk-aprt- cells with pVCOS-a and DNA-mediated gene transfer (Fig. 4). Using the retrovirus system, we have demonstrated that ( I ) Torpedo a-protein was made in both mouse fibroblast cells and rat muscle cells, (2) it was recognized by polyclonal antisera directed against Torpedo a-subunit, and (3) it migrated with the same molecular mass as native Torpedo a-subunit, indicating that the protein was synthesized and processed correctly in both cell types. In addition, we have shown that Torpedo a-subunits can easily be distinguished from endogenous rat a-subunits. Thus, should it be necessary to use muscle cell lines in order to

11. ESTABLISHING A STABLE EXPRESSION SYSTEM

237

FIG.5 . lmmunoprecipitation of Torpedo a-protein stably expressed in mouse NIH3T3 fibroblast cells and rat L6 muscle cells. Cells were labeled with ["S]methionine (lanes 1-5) or ['Hlleucine plus ['H]isoleucine (1'-5'). Each lane represents approximately 7.5 x 10" cells labeled with 400 FCi of ["Slmethionine or 200 FCi each of ['H]leucine plus ['H]isoleucine and immunoprecipitated with anti-Torpedo a antiserum. Lane I ( I '1 is 3T3 cells; lanes 2 ( 2 ' ) and 3 (3') are two different clonal lines of 3T3 cells transduced with the retrovirus a cDNA recombinant; lane 4 (4') is a clonal line of L6 cells infected with the retrovirus vector not containing the a cDNA insert; lane 5 ( 5 ' ) is a clonal L6 cell line infected with the retrovirus a cDNA recombinant. The arrow indicates the position of the Torpedo a-subunit in the immunoprecipitation.

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TONI CLAUD10 ET AL.

obtain proper expression of a fully assembled and functional Torpedo AChR complex, then retroviral infection can be employed much more efficiently than DNA-mediated gene transfer for establishing the stable cell lines. G. Optimized Labeling and lmmunoprecipitation Conditions

The AChR is an integral membrane protein that forms an ionic channel. It would therefore be expected to be composed of several membranespanning domains which would be composed of amino acid residues containing hydrophobic side chains. The first predictions for the topography of the subunits about the membrane, based on the hydropathy profile of the y-subunit (Claudio et al., 1983), predicted four membrane-spanning domains for this subunit. By adding up the content of leucine plus isoleucine residues, one finds that they account for 20, 19, 20, and 16% of the amino acids of a, p, y, and 8, respectively, whereas methionine accounts for only 1.6-3.4% of the residues of each subunit. The content of leucine plus isoleucine in 1081 peptides and proteins listed in the Atlas of Protein Sequence and Structure (Doolittle, 1981) is only about 11%. For this receptor-channel protein with its large number of hydrophobic domains, radiolabeling with hydrophobic amino acids might therefore be a method of specifically increasing the amount of label incorporated into AChR subunits. Indeed, this appeared to be the case. In Fig. 5 , identical dishes of cells were labeled with 400 pCi of ['-'S]methionine (A) or 200 pCi each of [-'H]leucine and [3H]isoleucine(B), immunoprecipitated with polyclonal antisera, and processed for autoradiography. A comparison of Fig. 5A and B showed that labling AChR subunits with leucine and isoleucine followed by immunoprecipitation gave a much cleaner gel pattern than labeling with the same total number of microcuries of methionine. H. Partial Characterization of Torpedo a-Subunits in Fibroblasts and Muscle Cells

Pulse-chase experiments were conducted in order to determine the stability of an AChR subunit when the other subunits were not present and able to form stable a$y6 complexes. 314-confluent dishes of 3T3 cells were labeled for 10 min with [3SS]rnethionineand harvested immediately (0 min chase) or harvested after 10 min, 20 min, 40 min, 2 hr or 18 hr in media containing an excess of unlabeled methionine. As seen in Fig. 6, the half-life of Torpedo a-subunits in 3T3 cells was approximately 40 min. Although this is a fairly short half-life, it is similar to that observed by Merlie and Lindstrom (1983) for unassembled endogenous AChR a-sub-

11. ESTABLISHING A STABLE EXPRESSION SYSTEM

239

FIG.6. Pulse-chase experiment on 3T3 cells transduced with the retrovirus (I cDNA recombinant. Each lane represents approximately 7.5 x 10' cells labeled for 10 min with 200 $3 of I"S1methionine. Lane I . no chase: lane 2. 10-min chase: Lane 3. 20-min chase; lane 4. 40-min chase: lane S. 2-hr chase; lane 6 . 18-hr chase. Each plate was immunoprecipitated with anti-a antiserum but not all washes were included (See Section 1I.K) in order to see background bands. The arrow indicates the position of the Torpedo a-subunit in the immunoprecipitation.

units in BC,H-I muscle cells (about 30 min). This result demonstrated that Torpedo a polypeptides in mouse fibroblast cells were no less stable than unassembled mouse a polypeptides in their native muscle cell environment. We tested for cell surface expression of a-subunits by two different methods. Initially, we labeled intact cells with [ '251]a-B~TX but were unable to detect any toxin-binding activity. Because this assay requires that the subunit be expressed on the cell surface and be in the correct toxinbinding conformation, we also wanted to test for surface expression of

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subunits that might be incapable of binding toxin. This was accomplished by first incubating intact cells with polyclonal anti-a antisera followed by '251-labeledprotein A. The results of these experiments were also negative and we were still unable to detect cell surface expression of Torpedo asubunits in fibroblasts or muscle cells. These results are different from those obtained by Hess and his colleagues using a yeast expression system (Fujita et al., 1986; Chapter 10, this volume). It is not known if the different results are due to the different expression systems used or if they are due to differences in the method of assay. We were, however, able to detect some internal toxin binding activity to Torpedo a-subunits in both fibroblasts and muscle cells. We detected toxin binding to NIH3T3-a cells by either solubilizing the cells with nonionic detergents and incubating them with ['''I]a-BuTX or by denaturing the cells in SDS, running the extracts on SDS gels, transferring to nitrocellulose paper and probing with ['"IlaBuTX (Fig. 7). Undifferentiated L6-a cells (not expressing endogenous AChRs) were also probed using this blotting technique and found to be capable of binding [ "'IJa-BuTX. These results indicated that at least some of the Torpedo a-subunits being expressed in both fibroblasts and muscle cells are in a conformation that is capable of binding toxin. We have also begun to address the question of whether subunits form homooligomers as an intermediate step in the assembly of a2pyS pentamers (Anderson and Blobel, 1983). One of the difficulties people have had in the past with investigating this problem in cultured muscle cells is that all of the subunits are present, and it is therefore difficult to rule out the possibility that the presence of one of the subunits might be influencing the behavior of another subunit. The 3T3-a cell lines we have established are therefore ideal for such an analysis because there are no endogenous subunits present in these cells. These studies are performed by metabolically labeling cells with [3H]leucine, solubilizing the cells, running the extract on sucrose gradients, fractionating the gradient, and immunoprecipitating the fractions. From the position of the subunit in the gradient, we can determine if the subunit is migrating as a monomer or as an oligomer. We are simultaneously performing these same studies in muscle cells. In order to eliminate any possible influence of endogenous subunits on exogenous subunits, we prevent the expression of endogenous subunits by maintaining the cells as undifferentiated myoblasts. In addition to investigating the interactions between identical subunits with this system, we can also investigate the interactions between different subunits. Toward this goal, we have established 3T3 and L6 cell lines that stably express Torpedo p-subunits. We have used two methods for readily introducing the a- and p-subunits into the same cell such that the inter-

11. ESTABLISHING A STABLE EXPRESSION SYSTEM

24 1

FKi. 7 . [ ' " ~ ] ~ - B u T binding X to T C J ~ AChR ~ ~ J and ~ J NIH3T3-u cells. The AChR lane contained 4 ~g of Torpt.do AChR. I n the 3T3-a lane. eight 10-cm dishes of -5 x 10" NIH3T3a cells per dish were lysed. immunoprecipitated with anti-a antisera followed by protein ASepharose. Samples were run on SDS gels. transferred to Zetabind filters. and labeled with ('"llu-BuTX as described (Wilson e f d.,1984).

action between the two could be investigated. We can plate equal quantities of 3T3-a and 3T3-P cells together and then artificially fuse the membranes with polyethylene glycol once the cells are confluent. With the muscle cells, we can plate equal amounts of L6-(Yand L6-P cells together and then allow the cell membranes to fuse and form multinucleated myotubes by exposing t h e cells to starvation conditions. The apparent lack of interaction between endogenous and exogenous subunits that we have observed thus far in the L6 cell lines makes this type of experiment plausible. The technique of allowing the membranes of different muscle cell lines to fuse naturally or of fusing fibroblast membranes with polyethylene glycol in order to investigate subunit-subunit interactions will also be very useful as a method for quickly introducing the four different subunits into the same cell once we have expressed each subunit individually in separate cell lines (transient-stables).

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IV. DISCUSSION

Several expression systems are available which allow one to study the properties of acetylcholine receptors expressed from cDNA clones. The fastest and easiest system to use is to microinject in vitro transcribed mRNA into Xenopus oocytes and analyze the transiently expressed AChRs. This system has been used very successfully as a method for screening and partially characterizing new AChR isolates, hybrid AChRs, and in vitro mutated AChRs. For addressing many cell biological questions or studying the biosynthesis and assembly of AChR subunits, stable expression of AChRs in cultured cells has several advantages over the transient systems. In order to establish the stable system, we first needed to obtain the four AChR subunit cDNA clones. A T. californica electric organ cDNA library was constructed in AgtlO and the four full-length AChR subunit cDNA clones were isolated. When these clones were used as templates to make mRNA in vitro and the mRNA was microinjected into Xenopus oocytes, functional cell surface AChRs were formed. We presume, therefore, that all the information required to make a correctly synthesized, processed, assembled, and functional AChR is encoded in these clones. We used two methods for introducing the AChR subunit cDNAs into cultured cells: DNA-mediated gene transfer using calcium phosphate precipitation and retroviral infection. We chose these two methods because of properties peculiar to, but not necessarily unique to, the AChR. One consideration was the need to stably introduce four different cDNAs into the same cell; another was the goal of introducing the cDNAs into muscle cell lines. Using DNA-mediated gene transfer, we demonstrated that the cotransformation efficiency of introducing all four AChR cDNAs plus the selectable marker gene into the same Ltk-aprt- fibroblast cell was 80%. In addition, half of these cells took up approximately equal copy numbers of each cDNA. Thus, DNA-mediated gene transfer is a technique that will readily allow one to introduce multiple cDNAs into the genome of the same cell. We have also shown that Torpedo a-protein is expressed in Ltk-aprt- cells. The subunit migrated with the same molecular mass as native Torpedo &-subunit and it was recognized by polyclonal anti-a antisera, indicating that the protein had been correctly synthesized and processed. One clear disadvantage of the calcium phosphate precipitation method was that two muscle cell lines (L6 and BC,H-I) did not take up DNA well by calcium phosphate precipitation. We might have been able to introduce DNA into these cells using electroporation (Neumann et al., 1982), but this method was not tested. We chose instead, to use retrovirus recom-

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binants and viral infection as a means of stably introducing subunit cDNAs into muscle cell lines. With this method, both mouse fibroblast NlH3T3 cells and rat fusing L6 muscle cells were readily infected and new fibroblast and muscle cell lines were established that expressed Torpedo a-subunits. The use of transmissible retrovirus vectors is therefore a technique that can be used to efficiently introduce a single cDNA into a fibroblast or a muscle cell. As with the Ltk-aprt- cells, the Torpedo a-subunits expressed in 3T3 and L6 cells migrated with the same molecular mass and expressed at least some of the same antigenic determinants as native Torpedo asubunits. Although we were able to express very high levels of the protein in fibroblasts and muscle cells, individual subunits did not appear to insert into the plasma membranes of these cells. We were, however, able to detect some toxin binding activity with a-subunits in fibroblasts and muscle cells indicating that some subunits were made with the correct toxin binding conformation. We also investigated the stability of a single AChR subunit in fibroblasts. We demonstrated that the half-life of Torpedo a-subunits in mouse fibroblast cells was similar to that observed for unassembled asubunits in BC,H-1 cells. In fibroblasts, the Torpedo protein thus appeared to be no less stable than its mammalian muscle counterpart. As characterized thus far, the system we are establishing looks very promising for the stable expression of Torpedo AChRs in fibroblast and muscle cell lines. In addition, we have begun to study the process of AChR subunit assembly by investigating the possible interactions between identical subunits (homooligomers) and between different subunits (a-p interactions), It appears that we will, indeed, be able to address various cell biological questions with our system as well as investigate structure-function relationships intrinsic to the AChR and its subunits. ACKNOWLEDGMENTS We are especially grateful to Richard Mulligan for providing advise and materials for work dealing with the retrovirus recombinants and viral infection, and to Richard Axel, Stephen Goff, and Saul Silverstein for providing cell lines or vectors. This work was supported in part rt by grants #NS21714 (TC) and #NS21501 (FJS) from the National Institutes of Health. REFERENCES Anderson. D. J.. and Blobel. C. ( 1983). Identification of homo-oligomers a s potential intermediates in acetylcholine receptor subunit assembly. Proc. N o r / . Accrd. Sci. U . S . A . 80, 4359-4363. Anholt. R.. Lindstrom. J.. and Montal. M. (1984). The molecular basis of neurotransmission: structure and function of' the nicotinic acetylcholine receptor. I n "The Enzymes of Biological Membranes'' ( A . N. Martinosi. ed.). 2nd Ed.. pp. 335-401. Plenum. New York.

Baldwin. T. J.. Yoshihara. C. M.. Blackmer. K.. Kintncr. C. R.. and Burden. S. J. (1988).

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Regulation of acetylcholine receptor transcript expression during development in XenJ . Cell B i d . 106, 469-478. Boulter. J.. Evans. K.. Goldman. D.. Martin, G., Treco. D., Heinemann. S.. and Patrick, J. (1986). Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor a-subunit. Ntitrrrc fhJ/ldlinJ319, 368-374. Chirgwin. J. M.. Przybla. A. E.. MacDonald. R. J.. and Rutter. W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonucleases. Bbdrernisrry 18. 5294-5298. Claudio. T. (1986). Recombinant DNA technology in the study of ion channels. Trends f h r n i . Sci. 7 , 308-312. Claudio. T. ( 1987).Stable expression of transfected Torpedo acetylcholine receptor a-subunits in mouse fibroblast L cells. f r o c . Ntrtl. Acrid. Sci. U . S . A . 84, 5967-5971. Claudio. T.. and Raftery. M. A. (1977). Immunological comparison of acetylcholine receptors and their subunits from species of electric ray. Arch. Biochem. Biciplr.v.s. 181, 484-489. Claudio. T.. Ballivet, M.. Patrick, J.. and Heinemann, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor y subunit. Proc. N r / t / . A ~ t / dS. C ~U . . S . A . 80. I I 11-1 115. Claudio. T.. Green. W. N.. Hartman. D. S.. Hayden. D.. Paulson. H. L.. Sigworth. F. J.. Sine. S. M.. and Swedlund. A. (1987). Genetic reconstitution of functional acetylcholine receptor-channels in mouse fibroblasts. Science 238, 1688-1694. Davies. J.. and Jimenez. A. (1980). A new selective agent for eukaryotic cloning vectors. A m . J . Trop. M 4 . H y g . 29, Suppl.. 1089-1092. Davis. A. R.. Bos. T. J.. and Nayak. D. P. (1983). Active influenza virus neuraminidase expressed in monkey cells from cDNA cloned in simian virus 40 vectors. froc.. N u t / . Acctd. Sci. U . S . A . 80, 3976-3980. Dolly. J . 0.. and Barnard. E. A. (1984). Nicotinic acetylcholine receptors: an overview. B i ~ c h P n P/icirrntrc~d. ~. 33, 84 1-858. Doolittle. R. F. (1981). Similar amino acid sequences: chance or common ancestry'? Science 214, 149-159. Fujita. N.. Nelson. N.. Fox, T. D.. Claudio. T.. Lindstrom. J. M., and Hess, G. P. (1986). Biosynthesis of the Torpc~doccrlifiirnic~ccacetylcholine receptor a subunit in yeast. Sticwce 231. 1284-1287. Gething. M.-J.. and Sambrook. J. (19811. Cell-surface expression of influenza haemagglutinin from a cloned DNA copy of the RNA gene. Nrrtrrre (London) 293, 620-625. Gluzman. Y . (ed.) (1982). "Eukaryotic Viral Vectors." Cold Spring Harbor Lab., New York. Graham. R.. and van der Eb. A. (1973). A new technique for the assay of infectivity of human adenovirus 5 virus. Virologv 52, 456-467. 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. ffircger.v Arch. 391, 85-100. Hermans-Borgmeyer. I.. Zoph. D.. Ryseck, R.. Hovemann. B.. Betz. H.. and Gundelfinger. E. ( 1986). Primary structure of a developmentally regulated nicotinic acetylcholine receptor protein from Drosopliiltr. EMBO J. 5, 1503-1508. Hershey. N. D.. Noonan. D. J.. Mixter, K. S., Claudio, T.. and Davidson. N. ( 1983). Structure and expression of genomic clones coding for the &subunit of the Torpedo acetylcholine receptor. Cold Spring Hor-hor Syriip. Q i i r r n f . Biol. 48, 79-82. Hohn. B.. and Collins. J. (1980). A small cosmid for efficient cloning of large DNA fragments. Gc'ri(, I I . 291-298. ( J ~ U . Y /trc,iiv.

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Huynh, T.. Young. R. A., and Davis. R. W. (1985). Construction and screening cDNA lihraries in AgtlO and hgtl I . 1/7 "DNA Cloning. a Practical Approach" (D. M. Glover. ed.). Vol. I. pp. 49-78. IRL Press. Oxford. Washington, DC. Imoto. K.. Methfessel. C.. Sakmann. B., Mishina. M.. Mori, Y.. Konno, T.. Fukuda. K.. Kurasake. M.. Bujo. H.. Fujita. Y..and Numa. S. ( 1986). Location of a 8-subunit region determining ion transport through the acetylcholine receptor channel. N o t r t r ~fLorrdonJ 324, 670-674. Jorgensen. R. A,. Rothstein. S. J.. and Reznikoff. W. S. (1979). A restriction enzyme cleavage map of Tn5 and location of a region encoding neomycin resistance. M o l . Gen. Gent,/. 177. 65-12.

Karlin. A.. Kao. P. N.. and DiPaola. M. (19861. Molecular pharmacology of the nicotinic acetylcholine receptor. Trrtids P/i(rrm. S c i . 7, 301-308. Korman. A. J.. Frantz. J . D.. Strominger. J . L.. and Mulligan. R. C. (1987). Expression of human class I1 major histocompatibility complex antigens using retrovirus vectors. Proi.. N o t / . At.&. Sci. U.S.A. 84. 215(L2154. Laemmli. U. K. (1979). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nutrrre (London) 227, 680-685. LaPolla. R. J . . Mixter-Mayne. K.. and Davidson. N. (1984). Isolation and characterifation of a cDNA clone for the complete protein coding region of the 8 subunit of the mouse acetylcholine receptor. /'roc. Ntrrl. A[.od. Sci. U.S.A. 81, 7970-7974. McCarthy. M. P., Earnest, J . P.. Young. E. F.. Choe. S.. and Stroud. R . M. (19x6). The molecular neurobiology of the acetylcholine receptor. Aiinrr. R i v . Nc~rrrosc~i. 9. 383413. Maniatis. T.. Fritsch, E. F.. and Sambrook, J . (1982). "Molecular Cloning: A Laboratory Manual." Cold Spring Harbor Lab.. Cold Spring Harbor. New York. Mann, R.. Mulligan. R. C.. and Baltimore. D. (1983). Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell (Ctimbridgc, Mrrss.) 33, 153-159.

Maxam. A. M.. and Gilbert. W. (1980). Sequencing end-labeled DNA with hase-specific chemical cleavages. In "Nucleic Acids." Part I (L. Grossman and K. Moldave. eds.). Methods in Enzymology. Vol. 65, pp. 499-580. Academic Press. New York. Melton. D. A,. Krieg. P. A.. Rebagliati. M. R.. Maniatis, T., Zinn. K.. and Green. M. R . (1984). Efficient iti iitro synthesis of biologically active RNA and RNA hybridization prohes from plasmids containing a bacteriophage SP6 promoter. N r d e i c Acids Rt*.s. 12, 7035-7056. Merlie. J. P.. and Lindstrom. J. (1983). Assembly in i i i w of mouse muscle acetylcholine receptor: identification of an a subunit species that may be as assembly intermediate. C P / /fC(1/Jlhridge. Mt1.v.v.) 34, 747-757. Methfessel. C., Witzemann. V.. Takahashi. T., Mishina. M.. Numa. S.. and Sakmann. B . ( 1986). Patch clamp measurements on Xenoprrs laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. ~ f h r c g i w Arc+. 407, 577-588. Mishina. M.. Tobimatsu. T.. Imoto. K.. Tanaka. K.. Fujita. Y.. Fukuda. K.. Kurasaki. M.. Takahashi. H.. Morimoto. Y.. Hirose. T.. Inayama, S., Takahashi. T.. Kuno. M.. and Numa. S. (1985).Location of functional regions of acetylcholine receptor a-subunit hy site-directed mutagenesis. Nrrtrrri, (London) 313, 364-369. Nef. P.. Mauron. A.. Stralder. R.. Alliod. C.. and Ballivet. M. (1984). Structure. linkage. and sequence of the two genes encoding the 6 and y subunits of the nicotinic acetylcholine receptor. Pro(,. Ntrtl. Actrd, S c i . U.S.A. 81. 7975-7979.

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Neubig. R. R., Krodel. E. K., Boyd, N. D., and Cohen. J . B. (1979). Acetylcholoine and local anesthetic binding to T o r p c h nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc. Ntrtl. Actrd. Sc,i. U.S.A. 76, 690-694. Neumann. E., Schaefer-Ridder. M.. Wang, Y., and Hofschneider. P. H. (1982). Gene transfer into mouse lyoma cells by electroporation in high electric fields. E M B O J. 1. 841-845. Noda. M., Takahashi. H.. Tanabe, T.. Toyosato, M., Fusutani. Y.. Hirose. T.. Asai. M.. Inayama. S., and Numa. S. (1982). Primary structure of a-subunit precursor of TorpcJdo crrlifornirri acetylcholine receptor deduced from cDNA sequence. Nrrttrrc, (London) 299, 793-797. Noda. M., Takahashi. H.. Tanabe, T.. Toyosato, M., Kikyotani. S.. Hirose. T.. Asai. M., Takashima. H., Inayama, S. . Miyata. T., and Numa, S . (1983a). Primary structure of p- and 8-subunit precursors of T(irprdo c~rlifiwnicwacetylcholine receptor deduced from cDNA sequences. Ncrtrrrr (London) 301, 25 1-255. Noda. M.. Furutani. Y.. Takahashi. H.. Toyosato. M.. Tanabe, T.. Shimizu. S.. Kikyotani, S.. Kayano. T., Hirose, T.. Inayama. S., and Numa. S. (1983b). Cloning and sequence analysis of calf cDNA and human genomic DNA encoding a-subunit precursor of muscle acetylcholine receptor. Nrrtrrrr (London) 305, 818-823. Okayama. H.. and Berg, P. (1982). High-efficiency cloning of full-length cDNA. Mol. Ccll. B i d . 2, 161-170. Parker. B. A., and Stark, G . R. (1979). Regulation of Simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J. Virol. 31, 360-369. Penefsky. H. S. (1977). Reversible binding of P, by beef heart mitochondria1 adenosine triphosphates. J . Biol. Ckem. 252, 2891-2899. Popot. J.-L.. and Changeux, J.-P. (1984). Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein. Pliysiol. RPIJ.64, I 162-1239. Rabourdin-Combe, C.. and Mach, B. (1983). Expression of HLA-DR antigens at the sufiace of mouse L cells co-transfected with cloned human genes. Ntrtrrrc (Londori) 303, 670674. Rigby. P. W. J . . Dickmann. M.. Rhodes. C.. and Berg, P. (1977). Labeling in v i t r o by nick translation with DNA polymerase I. J. Mol. B i d . 113, 237-251. Schubert. D.. Harris. A . J.. Devine. C. E.. and Heinemann, S. (1974). ChardCleriTdtiOn of a unique muscle cell line. J . CrN Biol. 61, 398413. Sine. s.. and Taylor. P. ( 1979). Functional consequences of agonist-medialed state transitions in the cholinergic receptor. J. Biol. Ckern. 254, 33 15-3325. Southern. E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J . M o l . B i d . 98, 503-517. Temin. H. M. (1986). Retrovirus vectors for gene transfer: efficient integration into and expression of exogenous DNA in vertebrate cell genomes. I n "Gene Transfer" (R. Kucherlapati. ed.). pp. 149-187. Plenum, New York. White. M. M.. Mixter-Mayne, K., Lester. H. A.. and Davidson, N. (1985). MOUSe-Torpc,do hybrid acetylcholine receptors: functional homology does not equal sequence homology. Proc. N u / / . Acod. Sci. U.S.A. 82, 48524856. Wigler. M.. Silverstein. S.. Lee. L.-S.. Pellicer. A.. Cheng. Y.-C.. and Axel. R. (1977). Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cdl fCoinhridgr. Mrrss.) 11, 223-232. Wilson. P. T.. Gershoni. J. M.. Hawrot. E., and Lentz. T. L. (1984). Binding of a-bungarotoxin to proteolytic fragments of the a subunit of Torpedo acetylcholine receptor

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analyzed by protein transfer on positively charged membrane filters. Proc. Ncrrl. Acrrd. Sci. U.S.A. 81, 2553-2557. Yaffe, D. ( 1968). Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc.. Ntirl. Acrid. Sci. U . S . A .61, 477-483. Young. R. A,. and Davis. K. W.(19x3). Efficient isolation of genes by using antibody probes. Proc.. Ntitl. Aurd. Sc.i. U.S.A. 80, 1194-1 198.

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Part 111

Voltage-Sensitive Sodium Channels

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 33

Chapter 12

Molecular Characteristics of Sodium Channels in Skeletal Muscle ROBERT L. BARCHl Mahoney Institute of Neurological Sciences University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104

I. Introduction 11. Biochemistry of Skeletal Muscle Sodium Channels A. Channel Purification B. Chemistry and Stoichiometry of Subunits 111. Functional'Reconstitution of the Purified Sodium Channel A. Reconstitution into Phospholipid Vesicles B. Single Channel Measurements in Planar Bilayers C. Voltage Dependence in Population Flux Studies I V . Channel Primary Sequence V. Probing Channel Topography A. Synthetic Oligopeptides and Directed Antisera B. Monoclonal Antibodies as Structural Probes VI. Sodium Channel Subtypes VII. Summary References

1.

INTRODUCTION

The last 10 years have seen remarkable advances in the molecular characterization of ion channels in nerve and muscle. This is particularly true for the voltage-dependent sodium channel that controls the generation of action potentials in most excitable membranes. This transmembrane glycoprotein has now been purified from mammalian muscle (Barchi er af., 1980; Kraner et af., 1985) and nerve (Hartshorne and Catterall, 1981; Barhanin er d., 1983) as well as from eel electroplax (Agnew er d., 1978) and chick cardiac tissue (Lombet and Lazdunski, 1984). The purified proteins have been functionally reconstituted into vesicles and planar bilayers. 251 Copyright 0 198X by Academic Press. Inc All rights of reproduction in any form reserved.

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confirming that they indeed contain the machinery responsible for the voltage-dependent control of membrane sodium conductance (for reviews see Hartshorne e / a / . , 1986; Tanaka ef a / . , 1986; Agnew ef LA., 1986b). The primary sequences for the eel electroplax sodium channel and for several related sodium channels from rat brain have been deduced from their cloned DNA (Noda e/ a / . , 1984, 1986a). An impressive degree of sequence homology is apparent among these proteins. These advances have set the stage for the next phase of the molecular analysis of this complex protein. This phase will deal with the resolution of the channel’s secondary and tertiary structure, and with the correlation of these structural features with the complexities of channel function. This article will briefly review our current knowledge of the molecular aspects of the voltage-dependent sodium channel, concentrating mainly on those studies done with channels purified from mammalian skeletal muscle. Early results of work designed to probe the relationship of structure and function in this macromolecule will then be considered.

II. BIOCHEMISTRY OF SKELETAL MUSCLE SODIUM CHANNELS A.

Channel Purification

The sodium channel from rat and rabbit skeletal muscle membranes can be solubilized with nonionic detergents such as Nonidet-P40 (NP-40) or Lubrol-PX (Barchi et al., 1980; Kraner et al., 1985). After treatment with 1% NP-40, the channel is found in solution as mixed micelles with detergent and endogenous phospholipid. These micelles consist of 45% by weight detergent and phospholipid and 55% protein, have a partial specific volume of 0.83 ml/g, and an overall molecular weight of about 560,000 (Barchi and Murphy, 1980; Kraner e / al., 1985). Physical measurements of the solubilized channel from both rat and rabbit muscle suggest a molecular weight for the protein component of between 287.000 and 314,000, similar to values reported for the sodium channel from rat brain (Hartshorne et a / . , 1980). Skeletal muscle sodium channel can be purified to virtual homogeneity using the high-aftinity binding of saxitoxin or tetrodotoxin as an identifying marker. The proteins purified from rat and rabbit muscle contain a large glycoprotein subunit of 260,000 MW which runs as a diffuse band with anomalous migrating characteristics both on SDS-PAGE (Barchi, 1983) and on molecular sieve columns under denaturing conditions (Kraner e / al., 1985). This 260,000 MW a-subunit appears analogous in its physical properties to the a-subunit of the rat brain channel and to the high-

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molecular-weight glycoprotein that represents the only protein component of the eel sodium channel (Miller et d.,1983). Both of the mammalian skeletal muscle channels also contain stoichiometric amounts of a smaller p-subunit of 38,000 MW (Casadei rt d.,1986; Roberts and Barchi, 1987). while two smaller P-subunits have been reported in the channel preparations from rat brain (Hartshorne et a / . , 1982).

B. Chemistry and Stoichiometry of Subunits The a- and P-subunits of the mammalian skeletal muscle channel can be separated under denaturing conditions by chromatography on molecular sieve columns and the separated proteins have been subjected to detailed microchemical analysis (Roberts and Barchi. 1987). Both proteins are heavily glycosylated. When analyzed in a gas-phase sequenator, both subunits have proved to be N-terminally blocked. After enzymatic deglycosylation by N-acetylneuraminidase and endoglycosidase F, the psubunit demonstrates four stepwise increases in mobility on SDS-PAGE. reaching a limiting core molecular weight of 26,500. The a-subunit shows a larger relative increase in mobility following neuraminidase treatment than the P-subunit. and ultimately is reduced to an apparent molecular weight of 209,000 after treatment with endoglycosidase F. Both subunits of the rabbit sodium channel have been subjected to quantitative amino acid and carbohydrate analysis (Roberts and Barchi, 1987). The carbohydrate content of the a-subunit is 26.5% by weight, while the p-subunit is 29.4% carbohydrate by weight, both based on the absolute amino acid content determined directly on the samples analyzed. In both cases, these values are in reasonable agreement with the changes in molecular weight observed on SDS-PAGE following enzymatic deglycosylation. The predominant carbohydrates for both the a-and the p-subunits are N-acetylhexosamine and N-acetylneuraminic acid, as expected for Nlinked carbohydrate moieties (see Fig. I). The relative labeling efficiency of the two subunits with the detection system used for analysis on our SDS-polyacrylamide gels has been directly determined using the isolated a-and p-subunits calibrated for concentration by direct amino acid analysis. With our adaptation of the labeling procedure of Shing and Ruoho (1983), the ratio of labeling efficiency for a@ on a molar basis was found to be 0.23 (Roberts and Barchi, 1987). Using this direct calibration value, the stoichiometry of a-and p-subunits was then determined in channels purified from both rat and rabbit muscle (Casadei er al., 1986; Roberts and Barchi, 1987). In each case, the stoichiometry is clearly one p-subunit of 38,000 for each a-subunit of 260,000. The 38,000 band occasionally appears as a doublet on SDS-PAGE. This

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FIG. I . a- and p-subunits of the skeletal muscle sodium channel can be enzymatically deglycosylated to yield core proteins of 209.000 and 26.500 MW. respectively. Here. isolated rabbit skeletal muscle p-subunit was treated for 4 hr with neuroaminidase and then for increasing intervals with endoglycosidase F. Samples were removed at the indicated time and separated by SDS-PAGE. At least three discrete intermediates are seen with endoglycosidase F treatment of the p-subunit. consistent with the expected presence of three to four complex carbohydrate chains.

seems to be a result of variable glycosylation since, when present, both bands can be reduced to a common 26,500 core by treatment with endoglycosidases. Our data suggest that the mammalian skeletal muscle sodium channel consists of one 260,000 and one 38,000 subunit. Using the core molecular weight value obtained from SDS-PAGE after enzymatic deglycosylation and the carbohydrate content measured analytically, the total subunit molecular weights for (Y- and p-subunits are calculated to be 285,000 and 38,000, respectively. Assuming a 1 : 1 stoichiometry, the total molecular weight for the rabbit channel complex is predicted to be 323,000.

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Studies by Catterall and colleagues with the rat brain channel indicate a stoichiometry of 1 : 1 : 1 for a : p, : p2 in that preparation (Hartshorne and Catterall, 1984). The 38,000 subunit is closely associated with the larger a-subunit in mammalian muscle. Sodium channels can be immunoaffinity purified using either monoclonal or polyclonal antibodies directed specifically against the 260,000 a-subunit (Casadei et al., 1986). These antibodies show no crossreactivity with the 38,000 subunit. The protein complex purified with these antibodies, however, invariably contains both the 260,000 and the 38,000 subunits and demonstrates the 1 : 1 stoichiometry between the two found in sodium channel purified with traditional biochemical methods. Studies with purified, reconstituted, eel sodium channel (Agnew et al., 1986a),as well as with the rat brain sodium channel a-subunit expressed in vitro from its isolated message (Noda et al., 1986b),confirm that most, if not all, of the functional properties usually associated with the voltagedependent sodium channel are intrinsic to the a-subunit. The role of the p-subunit in the mammalian channels is not clear. This small subunit might play an additional modulatory role in channel function or, alternatively, could be involved in the association of the channel with cytoskeletal elements in these tissues. This point remains to be clarified. 111.

A.

FUNCTIONAL RECONSTITUTION OF THE PURIFIED SODIUM CHANNEL

Reconstitution into Phospholipid Vesicles

Sodium channels from rat and rabbit skeletal muscle, like their counterparts from the rat brain and eel electroplax, have been functionally reconstituted into both lipid vesicles (Weigele and Barchi, 1982; Tanaka et al., 1983) and planar bilayers (Furman et al., 1986). In lipid vesicles it can be demonstrated that these purified, reconstituted channels gate the movement of monovalent cations in response to pharmacologic activation by veratridine (VER) and batrachotoxin (BTX), and that these fluxes are specifically blocked by saxitoxin (STX) and tetrodotoxin (TTX). After reconstitution into lipid vesicles, skeletal muscle sodium channels insert randomly with about 50% of their high-affinity STX binding sites accessible to this polar toxin on the outside of the intact vesicles. BTX, due to its high lipid solubility, is able to partition into the vesicle membrane and activate channels facing in either direction. As expected, half of the BTX-activated fluxes in these vesicles can be inhibited by external STX; all activated flux can be blocked by the inclusion of STX within the vesicles

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in addition to its external application. All of the purified, reconstituted channels that retain the capacity to gate cation flux in response to BTX activation also contain the high-affinity site for STX binding. The concentrations of BTX and VER required to activate the purified reconstituted channels, as well as those of STX and TTX needed to block this activated current flux, each correspond to the apparent dissociation constants for these compounds measured with sodium channels in normal muscle membranes (for review see Tanaka et al., 1986) (see Fig. 2). A

n 3

I

0 2

4

6

L

z

Control (-BTX) B

10 12 Seconds

14

16

18

+ 0

30

10-7

10-6

[ Bahachaloxin]

0-5

IM)

C

2

4

6 8 Time lsec)

1 0 1 2

FIG.2. Purified rabbit skeletal muscle sodium channels can be functionally reconstituted into phosphatidylcholine vesicles. ( A ) Vesicles containing the purified channel demonstrate that is a rapid influx of monovalent cations in response to activation by batrachotoxin (0) not present in the absence of this toxin (A). This specific influx can be blocked by TTX and STX. (B) Pharmacological activation and blocking of channel activity occurs at concentrations of ligand corresponding to those effective in v i w . For example. activation by BTX ( 0 )exhibits an apparent K, of 1.5 pM.(C) The reconstituted channel exhibits selectivity among monovalent cations. as demonstrated by the relative rate of uptake of each cation into vesicles containing the purified channel protein. The rate of influx for Na' (not shown) is at least &fold higher than that for K' ( 0 )when examined with quenched-flow techniques.

12. MOLECULAR CHARACTERISTICS OF Na CHANNELS

257

Using quenched-flow techniques, cation selectivity ratios have been measured directly for purified channels from rat and rabbit skeletal muscle reconstituted into phosphatidylcholine vesicles (Tanaka el al., 1983; Kraner et al., 1985). With this approach, the initial influx rates for K' , Rb' , and Cs' could be measured directly and an upper limit could be assigned to the influx rate for Na'. Based on these values, relative selectivity ratios for the monovalent cations were then calculated. The purified reconstituted channels from both rat and rabbit skeletal muscle exhibit cation selectivities following BTX activation which closely parallel those reported for these channels in their native membrane environment. VER-activated reconstituted channels are considerably less selective among cations, and activated cation fluxes have a much higher activation energy then comparable values with BTX (BTX = 6 kcal/mol; VER = 30 kcal/mol). Some of this apparent difference in cation selectivity may reflect the differing actions of each toxin on the channel's selectivity mechanism, but much is due to their different effects on the probability of channel opening as reflected in this assay system. BTX-activated channels are opened nearly continuously at 0 mV (Huang et a/., 1982) while VER-activated channels open infrequently, but once opened, may remain open for periods long enough for cationic equilibration to occur in a given vesicle. Thus, with the small vesicles used for these studies, the rate of cation uptake will more closely relate to open-channel conductance with BTX, while similar measurements after VER activation will depend more on the probability of channel opening.

B. Single Channel Measurements in Planar Bilayers Purified skeletal muscle sodium channels have also been incorporated into planar lipid bilayers by first forming vesicles containing the purified channel and then allowing these vesicles to fuse with preformed bilayers prepared from 80% phosphatidylethanolamine (PE)/20% phosphatidylcholine (PC) in decane. With the purified channel from rabbit muscle, single channel events with an average conductance of 20 pS were observed with this approach (Furman P I ul., 1986) (see Fig. 3). Channel activation in the presence of BTX was steeply voltage dependent with 50% opening points between -95 and - I I6 mV. Application of TTX to the cis-chamber which contained the intracellular domain of a reconstituted channel had no effect on channel activity at -77 mV, but application to the trans (extracellular) side resulted immediately in >99% block of single channel open events at the same potential. The average gating charge movement calculated from the relationship between channel open probability and voltage was approximately 4.

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ROBERT L. BARCHI

A

-68mV

-c - 0

B

- 8 8 mV

IpA

I

*oGS

-c -0

-3

-2

PA

-I

0

c

-94mV

-c -3

-2

-0

-I

-c -0

E

-123mV

i

-3

-2

-I

0

0

DA

FIG. 3. Single channel recordings of purified rabbit skeletal muscle sodium channels incorporated into a planar lipid bilayer. After insertion into the bilayer. channels exposed to BTX exhibit voltage-dependent activation, with 50% opening in this example at about -90 mV. Average single channel conductance for reconstituted rabbit channels i n 500 mM/ 200 mM Na' was 20 pS. The left-hand figures are current histograms and the right-hand figures are representative recordings at each of the indicated membrane potentials. Planar bilayer formed with 80% PE/20% PC in decane. (From Furman t ~ 01.. f 1986.)

12. MOLECULAR CHARACTERISTICS OF Na CHANNELS

259

Single channel measurements have also been made with purified reconstituted sodium channels from rat skeletal muscle (Barchi et ul., 1984). For these studies, vesicles containing the purified protein were frozen and thawed, and blebs on the resulting multilamellar liposomes were patch clamped using the method of Tank et d.(1982). Although technically more difficult to carry out. these experiments produced results comparable to those found for skeletal muscle in planar bilayers. The average single channel conductance for the purified rat channel was 19 pS.

C. Voltage Dependence in Population Flux Studies Measurements with planar lipid bilayers indicate voltage-dependent activation in single isolated channels. We have been able to demonstrate that this voltage dependence is also characteristic of the population of purified channels as a whole (Furman et al., 1986). Oriented populations of reconstituted channels were created in lipid vesicles by the inclusion of high concentrations of internal STX during vesicle formation, thus blocking all the inward-facing channels. Subsequent removal of external STX by gel exclusion chromatography produced a functionally homogeneous population of normally oriented channels. A membrane potential was subsequently created by the formation of a [K'] gradient in the presence of valinomycin. Using a quenched-flow device, the external [K'] was then rapidly changed to produce a shift in membrane potential. Sodium fluxes into vesicles containing BTX-activated channels were monitored as a function of the calculated K' Nernst potential. We found that all sodium channels that were activated by BTX at 0 mV and blocked by TTX could also be blocked by hyperpolarization, and that the range of calculated Nernst potentials over which this progressive block was seen corresponded approximately to that expected for the known relationship between BTXmodified channel activation and membrane voltage (Huang et al., 1982).

IV. CHANNEL PRIMARY SEQUENCE A major advance in the structural characterization of these sodium channel proteins has been the deduction of the complete primary sequence of the eel sodium channel (Noda et al., 1984), as well as the sequences of two related sodium channels and a part of a third from rat brain (Noda el al., 1986a), through the cloning of DNA complementary to their respective mRNAs. In each case, the nucleotide sequence for a message of approximately 6800 base pairs was obtained with an open reading frame for 1820 amino acids in the eel sequence and 1900 and 1920 amino acids

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for the two complete brain channel sequences. Computer-assisted analysis of these linear sequences has demonstrated the presence of four large homologous domains within the linear structure of each channel (Noda et ul., 1984, 1986a). Interspecies sequence homology is highest within these domains, in some areas approaching 90%. while regions between domains exhibit lower homology and contain large stretches, especially between the first and second repeat domains, that are present in brain but absent in the eel channel. These observations suggest that the structural features of the channel most important for their common functional characteristics will be contained within the four homologous domains. The extended linear sequence of the sodium channel can be conceived of as containing four homologous subunits incidentallyjoined within the same primary sequence, a situation functionally similar to the separate homologous subunits that comprise the acetylcholine receptor. It is tempting to speculate that the large insert between domains 1 and 2, unique to the mammalian channel, is related in some way to interactions with the p-subunit. V.

PROBING CHANNEL TOPOGRAPHY

The availability of primary sequence information for the sodium channel has lead to a number of models predicting the secondary and tertiary structures of the protein (Noda et ul., 1986a; Guy and Seetharamulu, 1986; Greenblatt ef al.. 1985; Kosower, 1985). Three of these models propose that the channel contains four homologous domains which are symmetrically organized to share in the formation of a common central pore or ion channel. Implicit in these models is the assumption that the basic folding patterns within the four domains will closely resemble each other. The models differ in placing certain putative helices either within or outside of the lipid bilayer, and in their speculations concerning the role of an amphipathic helix from each domain in the formation of the actual ion channel lining. Although all of these models remain highly speculative, they can now serve as a starting point for further studies probing the topographical organization of the channel in the membrane. A. Synthetic Oligopeptides and Directed Antisera

We have set out to probe channel topography in several ways. In the first approach, discriminate stretches of eel channel primary sequence that are predicted in various models to be either intracellular or extra-

12. MOLECULAR CHARACTERISTICS OF Na CHANNELS

26 1

cellular have been identified and synthesized. Polyclonal antibodies raised against these synthetic oligopeptides are then used to identify their topography in intact tissue or in vesicles containing oriented channels. On the one hand, these experiments can provide independent information concerning sodium channel topography while, on the other, they provide additional constraints for the development of channel models. Most models of channel structure postulate that the C-terminus of the protein is on the cytoplasmic side of the membrane. In order to test this hypothesis, we synthesized a I3-amino acid oligopeptide corresponding to residues 1783-1794 at the C-terminus of the eel primary channel sequence (Gordon et al., 1987). Antisera were raised against the purified peptide in rabbits. These antisera, as well as IgG fractions immunoaffinity purified from them using the immobilized synthetic peptide, recognized the peptide in a solid-phase radioimmunoassay and specifically identified the 260,000 MW sodium channel protein of the eel on Western blots prepared from crude eel vesicle membrane proteins separated by SDS-PAGE. Binding of the antisera to this C-terminal peptide, designated R-8, was studied in oriented vesicles prepared from eel electroplax using a modification of methods previously described by Miller et al. (1983). The rightside-out orientation of these vesicles was confirmed by measurement of ['HISTX binding before and after permeabilization of vesicles with 0.05% saponin. In preparations where >95% of the total STX binding sites was accessible prior to permeabilization, only low levels of antibody binding were detectable. After permeabilization, however, specific binding of R8 increased 8- to 10-fold, suggesting that the epitope recognized by this antiserum resides on the cytoplasmic surface of the plasma membrane (Gordon et al., 1987). This localization was subsequently confirmed with immunocytochemical techniques. It is known from previous electrophysiological experiments that voltage-dependent sodium channels are confined largely to the innervated face of the electroplax cells (Keynes and Martin-Ferreira, 1953). At the light microscopic level, R-8 bound specifically to the innervated face of these cells. No significant binding was detected to the uninnervated face, and the labeling of the innervated face was specifically blocked by preincubation of the serum with 10 pM of the purified peptide. Electroplax tissue, labeled with the C-terminal antibody after disruption by freezing, was then examined at the electron microscopic level. Bound R-8 was detected by colloidal gold-labeled second antibody. Colloidal gold particles were found almost exclusively in association with the surface membrane of the innervated face of the cell, bearing out the observations made at the light microscopic level. Further examination indicated that virtually all of these colloidal gold particles were present on the cyto-

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ROBERT L. BARCHI

plasmic side of the bilayer (Gordon ef al., 1987), in agreement with the localization inferred from antibody-binding studies in oriented vesicles (see Fig. 4). From these experiments we conclude that the C-terminus of the eel sodium channel, including a region containing a potential N-glycosylation’s group site (residue 1806), resides on the cytoplasmic side of the plasma membrane. If the four internal repeat domains are, in fact, homologous pseudo-subunits which are organized comparably to each other in the

FIG.4. Topographical localization of the C-terminus of the eel sodium channel. Antiserum was prepared against a synthetic peptide corresponding to residues 1783-1794 of the eel sequence. and specific IgG immunoaftinity purified using the immobilized peptide coupled to Sepharose. Binding of this IgG fraction to eel electroplax membrane containing the channel was localized with colloidal gold-labeled second antibody. Labeling is confined exclusively to the cytoplasmic face of the membrane (original magnification: 15.200 x ). (From Gordon CI

d.,1987.)

12. MOLECULAR CHARACTERISTICS OF Na CHANNELS

263

membrane, then the backbone of the protein must cross the membrane an even number of times in each domain. This would lead to the extended prediction that, since the C-terminus is cytoplasmic, then the N-terminus and the interdomain regions should be cytoplasmic as well. The cytoplasmic location for all of these domains would be in agreement with that proposed in the folding models of Numa (Noda er al., 19841, Guy and Seetharamulu (1986). and Greenblatt c’r NI. (1985). Analysis of a second peptide corresponding to amino acid residues 93 1956, which lies in the primary sequence between homologous domains 2 and 3, has also been completed (Gordon et al., 1988). As with the Cterminal antiserum R-8, the antiserum raised against this peptide (designated antiserum R-6) recognizes its specific peptide in a solid-phase RIA and immunoreacts exclusively with a 260,000 band on Western blots of eel membrane protein. This antiserum also specifically labels the innervated face of the eel electroplax at the light microscopic level. Electron microscopy suggests that the epitope recognized by this antibody is also cytoplasmic, again consistent with the general folding models discussed above. Localization studies are now underway with a number of other synthetic peptides that should help to define further the folding pattern of this complex protein. 6. Monoclonal Antibodies as Structural Probes

A second approach to probing the structure of the sodium channel involves the use of monoclonal antibodies. We have prepared monoclonals to the purified channel in its mixed micellar form with phospholipid and detergent (Casadei et al., 1984). Since the channel is capable of functional reconstitution in this form, it should retain many of its native secondary and tertiary structural features. A panel of 29 monoclonal antibodies against the purified rat channel has been characterized (Casadei and Barchi, 1987). Of these, 6 appear to recognize determinants containing carbohydrate residues as evidenced by the competitive inhibition of their binding by various monosaccharides. We have mapped out the binding interactions of these monoclonals in the hope of identifying regions adjacent in the channel tertiary structure which can subsequently be localized within its known linear structure. Individual monoclonal antibodies were biosynthetically labeled and competitive binding studies were then carried out with each of the remaining unlabeled antibodies in the panel (Casadei and Barchi, 1987) (see Fig. 5). Using this approach, we have defined two groups of interacting sites which together contain the epitopes for 21 of the 29 antibodies in this panel. The largest group includes 16 antibodies linked together through

264

ROBERT L. BARCHI

M Ab Families

FIE4 llE3 GIG8 GIG9

&w \

LID3

C/H3

FIG6 DIE6

8/44 FlH4 AtClP

Cross- react with rabbit

Binding blocked by sialic acid

FIG.5 . Map of monoclonal antibody-binding domains on the rat skeletal muscle sodium channel. A panel of 21 monoclonal antibodies against the channel 260,000 subunit was tested for mutual competitive interaction of binding in an RIA. Each circle represents the binding region for one antibody or group of antibodies that exhibit identical characteristics and that are capable of complete mutual inhibition of binding. Regions showing no interaction between their member antibodies do not overlap. Major overlap indicates complete inhibition of the binding of antibodies in one region by those in another, whereas minor overlap indicates partial inhibition. Shaded regions indicate groups that cross-react with the rabbit skeletal muscle channel and broken circles indicate domains that contain N-acetylneuraminic acid as determined by competition studies. For domains recognized by more than one antibody, the group members are given in the upper right of the figure. (From Casadei and Barchi, 1987.)

mutual competitive interactions. This immunogenic domain appears to be on the external surface of the sodium channel since one group of related antibodies recognizes an epitope that contains carbohydrate. Subsequent localization of these and other interactive monoclonal antibody-binding sites in the channel’s primary sequence should shed additional light on the folding pattern for the protein as a whole.

12. MOLECULAR CHARACTERISTICS OF Na CHANNELS

VI.

265

SODIUM CHANNEL SUBTYPES

In addition to providing a valuable approach to the study of common features of sodium channel structure, these monoclonal antibodies have served to emphasize the existence of closely related sodium channel subtypes in mature skeletal muscle. Although all our monoclonal antibodies recognize the 260,000 a-subunit of the rat sodium channel on Western blots, they can be separated into several distinct groups on the basis of their patterns of immunocytochemical reactivity in intact muscle (Haimovich et al., 1987). Most of these antibodies recognize sodium channels on the surface membrane of both fast and slow muscle fibers in frozen cross-sections of adult rat skeletal muscle (see Fig. 6). Interestingly, these antibodies identify high concentrations of sodium channels at the synaptic regions; at the electron microscopic level this immunoreactivity proves to be distributed throughout the postsynaptic membrane both at the surface and in the depths of the postjunctional folds (Haimovich ef al., 1987).This concentration of sodium channels in the end plate region corresponds to that recently described with electrophysiological techniques (Bean e t trl., 1985). A few of the monoclonal antibodies show a different labeling pattern. These antibodies specificallylabel a repeating internal array of transverse bands in slow muscle fibers that correspond in their location at the A-I junction to that of the T-tubular network in this species (Ishikawa, 1983). At the electron microscopic level it can be demonstrated that these antibodies label T-tubular elements but not elements of the sarcoplasmic reticulum or contractile proteins. However, these antibodies do not label the T-tubular system of fast fibers (see Fig. 7). Thus there appear to be at least three subtypes of sodium channel in adult innervated skeletal muscle: those common to the surface membranes of fast and slow fibers, those found in slow fiber T-tubular membranes, and those in the T-tubular membranes of fast fibers. In addition to these sodium channels subtypes in adult muscle, all of which appear to be sensitive to TTX, several other channel subtypes have been defined through a combination of pharmacological and physiological techniques. For example, after denervation, mature skeletal muscle expresses a sodium channel that is resistant to block by TTX (Harris and Thesleff, 1971) and has kinetic channel properties slightly different from those of the 'ITX-sensitive form (Pappone, 1980). The appearance of these channels is prevented by the presence of inhibitors of protein synthesis (Gramp et al., I97I), and is accompanied by a reciprocal fall in the number of TTX-sensitive channels present (Barchi and Weigele. 1979). Recently,

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FIG. 6. Sodium channels are concentrated at the neuromuscular junction. Consistent with recent electrophysiological studies. monoclonal antibodies to the rat sodium channel a-subunit show intense specific staining of the postsynaptic membrane. (A. D) Phase-contrast images of the neuromuscular junction in a skeletal muscle fiber cross-section (original magnification: 450 x ): ( 8 , E) same sections labeled with antibody against t h e sodium channel; (C. F) localization of acetylcholine receptors in these sections labeled with fluorescentconjugated a-bungarotoxin: ( G )immunocytochemical localization of sodium channels in the postjunctional membrane at the E M level indicates distribution throughout the secondary synaptic folds. unlike the localized superficial distribution of acetylcholine receptors.

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FIG.7. Monoclonal antibodies raised against the purified rat skeleial muscle sodium channel identify subtypes with differing distribution in mature skeletal fibers. Here, a monoclonal (AB2) labels channels in the T-tubular system of slow fibers (A). In the same section, other antibodies label only sodium channels in the surface membrane ( B ) .

we have found a 3-fold increase in sodium channel mRNA in skeletal muscle 3 days after denervation, possibly associated with the transcription of the genomic message for this new channel subtype (Cooperman et d., 1987). In developing muscle, there is a similar transition from the expression of TTX-insensitive sodium channels to mature TTX-sensitive channels. Although it is potentially feasible to isolate each of these channel subtypes and delineate their differences by direct chemical analysis, it is likely that such information will be obtained more quickly and directly by the cloning of the messages encoding these proteins, and the assignment of appropriate sequences to each subtype through experiments that trace their parallel expression after physiological or ontological transitions. Using S, nuclease techniques, it should be possible to detect differences as small as several base pairs between these messages. VII. SUMMARY

An exciting new phase of work on the voltage-dependent sodium channel is now beginning. Using the wealth of physiological data available from decades of biophysical research in conjunction with more recent biochemical and structural information, new models of sodium channel structure are being proposed, tested, and refined. In addition to unraveling the common structural themes that underly the general aspects of sodium channel function, increasing attention is also being directed toward more subtle differences in structure which may be responsible for the various subtypes of sodium channels expressed in normal nerve and muscle. The

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next decade should be marked by rapid advances in our understanding of both the relationships of structure to structure-function in this important membrane ion channel and of the various regulatory factors which control its expression. ACKNOWLEDGMENTS Work from the author's laboratory cited in this chapter was supported in part by NIH grants NS-18013 and NS-08075 as well as by a grant from the Muscular Dystrophy Association of America. The secretarial assistance of Ms. Gail Heimberger in the preparation of this manuscript is greatfully acknowledged.

REFERENCES Agnew, W. S., Rosenberg. R. L., and Tomiko, S. A. (1986a). Reconstitution of the sodium channel from E/ecfropliorlis elecfric~rr.s.I n "Ion Channel Reconstitution" (C. Miller, ed.). pp. 307-336. Plenum. New York. Agnew, W. S., Tomiko, S. A.. Rosenberg, R. L.. Emerick, M. C.. and Cooper. E. C. ( 1986b). The structure and function of the voltage-dependent sodium channel. Proc. Nritl. Acrid. Sci U . S . A . 479, 238-256. Barchi, R. L. (1983). Protein components of the purified sodium channel from rat skeletal muscle sarcolemma. J. Netrrocliem. 40, 1377-1385. Barchi, R. L., and Murphy, L. E. (1980). Size characteristics of the solubilized sodium .~. channel saxitoxin binding site from mammalian sarcolemma. Bicdzirn. B i ~ ~ p h yActti 591, 391-398. Barchi. R. L., and Weigele. J . B. (1979). Characteristics of saxitoxin binding to the sodium channel of sarcolemma isolated from rat skeletal muscle. J. Pliysiol. (London) 295, 383396. Barchi. R. L., Cohen. S. A.. and Murphy, L. E. (1980). Purification from rat sarcolemma of the saxitoxin-binding component of the excitable membrane sodium channel. Proc. N u t / . Acrid. Sci. U.S.A. 71, 1306-1310. Barchi. R. L., Tanaka. J . C., and Furman, R. F. (1984). Molecular characteristics and functional reconstitution of muscle voltage-sensitive sodium channels. J . Cell. Bioc.he/n. 26, 135- 146. Barhanin, J., Pauron, D.. Lombet, A.. Norman, R. 1.. Vijverberg. P. M., Giglio. J . R.. and Lazdunski. M. ( 1983). Electrophysiological characterization, solubilization and purification of the Tifvirs toxin receptor associated with the gating component of the Na' channel from rat brain. EMBO J . 2, 915-920. Bean. K. G., Caldwell. J. H.. and Campbell, D. T. (1985). Sodium channels in skeletal muscle concentrated near the neuromuscular junction. Nufrrre (London) 313, 588-590. Casadei, J. M.. and Barchi, R. L. (1987). Monoclonal antibodies against the voltage-sensitive sodium channel from rat skeletal muscle: mapping antibody binding sites. J. Nerrrochrwt. 48, 773-778. Casadei. J. M.. Gordon. R. D.. Lampson. L. A., Schotland. D. L., and Barchi. R. L. (1984). Monoclonal antibodies against the voltage-sensitive Na' channel from mammalian skeletal muscle: analysis of subunits. Pro(.. Natl. Acud. Sci. U . S . A . 81, 6227-6231. Casadei. J. M..Gordon, R. D.. and Barchi. R. L. (1986). lmmunoaffinity purification of the voltage dependent sodium channel from mammalian skeletal muscle. J . B i d . Cltrrii. 261, 4318-4323.

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Cooperman. S.S.. Grubman. S. A,. Barchi. R. I - . . Goodman. R. H.. and Mandel. G . (1987). Modulation of sodium channel mRNA levels in rat skeletal muscle. Proc. Nnfl. Actrd. S C ~U.S.A. . 84, 8721-8725. Furman. R. E.. Tanaka. J. C.. Mueller. P.. and Barchi, R. L. (1986). Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes. Proc. N o r / . A c i d . Sci. U.S.A. 83, 488-492. Gordon, R. D., Fieles. W. E.. Scholland. D. L.. Hogue-Angeletti. R.. and Barchi. R. L. ( 1987). Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Elrc.frophorrrs e/twric.tts using antibodies raised against a synthetic peptide. Proc. Nitrl. A i d . Sci. U.S.A. 84, 308-313. Gordon. R. D.. Li. Y.. Fieles. W. E.. Schotland. D. L.. and Barchi. R. L. (1988). Topological localization of a segment of the eel voltage-dependent sodium channel primary sequence that discriminates between models of tertiary structure. J. Ncirrosci. (in press). Cramp. W.. Harris. J. B.. and Thesleff. S. (1971). Inhibition of denervation changes in skeletal muscle by blockers of protein synthesis. J. P/iy.siol. fLondon) 221, 743-754. Greenblatt. R. E.. Blatt, Y.. and Montal, M. (19x5). The structure of the voltage-sensitive sodium channel. FEBS L e t f . 193, 125-134. Guy, H. R . . and Seetharamulu. P. (1986). Molecular model of the action potential sodium channel. Proc. Nritl. Acitd. Sci. U . S . A . 83. 508-512. Haimovich. B. G . . Fieles. W.. Schotland, D.. and Barchi, R. (1987). Localization of sodium channel subtypes in adult rat skeletal muscle using channel-specific monoclonal antibodies. J. Nrrtrosci. 7, 2957-2966. Harris. J. B.. and Thesleff, S. (19711. Studies on tetrodotoxin resistant action potentials in denervated skeletal muscle. Acrci Pliysiol Scitnd. 83. 382-388. Hartshorne. R. P.. and Catterall. W . A. (1981). Purification of the saxitoxin receptor of the sodium channel from rat brain. Proc. N u l l . Actid. Sci. U.S.A. 78, 4620-4624. Hartshorne, R. P.. and Catterall. W. A. (1984).The sodium channel from rat brain: purification and subunit composition. J. Biol. Chon. 258, 1667-1675. Hartshorne. R. P.. Coopersmith. J.. and Catterall, W . (1980). Size characteristics of the solubilized STX receptor of the voltage-sensitive sodium channel from rat brain. J . B i d . Chrtn. 225, 10. 572-575. Hartshorne. R . P., Messner, D. J.. Coopermith. J . C.. and Catterall. W. A. (19x2). The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical b subunits. J. B i d . C i i m 257, 13888-13891. Hartshorne. R. P.. Tamkun. M.. and Montal. M. (1986). The reconstituted sodium channel from brain. I n "Ion Channel Reconstitution" (C. Miller, ed.). pp. 337-362. Plenum. New York. Huang. L . M.. Moran. N.. and Ehrenstein. G . (1982). Batrachotoxin modifies the gating kinetics of sodium channels in internally perfused neuroblastoma cells. Pro(.. Ntrfl. Acrid. Sci. U.S.A. 79, 2082-2085. Ishikawa. H. (1983). Fine structure of skeletal muscle. Cc4 Miisi.le Mofil. 4, 1-84, Keynes. R. D.. and Martin-Ferreira. H. (1953). Membrane potentials in the electroplates of the electric eel. J. Plivsiol. (London) 119, 3 15-35 I . Kosower. E. M. (1985). A structural ilnd dynamic molecular model for the sodium channel of Elrc~froplwrrtselrcrriciis. FEBS Lrrf . 182, 234-242. Kraner. S. D., Tanaka, J. C . . and Barchi. R. L. (1985). Purification and functional reconstitution of the voltage-sensitive sodium channel from rabbit T-tubular membranes. J. Biol. Clrrtn. 260, 6341-6347. Lombet. A,. and Lazdunski, M. (1984). Characterization. solubilization. affinity labeling. and purification of the cardiac sodium channel using Titius toxin gamma. Ettr. J. Bioc.hcvfi. 141, 651660.

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Miller, J. A., Agnew. W. S.. and Levinson, S. R. (1983). Principal glycopeptide of the tetrodotoxin binding protein from Electrophoriis electricus: isolation and partial chemical and physical characterization. Biochivnistr?, 22, 4 6 2 4 7 0 . Noda. M., Shimizu. S., Tsutomu. T.. Takai. T., Kayano, T., Ikeda, T.. Takahashi. H.. Nakayama, H., Kanaoka. U.. Minamino, N., Kangawa, K.. Matsuo. H.. Raftery. M. A.. Hirose, T., Inayama. S.. Hayashida, H.. Miyata, T.. and Numa, S. (1984). Primary structure of Electropkorcts electricus sodium channel deduced from cDN A sequence. Nutiire (London) 312, 121-127. Noda. M., Ikeda. T.. Suzuki, H., Takeshima, H., Kurasaki. M., Takahashi. H., and Numa, S. (1986a). Existence of distinct sodium channel messenger RNAs in rat brain. Nature (London) 320, 188-192. Noda. N . , Takajuki. I.. Suzuki. H., Takashima, H.. Takahashi. T.. Kuno. M.. and Numa. S. (1986b). Expression of functional sodium channels from cloned cDNA. Ntrtrrrt, (London) 322, 826-828. Pappone, P. (1980). Voltage-clamp experiments in normal and denervated mammalian skeletal muscle fibers. J . Pliysiol. (London) 306, 377410. Roberts, R., and Barchi. R. (1987). The voltage-sensitive sodium channel from rabbit skeletal muscle: chemical characterization of subunits. J . B i d . Chem. 262, 2298-2303. Shing. T., and Ruoho, A. (1983). A method for detecting nanogram amounts of proteins on SDS-polyacrylamide gels. A n d . Biochem. 110, 171-174. Tanaka, J. C.. Eccleston. J. F., and Barchi, R. L. (1983). Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma. J. B i d . Chcm. 258, 75 19-7526. Tanaka, J . C.. Furman. R. E., and Barchi. R. L. (1986). Skeletal muscle sodium channels: isolation and reconstitution. In “Ion Channel Reconstitution” (C. Miller, ed.), pp. 277305. Plenum, New York. Tank, D. W., Miller, C.. and Webb, W. W. (1982). Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo elecwopltix. Proc. Nrctl. Acad. Sci. U.S.A. 79, 7749-7753. Weigele. J . B.. and Barchi. R. L. (1982). Functional reconstitution of the purified sodium channel protein from rat sarcolemma. Proc. Nut/. Acud. Sci. U.S.A. 79, 3651-3655.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 33

Chapter 13 Electrical Recordings from Cloned Sodium Channels Expressed in Xenopus Oocytes WALTER STUHMER Max Planck Institute for Biophysical Chemistry 3400 Gottingen Federal Republic of Germany

Oocytes from Xenopus luevis provide an efficient translation system for expressing ionic channels under in vivo equivalent conditions (Gundersen et a/., 1984). This transcription of the genetic information starts by microinjection of mRNA that has been either extracted directly from cells or derived from cDNA. The elucidation of the cDNA encoding for ionic channels such as acetylcholine receptors (Mishina et ul., 1984) and sodium channels (Noda et ul., 1984, 1986a) from different origins has led to investigations of the structure-function relationship of these channels. This is done by either assembling multisubunit channels with elements from various species or by introducing artificial mutations. This approach has been used to assign specific functions to individual subunits of the nicotinic acetylcholine receptor (Sakmann et ul., 1985) and to analyze functional properties of acetylcholine receptor mutants altered by sitedirected mutagenesis, enabling localization of functional regions within a subunit (Mishina et ul., 1986). It should be emphasized here that this was made possible by application of the patch-clamp technique developed originally by Neher and Sakmann (Hamill et ul., 1981). cDNA-derived mRNA encoding for the type I1 rat brain sodium channel has been used to express functional channels in Xenopus oocytes (Stuhmer et a!., 1987). It is obvious that the procedure used for studying the structure-function relationship of the acetylcholine receptor channel can and will also be applied to the sodium channel. Before this can be accomplished, however, the rat brain sodium channel has to be characterized as it is expressed in Xenopus oocytes. 271 Copyright 0 1988 by Academic Prec\. Inc. All rights of reproduction in any form reserved.

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For this purpose, mRNA specific for rat brain sodium channel I1 was transcribed in vitro with the SP6 system using the recombinant plasmid pRII-1 (Green et at., 1983; Melton et al., 1984; Noda et al., 1986b). Stage V Xenopus oocytes were injected with 50 nl of a 0.125 pg/pI mRNA solution and then incubated for periods of up to 12 days. After 5 days, tetrodotoxin (TTX) blockable sodium currents would appear as measured either by a two-electrode voltage clamp or by patch-clamp recording using rather large (up to 5 pm) and low resistance patch pipets (down to 0.8 M a ) in the cell attached configuration (Leonard et al., 1986). Peak sodium currents of up to 3 nA could be recorded through these “macro patches.” (For details of the method see Stiihmer et al., 1987.) Figure 1 shows a family of curves obtained in response to step depolarizations from -50 to 60 mV in steps of 10 mV applied to the oocyte membrane with the patch pipet. A rather large hyperpolarization of - 120 mV was maintained between the test pulses since the sodium currents showed a pronounced potential-dependent slow inactivation process with time constants in the range of minutes (Simoncini et al., 1987; Ruff et al., 1987). The temporal resolution of the currents is high (better than 100 psec) due to the fact that only a small area of the oocyte membrane is clamped through a relatively low resistance pathway. Figure 2 shows the peak inward current versus voltage relation for the current records shown in Fig. 1. The smooth line through the data points represents a least squares fit to the data according to Eq. ( l ) ,

yielding activation parameters of v “ / 2 = - 30 mV and a‘ = 7.3 mV, with G = 12 nS and V,,, = 64.5 mV. The sodium currents can be inactivated in a potential-dependent manner as shown in Fig. 3. For this purpose, a 36-msec prepulse is applied in the range of - 150 to - 30 mV in steps of 5 mV. At the end of this conditioning prepulse, the inactivated channels are tested by a step depolarization to - 10 mV, a potential at which the current is nearly maximal. As the prepulse potential becomes less negative, the sodium channels become quickly inactivated and conduct less current (see Fig. 3). The peak sodium current can be plotted as a function of the conditioning prepulse potential (VJ as shown in Fig. 4. The data can be fitted by the function shown in Eq. (2),

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1 ms F I G . I . Family of currents elicited in response to step depolarizations from -SO to 60 mV in steps of 10 mV, from a resting potential of - I20 mV. Duration of the pulses; 5 msec: temperature, IS.3"C. The current signals were corrected for capacitive transients with a combination of analog and digital techniques and filtered with a low-pass Bessel filter at 10 kHz. Each record represents the average of eight consecutive responses.

h, I

+ exp(""

Uh

which characterizes the fast inactivation by V''I/z, the potential at which half of the channels are inactivated, and ah,the steepness of this potential dependence. It is interesting to compare steady-state parameters gained from experiments as described above with other excitable tissue from rats.

FIG.2. Peak inward current versus test potential relation of the current records shown in Fig. I . The smooth line is a least squares fit to Eq. ( I ) . The maximal peak inward current of 8.52 pA flows at - 14 mV.

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FIG.3. Family of current responses obtained at - 10 mV after a 36-msec prepulse between IS0 and -30 mV in steps of S mV. Holding potential, - 122 mV; temperature. 15.3"C.

Neumcke and Stampfli (1982) report activation parameters from rat peripheral nerve which are close to our results within experimental error. However, the potential of half-maximal inactivation reported by them is lower by more than 10 mV. This would result in a significant overlap of the activation and inactivation relation in our case. We have one more line of evidence which suggests that this is the case. When recording from smaller patches with only a few channels, we consistently see noninactivating channel activity at low depolarizations. This behavior in brainderived tissue has also been described by other groups, including French and Gage (1985) and Hamill et al. (1986). At low depolarizations this noninactivating component gives rise to a persistent current for times when

-8-

-120

-90

-60

.

-30

I

0

FIG.4. The normalized peak inward sodium currents of Fig. 3 are plotted as a function of prepulse potential. The smooth line through the data points is a least squares fit to Eq. (2).

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sodium channels from other tissues would be completely inactivated. In Fig. 1, the responses to the first depolarization steps show some indication of this. Point mutations and alterations of the amino acid sequence are going to be a tool in elucidating the relationships between the sodium channel structure and its function. One problem encountered with this approach is the practically infinite number of possibilities for such mutations. The number of theoretically possible mutants of a polypeptide with 2000 amino acids, such as the sodium channel, is 20 elevated to the 2000th.This is truly an astronomically large number. As a comparison, the volume of the present universe is believed to be smaller than 10 to the 110Ih cubic Angstrom. Even considering that most of all possible mutants are going to be nonsense proteins, there still remain far too many possibilities. This makes it clear that it is essential to have theoretical models serving as guides in considering what mutations to make. Even then, the task is going to be a difficult one, since most mutants will either behave very similarly to the wild type, or not function at all. The aim is to find sites that, when altered minimally, produce a maximally modified (but functional) behavior that hopefully concerns mainly only one parameter. Even then, it cannot be said that this particular site is “responsible” for that specific behavior, since conformational changes are strongly allosterically coupled. Another difficulty is that the mutants are only going to be screened for large deviations in the main parameters describing the behavior of sodium channels. This is because every mutant cannot be studied as thoroughly as the wild type in times comparable to the generation of a new mutant. It seems clear that the two p-subunits associated with the main chain that are found in rat brain (Hartshorne and Catterall, 1981) and rat skeletal muscle (Barchi, 1983) must have some function. We have just not had enough time to associate a function with these subunits. In conclusion it can be said that the combination of two rather new techniques has provided us with exciting new ways to probe the molecular properties of ionic channels. This can be considered a major breakthrough, but it still might take a long time until we find the answer to the urgent question: How does a channel work? REFERENCES Barchi, R. L. (1983). Protein components of the purified sodium channel from rat skeletal muscle sarcolemma. J . Neurochem. 40, 1377-1385. French, C . R., and Gage, P. W. (1985). A threshold sodium current in pyramidal cells in rat hippocampus. Neurosci. Lett. 56, 289-293. Green. M. R.. Maniatis, T., and Melton, D. A. (1983). Human P-globin pre-mRNA synthesized 32, in vitro is accurately spliced in Xenoprts oocyte nuclei. Cell (Cambridge. MCJSS.) 681-694.

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Gundersen, C. B., Miledi, R., and Parker, L. (1984). Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopus oocytes. Nature (London) 308, 421-424. 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. Pfluegers Arch. 391,85-100. Hamill, 0. P.. Huguenard, J. R., Enayati, E. F.. and Prince, D. A. (1986). Single channel currents underlying slow threshold Na' conductances in rat neocortical neurones. Neur o d . SOC.Abstr. No. 261.5. Hartshorne, R. P., and Catterall, W. A. (1981). Purification of the saxitoxin receptor of the sodium channel from rat brain. Proc. Nail. Acud. Sci. U . S . A . 78, 46204624. Leonard, J . , Snutch. T., Lubbert. H.. Davidson, N., and Lester, H. A. (1986). Macroscopic Na currents with gigaohm seals on mRNA-injected Xenopus oocytes. Biophys. J . 49, 386a. Melton, D. A., Krieg, P. A,, Rebagliati, M. R., Maniatis, T., Sinn, K., and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12,7035-7056. Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto, Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom, J., Takahashi, T.. Kuno, M.. and Numa, S. (1984). Expression offunctional acetylcholine receptor from cloned cDNAs. Nature (London) 307, 604-608. Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel. C., and Sakmann, B. (1986). Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature (London) 321, 40641 I . Neumcke, B., and Stampfli. R. (1982). Sodium currents and sodium-current fluctuations in rat myelinated nerve fibres. J. Physiol. (London) 329, 163-384. Noda, M., Shimizu, S., Tanabe, T., Takai, T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama, H., Kanaoka, y., Minamino, N., Kangawa, K., Matsuo, H., Raftery, M. A,, Hirose, T., Inayama, S., Hayashida, H.. Miyata, T., and Numa, S. (1984). Primary structure of Elecfrophorus electricus sodium channel deduced from cDNA sequence. Nature (London) 312, 121-127. Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi. H., and Numa, S. (1986a). Existence of distinct sodium channel messenger RNAs in rat brain. Nuture (London) 320, 188-192. Noda, M., Ikeda, T., Suzuki, H.,Takeshima, H., Takahashi, T., Kuno, M., and Numa. S. (1986b). Expression of functional sodium channels from cloned cDNA. Nuture (London) 322, 826828. Ruff, R. L.. Simoncini. L., and Stiihmer. W. (1987). Comparison between slow sodium channel inactivation in rat slow- and fast-twitch muscle. J. Pliv.sio1. (London)383, 339348. Sakmann, B., Methfessel, C.. Mishina. M.. Takahashi, T., Takai, T., Kurdsaki. M.. Fukuda, K., and Numa, S. (1985). Role of acetylcholine receptor subunits in gating of the channel. Nutiire (London) 318, 538-543. Simoncini, L.. and Stiihmer. W. (1987). Slow sodium channel inactivation in rat fast-twitch muscle. J. Physiol. (London) 383, 327-337. Stiihmer, W.,Methfessel. C.. Sakmann, B.. Noda. M., and Numa. S. (1987). Patch clamp characterization of sodium channels expressed from rat brain cDNA. Eiir. Biophys. J . 14, 131-138.

CURRENT TOPICS IN MEMBRANES A N D TRANSPORT. VOLUME 33

Chapter 14 Tissue-Specific Expression of Genes Encoding the Rat Voltage-Gated Sodium Channel SHELLEY A . GRUBMAN, SHARON S.COOPERMAN, MARY P . BEGLEY, JOSHUA L . WEINTRAUB, RICHARD H . GOODMAN, A N D GAIL MANDEL Division of Molecular Medicine, Department of Medicine Tufts-New England Medical Center Boston. Massachusetts 02111 1. Introduction 11. Ontogeny of Sodium Channel Type I and Type I1 in Rat Brain A. Synthesis of Oligonucleotide Probes Specific for Type I and Type I 1 mRNAs B. Northern Blot Analysis of Sodium Channel mRNA 111. Tissue-Specific Expression of Sodium Channel Type 11 A. Generation of an Antisense Probe Coding for Sodium Channel Type 11 B. RNase Protection Analysis of Sodium Channel Type 11 mRNA in Brain and Muscle IV. Discussion References

1.

INTRODUCTION

The voltage-gated sodium channel is the basis of electrical excitability in many tissues. It is of considerable interest, therefore, to elucidate the molecular events underlying regulation of this channel during development. The differentiation of excitable cells involves the expression of genes encoding voltage-gated ion channels, and synthesis and assembly of ion channel subunits into the cell membrane. Accordingly, regulation of excitability can occur at several levels, by transcriptional, posttranscriptional, translational, and posttranslational mechanisms. Specific pharmacological 277 Copyright C? 19811 by Academic P r a s . Inc. All nphls of reproduction in any form reserved.

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agents for the mammalian voltage-gated sodium channel have been useful for studying translational and posttranslational regulation of this channel in brain, skeletal muscle, and cardiac muscle. Recently, reagents have also become available for studying sodium channel gene expression. The cDNA and genomic clones that we and others have isolated are useful probes of the transcriptional events underlying the appearance of excitability in mammalian tissues. In this chapter, we describe the strategy and techniques we have used to begin to study expression of the rat sodium channel genes in different excitable tissues. The voltage-gated sodium channels in rat brain and skeletal muscle are composed of a large a-subunit ( M , approximately 260,000)and one or two smaller subunits (Barchi, 1983; Hartshorne and Catterall, 1984). Complementary DNAs encoding two distinct a-subunits in brain, termed types I and 11, have been isolated (Noda et al., 1986a). Independent studies from two laboratories have demonstrated that RNA transcribed from the asubunit cDNA is sufficient for expression of functional channels in Xenopus oocytes (Goldin et al., 1986; Noda et al., 1986b). Although the coding sequences of cDNAs I and I1 are highly homologous, the 5' untranslated (UT) regions are poorly conserved. The dissimilarities within the 5' UT regions of the cDNAs have enabled us to generate probes specific for each of brain sodium channels. The probes can be used in Northern blot or RNase-protection assays to determine the levels of mRNA coding for the a-subunits of the different sodium channels. II. ONTOGENY OF SODIUM CHANNEL TYPE I AND TYPE II IN RAT BRAIN

A. Synthesis of Oligonucleotide Probes Specific for Type I and Type II mRNAs

Two oligonucleotide primers, corresponding to 25 base pairs (bp) in the unique 5' UT regions of either sodium channel I or 11, were synthesized and end-labeled with '*P (Fig. 1). In the region selected, the sequences of the probes are completely different, and the probe specific for sodium channel gene I will not detect the mRNA coding for gene 11. 6. Northern Blot Analysis of Sodium Channel mRNA

Total cellular RNA was prepared from rat brains by a modification of the technique of Chirgwin et al. (1979). This procedure involves disruption of the tissue in the presence of guanidine isothiocyanate, followed by so-

279

14. TISSUE-SPECIFIC EXPRESSION OF GENES -161

-137

(I) C C T T C T T G C A A A A G G T G A C C A A G T G (11) A G C A G T C T C C A G T G C A G G T T T C T A G -130

-1 06

FIG. I . Oligonucleotide probes used in Northern blot analysis of sodium channel RNAs. The numbers refer to the nucleotide position within the untranslated region with respect to the translational start site.

lubilization of the tissue extract in guanidine hydrochloride. The extracted nucleic acids were purified and concentrated by several ethanol precipitations. The amount of total cellular RNA in each sample was quantitated and normalized by the ethidium bromide fluorescence of the rRNA subunits. The ontogeny of the two sodium channel mRNAs was studied by Northern blot analysis. RNA was purified from animals ranging in age from embryonic day 15 to adult (postnatal day 90). An equal amount of the total cellular RNA from each sample was resolved on a denaturing agarose gel and the RNA was transferred electrophoretically to a charged nylon membrane. The membrane was incubated with a "P-radiolabeled oligonucleotide probe specific for either sodium channel type 1 or type 11. Hybridization was detected by autoradiography as shown in Fig. 2. The results from this study indicated that there is significantly more mRNA coding for sodium channel type I1 than for type I in rat brain, at all stages of development. The mRNA levels of both sodium channels increase significantly between embryonic day 15 and embryonic day 20. A further gradual increase in mRNA levels is observed during postnatal development. The change in sodium channel mRNA levels detected by Northern blot assay correlates with previous measurements of sodium channel protein in developing rat brain. Lombet et d.(1983) have shown, for example, that the number of sodium channels, measured by binding to tetrodotoxin (TTX), increases dramatically between embryonic days 17 and 20, plateauing at postnatal day 20. 111.

TISSUE-SPECIFIC EXPRESSION OF SODIUM CHANNEL TYPE II

It was apparent from the Northern blot hybridization studies that sodium channel type I mRNA was present in rat brain at extremely low levels. Because we were interested in comparing levels of sodium channel mRNAs in tissues other than brain, where the level of sodium channel mRNAs I

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FIG.2. Northern blot analysis of rat brain RNA. Total cellular RNA was purified from rat brains of different developmental stages and was probed with oligonucleotides specific for sodium channel genes I or 11. Each lane contained 40 pg of RNA. Samples are as follows: El5 and E20, 15 and 20 days of embryonic development, respectively; 0, birth; 5. 9. and 20. postnatal days of development; and adult, postnatal day 90.The autoradiograph was developed after an overnight exposure with an intensifying screen.

and I1 might be even lower, a more sensitive assay was required. This increased sensitivity was achieved using an RNase protection assay (Zinn et al., 1983). In this technique, total cellular RNA is mixed with a radiolabeled probe (either DNA or antisense RNA) for a specific mRNA. After the RNA has been allowed to hybridize to the probe, nucleases are added which preferentially cleave single-stranded (i.e., nonhybridizing) nucleic acids. If the probe hybridizes to entirely complementary sequences,

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28 1

it will be resistant to nuclease digestion, and will be the same length before and after the nuclease reaction. On the other hand, regions of mismatch between the probe and mRNA will be susceptible to nuclease digestion, giving rise to smaller probe fragments. The sizes of very small ( 4 0 bp) protected probe fragments can be resolved precisely on a denaturing sequencing gel. Under appropriate hybridization conditions, a single nucleotide difference between two closely related mRNAs can be detected using this technique.

A.

Generation of an Antisense Probe Coding for Sodium Channel Type II

Although RNase protection assays can be performed using oligonucleotide probes, the short length of the oligonucleotides and the inability to incorporate radioactivity at more than one site, limits the usefulness of these probes for optimizing assay sensitivity. A genomic probe offers two advantages over an oligonucleotide probe: First, the genornic clone is longer, permitting hybridization under more stringent conditions. Second, the genomic DNA can be used to generate antisense RNA. The latter property results in a probe which has higher specific activity and which forms more stable hybrids with mRNA than oligonucleotide probes. A rat genomic clone was isolated that contained 5' UT and coding sequences unique to sodium channel gene 11. The strategy used to obtain the genomic clone was to screen a rat genomic library with mixed oligonucleotide primers. To screen the library, a 30-nucleotide oligonucleotide probe (GMI) was synthesized that was complementary to 30 bp in the 5' coding region of sodium channel I and 11 (Fig. 3). Because genomic libraries contain large inserts (approximately 10-15 kb), we assumed that hybridizing recombinant phage would contain 5' UT sequences, in addition to the coding sequences. Two positive recombinants, out of 10' phage screened, were identified with the probe. To confirm that the phage contained sodium channel gene sequences, the two positive phage were rescreened with asecond synthetic oligonucleotide primer(GM2), also within the coding region (Fig. 3), and were again found to be positive. The two phage were then plaque-purified. Phage DNA was isolated and analyzed by Southern blotting of restriction endonuclease fragments to identify the sodium channel gene sequences. Restriction fragments which hybridized to the oligonucleotide probes (Fig. 4) were individually subcloned into a plasmid expression vector (Gemini, Promega Biotech) for sequence analysis and generation of antisense RNA. Nucleotide sequence analysis revealed that one clone contained sodium channel type I gene sequences, and the other clone contained sodium channel type I1 sequences. Both

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I Coding IL2

*1 2

GM 1 GM2

1

C A C G A (C/A) C A T G G (A/T) G G T C C T G G A C T G T C G A A G G A T T C (C/T) C T G G T (A/G) A A G A

t

*45

FIG.3 . Oligonucleotide probes used in isolating genomic clones encoding sodium channels I and 11. GMI is 30 nucleotides in length and is complementary to a region beginning 12 bp downstream from the translational start site. GM2 is 16 nucleotides in length and is complementary to a region 4 bp downstream ( # 4 3 from GMI. The proximity of the oligonucleotide sequences to the untranslated region is diagrammed above the sequences. I , GM I; 2, GM2. Genomic clones isolated with these probes contained the coding, untranslated, and intervening sequences.

subclones contained portions of intervening sequences (IVS), as well as 5' UT and coding segments of the gene. One hundred and eighty eight base pairs of the sodium channel I1 gene fragment was subcloned into the plasmid expression vector Gemini. Sixty base pairs of the insert corresponds to IVS sequences, 54 bp corresponds to 5' UT sequence, and 74 bp represents coding sequence (Fig. 5 ) . The gene I1 recombinant was used to generate radiolabeled antisense RNA according to standard techniques (Promega Biotech). The antisense probe was then used in RNase protection assays with RNA isolated from different rat tissues. B. RNase Protection Analysis of Sodium Channel Type II mRNA in Brain and Muscle

If the 188-bp antisense probe for sodium channel I1 is used in an RNase assay with samples containing sodium channel type 11 mRNA, 128 bp of the probe should be protected from digestion (Fig. 5 ) . RNA purified from tissues that do not contain sodium channel I1 mRNA should be completely digested. The RNase protection assay was performed as follows: 0. I pmol of antisense probe, labeled with "P-UTP, was coprecipitated with test RNA and carrier tRNA (20 c1.g total). The RNAs were resuspended in a hybridization buffer consisting of 80% formamide, 40 mM piperazine-N,N'bis(2-ethanesulfonic acid (PIPES) buffer (pH 6.71, 0.4 M NaCI, and 1.0 mM ethylenediaminetetracetic acid (EDTA), heated briefly to 90°C to denature, and incubated overnight at 50°C. The resulting hybrid was then

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FIG.4. Southern blot analysis of bacteriophage containing sodium channel gene I and gene I1 sequences. The left panel is a photograph of an ethidium bromide-stained gel showing an EcoRI digest of two positive recombinant phages. The right panel is an autoradiograph of a Southern blot of the same gel. Arrows indicate the position of the EcoKI fragment5 that hybridize to an end-labeled oligonucleotide probe specific for sodium channel type I (lane C ) and type I1 (lane B). Lane A. molecular size standards; lane B, DNA from a recombinant phage containing gene 11 sequences; lane C, DNA from a recombinant phage containing gene 1 sequences; lane D. hybridizing DNA fragment from the digest shown in lane B; lane E, hybridizing DNA fragment from the digest shown in lane C.

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1-4

Probe (188 bp) 1 4 -

Protected Fragment (128 bp)

FIG.5. Schematic representation of the antisense probe for sodium channel gene 11. The 188-bp probe contains 60 bp of intervening sequence (IVS). 54 bp of 5' untranslated (UT) sequence. and 74 bp coding (CODING) sequence. In the presence of sodium channel type 11, mRNA, 128 bp, corresponding to UT and coding sequences, should be resistant to digestion with RNases A and TI.

treated with RNase A (6 pg/ml) and RNase TI (20 units/ml) for 30 min at 37°C. The reaction was terminated by phenol extraction followed by ethanol precipitation. The final pellet was resuspended in 5 ~1 of sequencing buffer containing 80% formamide, 1 mM EDTA, and 0.14% dyes, heated to 90°C for 3 min, and then loaded on a 6% denaturing gel. Figure 6 shows the result of an RNase protection experiment, using the antisense probe for gene I1 and RNA prepared from adult rat brain. A single band of 128 bp is detected in rat brain, indicating the presence of type I1 mRNA using the conditions described above. The assay can detect as little as 50 fg of sodium channel type I1 mRNA. Figure 7 shows an RNase protection analysis of sodium channel type I1 RNA in skeletal muscle, cardiac muscle, and brain of different stages of development. Sodium channel type I1 mRNA is expressed at detectable levels only in the rat brain samples. IV.

DISCUSSION

The voltage-gated sodium channel is responsible for the generation of the action potential in nerve and muscle cells. Previous comparative studies of the functional properties of sodium channels have indicated that sodium channels in these two tissue types are very similar. Recent studies, however, have concluded that sodium channels in nerve and muscle differ in significant ways. For example, investigators have shown that the sodium channels of skeletal muscle differ from the sodium channels in nerve and cardiac muscle in their affinities for the peptide sodium channel blocker, p-conotoxin (Cruz et a / . , 1985; Moczydlowski et al., 1986). Additionally, Yoshikami ( 1986) has demonstrated that mRNA purified from brain and muscle produces sodium channels that are both functionally and pharmacologically distinguishable in Xenopus oocyte membrane. Results from molecular cloning experiments indicate that the voltage-gated sodium channel is encoded by a multigene family. Two distinct cDNAs encoding

FIG.6. RNase protection analysis of sodium channel gene I1 mRNA in rat brain. Autoradiograph of an RNase protection assay, using 0. I pmol of probe and 10 pg of adult rat brain RNA. The experiment was performed in duplicate. The two left lanes contain size standards. The intact sodium channel antisense probe i s 188 bp in length: the protected fragment is 128 bp in length. The autoradiograph was developed after an overnight exposure using an intensifying screen.

FIG.7. RNase protection analysis of sodium channel gene I1 mRNA in rat skeletal and cardiac muscle. Autoradiograph of an RNase protection assay. using 0. I pmol of probe and 20 pg of RNA. Lane C contains cardiac muscle RNA; lane G contains human gastrinoma RNA: lane I contains innervated rat muscle RNA; lane D contains denervated rat skeletal muscle RNA; lanes 0. 9 and A contain 10 pg of rat brain RNA isolated from newborn. 9day-old. and adult rats. respectively. A 128-bp fragment of the antisense probe is resistant to RNases A and TI digestion only in the presence of rat brain RNA. The autoradiograph was developed after an overnight exposure using an intensifying screen.

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the a-subunits in brain have already been sequenced (Goldin el al., 1986; Noda et al., 1986a). Because the cDNAs are so highly conserved, the available cDNA probes are not ideal for distinguishing among the different sodium channel genes. Therefore, we have synthesized oligonucleotide probes and isolated genomic clones containing sequences in the 5' UT region that are unique to sodium channels types 1 and 11. The results of our Northern blot analyses and RNase protection assays indicate that type 11 mRNA is much more abundant in brain than type I mRNA. Sodium channel I1 is detectable in brain, but not in skeletal muscle (either innervated or denervated) or in cardiac muscle. We have rescreened the rat genomic library with a probe specific for sodium channel type I, and are currently characterizing clones that are, by sequence analysis, distinct from gene I and gene 11. The clones will be analyzed by RNase protection assay to determine where they are expressed. By pursuing this strategy, we hope to be able to identify all the sodium channel genes expressed in rat excitable tissue. These probes will be useful for elucidating the events underlying regulation of expression of sodium channel excitability. REFERENCES Barchi, R. L. (1983). Protein components of the purified sodium channel from rat skeletal muscle sarcolemma. J . Neurochem. 40, 1377-1385. Chirgwin, J. M.. Przybyla, A. E.. MacDonald, R. J., and Rutter. W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. Couraud, F.. Martin-Moutot. N.. Koulakoff. A.. and Benvald-Netter. Y. (1986). Neurotoxinsensitive sodium channels in neurons developing in vivo and in viiro. J. Neurosci. 6. 192- 198. Cruz, L. J., Gray, W. R.. Olivera, B. M., Zeikus. R. D.. Kerr. L.,Yoshikami. D., and Moczydlowski, E. ( 1985). Conus geogruphus toxins that discriminate between neuonal and muscle sodium channels. J. Biol. Chem. 260, 9280-9288. Goldin, A. L., Snutch, T., Lubbert, H.. Dowsett, A., Marshall, J., Auld, V.. Downey, W.. Fritz. L. C., Lester, H. A.. Dunn, R., Catterall. W. A., and Davidson, N. (1986). Messenger RNA coding for only the alpha subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus oocytes. Proc. Nut/. Acud. Sci. U.S.A. 83, 7503-7507. Hartshome. R. P., and Catterall, W. A. (1984). The sodium channel from rat brain: Purification and subunit composition. 1. B i d . Chem. 259, 1667-1675. Lombet. A., Kazazoglou. T., Delpont, E., Renaua. J. F.. and Lazdunski, M. (1983). Ontogenic appearance of Na' channels characterized as high affinity binding sites for tetrodotoxin during development of the rat nervous and skeletal muscle systems. Biochcm. Biophys. Res. Commun. 110, 894-901. Moczydlowski. E.. Olivera. B. M.. Gray, W. R., and Strichartz, G. R. (1%). Discrimination of muscle and neuronal Na-channel subtypes by binding competition between ['Hlsaxitoxin and )I conotoxins. Proc. Nut/. Acud. Sci. U.S.A. 83, 5321-5325.

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Noda. M.. Ikeda. T.. Kayano. T.. Suzuki, H.,Takeshima. H.. Kurasaki, M..Takahashi, H.. and Numa. S. (1986a). Existence of distinct sodium channel messenger RNAs in rat brain. Nutitre (London) 320. 188-192. Noda. M..Ikeda. T.. Suzuki. H.. Takeshima. H.,Takahashi. T..Kuno. M..and Numa, S. (Iy86b). Expression of functional sodium channels from cloned cDNA. N(triirc. fLondonJ 322. 82-28. Yoshikami, D. (1986). Sodium channels expressed by Xenoprrs oocytes i i e c t e d with mRNA from brain differ from those expressed by oocytes injected with mRNA from muscle. Soc. Neirrosci. Ahsrr. 12, 42.

Zinn. K.. DiMaio. D.. and Maniatis. T. (1983). Identification of two distinct regulatory regions adjacent to the human f3-interferon gene. Cell 34, 86S-879.

CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLLIME 33

Chapter 15 A Model Relating the Structure of the Sodium Channel to Its Function H . ROBERT GUY Laboratory of Mathematical Biology National Cancer Institute National Institutes of Health Bethesda. Maryland 20205

I. 11. 111.

IV. V.

Introduction A. Voltage-Clamp Measurements B. Membrane Protein Structure Model of Sodium Channel Transmembrane Segments Tertiary Structure A. General Concepts B. Positions of a-Helices C. Selectivity Filter D. Activation Gating E. Inactivation F. Pharmacology Experimental Tests Conclusions References

1.

INTRODUCTION

A. Voltage-Ciamp Measurements A number of methods have been developed to measure kinetics of voltage-dependent channel opening and closing and ion-conducting properties of open channels. Most of these methods involve “clamping” the membrane voltage at a given potential and measuring either the ionic current that flows through many channels, the ionic current that flows through only one channel, o r the “gating” current that is due to movement of charged groups of the voltage-sensing mechanism responsible for channel 289

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activation (reviewed in Hille, 1984; Armstrong, 1981; Aldrich, 1986; Bezanilla, 1985; French and Horn, 1983). A major goal of voltage-clamp studies is to understand how channels function; i.e., how they open and close, how ions move through the channel, why some ions are more permeant than others, and how various drugs and toxins alter these processes. Although data gained through voltage-clamping experiments are essential, they are not sufficient to understand precise molecular mechanisms by which channels function. To do that, one must know something about the protein structure of the channel and how that structure depends on the membrane voltage. B. Membrane Protein Structure

The classic way to determine the structure of a protein is to crystallize it and determine its structure by X-ray diffraction. This method is time consuming and is difficult to use on large integral membrane proteins. The photosynthetic reaction center is the only large integral membrane protein for which the structure has been determined by X-ray diffraction (Deisenhofer et al., 1985). Structures of smaller peptides such as alamethicin (Fox and Richards, 1982) and gramicidin (see Chapter 3, this volume) that form ionic membrane channels have been determined by X-ray diffraction; however, conformations of these peptides and/or aggregates formed by these peptides in the crystals are not the same as those of the ionic channels in the membrane. If the sodium channel protein structure can be determined by X-ray diffraction and if that structure corresponds to a functional ionic channel conformation, a great deal of theoretical and experimental analysis will still be required to understand completely the molecular processes underlying channel conductance, gating, and pharmacology. Another approach is to develop models of the channel protein structure based on its amino acid sequence, and then to experimentally test aspects of the models by a variety of methods. This approach is complementary to X-ray diffraction because information gained in the studies may help determine whether a crystal structure is the same as the membrane structure and, if so, may aid in understanding how the structure functions. It should be considered as an alternative approach only if the sodium channel structure cannot be determined by X-ray diffraction. The model-building approach is made difficult by our inability to predict accurately known structures of large soluble proteins and by our inability to test methods for predicting membrane channel protein structures on known structures. There are many reasons why efforts to predict threedimensional structures of large soluble proteins from their amino acid sequences have generally failed. One of the main reasons is that in most

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soluble proteins most amino acid residues do not have a regular repeating secondary structure of a-helices and @-sheets.These less regular structures, which are sometimes called random coils, are much more difficult to model than a-helices or @-sheets.Even when a-helices occur, they may pack next to each other in an apparently irregular manner that is difficult to predict. The structure of the photosynthetic reaction center suggests that the transmembrane portion of membrane proteins may be much easier to model. It is composed of four subunits; a cytochrome subunit on the extracellular surface that does not interact with the membrane lipid, an H subunit with one transmembrane a-helix and a large cytoplasmic region, and two homologous subunits, L and M, that each contain five transmembrane a-helices (see Fig. I). The extracellular and intracellular portions of the protein appear very complex with large “random coil” segments connecting a few a-helices and @-strands.In contrast, all segments in the transmembrane region are a-helices that each contain a segment of 19 or more consecutive noncharged residues. These helices can be located within the sequence by hydrophobicity plots that scan the sequence looking for segments of about 20 residues that have primarily hydrophobic side chains (Rose and Roy, 1980; Von Heijne, 1981; Engleman and Steitz, 1981; Kyte and Doolittle, 1982; Guy, 1984). All helices within a subunit are approximately parallel or antiparallel to each other and the hydrophobic portions of each helix appears to begin and end on about the same plane. Each a-helix appears to be tilted relative to its neighbor by a small positive angle predicted by “3-4 ridges into grooves” packing (see Section 111,D). This packing is more regular than in most soluble proteins and is easier to predict. The two homologous subunits are arranged with a pseudotwofold symmetry and have almost identical backbone structures in spite of the fact that only 26% of the residues are identical. The assumptions that homologous subunits or domains have similar backbone structures and are arranged in a symmetrical manner are very useful in developing models. The assertion that membrane proteins are easier to model than soluble proteins does not mean they are easy. The first step in developing a structural model of a membrane protein is to postulate which segments cross the membrane and what their secondary structures are. Although hydrophobicity plots may be useful for identifying segments that form transmembrane a-helices with little or no contact with water, it is clear that they will not identify all transmembrane segments in every membrane protein. Membrane channel proteins such as a-toxin (Tobkes et al., 1985) from Staphylococcus and porins from the outer membranes of gram-negative bacteria (Kleffel et al., 1985) and mitochondria (Forte and Guy, 1986)

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FIG. I . Structure of the photosynthetic reaction center. Large cylinders represent ahelices, ribbons with arrows are p-strands, and small tubes are random coils. Transmembrane a-helices are shaded. Structure determined by Deisenhofer cr nl. (1985). Drawing by Jane S. Richardson.

contain no long hydrophobic segments but d o contain many segments in which every other side chain is hydrophobic. These proteins appear to have much more p-sheet structure than a-helix. The pore may be formed through large p-barrels that are lined with primarily hydrophilic side chains and that have an hydrophobic exterior in contact with the lipid (Paul and Rosenbusch, 1985; Forte and Guy, 1986).

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Water-filled pores of sodium channels and nicotinic acetylcholine receptors (AChRs) are much smaller than those of a-toxin and the porins. Sequences of these proteins contain several hydrophobic segments that could span a membrane as a-helices (Claudio et d., 1983; Noda el d., 1983, 1984: Devillers-Thiery e r d.,1983). It is not yet clear whether only these segments cross the membrane or whether they form an outer layer that surrounds more hydrophilic segments that line the pore (Finer-Moore and Stroud. 1984; Guy, 1984). The strong voltage dependency of sodium channel activation suggests movement of substantial charge in the transmembrane region. Thus, if one develops models from only the hydrophobic segments, the most interesting portions that control the channel gating and ion selectivity may be excluded. If segments that form the channel lining and/or the voltage-sensing mechanism contain a substantial number of charged side chains, then these segments may be difficult to distinguish from similarly charged segments that comprise intracellular and extracellular portions of the protein. One hypothesis is that channel linings can be formed by amphipathic a-helices that have hydrophobic side chains on one face and hydrophilic side chains on the other face (Dunker and Zaleske, 1977; Guy, 1983, 1984; FinerMoore and Stroud, 1984) or by amphipathic @strands that have alternating hydrophobic and hydrophilic side chains. This criterion is, however, neither necessary nor sufficient for identifying possible channel lining segments. Many a-helices and @-strandsin soluble proteins are amphipathic, and thus identification of an amphipathic pattern does not indicate that it is part of the channel lining. The channel lining of the sodium channel model presented here is formed primarily by long positively charged and short negatively charged a-helices; however, these segments would not be classified as good candidates to be channel lining amphipathic a-helices by most methods, since the positive charges spiral around the long helices and the negatively charged a-helices can span only about half of the membrane. Not only are some transmembrane segments difficult to identify, but the criteria for deciding their secondary structure are weak. Methods to predict secondary structure based on statistical analyses of soluble proteins (Chou and Fasman, 1978; Gamier et a/., 1978) have less than 60% accuracy (Kabsch and Sander, 1983) for soluble proteins and appear to have no predictive value for deciding (Y and @ content of membrane proteins (Wallace er al., 1986). If these methods are used, they should be applied only to those segments that interact only with water and other protein segments and do not interact with the lipid. Examining the periodicity of hydrophobic and hydrophilic residues is helpful in identifying potential amphipathic ahelices and @-strands;however, many transmembrane strands are not am-

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phipathic and sequences that could from amphipathic a-helices or @strands do not always form those structures in soluble proteins. Long hydrophobic segments appear to favor a-helical conformations in membranes; however, so few membrane protein structures are known that it is difficult to exclude the possibility that a long hydrophobic segment crosses the membrane twice as two P-strands instead of once as an a-helix. It thus appears difficult to predict from a single sequence alone which segments cross the membrane. Comparison of homologous domains or subunits within a single protein and of homologous sequences from different organisms or tissues is useful in identifying possible transmembrane segments and their secondary structure. Segments responsible for crucial functions such as gating and ion selectivity should be well conserved among all sodium channels. Also, a clue to the secondary structure may be obtained by examining the periodicities of conserved and nonconserved residues in addition to those of hydrophobic and hydrophilic residues (see discussion of S l , S2, and S3 helices in Section 111,B). II. MODEL OF SODIUM CHANNEL TRANSMEMBRANE SEGMENTS

Amino acid sequences for the major or a-subunit of the sodium channel have been inferred from cDNA sequences from the electric organ of the electric eel (Noda et al., 1984) and rat brain (Noda et al., 1986a). These proteins are very large, ranging in size from 1820 residues for eel to 2009 for the larger rat brain sequence. Two additional subunits have been identified in some tissues (Catterall, 1984); however, they have never been found in eel and are not required for expression of functional sodium channels in Xenopus oocytes (Noda et al., 1986b). Several models (Noda et al., 1984, 1986a; Kosower, 1985; Greenblatt ef al., 1985; Guy and Seetharamulu, 1986) have been developed for the transmembrane topology of the sodium channel. A protein of this size would be difficult to model were it not for the fact that the sequence contains four homologous domains and all of the long hydrophobic segments are located within these domains. All these models except for that of Kosower assume that all of the transmembrane segments are located within these domains, that each domain has essentially the same folding pattern, and that the amino- and carboxy-termini and segments linking the homologous domains are on the cytoplasmic side of the membrane. When they published the sequence of the eel sodium channel Noda et al. (1984) noted that each homologous domain has two very hydrophobic segments (S6 and S8 in Fig. 2A) that are long enough to span the membrane as ahelices, three relatively hydrophobic segments (Sl, S2, and S3) that could

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also span a membrane as a-helices but that have a few charged side chains on one face of the helices, and one segment (S4) that has positively charged side chains (mostly arginines) at every third position and relatively hydrophobic side chains at the other positions. They postulated that most or all of the hydrophobic segments were transmembrane a-helices and that S4 segments form the voltage-sensing mechanism that opens the channels. They suggested that S4s could be either on the cytoplasmic surface (Noda et at., 1984) or transmembrane (Noda et al., 1986a). If S4s are transmembrane as they probably should to respond to changes in the membrane voltage, then the transmembrane region should contain enough negative charges to neutralize the positive charges of the S4s and to make the channel selective to cations. Kosower postulated that 54 segments were 3," helices that have three residues per turn and that they are neutralized by very negatively charges segments between the B and C domains that also formed transmembrane 3,,-helices. Guy and Seetharamulu ( 1986) and Greenblatt et al. (1985) reasoned that S4 segments were more likely to be transmembrane a-helices (see Section 1II.D) and that they could be neutralized by negative charges on S7 segments that may form short amphipathic a-helices in the first three domains and a triple-stranded p-sheet in the last domain and by negative charges on S1, S2, and S3. S7 segments are preceded in the sequence by S6 segments that may form short hydrophobic a-helices of about the same length as the S7 a-helices. The scheme in Fig. 2A represents these latter models and differs from the model of Noda el al. (1986a) only by having S6 and S7 as transmembrane segments instead of on the extracellular surface. The S6 and S7 segments are very well conserved between eel and rat whereas the negatively charged segments, proposed by Kosower to cross the membrane, contain deletions and several substitutions. 111. A.

TERTIARY STRUCTURE

General Concepts

The problem of predicting which segments cross the membrane is relatively simple compared to the problem of developing a three-dimensional model from those segments. At first glance, models of the tertiary structure may appear to have little value because they are so speculative; however, this is deceptive. It is true that our ability to predict three-dimensional structures of proteins is still crude and it is unlikely that all features of a three-dimensional model are correct; however, there are several reasons for developing them. (1) The goals of developing these models are to better

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understand how the protein functions and to develop experimental tests of the models. It is very difficult to understand how a membrane protein functions from its transmembrane topology alone: you need to have some idea of its three-dimensional structure. (2) Some experiments can be designed to test a model of the membrane topology; however, many more experiments can be designed to test a more precise three-dimensional model. This is especially true of mutagenesis experiments in which one tries to test a model by altering the protein sequence. (3) All models of transmembrane topology should be considered as speculative and one can often envision several topologies. One way to constrain the transmembrane topologies is to favor those for which a three-dimensional model can be constructed that is consistent with known principles of protein structure and that can explain how the protein functions. B. Positions of a-Helices

Figure 2B shows a computer graphics representation of the positions of the transmembrane strands postulated here. The tertiary structure is based primarily on the following concepts:

(1) Very polar residues in transmembrane segments should be exposed to water inside the channel and most charged side chains should be able to form salt bridges. These criteria are satisfied by postulating that negatively charged faces of S7 segments form the narrowest portion of the FIG. 2. ( A ) Model o f the transmembrane folding scheme. The structure has four homologous domains that each contain eight (SI-S8) transmembrane a-helices, except in the D domain in which the S7 segment i s a triple-stranded p-sheet. Putative extracellular and cytoplasmic domains are white. Domains are labeled A-D i n the cytoplasmic regions.Negatively charged side chains conserved between eel and rat are red, positively charged ones are blue. (B) Stereo computer graphics of transmembrane a-helices and pstrands. Only positions o f the a carbons are shown. Segments are colored as in (A). View i s from the inside. ( C ) Stereo drawing of the channel lining segments. S4s are yellow. S7s white, positively charged groups blue, negatively charged carboxyls red. van der Waals surfaces have been added to carboxyl groups and tyrosine hydroxyl groups of the channel lining. View i s from the outside. All atoms except hydrogens are shown. Dimensions of the channel opening are approximately 3 x 5 A. (D) Schematic representation of “helical screw” gating mechanism. Ridges on S4 helix (yellow) are formed by every third side chain; those on S7 helices (white) are formed by every fourth side chain. Blue spheres are positively charged side chains of S4s; red spheres are negatively charged side chains belonging to segments indicated by the numbers in the spheres (see text for details). View is from inside the channel. (E) Stereo drawing of S4 segment positioned between two S7 segments. Colors are the same as in (D). The central three arginine side chains have been given three conformations; the downward navy blue and upward green conformations differs from the central light blue-green conformation by 120” rotations about the p-y and y-6 side chain bonds. View i s from inside the channel.

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channel lining, that positively charged side chains of S4 segments extend below, between, or above S7 segments, and that charged residues on SI, S2, and S3 are exposed to water and form salt bridges with positively charged side chains near the ends of S4 segments. This arrangement simultaneously satisfies the requirements that the narrowest portion of the channel be electronegative, that positive charges on S4 segments must be able to move when the channel opens and closes, and that the open channel have a “funnel-like’’ shape with a large cytoplasmic entrance and a smaller, negatively charged region near the extracellular surface (see Sections III,C, D, and F). (2) When given an a-helical conformation, SI, S2, and S3 have faces that are well conserved among all four domains. These faces are probably conserved to maintain a specific packing arrangement. If so, it is unreasonable that a highly conserved a-helical face will pack next to one that is not well conserved. I n the model, the highly conserved faces of these helices pack next to each other and interact with the putative gating helix S4. (3) SI, S2, and S3 are the only putative a-helices that have hydrophobic faces that are not well conserved between eel and rat. These faces are on the opposite sides of the helices from the faces that are well conserved among the four domains. The poorly conserved faces should be exposed either to other poorly conserved faces or, more likely, to lipid. The last assumption appears valid for the photosynthetic reaction center (personal observation). The bundles of four a-helices were positioned so that poorly conserved faces of SI, S2, and S3 are on the exterior of the structure where they can be in contact with lipid. SI, S2, and/or S3 were not considered good candidates for forming the negatively charged selectivity fiIter because, with the number of transmembrane segments proposed in most models, the innermost a-helices cannot be exposed to lipid and because by our calculations the central regions of these helices are too hydrophobic to form the lining of a water-filled channel. All faces of S5 and S8 segments are well conserved between eel and rat. In the present model, S5 and S8 are buried except for some nonconserved residues near both N-termini. (4) Transmembrane segments that are adjacent to each other in the sequence and have short connecting segments (SI-S2, S2-S3, S3-S4. S6S7) were packed next to each other. Antiparallel helix interactions were favored because of dipole interactions between helices (Hol ut al., 1981; Wada and Nakamura, 1981). ( 5 ) Most adjacent a-helices cross each other at an angle of about 20” as predicted by “3-4 ridges into grooves” (Chothia et af., 1981) helix packing (see Section 111,D). (6) Helices were arranged so that the four domains could form a channel

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with similar backbone structures of homologous regions, with minimal surface exposed to lipid, and with apolar portions of outermost a-helices beginning and ending near membrane surfaces. These constraints are sufficient to develop the general model shown in Fig. 2B without specifying to which domain each helix belongs and the exact position and orientation of each helix. A tentative assignment of these more precise interactions has been made based on the following considerations. (1) Preference was given to structures with more disulfide bridges, salt bridges, hydrogen bonds, and apolar interactions. (2) Structures that allow conserved residues of one helix to pack next to complimentary conserved residues of another helix and nonconserved residues to pack next to complimentary nonconserved residues were favored. (3) A transmembrane scheme consistent with the model of the cytoplasmic portion shown in Fig. 3 was favored. (4) A model in which the putative helix connecting S4 and S5 is placed over the short S6 and S7 segments was selected. C. Selectivity Filter

Models that have been proposed differ most by which segments are postulated to form the negatively charged portion of the channel lining that selects for sodium over other ions. We postulate that S7 segments form the selectivity filter, Greenblatt et af. (1985) chose S3 segments, Noda et af. (1986a) suggested S2, and Kosower (1985) postulated segments between the B and C domains. Several of the reasons we favor S7 segments and do not favor S 1 , S2, or S3 segments were discussed above. A selectivity fdter formed by S7 segments allows (1) the channel to have a “funnel shape” with a large entrance on the cytoplasmic surface, (2) arginine side chains in the central region of S4s to be neutralized and partially exposed to water, (3) charged side chains near the ends of S l , S2, and S3 to be exposed to water and form salt bridges with positively charged side chains near the ends of S4 segments, (4) positively charged side chains of S4 segments to move during gating (see Section IIl,D), and (5) construction of a selectivity filter with dimensions consistent with permeability studies and in which all side chains are conserved between eel and rat. Based on his studies of the permeability of sodium channels to various organic cation, Hille (1971) postulated that the narrowest portion of the channel that selects for sodium is lined with oxygens and has an rectangular opening of about 3.1 by 5.1 A.S7 and S4 helices were packed in a way consistent with “ridges into grooves” packing and that allowed the postulated activation gating mechanism to function (see Section 111,D). A computer

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graphics representation of this arrangement is shown in Fig. 2C. The narrowest portion of the channel in this model is lined with carboxyl groups and one tyrosine hydroxyl group and has dimensions near those proposed by Hille. These dimensions are affected greatly by the postulate that S7D is a triple-stranded @-sheetinstead of an a-helix as in the other domains. All of the negatively charged S7 side chains and positively charged S4 side chains postulated to interact with S7 side chains are identical in eel and rat sequences. One would expect the residues that control the channel selectivity to be highly conserved.

D. Activation Gating

All groups that have modeled the sodium channel from its sequence postulate that movement of positive charges on the S4 segments in the transmembrane region leads to activation of the channel. Before the sodium channel sequence was determined, Armstrong (1981) postulated that the voltage-sensing mechanism of the activation gate could be a string of negative charges associated with a string of positive charges and that one of these strings moves relative to the other when the channel opens. If the string of charges that moves is the positive charges on S4 then how does this movement occur and why are positive charges located on every third residue? One explanation for the periodicity is that S4 segments are 3,,, helices that have three residues per turn (Noda et ul., 1984; Kosower, 1985). The problem with this model is that long 3," helices have never been observed in proteins. When given a choice between postulating a structure that has never been observed (long 3,, helix) and one that is frequently observed (ahelix), the frequently observed structure should be favored if it can be used to develop a reasonable model. A clue to why every third residue is charged may be given by the "ridges into grooves" a-helix packing theory (Chothia ei ul., 1981). Four ridges that spiral around an a-helix in a right-handed manner are formed by side chains that are four residues apart, and three ridges that spiral around an a helix in a left-handed manner are formed by side chains that are three residues apart. In soluble proteins most adjacent helices pack next to each other so that their ridges pack between each other. Ridges formed by every third residue on one helix can fit between those formed by every fourth residue on an adjacent ahelix if these helices cross each other at an angle of about 20" (see Fig. 2D). If one assumes that charge movement is due to movement of the S4 helices and not due to conformational changes of the arginine side chains, then the only way this movement can occur and maintain the "ridges into grooves" packing is for the helix to rotate about its axis as it moves along

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its axis. A translation of 4.5 A and a rotation of 60”results in the movement of each positive charge on the S4 helix to the position occupied by the side chain that preceded it by three residues before the movement (see Fig. 2D). This movement for one S4 helix results in the equivalent movement of one charge and three residues across the membrane. The voltage dependency of activation indicates that about four charges move across the membrane when the sodium channel opens (Bezanilla, 1985). The charge movement could be due to one of these movements for each of the four S4 helices or several of these movements by one S4 helix. This mechanism is called the “helical screw” gating mechanism because the motion is “screwlike,” and ridges formed by every third residue on S4 can be thought of as the threads on a screw, and the ridges on the surrounding a-helices can be considered the matrix through which the screw moves. Arginine side chains are very bulky and are the most hydrophilic side chains in proteins. It would be energetically difficult for arginine side chains to move from one side of an adjacent a-helix to the other side because of their size. It would also be energetically unfavorable to bury these side chains between a-helices where they have no contact with water. These difficulties can be overcome if the S7 segments are short enough to allow the arginine side chains near the ends of S4 a-helices to extend above and below the S7 segments and to move under and over these segments during the gating process. Note that the conformation of the protein in the region of the selectivity filter may be essentially the same in both conformations since one arginine side chain is replaced by another arginine side chain. The conformation of the segments that connect S4 to S3 on the outside and to S5 on the inside will be affected. Blockade of open sodium channels by a variety of drugs from the cytoplasm suggests that the actual activation gate is on the cytoplasmic surface (see Hille, 1984, for review). The gate could correspond to the segments at the C-termini of S4 helices since by this mechanism essentially three residues are removed from the inside of the membrane and placed on the outside. The assumption that the arginine side chains do not change their conformations when the membrane voltage changes may be invalid. Positive charges that sense the voltage are located on the ends of relatively flexible alkyl chains which may well undergo conformational changes. Figure 2E shows that almost as much charge movement can be caused by simple 120” rotations in the alkyl chains as by the helical screw mechanism of Fig. 2D. The hypothesis that local anesthetics can enter and block the channel from the cytoplasmic side and be trapped in the channel when it closes (Hille, 1984; Starmer et al., 1986) suggests, however, a more substantial conformationalchange than simple side chain rotations. This con-

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clusion is also supported by pressure and temperature studies (Conti, 1986). It seems plausible that activation involves both conformational changes of the arginine side chains and movement of at least some S4 helices. The movement of S4 helices, however, could be less than that required by the helical screw mechanism, and more consistent with smaller conformational changes between adjacent a-helices that have been observed in soluble proteins (Lesk and Chothia, 1984). If both side chain and helix movements are involved, activation kinetics may be too complex to express in simple kinetic schemes that have only a few conformational changes.

E. Inactivation When the membrane is depolarized, activation is followed by inactivation. Measurements of sodium currents (Bezanilla and Armstrong, 1978; Goldman and Schauf, 1972; Goldman and Kenyon, 1982),gating currents (Armstrong and Bezanilla, 1977),and single channel recordings (Aldrich, 1986; Aldrich et al., 1983; Horn and Vanderberg, 1986) all indicate that inactivation does not occur independent of activation. It appears that the probability of inactivation occurring is much greater when the channel is open but that the channel does not have to be in an open conformation for it to enter or leave the inactivated conformation. Coupling of inactivation to activation is supported by the observation that inactivation immobilizes about two-thirds of the activation gating charge. Inactivation can be eliminated by proteotytic cleavage and by various chemical rnodifying reagents on the cytoplasmic side of the membrane (see Hille, 1984, for review). Armstrong and Bezanilla ( 1977) postulated that a positively charged inactivation gate plugs the open channel from the inside. Noda et al. (1986a) noted that a positively charged segment following S8C and a negatively charged segment following SSD are well conserved between eel and rat and suggested that these may be involved in activation. Although most portions of the putative cytoplasmic segments are not well conserved between eel and rat, segments (Re I ) that precede S1 segments and segments (Post 8) that follow the S8 segments are relatively well conserved. These segments appear to be distantly related, but the homology among domains is less than within the putative transmembrane regions. Analyses of amphipathicity, homology periodicity, and secondary structure predictions (Garnier et a l . , 1978) suggest that these segments form a-helices. Examination of these helices indicates that several energetically favorable salt bridges can form between the putative Pre I and Post 8 a-helices if they stack next to each other in an antiparallel manner. One way these segments could form the cytoplasmic entrance to the channel is shown

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in Fig. 3. In this scheme, inactivation is caused by movement of the absolutely conserved Post 8C segment and an extended strand that links it to Pre ID. In the inactivated conformation, a series of salt bridges are formed between positive charges on Post 8C and negative charges on Post 8D and between positive charges of the extended segment following Post 8D and negative charges on Post 8A and Post 8B. There is a large segment

FIG.3. Schematic representation of cytoplasmic entrance to the channel illustrating possible activation and inactivation mechanisms. Segments that follow S8D and S8C are assigned a-helical conformations and positioned near the channel entrance. Segments that follow S4 are shown as loops. Circles represent positively and negatively charged side chains; if half of the circle is black. the side chain is not charged in eel (left) or rat (right). The channel is postulated to go from the resting to open conformation when S4s (not shown) of some or all four domains undergo a "helical screw" conformational change. This removes an occlusion caused by loop segments near the C-termini of S4s. An additional helical screw movement of S4D and possibly S4C then allows the Post 8C segment to move over the channel entrance when the channel inactivates. Note interactions between positive charges on Post 8C and negative charges on Post 8D in the inactivated conformation. The arrows represent a series of conformational changes.

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between S8B and Post 8B that contains three very negatively charged segments. These segments may neutralize the positive charges on Post 8C when the channel is open and help make the channel cation selective. Figure 4 illustrates how activation and inactivation may be coupled. In this scheme activation is caused by helical screw conformational changes of some or all S4 helices. This removes protein and positive charge from the cytoplasmic entrance of the channel and leaves it open. S4D is postulated to undergo at least one more helical screw conformational change that removes more positive charge and protein from the cytoplasmic entrance. This additional step is possible because the pattern of positive charges repeats more times for the S4D helix and the segment that connects it on the outside to the S3D helix is longer and better conserved than in the other domains. A transmembrane conformational change associated with inactivation is suggested by modulation of inactivation by a scorpion and sea anemone toxins (see Section 111,F).The additional movement of

FIG.4. Schematic representation of possible activation and inactivation mechanisms and binding sites of drugs and toxins. General mechanism for activation and inactivation i s the same as in Fig. 3. Plausible binding sites for putative channel blockers TTX. p-conotoxin (pCTX). local anesthetics (LA). pancuronium (pan). batrachotoxin (BTX). and a scorpion toxin (aScTX) are shown in black.

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S4D in turn allows the positively charged inactivation gate, Post 8C, to move into the channel entrance. When the channel is inactivated, S4D and possibly some of the other S4 segments cannot return to their closed conformations and thus their gating charges are immobilized. The channel could go between the resting and inactivated conformations without passing through the open conformation if some of the S4 helices, say S4A and S4B, can be in closed conformations when the channel is inactivated. F. Pharmacology

Sodium channels are affected by a variety of drugs and toxins (for reviews see Hille, 1984; Catterall, 1984). Tetrodotoxin (TTX) and saxitoxin (STX) inhibit the channel from the outside. These toxins bind to all conformations of the protein. The voltage dependency of binding is the same for TTX and STX even though STX has two charges and TTX has only one charge. This indicates that they do not transverse a substantial portion of the membrane before binding. One hypothesis is that they block the outer entrance of the channel; however, the inhibitory effect could be allosteric (Chapter 16, this volume). Figure 4 illustrates that a binding site at the outer entrance would be negatively charged and would not change conformations greatly during activation and inactivation. This region involves segments near the C-termini of S7 segments. The p-conotoxin from cone snails is a very positively charged peptide that competes with TTX and STX binding in some sodium channels (Moczydlowski et al., 1986). It is substantially larger than TTX and STX and would probably bind to additional segments not involved in TTX and STX binding. Segments connecting S7D to S8D and S7C to S8C are more negatively charged in eel, which is blocked by p-conotoxin, than in rat brain, which is not affected. Sodium channels can be blocked from the inside by a variety of positively charged local anesthetics. Analyses of the effects of these drugs suggest that they can enter and leave the channel only when it is open, but can be trapped in resting and inactivated channels (Hille, 1984; Starmer et al.. 1986). A variety of larger positively charged toxins such as pancuronium and strychnine also block sodium channels from the inside; however, once the channel is blocked it cannot go to a resting or inactivated conformation, and all gating charge is immobilized. Mimicry of inactivation by these drugs was part or Armstrong and Bezanilla’s (1977) rationale for postulating the positively charged inactivation plug. Figure 4 illustrates how, in this model, local anesthetics may bind near the N-termini of S7 segments and the larger drugs may interact with this region and also with the activation and inactivation gates.

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Batrachotoxin (BTX), veratridine, aconitine, and grayanotoxin inhibit inactivation and alter activation kinetics and channel selectivity. These toxins are can readily cross a lipid membrane. It is thus difficult to say where they bind. Sea anemone and a scorpion toxins (aScTX) inhibit inactivation from the outside and potentiate the binding of BTX (Catterall, 1984). The binding of aScTX decreases as the membrane is depolarized with a voltage dependency similar to that of activation. Inhibition of inactivation from the outside implies a transmembrane conformational change associated with inactivation. One possibility suggested by the model is that aScTX binds near the N-terminus of S4D to prevent the last helical screw step postulated to precede inactivation, and that BTX binds near the C-terminus of S4D in a way that prevents the same conformational change, that alters the kinetics S4 movements during activation, and that alters the conformation of some S7 segments. In this scheme, both aScTX and BTX should bind to the inactivated conformation with low affinity. The binding of one toxin would potentiate the binding of the other since binding of either would inhibit formation of the inactivated conformation. IV.

EXPERIMENTAL TESTS

Models of the transmembrane topology can be tested by determining (1) to which side of the membrane antibodies to specific segments bind, (2) which sites are glycosylated on the outside or phosphorylated on the inside, (3) which segments bind toxins of drugs known to bind from only one side of the membrane, o r (4) which segments are accessible to proteolytic o r chemical modifying reagents that are applied to only one side of the membrane. Antibody studies have indicated that the C-terminus and a segment in the putative B-C cytoplasmic region (Gordon et al.. 1987; Chapter 12, this volume) are on the cytoplasmic side of the membrane as predicted by the model. The three-dimensional structure, gating mechanisms, and ion permeation mechanisms are more difficult to test. Sitedirected mutagenesis combined with single channel recording methods now make it possible to observe how altering the sequence affects the conductance properties, gating kinetics, and pharmacology of a single channel protein (Chapter 13, this volume). Models are very important to this approach because they suggest which of a virtually infinite number of possible mutants should be made, which experiments should be done to test these mutants, and how results of these experiments should be interpreted. Less model-dependent approaches, such as determination of the binding sites of drugs and toxins and locations of disulfide bridges, are also very important in testing models.

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306 V. CONCLUSIONS

The model presented here is very appealing in that it explains how a structure consistent with known principles of protein structures can account for the selectivity, activation gating, and inactivation of a channel. Our enthusiasm should be tempered, however, by the realization that prediction of protein structures from their sequences is not a highly developed science. A great deal of effort is currently being made to improve theoretical predictions of protein structures. The situation is sure to improve as more membrane structures are determined experimentally, as theoretical methods and parameters are improved, and as computational power increases. The encouraging aspect is that even now the model suggests many experiments, especially with mutagenesis. It will be very interesting to see whether these models live up to their promise and whether other voltage-activated channels are homologous and can function with similar mechanisms. REFERENCES Aldrich, R. W. (1986). Voltage-dependent gating of sodium channels: towards an integrated approach. Trends Neurosci. 9, 82-85. Aldrich, R. W., Corey, D. P., and Stevens, C. F. (1983). A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature (London) 306, 436441.

Armstrong, C. M. (1981). Sodium channels and gating currents. Physiol. Rev. 61, 644-683. Armstrong, C. M., and Bezanilla, F. (1977). Inactivation of the sodium channel 11: Gating current experiments. J . Gen. Physiol. 70, 567-590. Bezanilla, F. (1985). Gating of sodium and potassium channels. J . Membr. Biol. 88,97-112. Bezanilla, F., and Armstrong, C. M. (1977). Inactivation of the sodium channel I: Sodium current experiments. J . Gen. Physiol. 70, 549-566. Catterall, W. A. (1984). The molecular basis of neuronal excitability. Science 223, 653-661. Chothia, C., Levitt, M., and Richardson, D. (1981). Helix to helix packing in proteins, J . Mol. Biol. 145, 215-250. Chou, P. Y., and Fasman, G. D. (1978). Prediction of the secondary structure of proteins from their amino acid sequence. Adw. Enzymol. Relur. Areas Mol. Biol. 47, 45-148. Claudio, T., Ballivet, M.,Patrick, J., and Heineman, S. (1983). Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor h subunit. Proc. Narl. Acad. Sci. U . S . A . 80, 1111-1115. Conti, F. ( 1986). The relationship between electrophysiological data and thermodynamics of ion channel conformations. I n “Ion Channels in Neural Membranes” ( J . M. Ritchie. R. D. Keynes, and L. Bolis, eds.), pp. 2 5 4 1 . Alan R. Liss, New York. Mike, K., Huber, R., and Michel, H. (1985). Structure of the Deisenhofer, J., Epp, 0.. protein subunits in the photosynthetic reaction centre of Rhodopseudomonas viridis at 3 A resolution. Nature (London) 318, 618-623. Devillers-Thiery. A., Giraudat, J., Bentaboulet, M., and Changeux. J.-P. (1983). Complete mRNA coding sequence of the acetylcholine binding o subunit of Torpedo marrnoraru

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acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain. Proc. Nutl. Acud. Sci. U . S . A . 80, 2067-2071. Dunker, K . , and Zaleske, D. J . (1977). Stereochemical considerations for constructing (I helical protein bundles with particular application to membrane proteins. Biochem. J. 163, 45-57. Engleman, D. M., and Steitz. T. A. (1981). The spontaneous insertion of proteins into and across membranes: the helical hairpin hypothesis. Cell (Cumbridge. Muss.) 23, 41 I422. Finer-Moore, J.J.. and Stroud, R. M. (1984). Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor. Proc. Nutl. Acud. Sci. U . S . A . 81, 155159. Forte, M., and Guy, H. R. (1986). Isolation of the cDNA coding for the voltage-dependent anion channel of the outer mitochondria1 membrane of yeast. Biophys. J. 49, 41 la. Fox, R. 0.. J r . , and Richards, F. M. (1982). A voltage-gated ion channel model inferred from the crystal structure of alamethicin at I S-A resolution. Nuture (London) 300,325330. French, R. J.. and Horn, R. (1983). Sodium channel gating: models, mimics, and modifiers. Annu. Rev. Biophys. Bioeng. 12, 319-356. Gamier, J., Osguthorpe, D. J . , and Robson, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J . M o / . Biol. 120, 97-120. Goldman, L.. and Kenyon, J . L. (1982). Delays in inactivation development and activation kinetics in Myxicolu giant axons. J. Gen. Physiol. 80, 83-102. Goldman, L., and Schauf, C. L. (1972). Inactivation of the sodium channel in M ~ X ~ C O ~ U giant axons: evidence for coupling to the activation process. J. Gen. Physiol. 59, 659675. Gordon, R. D.. Fields, W. E.. Schotland, D. L., and Barchi, R. L. (1987). lmmunochemical testing of current molecular models of the voltage-dependent sodium channel from E/ectrophorris rlec.trii.ris. Biophvs. J . 51, 437a. Greenblatt, R. E., Blatt, Y., and Montal, M. (1985). The structure of the voltage-sensitive sodium channel: Inferences derived from computer-aided analysis of the Electrophorus electricus channel primary structure. FEBS Left. 193, 125-134. Guy. H. R. (1983). A model of colicin El membrane channel protein structure. Biophys. J . 41, 363a. Guy. H. R. (1984). A structural model of the acetylcholine receptor channel based on partition energy and helix packing calculations. Biophys. J. 45, 249-261. Guy. H. R.. and Seetharamulu, P. (1986). Molecular model of the action potential sodium channel. Proc. Nutl. Acud. Sci. U . S . A . 83, 508-512. Hille. B. (1971). The permeability of the sodium channel to organic cations in myelinated nerve. J. Gen. Physiol. 58, 599-619. Hille, B. ( 1984). “Ionic Channels of Excitable Membranes.” Sinauer. Sunderland, Massachusett s. Hol, W. G. J . . Halie, L. M., and Sander, C. (1981). Dipoles of the a-helix and p-sheet: their role in protein folding. Nuture (London) 294, 532-536. Horn, R.. and Vandenberg, C. A. (1986). Inactivation of single sodium channels. In ”Ion Channels in Neural Membranes” (J. M. Ritchie. R . D. Keynes, and L. Bolis, eds.), pp. 71-84. Alan R. Liss. New York. Kabsch. W., and Sander, C. (1983). How good are predictions of protein secondary structure? FEBS Lett. 155, 179-182.

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Kleffel, B., Garavito, R. M., Baumeister, W., and Rosenbusch, J. P. (1985). Secondary structure of a channel-forming protein: porin from E. coli outer membranes. EMBO J . 4, 1589-1592. Kosower, E. M. (1985). A structural and dynamic molecular model for the sodium channel of Electrophorus electricus. FEES Lett. 182, 234-242. Kyte. J., and Doolittle. R. F. (1982).A simple method for displaying the hydropathic character of proteins. Biochim. Biophys. Actu 233, 420-433. Lesk. A. M . . and Chothia. C. (1984). Mechanisms of domain closure in proteins. J . Mol. B i d . 174, 175-191. Moczydlowski, E., O h e r a , B. M., Gray, W. R., and Strichartz, G. R. (1986). Discrimination of muscle and neuronal Na-channel subtypes by binding competition between ['Hlsaxitoxin and p-conotoxins. Proc. Natl. Acad. Sci. U.S.A. 83, 5321-5325. Noda, M., Takahashi, H., Tanabe. T., Toyosato, M., Kikyotani, S., Furutani, Y.,Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983). Structural homology of Torpedo californica acetylcholine receptor subunits. Nature (London) 302,528-532. Noda, M., Shimizu. S . , Tanabe, T., Takai. T., Kayano, T., Ikeda, T., Takahashi, H., Nakayama. H.. Kanaoka, y., Minamino, N., Kangawa, K., Matsuo, H., Raftery. M. A., Hirose, T.. Inayama, S., Hayashida, H., Miyata, T., and Numa, S. (1984). Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. Nature (London) 312, 121-127. Noda. M.. Ikeda. T., Kayano, T., Suzuki, H., Takeshima, H., Kurasaki, M., Takahashi, H., and Numa, S. (1986a). Existence of distinct sodium channel messenger RNAs in rat brain. Nature (London) 320, 188-192. Noda, M., Ikeda, T., Kayano, T., Suzuki, H., Takeshima, H., Takahashi, T., Kuno, M., and Numa, S. (1986b). Expression of junctional sodium channels from cloned cDNA. Nature (London) 322, 826-828. Paul, C., and Rosenbusch, J. P. (1985). Folding patterns of porin and bacteriorhodopsin. EMBO J . 4, 1593-1597. Rose, G . D., and Roy, S. (1980). Hydrophobic basis of packing in globular proteins. Proc. Natl. Acad. Sci. U.S.A. 77, 46434647. Starmer, C. F., Yeh. J. Z., and Tanguy, J. (1986). A quantitative description of QX222 blockade of sodium channels in squid axons. Biophys. J . 49, 913-920. Tobkes, N.. Wallace, B. A., and Bayley, H. (1985). Secondary structure and assembly mechanism of an oligomeric channel protein. Biochemistry 24, 1915-1920. Von Heijne, G. (1981). Membrane proteins: the amino acid composition of membrane-penetrating segments. Eur. J . Biochem. 120, 275-278. Wada, A., and Nakamura, H. (1981). Nature of the charge distribution in proteins. Nature (London) 293, 757-758. Wallace, B.. Cascio, M.. and Mielke, D. L. (1986). Evaluation of methods for the prediction of membrane protein secondary structures. Proc. Natl. Acad. Sci. U . S . A . 83, 94239427.

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

Chapter 16 Sodium Channels in Lipid Bilayers: Have We Learned Anything Yet? CHRISTOPHER MILLER A N D SARAH S . GARBER Graduate Department of Biochemistry Brandeis University Waltham, Massachusetts 02254 I . Introduction 11. The Method 111. Interaction of Guanidinium Toxins A. Classical Electrophysiology B. Model Membranes Enter the Picture IV. Fixed Surface Charge V . Two Not Totally Speculative Proposals A. Guanidinium Toxin Binding: Physical Occlusion or Conformational Change'! B. Where Is the Sialic Acid'? References

1.

INTRODUCTION

During the past decade, two methods have been developed for observing the behavior of ion channel proteins at the single-molecular level: patch recording, and reconstitution into model membranes. Both of these techniques exploit the unique property of ion channels: the extremely high rate of ion movement through the open channel; the experimenter can literally watch individual channel molecules opening, closing, and conducting ions. The results obtained by these methods provide high-resolution functional information at the molecular level: What does the channel do? This information nicely complements the structural work provided by the biochemists and molecular biologists: How) is the channel built? We must be able to answer both these types of questions before we can attack the real question: How does the channel work.? 309

Copyright

KJ

IYXX hy Academic Pre\.r. Inc

All right\ of reproduction in m y form re\erved

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CHRISTOPHER MILLER AND SARAH S. GARBER

The Na’ channel, the first child of modern electrophysiology, has been under study for about 40 years. From the earliest days of voltage clamping, through the development of noise analysis and gating current methods, to the recording of single channels on intact cells, an enormous number of “what-does-it-do” type of experiments have been performed on the Na’ channel. Presently the “how-is-it-built” experiments are just beginning, as we are treated to the purification, cloning, and sequencing of the channel protein. In this chapter, we discuss the use of model membranes to study functional questions about Na’ channels. Nearly all such studies have been carried out in planar lipid bilayer membranes, and this raises a serious question: Why should we do this work at all? Given that direct electrophysiological methods are of higher time resolution, and that so many aspects of Na’ channel behavior have been studied for so long, what can we gain by watching Na’ channels open and close in “artificial” membranes? As it turns out, there are several classes of questions about the molecular character of the Na’ channel which are best answered by reconstituting the channel into planar bilayers, and it is our purpose here to summarize what has been learned by this approach. We do not mean to review planar bilayer work on Na’ channels comprehensively, but rather to focus on two specific topics which have yielded quite naturally to the advantages of model membranes, while avoiding the disadvantages. These topics are (1) the interaction of guanidinium toxins with Na’ channels and (2) the influence of surface charge on Na’ channel behavior. As we will see, there are several apparently contradictory results here, and we will offer a hypothesis which attempts to resolve some of this controversy. It will not be long, we hope, before the sorts of conclusions which these functional studies allow us to draw about the molecular nature of the channel will be able to mesh with biochemical results on the structure of the Na’ channel. II. THE METHOD

In all the work discussed here, a single method has been used: the insertion of channels into planar lipid bilayer membranes, an approach which has been reviewed in detail (Miller, 1984). A planar lipid bilayer is formed on a hole (0.1-0.3 mm diameter) in a septum separating two aqueous chambers (Fig. 1). Na’ channels are then inserted into this model membrane by fusing membrane vesicles prepared from some excitable tissue of interest. Skeletal muscle transverse tubule membranes or brain plasma

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31 1

Muscle T-tubule

1 Planar lipid

FIG. I . Diagram of the planar lipid bilayer system. The figure depicts the essential aspects of the experimental system: two aqueous chambers separated by a phospholipid bilayer. and connected to a low-noise current-measuring circuit. For measurements on Na + channels from rat skeletal muscle transverse tubule membranes, the side to which vesicles are added is topologically equivalent to the external side of the channel.

membranes are particularly convenient sources of Na' channels for planar bilayer work. The membrane is voltage clamped, and transmembrane current is monitored at a high gain. Usually, conditions are rigged such that only a small number of channels are inserted into the bilayer, so that single channel fluctuations may be observed. The main disadvantage of the method is the inherently low time resolution involved. Because of the large membrane capacitance, signal-tonoise characteristics dictate that Na' channel data must be filtered heavily, and events occurring faster than 10 msec are lost. The advantage of the method is that the bilayers are stable over time and have very high "seal" resistance (in the order of teraohms), so that single channel data can be collected for many hours. Furthermore, the lipid composition of the bilayer can be controlled at will, and the ionic composition of the aqueous media can be easily vaned over an enormous range. These characteristics dictate that this method is best used for studying slow processes. 111. A.

INTERACTION OF GUANlDlNlUM TOXINS

Classical Electrophysiology

Narahashi and colleagues (1964) originally found a magic bullet for the Na' channel: a potent and specific inhibitor, tetrodotoxin (TTX), a small organic molecule which binds with nanomolar affinity to the externally facing side of the channel and shuts down its operation. A variety of toxins with similar actions have since been discovered, saxitoxin (STX) being

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CHRISTOPHER MILLER AND SARAH S.GARBER

among the most potent. These toxins share a common site. as is known from competition studies, and they all share a common chemical feature: a guanidinium group. The simplest idea to explain the inhibition of Na’ channels by these “guanidinium toxins” is that they bind to a site near the entryway of the channel and physically block the passage of Na’ ions (Kao and Nishiyama, 1965; Hille, 1971), as illustrated in Fig. 2. This idea is plausible since Na’ competes with the toxins, and since the guanidinium ion itself is a permeant cation. In fact it has been proposed that the guanidinium toxins bind at the “selectivity filter” of the channel, the site within the channel where Na’ is selected over other cations for permeation (Hille, 1975; Reed and Raftery, 1976). A further piece of evidence supporting this physical “plugging” mechanism is that the monovalent cations compete with toxin binding in a “Na-like” sequence, where Li’ is the most effective competitor and Cs’ the least (Henderson et al., 1974). It is as though the guanidinium toxin binds at a site normally used for liganding a permeating cation. But there does not exist any truly compelling evidence in favor of the plugging hypothesis. The results above are the only indications in favor of this idea, and taken together, they make quite a weak case. An alternative “allosteric” picture-one in which the guanidinium toxin binds at a place far away from the entryway of the channel and causes the channel to close (Fig. 2 G i s also possible, and some results make it quite plausible. For instance, treatment of the Na’ channel with the methylating agent trimethyloxonium (TMO) renders the channel insensitive to toxin, but has no effect on its selectivity among cations (Spalding, 1980; Sigworth and

A

FIG.2. Alternative models of guanidinium toxin inhibition of Na’ channels. ( A ) An open Na’ channel; (B) physical plugging model, in which the guanidinium toxin binds within the ion conduction pathway and physically blocks it; (C) allosteric model, in which binding of toxin leads to a conformational change which prevents ion conduction.

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Spalding, 1980). The problem we wish to address is how to distinguish these two models of guanidinium toxin action, the physical occlusion picture or the allosteric model. 6. Model Membranes Enter the Picture

In all of the electrophysiological studies above, there is a big problem: the guanidinium toxins were always interacting with closed channels. The toxins require seconds or minutes to bind, while the channel itself can stay open only for a millisecond or so. However, the first successful observations of Na' channels in model membranes (Krueger et al., 1983) led to the direct demonstration of toxin binding to open channels. The low time resolution of the planar bilayer system made it clear that a trick would have to be used to observe single Na' channels; batrachotoxin (BTX), a n alkaloid poison which holds Na' channels open o n a time scale of hours, was added, and Na' channels were detected, as shown in Fig. 3. The channels remained open virtually all the time, but addition of low amounts of TTX induced the appearance of long-lived nonconducting intervals; these were subsequently shown to represent the binding of single toxin molecules to the single channel in the bilayer (French et d.,1984; Moczydlowski et al., 1984a). In these first experiments, two results were obtained right away: first, the binding of TTX and STX to conducting channels was directly demonstrated, and second, this binding was found, very unexpectedly, to be voltage dependent, with binding favored at negative voltages, as is evident in Fig. 3. Such a voltage dependence might seem to argue for the plugging hypothesis for toxin action, since the binding of a charged "blocker" within the electric field of the conduction pathway should be voltage dependent (Woodhull, 1973). But Moczydlowski and colleagues (l984b) found that this explanation was incorrect; they found that a variety of guanidinium toxins with electrical charges varying from 2 + to 0 all gave the same voltage dependence of binding (Fig. 4). The voltage dependence of toxin binding, therefore, does not reside in the toxin molecule. but was proposed to reflect a voltage-driven conformational equilibrium between a form of the channel which allows toxin to bind and one which does not. Moczydlowski and colleagues (1984a) also investigated the question of competition of TTX binding by Na', exploiting the capability of studying toxin binding t o open channels. T h e planar bilayer system makes it a straightforward experiment to ask whether Na' competes with TTX binding from both sides of the membrane o r from only one side. The answer was clear-cut: toxin binding is competed only by Na' added on the same side as the toxin, the external side of the channel. Na' ions flowing through

314

CHRISTOPHER MILLER AND SARAH S. GARBER

--

v J'

*'

-6OmV

0

FIG.3. Single Na' channel activated by BTX. A single Na' channel was inserted into a planar bilayer membrane, in the presence of symmetrical 200 mM NaCI. BTX (0.2 p.M) was presenl to hold the channel open, and 20 nM TTX was also present to induce the longlived closures apparent in the figure. Each closure represents the binding of a single TTX molecule to the single channel. Records were taken at the indicated voltages. (From Moczydlowski e r a / . , 1984b.3

the channel have no effect on toxin binding. This result showed that the guanidinium toxin-binding site is certainly not located deeply within the channel. It is consistent with both the allosteric model and a physical occlusion model where the toxin occludes the channel by binding at a shallow, or peripheral, site, "more like a lid on a funnel than a cork in a bottle" (Moczydlowski el al., 1984b). So once again, the data do not distinguish allosteric and physical occlusion mechanisms. A recent result further pointing to a peripheral location for the guanidinium toxin binding site concerns the action of a remarkable toxin, pconotoxin (Moczydlowski et al., 1986). By a combination of planar bilayer measurements and direct binding studies, it was shown that p-conotoxin shares a common site and similar mode of action with the guanidinium toxins; but p-conotoxin is a 22-amino acid peptide (containing two arginine residues), and physically is much larger than the classical guanidinium

16. Na CHANNELS IN LIPID BILAYERS

31 5

1000,

pC

100

L

L“ 10

1

-so

0

50

V, mV FIG.4. Voltage dependence of guanidinium toxins with differing charges. Apparent dissociation constants. K,, (app), were measured for various guanidinium toxins as a function of applied voltage. The three toxins shown, STX. T T X , and C2. a derivative of S T X with two sulfate groups. have net charges of 2 + , I + and Lero. respectively. Data taken from a larger collection o f toxins (Moczydlowski (’I c d . . 1984a).

.

toxins. It is difficult (though not impossible) to imagine a molecule of this size getting very far into the channel to occlude it. Recently, experiments in planar bilayers arguing for an allosteric model for guanidinium toxin inhibition were presented (Green et al., 1987a,b). These experiments were based on the fact that Zn” acts as a rapid openchannel blocker of the BTX-activated Na+ channel, i.e., the Zn” ion appears to bind within the pore in a rapid equilibrium and prevent Na’ conduction. But it was found that Zn” binding does not affect toxin binding, and so it was concluded that the Zn”-binding site cannot be near to the guanidinium toxin site. The authors favored an allosteric model, but it is clear that a “peripheral plugging” model would serve just as well. The arguments continue. Recently, Worley and colleagues ( 1986) examined the modification by TMO of Na channels in planar bilayers. A single channel was activated in a bilayer by adding BTX, and then TMO was added. Within a few minutes, a “single hit” was observed in which the channel suddenly changed its behavior. The clear result was that several characteristics of the channel always changed together as a result of a TMO hit. The channel conductance dropped by about 35%, and concomitantly the channel became insensitive to STX. At the same time, the channel was rendered insensitive to block by Ca”. The authors emphasized that all three of these changes occur together as though a specific

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TABLE I GUANIDINIUM TOXININHIBITIONOF SODIUM CHANNELS: AN EXPERIMENTAL SCORECARD Physical Plugging Model

Allosteric Conformational Model

Conduction by guanidinium. competition by Na' (Hille. 1975) Na'-like selectivity of competition (Henderson e/ d . . 1974) TMO modifies TTX binding but not ionic selectivity (Sigworth and Spalding. 1980) Coexistence of TTX-sensitive and insensitive conformations of open channel (Moczydlowski e / d . , 19846) Single hit by TMO changes both TTX binding and ionic conduction (Worley ef d..1986) No interaction between TTX and Zn2' block (Green e / d . , 1987a.b)

carboxylate group somewhere in the conduction pathway is involved in all three processes: Na' conduction, open-channel Ca" block, and guanidinium toxin binding. These results are most easily handled by the plugging model, although the allosteric model can also contort itself to accommodate them. We are left, then, in an uncomfortably vague situation. N o compelling argument can be advanced for or against either picture of toxin action (Table I). Planar bilayer studies have contributed to our understanding of how these inhibitors work, but they have not decided the important issue. A preference for one of the two models relies as much on personal aesthetics and intuition as it does on the experimental results. No really crucial experiments have been performed, but, as we shall see, there are two apparently unrelated results which do suggest a resolution of the question. To see this, we must turn to the next issue attacked in planar bilayer studies, that of surface charge effects.

IV. FIXED SURFACE CHARGE Surfaces carrying net electrical charge, such as membranes containing acidic phospholipids, profoundly influence the ionic composition immediately adjacent to the surface (McLaughlin, 1977). Near a negatively

16. Na CHANNELS IN LIPID B U Y E R S

317

charged membrane, an excess of cations and a deficit of anions arise, simply because of electrostatic forces. This “electrical double layer” has been understood for many decades, and the simple Gouy-Chapman theory applies disconcertingly well to phospholipid membranes (McLaughlin, 1977). Quantititively, the local ion concentration right next to a charged surface, [XI,, is given in terms of the electrical potential at the surface, the “surface potential,” ‘P,, and the bulk concentration, [XI,,: [XI,

=

[XI,,exp(- z N , / R T )

(1)

where z is the charge of the ion. The surface potential is a function of the surface charge density and the ionic strength (McLaughlin, 1977). In model membranes, it is possible to ask directly to what extent the surface charge of the lipid membrane influences the behavior of the channel dwelling in it. For example, is the conductance of a cation-selective channel higher, or that of an anion-selective channel lower, in a negatively charged membrane than in an uncharged one? Do open-channel blockers, or charged ligands which activate the channel, feel the influence of the lipid surface charge? Experiments designed to answer these kinds of questions quantitatively by systematically varying the lipid composition of the model membrane have been performed with CI- channels (White and Miller, 198l), K’ channels (Bell and Miller, 1984), Ca’+-activated K’ channels (Moczydlowski et al., 1985), Caz+channels (Coronado and Molter, 1986), and, most recently, Na’ channels (Green et al., 1988b; Worley et ul., 1988). The conductance of the Na’ channel, which is a function of the Na’ concentration in the aqueous medium immediately adjacent to the channel, is not sensitive to the lipid surface charge; channel conductance is the same whether measured in neutral membranes or in membranes containing the negatively charged lipid phosphatidylserine (Worley et al., 1988; Green et ul., 1987a). The entryway to the channel does not appear to sense the electric field due to the lipid charges. This is not in the least surprising, since the channel is known to be a large protein which may extend far into the aqueous phase such that the entryway for permeating ions is many Debye lengths removed from the membrane. But what about surface charges carried by the protein itself? It is known, for example, that the Na’ channel protein carries an unusually large amount of negatively charged carbohydrate on its outer surface. If the channel’s entryway were located close to this forest of negative charge density, then we might expect that the local Na’ concentration near this region would be higher than in the bulk solution. This would tend to enhance the Na’ conductance of the channel over that expected if there were no fixed negative charge near the channel “mouth.” But how are we to decide whether the negative charges on the protein surface actually

318

CHRISTOPHER MILLER AND SARAH S. GARBER

do influence Na' conduction through the channel? What experiments can we do to attack the question? The crucial experiment is to embed a channel in an uncharged lipid membrane and to measure the variation of Na' conductance as a function of Na' concentration, where NaCl is the only salt in solution (not counting the small concentration of a buffer ion which should be present). In other words, by letting ionic strength vary along with Na', we can tell whether a surface charge effect is present. The reason for this is that when NaCl concentration is increased, two things happen: first, the bulk Na' concentration increases, and second, the local surface potential, due to the

A zoo0

PE 4L

3

1600

1200

u)

n

z

000

400

L

so0

tNaC13

790

1000

mM

FIG.5 . Effects of negative surface charge on channel-mediated conduction. Expectations of the Gouy-Chapman theory of the electrical double layer are shown (McLaughlin, 1977). An infinite plane membrane of negative charge density faces an aqueous solution of I : I univalent electrolyte, NaCl in this example. Calculations done as in Bell and Miller (1984). ( A ) The surface isotherm. Local concentration of Na' or Cl-[X],,,, at the membrane surface i s plotted versus the bulk NaCl concentration. The line of equal surface and bulk concentrations. expected with no surface charge present, i s displayed as a dashed line. (9)A Na'selective channel is assumed to be embedded in the membrane, and i s assumed to follow a rectangular hyperbola with 20 pS maximum conductance and apparent dissociation constant K,,. as indicated. Zero-voltage conductance i s plotted versus [Na] with no surface charge (solid curve). or for a negative surface charge, u = 0. I chargeshm' (dashed curves). (C) Calculations as in B. but with a fixed K,, (10 mM) and varying u as indicated.

16. Na CHANNELS IN LIPID BILAYERS

319

1s v)

.

n

x

10

s

20

1s

10

S

- 1

25

so

7s

100

320

CHRISTOPHER MILLER AND SARAH S.GARBER

negatively charged groups on the protein, drops. The local Na' concentration near the fixed negative charge is determined by these two compensating effects. Thus, at very high NaCl, the local surface potential is small, and the local Na' concentration is close to the bulk concentration; at low NaCl, however, the surface potential gets very negative, and so the local Na' concentration remains high, according to Eq ( I ) . Figure 5A plots the local concentration of Na' at the mouth of a channel embedded in an infinite sheet of fixed negative charge, as a function of bulk Na' concentration. For a channel operating by a simple ion conduction mechanism, the conductance is expected to vary as a rectangular hyperbola with the ion concentration at the mouth of the channel (Hille, 1975). But with a negative surface charge present, the cation concentration near the mouth varies as described above. The net result of all this is that the conductance of a channel carrying significant fixed charge should not follow a rectangular hyperbola, but rather should vary as in Fig. 5B and C. These two figures

Vf

3.8mM

FIG.6 . Single veratridine-activated Na' channels: effect of Na'. Single Na' channels activated by veratridine (35 FM) were examined at varying symmetrical concentrations of Na' as indicated (see description in Garber and Miller, 1987).

16. Na CHANNELS IN LIPID BILAYERS

321

demonstrate that, in the presence of negative surface charge, the conductance-concentration relationship should depart dramatically from a rectangular hyperbola, regardless of how vigorously one adjusts the two parameters, surface charge density and dissociation constant for Na' . Different geometries for the channel mouth and disposition of surface charge do not qualitatively affect this kind of behavior, since the universal essence of the idea of surface charge is that the local counterion is accumulated near to the area of fixed charge density. What actually happens for the Na' channel? Garber and Miller (1987) measured the conductance of Na' channels activated either by BTX or by a similar alkaloid, veratridine (VT), over a wide range of Na' concentrations. Remarkably, the channels are well behaved even in very extreme salt conditions (Fig. 6). The result was clear: no surface potential effect is present, as a rectangular hyperbolic relationship was followed down to about 3 mM Na' (Fig. 7).

20

--

+A

BTX

I

VT f

1

50

100

150

200

2so

FIG.7. Conductance-concentration curves for alkaloid-activated N a channels. Channel conductance as a function of N a ' concentration was measured for N a ' channels activated by BTX or VT. as indicated. Solid curves are rectangular hyperbolas, with K , , of 6.5 and 5.5 m M , and maximum conductance of 21 and 10 pS, for BTX and VT. respectively. +

322

CHRISTOPHER MILLER AND SARAH S. GARBER

As Figs. 5 and 7 show, if a surface charge effect were present, we would have detected it readily, since we were able to follow this curve down to very low ionic strength. With negative surface charge present, the conductance-concentration relation always falls far above a rectangular hyperbola; the reason for this is that, even at low Na concentration, the local concentration of Na, and hence the conductance, is still high, because of the high negative surface potential. We conclude that the entryway for Na ions in the Na’ channel (from rat muscle, at least) does not carry any detectable negative charge density. Any patch of negatively charged residues, for example, the sialic acid groups, must be located far away (more than a Debye length at 3 mM, or farther than 30 A) from the channel’s opening for Na’ . We should point out that there is controversy surrounding this last conclusion. Green and co-workers ( 1987a) performed similar experiments on Na’ channels from canine brain, and came to exactly the opposite conclusion: that the channel’s entryway senses a substantial surface charge. The channel’s properties were not examined at ionic strengths lower than 30 mM NaCI. and the conclusion was based on the fact that the channel conductance slowly increases, or “creeps,” over the range 300 mM-3 M NaCI. However, it seems to us that this conclusion could be invalid, since it is based mainly on conductance data taken in the high ionic strength range. As Fig. 5 illustrates, it is dangerous to use data at high salt to make conclusions about local surface potentials. At high salt, surface potential effects are necessarily small, and other explanations for a “creep” in Na’ conductance may be naturally found (such as multiple ion occupancy of the channel at high Na’ concentrations). It is in the low ionic strength range where surface potential effects, if present, are unmistakable. But it is precisely this range that Green ef ul. did not examine, and which we relied upon heavily (Garber and Miller, 1987). We emphasize that the experimental data of the two groups are not in disagreement, at least in the range of concentration employed by both groups, only the interpretation of the results. We consider that the results relying on the lower ionic strength data are more germane to the issue, and provide very strong evidence that the entryway of the Na’ channel from rat muscle does not carry net negative charge. Green and co-workers (1987b) went on to examine the possibility that the guanidinium toxin-binding site is influenced by local fixed charges. These experiments studied the competition of Na’ for the guanidinium toxin-binding site, as in planar bilayer experiments described above (Moczydlowski et al., 1984a). But in this case, attention was paid to the dijferences in competition of Na’ with TTX and STX. This experiment is pertinent because TTX is monovalent, while STX, with two guanidinium groups, is divalent. If a negative charge exists near the guanidinium toxin-

323

16. Na CHANNELS IN LIPID BILAYERS

binding site, then as ionic strength changes with increasing Na', the divalent STX should be more strongly inhibited in its binding than the monovalent TTX; this is a simple consequence of Eq ( I ) : the local concentration of divalent STX drops much more rapidly as ionic strength increases than does that of the monovalent TTX. The results of Green et a / . (1987b) show clearly that such a surface charge effect is at work in the Na' channel; STX block is much more strongly inhibited by increasing NaCl than is TTX block (Fig. 8). Quantitatively, the effect operates as if the guanidinium toxin-binding site is located in a forest of charge density of 0.3 chargeshm'. I00

0.1

0.5

I.o

(NaCI), M FIG.8 . Competition o f T T X and STX by N a ' . Apparent dissociation constants. K,,. for T T X and STX were measured as a function o f symmetrical Na ' concentration. using BTXactivated N a ' channels i n uncharged phospholipid bilayers. A surface charge effect i s indicated by the fact that N a ' competes with the divalent STX more effectively than the monovalent T T X . The ratio o f the two K , , values i s shown as a dashed curve. which i s that theoretically expected for equal intrinsic affinity for the two toxins, but in the presence o f a negative surface charge density o f 0 . 3 chargeshm'. (Data replotted from Green P I ( I / . ,

1987b.)

324

CHRISTOPHER MILLER AND SARAH S. GARBER

V. TWO NOT TOTALLY SPECULATIVE PROPOSALS A.

Guanidiniurn Toxln Binding: Physical Occlusion or Conforrnational Change?

Where has this flurry of bilayer activity around the Na' channel led us? Have we learned anything as a result of looking at these channels in planar bilayers? We believe that we have, and that the advantages of the bilayer system were essential for gaining this knowledge. The experiments on guanidinium toxin binding have not led directly to a decision about the two main contenders for toxin action: direct plugging versus allosteric inhibition. But we argue that the surface charge experiments do lead to a strong conclusion in this murky area. First, our own experiments on the rat muscle channel (Moczydlowski et d.,1984b; Garber and Miller, 1987) demonstrate that the channel's entryway to Na' does not feel the presence of any significant net charge density. Second, the experiments of Green et al. (1987a) on the dog brain channel show that the guanidinium toxinbinding site is located close to a region of high negative charge density. It seems to us that the conclusion is inescapable: The guanidinium toxinbinding site is located far away from the entrywuy for N u + . Such a conclusion is incompatible with a plugging model. It is a strong argument in favor of an allosteric model for toxin action. This is the first strong evidence of any kind addressing the question of the mechanism of guanidinium toxin action. Clearly, the surface charge experiments should all be repeated together on channels from the same source (brain or muscle). But assuming that these experiments verify the results obtained so far, we can say that the action of TTX and related toxins cannot be described as a physical occlusion of the mouth of the Na' channel. Rather, some sort of toxininduced conformational change in the protein must be involved. B. Where Is the Sialic Acid? As we have seen, the fact that the conductance-concentration relationship of the Na' channel (Fig. 7) is rectangular hyperbolic down to very low ionic strength argues that the channel mouth is far away from any negative surface charge density. Our data place an upper limit on this effective surface charge density of 0.01 chargeshm' (Fig. 5). This is equivalent to the charge density of a lipid bilayer containing only 0.5% negatively charged lipid and corresponds an average separation of net negative charges of about 10 nm. If the Na' channel is built anything like the nicotinic acetylcholine receptor, with the channel opening in the order

16. Na CHANNELS IN LIPID BILAYERS

325

of 10 nm in outer diameter, then we are forced to an amazing conclusion: that there is virtually no net negative charge density anywhere on the rim around the channel opening! So where are sialic acid residues that are known to abound on the externally facing side of the Na' channel? Why don't they make their presence known to Na' ions entering the channel'?Aside from proposing that the negatively charged sialic acids are just balanced by net positive charges on nearby regions of the surface of the protein, we think that there is only one way out: that the carbohydrate residues of the Na' channel are placed nowhere near the channel mouth or the rim around it. The cartoon of Fig. 9 shows what we have in mind here: that the channel protrudes a substantial distance into the external aqueous solution, and that the sialic

Fic;. 9. Cartoon of Na' channel structure. The cartoon emphasizes the points discussed here: the large distance separating the guanidinium toxin-binding site from the Na + entryway, and the forest of negatively charged sialic acid near the toxin-binding site, depicted as a cavern in the side of the protein.

326

CHRISTOPHER MILLER AND SARAH S. GARBER

acid is located well away-farther than 3 nm-from the ion permeation entryway, perhaps on the protein surface near to the phospholipid membrane itself. Perhaps the negative charge density experienced at the guanidinium toxin-binding site is actually provided by the sialic acid. This idea may be unjustifiably speculative, but at least it is not crazy, since the charge density estimated by Green P t a / . (1987a) corresponds to an average distance between the charged groups of about 2 nm, a distance consistent with the structure of sialic acid groups on a carbohydrate tree. So we might envision the gross structure of the surface of the channel to be something as is drawn in Fig. 9, with the “allosteric” guanidinium toxin site located really far away from the Na ion entryway. When occupied by TTX,for example, this locale of the channel would change conformation in such a way as to block ion conduction through the pore. The conclusions elicited from the studies discussed here required the use of a reconstituted membrane-channel system. Without such a system it would not have been possible to follow routinely single Na’ channels over many hours, to vary the ionic media over a huge range of ionic strength, or to explicitly set the lipid composition of the membrane playing host to the channel. To be sure, this approach has in no sense given us the structure of the channel. But it has yielded up gross features for which we should be searching in the future as structural studies begin to emerge: the positioning of carbohydrate residues in relation to the mouth of the channel, for example. And because of the work described here, once these structural features are discerned, their functional consequences will be immediately apparent. ACKNOWLEDGMENTS We are grateful to Drs. Olaf Andersen and William Green for providing us with their results before publication. We also thank Dr. Irwin Levitan for critical discussions and readings of the manuscript. This work was supported by NIH Grant No. AR-19826-10 and GM-3 1768-04. REFERENCES Bell. J. E.. and Miller. C. (1984). Effects of phospholipid surface charge on ion conduction in the K’ channel of sarcoplasmic reticulum. Biophvs. J . 45, 279-288. Coronado. R.. and Affolter. H. (1986). Insulation of the conduction pathway of muscle transverse tubule C a t ’ channels from the surface charge of bilayer phospholipid. J . Gcn. Plivsiol. 87, 933-954. French. R. J.. Worley, J. F.. and Krueger. B. K. (1984). Voltage-dependent block by saxitoxin of sodium channels incorporated in planar lipid bilayers. Biophys. J . 45, 301-3 10. Garber. S. S .. and Miller, C. (1987). Single Na’ channels activated by batrachotoxin and veratridine. J . Gcn. Plivsiol. 89, 459-480. Green, W. N.. Weiss, L. B.. and Andersen, 0. S. (1987a). Batrachotin-modified sodium channels in planar lipid bilayers. Ion permeation and block. J . C c n . Physiol. 89, 841872. d

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Green. W. N.. Weiss. L. B.. and Andersen, 0. S. (I987bl. Batrachotoxin-moditied sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures. J. Geri. Pliysiol. 89. 873-903. Henderson. R.. Ritchie. J. M.. and Strichartz. G . R. (1974). Evidence that tetrodotoxin and saxitoxin act at a metal cation binding site in the sodium channel o f nerve membrane. Proc. Nu//.Aurd. Sci. U . S . A . 71. 3936-3940. Hille. B. (1971). The permeability o f the sodium channel to organic cations in myelinated nerve. J. Geti. Physiol. 58, 599-619. Hille. B. ( 19751. The receptor for tetrodotoxin and saxitoxin: A structural hypothesis. Biciplrys. J . 15, 615-619. Kao. C. Y.. and Nishiyama. A. (1965). Action o f saxitoxin on peripheral neuromuscular systems. J . Physiol. (Lotidon) 180, 50-66. Krueger. B . K . . Worley. J. F.. and French. K. J . (1983). Single sodium channels from rat brain incorporated into planar lipid bilayer membranes. Nrr//irc~(Loridmi)303, 172-175. McLaughlin. S . (1977). Curr. T o p . Mrruhr. Trtrtrsp. 9, 71-144. Miller. C. ( 1984). Integral membrane channels: studies i n model membranes. P/iy.siol. R(,ij. 63, 1209- 1239. Moczydlowski. E.. Hall. S.. Garber. S. S. . Strichartz. G. R . . and Miller, C. (1984a). Voltagedependent blockade o f muscle Na' channels by guanidinium toxins. Effect of toxin charge. J. Gcn. Plrysiol. 84, 687-704. Moczydlowski. E . . Garber. S . S.. and Miller. C. (IY84b). Batrachotoxin-aclivated N a channels i n planar lipid bilayers. Competition o f tetrodotoxin block by N a * . J. Geti. Pliysiol. 84. 665-686. Moczydlowski. E.. Alvarez. 0.. Vergara. C . . and Latorre. R. (1985). Effect o f phospholipid surface charge on the conductance and gating of a Ca ' ' -activated K ' channel in planar lipid bilayers. J. Metnhr. B i d . 83, 273-282. Moczydlowski. E.. Olivera. B. M.. Gray. W. R.. and Strichartz. G. R. (1986). Discrimination of muscle and neuronal subtypes by binding competition between [%]saxtoxin and pconotoxins. /'roc.. Nu//.A c d . S c i . U . S . A . 83. 5321-5325. Narahashi. T.. Moore, J., and Scott. W. R. (1964). Tetrodotoxin blockage o f sodium conductance increase in lobster giant axons. J. G ~ t iPIiysiol. . 47, 965-974. Reed, J. K.. and Raftery. M. A. (1976). Properties o f the tetrodotoxin binding component in plasma membranes isolated from Eloc/ropliorrrs elc,c,/ric.rr.s. Bioc/ieuii.s//:v 15, 944953. Sigworth. F. J.. and Spalding. B . C. 1980). Chemical modification reduces the conductance of sodium channels in nerve. Ncrliire ( L o t i h i ) 283. 293-295. Spalding. B . C. ( 1980). Properties oftoxin-resistant sodium channels produced by chemical modification in frog skeletal muscle. J . Plivsiol. (Loiidon) 305, 485-500. White, M. M . . and Miller. C. (1981). Probes o f the conduction process o f ii voltage-gated CI channel from Torpedo electroplax. J . Geti. Plrysiol. 78, 1-18. Woodhull. A. M. (1973). Ionic blockage of sodium channels i n nerve. J. &ti. P/iysio/. 61, 687-708. Worley. J. F.. French. R. J . . and Krueger. B. K. (1986). Trimethyloxonium modification o f single batrachotoxin-activated sodium channels in planar bilayers. Changes i n unit conductance and i n block by saxitoxin and calcium. J . Gtw. Ph>~sio/. 87, 327-349. Worley. J . F.. French. R. J . . and Krueger. B. K. (1988). Effects o f membrane surface charge and divalent cations on ion permeation through single Na' channels from rat brain. J . Gui. Plrysiril. (in press).

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CURRENT TOPICS IN MEMBRANES AND TRANSPORT. VOLUME 33

Chapter 17

Voltage-Sensitive Sodium 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. Ill.

IV.

V.

Na Channels as Proteins A. The Question of Mechanism B. Chemical Isolation and Characterization of Na Channel Protein from Eel Electroplax C. Amino Acid Sequence and Protein Structure Protein Structure and Channel Gating A. Activation Gating B. Inactivation Gating Function of the Purified Protein A. Reconstitution B. Single Channel Recording Experiments: Events Measured in the Absence and Presence of Neurotoxins Chemical Modifications That Alter Regulation of Ion Conductance A. inactivation Gating B. Biochemical Modification of the Reconstituted Protein Conclusion References

1.

Na CHANNELS AS PROTEINS

A. The Question of Mechanism

Voltage-sensitive Na channels are conformationally regulated molecules (Agnew, 1984; Armstrong, 1981). Classical conformational mechanisms have involved allostery in enzymes and in binding proteins such as hemo329 Copyright * 1 IYXX hy Academic P r o , . Inc. All right, of reproductiun in m y liimi r c w v c d .

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WILLIAM S.AGNEW ET AL.

globin. Allosteric mechanisms have been historically studied in symmetrical oligomeric proteins, in which the binding of a substrate or effectorligand causes a cooperative change in conformation, and a resultant shift in catalytic efficiency or binding affinity (Koshland, 1970; Monod et al., 1965). Na channels are, in several respects, distinct from such allosteric proteins. First, the Na channel is not a symmetrical oligomeric structure. The functional portion of Na channels is a large polypeptide apparently folded into pseudo-subunit domains (Noda et al., 1984, 1986; Salkoff et al., 1987). While these pseudo-subunits are similar, they are nonidentical and thus no axis of symmetry can exist. Secondly, Na channels respond, normally, not to the binding of chemical ligands, but to transmembrane voltage. Even in this the molecule is novel, because functionally it is not voltage per se that is important, but rather changes in voltage. Thefunction of the Na channel is to open a Na’ selective path across the membrane through which Na ions pass at near-diffusion-limited rates (- 10’ ions/second). At “equilibrium” (i.e., time 4 w), Na channels are most likely to be closed at all potentials. Only when the membrane passes from a sufficiently negative resting level (i.e., - 70 to - 90 mV) rapidly to a depolarized level does the conductance pathway transiently open, and then close (Bezanilla, 1985). Thus, if transient potential changes are considered electrical “signals,” the Na channel detects such signals, and amplifies and propagates them. Further distinguishing the Na channel from classical allosteric proteins, it is the kinetics of the conformational changes, the activation and inactivation mechanisms, which are crucial for the protein function and which confer the signaling properties of the membrane. The kinetic paths between various resting, opened, inactivated, and desensitized states are cooperative and precisely voltage dependent, and these delicate mechanisms appear to have been conserved over at least 600 million years of evolution (Saikoff et d., 1987, 1988). Briefly, then, the object of our studies is a large integral membrane protein which forms a transient, Na’-selective pathway that conducts picoampere scale currents, in response to changes in membrane voltage, in a complex conformational activity cycle of 1-5 msec duration. Needless to say, resolution of the mechanism of such a molecule presents nontrivial challenges to the protein chemist, and in fact demands an interdisciplinary approach. Our laboratory has investigated extensively the properties of a voltagesensitive Na channel from the electroplax of Electrophorus electricus, the Amazonian electric eel. The knowledge we have gained follows from first isolating the protein in chemically pure form (Agnew et al., 1978, 1980, 1983; Miller et al., 1983). These studies have provided important information about the structure of the protein and have led others (Noda et al., 1984) to clone the corresponding cDNA and thereby deduce the pri-

17. VOLTAGE-SENSITIVE CHANNELS

33 1

mary amino acid sequence of the peptide portion of the molecule. Beyond this, we have been able to reassemble the pure protein into artificial membranes and recover at least some of the functional properties, both biophysical and pharmacological, attributed to Na channels in natural membranes (Rosenberg et a / . , 1984a,b; Agnew et a / . , 1986). Further, we have now begun to use chemical modification to probe conformational mechanisms in the molecule (Cooper et a / . , 1987). B. Chemical Isolation and Characterization of Na Channel Protein from Eel Electroplax

The electric organs of the freshwater eel E. electricus are specialized muscle cells that have lost their ability to contract; these cells nevertheless have developed an exaggerated ability to undergo electrical excitation (Fig. I). About 2% of the caudal, innervated face of the cell is covered with cholinergic synapses, bearing nicotinic acetylcholine receptors in nearly crystalline densities of 5.000-10,000 Fm-' (Popot and Changeux, 1984). The rest of the surface is electrically excitable, containing an estimated 500-1,OOO voltage-sensitive Na channels per Fm' (Levinson, 1975). The Na channel protein thus represents about 5-10% of the integral membrane protein on this surface of the cell. The membrane is devoid of the laterectifying K channel which is important in recovery of the action potential in nerves, but is enriched in the anomolously rectifying K channel which sets the resting membrane potential (Nakamura et a / . , 1965). The main electric organ, 500 to 750 g per animal, is thus a relatively homogeneous tissue and is the richest known source of Na channels for preparativescale biochemistry. For isolation, Na channels a r e quantified by the binding of [3H]tetrodotoxin (TTX), a specific toxin that binds stoichiometrically to the extracellular surface to block Na' currents. While the mechanism of blockade by TTX and its pharmacological cousin saxitoxin (STX) is not known, it appears to involve conformational mechanisms (Chapter 16, this volume; Green et a / . . 1987a,b). Binding is reversible, of high affinity ( K , -lO-'M), and very specific (Ritchie and Rogart, 1977). Preparative studies begin with isolation of membranes, usually unfractionated. The protein is solubilized with the nonionic detergent Lubrol-PX. The solubilized molecule has a dramatic dependence on having small amounts of phospholipids, either phosphatidylcholine or phosphatidylethanolamine, to prevent denaturation. In the range below I lipid per 15 detergent molecules, the apparent first-order rate constant for denaturation depends on the lipid-to-detergent ratio raised approximately to the fortieth power (Agnew and Raftery, 1979). Failure to recognize this requirement for sta-

WILLIAM S. AGNEW ET AL.

332 +

Caudal I

I

Na,K ATPase

Na Ch K Ch, an

nAcChRext.jnx

nAcChRjnx

I

Excitable

Nonexcitable

FIG. I . Schematic of electroplax indicating distribution of excitability and cholinergic synapses, as well as the Na.K ATPase, Na channels, anomalous rectifier K channels. and extrajunctional and junctional nicotinic acetylcholine receptors.

bility delayed purification of Na channel molecules for more than 6 years (Henderson and Wang, 1972; Agnew ef al., 1978). The solubilized molecule has several distinguishing characteristics that permit its purification. In retrospect, we can now attribute all of these to extraordinary carbohydrate constituents. First, the molecule is extremely acidic, carrying about six times the net negative charge found in the comparably sized (molecular weight) nicotinic acetylcholine receptor (Agnew et al., 1980). Partly because of this, anion-exchange chromatography on DEAE-Sephadex provides about 40-fold purification. In addition, the protein has an extremely large hydrodynamic radius, effectively 90-95 A. This is far larger than the Triton X-100 solubilized acetylcholine receptor

333

17. VOLTAGE-SENSITIVE CHANNELS

(65 A), and indeed larger than most proteins extracted from the electroplax membranes. Thus, gel filtration provides an adequate second and, if needed, third step for purification. Consequently, via a two- or three-step fractionation, it is possible to purify the protein essentially to chemical purity. The Lubrol-PX solubilized protein is in the form of a mixed proteindetergent-lipid micelle of -500,000 Da. The protein portion is approximately 300,000 Da, with the remaining mass due to bound detergent and lipid (Agnew er al., 1988) (Table I). The purified molecule was observed to have a completely unanticipated peptide composition. SDS gels run on a purified Na channel molecule are illustrated in Fig. 2. Unlike the nicotinic acetylcholine receptor, a pentameric oligomer of four types of peptides of apparent molecular weight 40,000-65,000, the Na channel was a single peptide of nearly 300,000, which ran as a very diffuse band in SDS gels. Indeed, the anomalous behavior of this protein in electrophoresis, coupled with the unexpectedly large size, made us initially suspicious that aggregation of smaller subunits had occurred (see Chapter 16, this volume, for discussion). This turned out not to be the case. The amino acid composition of pure Na channel protein was remarkably similar to that of the nicotinic acetylcholine receptor, characteristic of a slightly more polar than average integral membrane protein (Miller ei al., 1983; Noda er al., 1984) (Table 11). This provided no explanation of the acidity of the molecule. However, direct carbohydrate analysis revealed an enormous carabohydrate constituent of about 30% by mass (Table 111). This carbohydrate was enriched particularly in N-acetylhexosamines and sialic acids. We now recognize that the sialic acid contributes about 110130 net charges to the molecule, accounting for the acidity of the molecule.

TABLE I COMPARISON OF CARDIAC AND SKELETAL MUSCLEC A L C ~ UCHANNELS M M, of proteindetergent-lipid complex

of complex fl of detergent-lipid micelles fl of glycopeptide M, of native protein Mass of bound detergent per gram protein Stokes radius ’20,w ”20. w fl

flL M, denataured glycopeptide

592.00 0.847 g/cm’ 0.9% gkm’ 0.73 g/crn’ 330,000 0.79 g/g protein

wA 8.9s 2.4F I .s 295 ,OOO

334

WILLIAM S. AGNEW ET AL.

FIG. 2. SDS-gel on a purified Na channel preparation. Diffuse bands at the top of the gel represent the heavily glycosylated Na channel peptide.

The carbohydrate composition is currently the object of some interest in our laboratory. Recently, Dr. William James (James and Agnew, 1987a,b) discovered that there are at least two classes of oligosaccharides on the protein. Of the possibly 9 asparagine-linked groups, all appear to contain one- or two-armed cores. Some of these (perhaps half) are neutral, containing large quantities of mannose and N-acetylhexosamines, but no

335

17. VOLTAGE-SENSITIVE CHANNELS

TABLE 11 AMINO ACIDCOMPOSITION OF THE LAKGEPEalDE Of:THE ELECTKOPLAX Na CHANNEL." Amino acid

Miller

}

Asp Asn Thr Ser

5.0 7.1

Gln Pro GlY Ala CYS Val Met I le Leu TY r Phe His Trp LY s Arg

4.7 6.5 6.0 2.0 5.3 3.4 5.5 9.3 2.8 5.9 2.8 3.5 5.0 4.3

}

ct

ul. (1983)

10.1

Noda ct uf. (1984) 5'10 6.81 5.10 6.15

10.8

6.81 2.96 4.06 4.94 5.76 I .97 7.41 3.90 8. I3 10.38 3.39 6.97 0.93 I .70 5.10 4.17

}

11.9

}

9.77

"The amino acid composition arrived at by direct analysis (Miller cr ul., 1983)is compared with that deduced from the cDNA sequence of Noda d a / . (1984).

sialic acid. These must represent bulky surface substituents, the function of which is not known. In addition, a second class bears an almost unique form of sialic acid. Probably attached to one or two-armed cores of 8 to 10 sugars are large unbranched homopolymers of sialic acid which likely consist of >20 tandem sialic acids linked in cy-2.8-bonds. These TABLE 111 CAKBOHYDKATE COMPoSlTlON OF THE LAKGEPEPI'IDE OF THE ELECTKOPLAX Na CHANNEI." Glycoside

wt%

Fucose Mannose Galactose N-Acetylhexoseamines Sialic acids Total

? 0.2 1.4 2 0.3 1.5 ? 0.3 13.3 ? 0.4 11.8 2 1.2 29.5 ? 2.4

"From Miller ef cif. (1983). "Assuming a molecular weight of 295,000.

0.5

m o h o l protein" 92 39 ? 25 2 177 2 1132 363

3 8 5 5 II

336

WILLIAM S.AGNEW ET AL.

groups are capable of extending perhaps 150 A away from the protein surface, making the carbohydrate portion comparable, hydrodynamically, to the protein-detergent-lipid mixed micelle structure itself. This no doubt contributes to the disproportionally large hydrodynamic radius. By making the molecule both large and acidic, the carbohydrate has permitted the simple purification scheme described above to be developed. Now, more appreciative of the carbohydrate, we have adapted a sialic acid-specific lectin affinity purification procedure which makes it possible to isolate > I .O mg of protein, at high specific activity (i.e., undenatured) in a 5-hr procedure (James and Agnew, 1988a,c). This sialic acid is readily removed by treatment with neuraminidase or an endoneuraminidase isolated from bacteriophage of KI strain of Escherichia coli, with a remarkable reduction of apparent molecular weight (James and Agnew, 1987b). Thus, the electrophoretic microheterogeneity of the protein on electrophoresis could easily be explained by variations in polysialic acid chain length. It should perhaps be mentioned that these unusual oligosaccharides are rare in vertebrates, having previously been demonstrated in neurons (Edelman. 1983; Lyles et a / . , 1984; Rutishauser et al., 1985) and skeletal muscle (Regier et al., 1985) as portions of the neural cell adhesion molecules (N-CAMS) (Finne, 1985). They are also characteristic of weakly antigenic capsular polysaccharides of meningitis bacteria (Finne, 1985). However, James has demonstrated that antibodies to coat polysaccharides of Neisseria rneningitidis, and to K1 type E. coli, selectively react with the electroplax Na channel (James and Agnew, 1988b). Perhaps the failure of the immune system to mount a response to these organisms results from their masquerade as Na channels or N-CAMS. These observations underscore the need for caution in breaking tolerance to these oligosaccharide antigens. In addition to these odd characteristics, we discovered that the molecule had an extraordinarily high free mobility on SDS-PAGE gels (Miller et al., 1983; Agnew et al., 1983). This suggested the molecule must bind 35 times more SDS than conventional peptides, or up to 7 g SDS per gram of protein. Such an unusual effect, which must represent binding of micellar SDS, suggests either highly ordered protein domains for detergent (and lipid) binding, or of the attachment of extensive covalently associated lipid groups (Levinson et al., 1986). C. Amino Acid Sequence and Protein Structure

Further structural insight into the Na channel structure has emerged from cloning of the large Na channel peptides from electroplax (Noda et

17. VOLTAGE-SENSITIVE CHANNELS

337

al., 1984), and subsequently from mammalian brain (Noda et ul., 1986) and Drosophila (Salkoff ef ul., 1987). This has permitted important deductions to be drawn about possible secondary, tertiary, and “pseudoquarternary” organization of the molecule. A more extensive discussion of molecular modeling is to be found elsewhere (Guy and Seetharamulu, 1986; see Chapter 15, this volume). The electroplax glycopeptide has a molecular weight of -295,000. with variability due to the glycosidic moieties. The polypeptide core, of 208,32I Da, exhibits sequences suggesting how the protein might be organized, in at least general respects (Fig. 3A). Prominently, there are four stretches of amino acid that are significantly homologous to one another. These domains, comprising each about 30 kDa of the peptide backbone, contain amino acids of which 47 to 55% are identical or conservative substitutions. These are separated by unique sequences of variable length. Analysis of the polarity by hydropathy criteria reveals that there are, within each repeat, up to eight potentially membrane-spanning sequences. These sequences are hydrophobic, or display patterns of hydrophobicity that suggest they lie in or pass through the plane of the membrane bilayer if folded into a-helices or other configurations. Interestingly. neither the carboxyl or amino termini, nor the three intervening sequences are sufficiently hydrophobic to span the membrane. It therefore has been proposed that the carboxyl and amino termini, as well as the intervening sequences lie on the cytoplasmic face of the membrane. Thus, the impression left is that the protein may penetrate the bilayer only within the homologous repeats. These repeats, designated I-IV from the amino to carboxyl terminus, respectively, may thus fold to form pseudo-subunits. If this pattern were true, one would expect all consensus glycosylation sites to be present within the repeat domains. This is essentially true. In the electroplax protein there exist 10 consensus sites for asparagine-linked glycosylation (N, *, S/T), and nine of these are within domains I, 11, and 111. The tenth, in the carboxyl tail, is evidently not used (Chapter 12, this volume). The heavy glycosylation suggests all nine of the remainder are likely to be used. Similarly, should the protein be phosphorylated, one predicts that consensus phosphorylation sites (K/R, K/R,*, (*), S/T) would be in regions speculated to be cytoplasmic. We have discovered that the electroplax protein is readily phosphorylated by the catalytic subunit of protein kinase A, but not of protein kinase C, at at least three sites per molecule (Emerick and Agnew, 1988a,b). At most, one of these sites involves threonine as the phosphoamino acid, the remainder involve serine. (The peptide contains four serine- and four threonine-containing consensus phosphorylation sites.) The candidate sites are illustrated in Fig. 3A, and satisfy the simple interpretation of the protein conformation.

17. VOLTAGE-SENSITIVE CHANNELS

339

It is thus anticipated that the homology repeats fold into domains resembling subunits on a string. These, representing about 60% of the polypeptide mass, may be organized in a tetrameric arrangement, with the remaining mass in the cytoplasmically exposed domain (Fig. 3B). Because only the repeats seem certain to span the membrane, they most likely contain the domains forming the ion pathway, the permeation-selection mechanism, and the sites of interactions with a variety of neurotoxins and local anesthetics. This conclusion, however speculative, is supported by comparisons of the electroplax protein with comparable peptides cloned from two brain Na channels and that from Drosophila melunoguster. Briefly, it has been observed that, particularly in comparing rat and eel sequences, the homologies are not uniformly distributed. While the carboxyl and amino termini and intervening sequences are modestly conserved (-25-50%), the pseudosubunit domains are strongly conserved (generally 80-95%) (W. S. Agnew, unpublished observations). (Interestingly, the brain proteins contain a stretch not present in the electroplax protein, between domains I and 11, of about 190 amino acids. This -20kDa hydrophilic sequence contains several consensus phosphorylation sites, and may be the locus for neuromodulation not required of the electroplax protein.) The close homology of the repeats is further evidence of mechanistic importance given that Drosophila sequences, evolutionarily far separated from the vertebrates, are closely comparable to that found between eel and rat (Salkoff et al., 1987). II. PROTEIN STRUCTURE AND CHANNEL GATING Hypotheses about mechanisms for Na channel gating advanced on the strength of our partial understanding of the protein structure remain little more than suspicions at present. Kinetic descriptions of gating regulation, while adequate to fit a variety of data, are so complex that they do little to suggest structural hypotheses amenable to direct testing (Armstrong, 1981; Bezanilla, 1985). Until recently no theoretical explunation for the stiJeply voltage-dependent kinetics had been udvanced: current kinetic models ure strictly phenomenological in churacter. However, in two in-

stances observations do suggest possibly meaningful experimental approaches with which to perturb the conductance regulatory mechanisms.

FIG.3 . ( A ) Schematic of repeat domain structure of electroplax Na channel peptide. Cylinders represent hydrophobic stretches capable of penetrating or spanning membrane bilayer. P and G identify consensus phosphorylation and glycosylation sequences. respectively. ( B ) Schematic of possible tetrameric organization of the protein in the membrane bilayer. with extensive negative charges contributed to the protein surface by 110-130 sialic acids.

WILLIAM S.AGNEW ET AL.

340

A.

Activation Gating

The probability of Na channel gating opening increases e-fold for approximately 4 mV depolarization (Hodgkin and Huxley , 1952; Armstrong, 1981). Such behavior must involve the movement of the equivalent of -6 electron charges through the membrane field (Fig. 4A). Large translocations of protein domains through the membrane seem unlikely on energetic grounds. The conformational mechanism, in addition, is cooperative, and conservative models suggest six or more resting states with

FIG.4. (A) Depiction of a minimal gating cycle illustrating the following characteristics: inactivation is coupled to activation, inactivation immobilizes part of the gating charges associated with movement of activation voltage sensors, channels rarely pass through an opened state on recovery from inactivation. gating charges move normal to the membrane. all activation gating sensors seem likely to move in the same direction, inactivation mechanism is exposed to cytoplasmic surface. (B) Adaptation of mechanism hypothesized by Armstrong (1981) suggesting sliding columns of positive and negative charges. Minimal motion yields translocation of two net charges.

17. VOLTAGE-SENSITIVE CHANNELS

34 1

voltage-dependent transitions between, leading to activation. (Many more states associated with inactivation and desensitization are also required to describe gating.) Nevertheless, one hypothetical mechanism advanced by Armstrong (1981) finds an intriguing correlate in the structure of each pseudo-subunit, and has been found closely replicated in a recently cloned dihydropyridine receptor [the L-type calcium channel? (Tanabe et al., 1987)], and in voltage-activated A-type K channel subunits (Tempel et al., 1987; Schwarz er al., 1987). Armstrong hypothesized that charged columns of anionic and cationic residues, sliding past one another across the voltage gradient could explain large charge translocations with minimal movement (Fig. 4B). Within each pseudo-subunit there exist segments of peptide in which alternating patterns of two hydrophobic amino acids followed by lysine or arginine occur for extended lengths. If coiled into an a-helix, these would appear as a hydrophobic column bearing a helical array of positive charges. While no simple equivalent anionic neighbor is obvious, such groups are currently postulated to be formed by noncontiguous regions designated S7 of the peptide. These segments, designated by Numa as "S4," could constitute a voltage sensor not unlike that predicted by the Armstrong hypothesis. Of course, the mechanism for coupling such movements to channel opening is undistinguished speculation. However, because these sequences are prominent (four repeats in domain 1, five each in I1 and 111, and eight in IV), and because they are precisely conserved in all Na channels so far sequenced, they invite close study by mutagenesis studies (see Chapter 13, this volume).

B. Inactivation Gating Inactivation, evaluated from voltage dependence of mean channel open times in single channel studies, appears to be weakly voltage sensitive (Armstrong and Bezanilla, 1977; Nonner, 1980), but strongly coupled to activation (Armstrong, 1981; Bezanilla, 1985; Aldrich et al., 1983; Vandenberg and Horn, 1984). Once activated, channels appear to close via this mechanism, and inactivation is associated with the immobilization of about one-third of the charge which moves during activation (Armstrong, 1981 ; Bezanilla, 1985). Recovery from inactivation rarely occurs until part of the activation mechanism has reversed, under influence of repolarization of the membrane, so that the channel does not reopen. Thus, channel gating appears to be cyclical rather than simply reversible, as shown in Fig. 4A. No structure of the protein sequence compellingly suggests an inactivation mechanism. However, chemical modification studies conducted in perfused native membranes suggest that the structure involved

342

WILLIAM S. AGNEW ET AL.

is exposed at the internal surface of the protein. Regions of the protein involved in inactivation thus may be amenable to identification in biochemical studies of reconstituted Na channel molecules. 111.

FUNCTION OF THE PURIFIED PROTEIN

A. Reconstitution

The electroplax protein is purified in a suspension of mixed lipid-detergent micelles. When supplemented with exogenous lipids, followed by adsorption of the detergent from the lipid-protein-detergent suspension, the protein is spontaneously reincorporated into lipid vesicles. These vesicles may then be examined for recovery of channel function. Radiotracer (Rosenberg et al., 1984a,b; Cooper et al., 1987) or fluorescence flux assays (Tomiko et al., 1986) can be used to monitor the conductance state of the protein. Neurotoxins that perturb the gating mechanisms of the channel can be used to force the protein into a chronically opened state, or into an opened but blocked state. In experiments such as illustrated in Fig. 5 , vesicles containing the reconstituted electroplax Na channels are incubated either alone, or with

BTX/TTX

B T X R T X & QX-314

control

Time, sec

FIG.5 . Reconstituted vesicles were diluted 10-fold into Tris-sulfate buffer containing "Na' and uptake measured at the indicated times. A weak uptake was observed when but a large signal was observed in vesicles were preincubated with I% ethanol carrier (0). vesicles treated with 2.5 pM BTX (A). This signal was blocked by approximately 60% by I pM external TTX (A),and completely when I p M TTX and 3 m M QX-314 were added ( 0 ) . Partial blockade is observed with 3 m M external QX-314, and complete blockade is observed with internal and external 3 mM QX-314 or I p M TTX (not shown).

17. VOLTAGE-SENSITIVE CHANNELS

343

the activating neurotoxin batrachotoxin (BTX) (or veratridine), and the rates of "Na' influx are measured. Under these conditions, a marked stimulation of ion flux to the vesicle interior is produced by BTX (cf. Fig. 51, or by veratridine (not shown). This flux can be selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX). Externally added TTX blocks about half the influx, while 'ITX equilibrated with both sides of the membrane completely suppresses influx (not shown). Thus, the protein is incorporated with approximately random orientation in the liposomes, and blockade by external TTX distinguishes the signal due to outside-out oriented molecules. Similar results are obtained with veratridine as the activator. To complement TTX, local anesthetics also may be used to block the opened channel. Membrane-permeant local anesthetics (Cahalan, 1981) such as tetracaine or dibucaine block all of the signal. Other local anesthetics, such as QX-222 and QX-314, block only about half of the flux signal when added to the vesicle exterior. These are permanently charged, quaternary ammonium derivatives (Cahalan, 1981) that permeate liposomes poorly. Normally, these compounds act only from the cytoplasmic membrane surface. When added only to the vesicle exterior, QX-314 suppresses only about half the flux. As shown in Fig. 5. addition of TTX and QX314 together suppresses all of the signal stimulated by BTX. The significance of these observations to the studies described below is that the blockade by TTX and local anesthetics provides a probe of the vectorialify of the channel molecules contributing to the flux signal. The reconstituted protein also retains its ion-selectivity mechanism. In a series of double-label experiments, influx of Na', K', Rb', and Cs' has been measured. As clearly indicated by the time courses shown in Fig. 6, the permeability sequence expected for the Na channel is retained. Estimated from the influx of each ion relative to Na' in 1-min uptake experiments, the relative selectivity of the channel is PNa = 1.00, P, = 0.22, P,, = 0.06, and Pcs = 0.04. In other studies, we have used voltage-sensitive dyes to extend the permeability selectivity of the reconstituted protein (Tomiko and Agnew, 1987, 1988). The dye di-S-C,-(5) is a so-called "slow dye" that partitions across the membrane in a time scale of seconds depending upon transmembrane potential (Hoffman and Laris, 1974; Sims et al., 1974).In these experiments, the vesicles are filled with high concentrations of Na,SO,, followed by removing external permeant cations by ion-exchange chromatography over Dowex cation-exchange resin. The voltage-sensing dye added to this suspension shows a very small fluorescence quench, suggesting that at most a small membrane potential has developed. However, if BTX is added to open the channels in the vesicle suspension, Na' ions

344

WILLIAM S. AGNEW E T AL.

"R b I=

'"CS

60 120 Time. seconds

FIG.6. Ion selectivity of BTX-stimulated uptake. Vesicles were diluted I0-foldinto Trissulfate buffer containing pairs of radiotracers: 0 , "Na; 0. 4'K; A, wRb; and A. "'Cs. Low weakly selective uptake by control samples was subtracted and net uptake stimulated by 2.5 fiM BTX is shown for each ion. Described in detail in Cooper ef d.,(1987).

tend to diffuse out of the vesicles. Retention of the impermeant Sod2anion produces a diffusion potential that induces a substantial quenching of the fluorescence signal. Subsequently, adding isoosmotic solutions of external permeant cations produces a collapse of membrane potential measured as a recovery of fluorescence. This recovery proceeds more or less rapidly, depending on the permeability of the channel to the ion. IIlustrated in Fig. 7A are time courses with the ions Na+, K', and Cs'. Again, the expected permeability sequence is identified. This method, which does not depend on radiotracers, can be used to rapidly screen a variety of ions for their ability to pass through the activated channel. As illustrated in the histogram in Fig. 7B, the protein exhibits a range of permeabilities to both metal and nonmetal polyatomic cations. The selectivity of the BTX-activated reconstituted channel is characteristic of BTX-activated Na channels found in native membranes studied electrophysiologically, but significantly different from that for channels activated by voltage alone (Hille, 1971, 1972; Khodorov, 1978). Thus, in the reconstituted preparation, BTX apparently not only forces the channel from a closed to a chronically opened state, but alters characteristically the selectivity, suggesting that the site of interaction with BTX is intact and functional. In the reconstituted membranes the channel is normally closed, presumably in an inactivated or slow-inactivated state. BTX induces the observed flux by removing inactivation, although it also alters the activation

A sample +dye

+cation ilOmM)

u1

c _

50sec

Ii

I

k

ri N( Li NH4 Fo GU K Rb Cs Ma

FIG.7. (A) Rate at which diffusion potential collapses vary with the species of depolarizing cation added to external solution. Vesicles containing purified Na channels, and filled with 84 mM Na2S0,. were incubated with 2.5 FM BTX. stripped of external Na'. and exposed to 5.07 mM Na,SO, external. Dye [di-S-C1-(5)1was added and fluorescence measured (single arrow) for -5.9 min. Then 10 mM of NaCI. KCI, or CsCl was added (arrows). (From Tomiko and Agnew. 1987.) (B) Selectivity sequence for monovalent cations through BTX-modified reconstituted Na channels. Data from experiments as in A were normalized to the level of fluorescence before depolarization. The change in fluorescence 100 sec after addition of each salt was determined and computed relative to that produced by Na'. Initial [Na + ,.,1,, equalled 5.07 or 0.05 mM Na,SO,. Error bars denote SEM. and number of experiments for each cation was TI' , 10; Na'. 12; Li' 8; NH i . 10; formamidine 4: guanidine + 8: K ' 12; Rb'. 9; Cs'. 6: methylamine'. 8. (From Tomiko and Agnew. 1987.)

.

+,

~

.

346

WILLIAM S. AGNEW ET AL.

gating kinetics and voltage dependence and produces subtle changes in the conductance, selectivity, and in the interaction of the channel with TTX and STX. The slow-time resolution flux assays are not suited for measuring the gating transitions of the molecule in the physiological (millisecond) time scale, nor under the influence of voltage. To do this, single channel recording techniques have been applied to the protein reconstituted into liposomes. B. Single Channel Recording Experiments: Events Measured in the Absence and Presence of Neurotoxins

In initial studies carried out by Rosenberg, liposomes containing reconstituted Na channels were expanded by freezing and thawing, yielding large ( 10-50 pm) multilamellar structures. Thin, unilamellar blebs from these structures may be used for patch-clamp recording (Rosenberg et al., 1984b). With patches excised from such liposomes, it has been possible to record unitary conductance events that are activated by voltage even in the absence of alkaloid neurotoxins such as BTX and veratridine. As shown in Fig. 8, depolarizing such a patch from - 100 to -25 mV elicited events of 11 pS (90 mM external NaCI, 10 mM internal NaCI). These events cluster toward the beginning of the depolarizing epochs. This is illustrated by summing a series of such records to produce the equivalent of a macroscopic current. These ensemble currents show clearly that the channels are activated by voltage, and the currents decline progressively after initial depolarization. Thus, the reconstituted currents show unmistakable evidence for inactivation. In other experimental recordings made at higher ionic strength (250 mM NaCI), the conductance of the events was 16-17 pS, close to the values of 15-18 pS seen in most native membranes. In studies where conductance through opened channels was measured at several different voltages in the presence of asymmetric concentrations of NaCl and KCI, the reversal potential was estimated from the single channel current versus voltage curves, indicating that the conductances were selective for sodium over potassium by about 7-fold. At the concentration of internal potassium used (90 mM), this is the ratio expected for sodium channels (Cahalan and Begenisich, 1976; Ebert and Goldman, 1976). Further analysis of the single channel data indicated that open times were distributed mono-exponentially and the mean channel lifetime was about 1.9 msec. This is in the range of 0.5-3 msec seen for single sodium channel events in native membranes. Collectively, these data indicated that at least some of the reconstituted,

17. VOLTAGE-SENSITIVE CHANNELS

B

347

t

W U 5 msec F a . 8. Single channel currents of an excised patch of reconstituted membrane containing the purified Na channel. The patch was held at - 100 mV and depolarizations to -2s m V lasting 30 msec were made at 0.7.5-sec intervals. The bath solution was 10 mM NaCI/XO mM KCVS mM MgCI,/IO mM HEPEYKOH, pH 7.4. and the electrode solution was 90 mM NaCM mM MgCI,/IO mM HEPESNaOH, pH 7.4. (A) Sixteen representative records from this patch. Onset and end of depolarization are indicated by the upward and downward arrows. Offset, leakage. and capacity currents were subtracted. Channel opening is a downward deflection. The mean channel current was -0.67 PA. ( B ) Summated currents of 62 individual records. scaled arbitrarily. Filter. loo0 Hz; temperature, 23°C. (From Rosenberg et cri.. 1984b.3

348

WILLIAM S. AGNEW ET AL.

purified sodium channel molecules are capable of functioning as normal, voltage-activated, inactivating sodium channels. However, these experiments have proven difficult to conduct simply because the encounter frequency of active Na channels is typically low when the experiments are carried out as initially described. A priori this could be due to the fact that few of the channels are “healthy” enough to function properly. However, flux studies suggested that under the influence of batrachotoxin or veratridine, nearly all of the proteins are functional. The second possibility is that channels are infrequently in a state which can be activated by voltage alone, but the powerful neurotoxins can force these proteins into activity. A third possibility is that of uneven surface distribution of the protein. In the described procedures, reconstituted vesicles containing high densities of Na channels are fused with exogenous liposomes to form the multilamellar structures. Failure of the protein to uniformly redistribute could reduce the encounter frequency. A. M. Correa and J. Y. Zhou, in our laboratory, have begun to successfully address these problems. These studies will be reported in more detail elsewhere (Correa et ul., 1988a,b). Lectin affinity chromatography procedure with the sialic acid-specific lectin from Limax flavus permits a two-step purification involving ion-exchange fractionation followed by lectin affinity purification. Because the procedure requires only a few hours, there is minimal denaturation and it is readily possible to prepare purified protein at > I mg/ml. Thus, reconstitution can be carried out at almost arbitrarily high protein : lipid ratios. We have found the optimal ratios to be 2- to 3-fold higher than those used by Rosenberg ef a f . in the initial studies. The second modification has been to mix the reconstituted liposomes with exogenous liposomes in a brief but vigorous sonication step before freeze-thawing. Subsequently, the vesicles are swollen by lower osmotic strength buffers to create large (-50 pm diameter) uniform unilamellar structures rather than the multilamellar structures described above (Fig. 9A). These membranes readily yield seals of 5-20 GR. Excised patches from these liposomes have been studied in the absence and presence of BTX. In the absence of BTX, single channel events have FIG.9. (A) Appearance of frozen. thawed, sonicated, (FTS) vesicles containing the purified eel Na channel, before ( A l ) and after (A2) dilution into a hypoosmotic medium. Scale bar: 10 pm; magnification: 2 0 0 ~ (.B ) Single channel currents from reconstituted purified channels in the presence of BTX. FTS vesicles containing lectin-affinity purified channels were diluted in a medium containing 2.5 p M BTX. Recordings were made in an excised patch in 250 mM NaCVIO mM HEPES/NaOH. pH 7.4. symmetrical solutions. The figure shows an array of sweeps of the raw data at the indicated imposed voltages. The traces reveal multiple channels ( 3 4 in the patch. voltages follow the right-side-out (cell) convention. 1988a.) Filter. 1000 Hz; temperature, 23°C. (From Correa ef d,,

349

17. VOLTAGE-SENSITIVE CHANNELS

mV 100 ms

+loo

+90 -0

0 -c

9 -60 -C

-0

-70

4 -100

350

WILLIAM S. AGNEW ET AL.

been observed which were activated by voltage alone. The detailed characterization of these events is not yet complete. While the encounter of purely voltage-activated events is relatively infrequent, it is remarkable that if BTX is added, virtually every patch has 1 - 4 channels in it. An example of a patch containing three BTX-activated channels is shown in Fig. 9B. These results are highly reproducible, and suggest that most of the channels are in a quiescent state. Knowing that the channels are present in the patches permits more thorough study of the conditions which are required to elicit nontoxin-modified activity. These results underscore the fact that demonstration of channel activity with neurotoxins is not a sufficient basis for claiming recovery of fully functional Na channel proteins. Identifying the conditions for recovery of normal function is a key technical problem under study in our laboratory. Despite these incompletely resolved questions, we have begun to examine the reconstituted preparations to learn whether chemical modifications can be introduced which affect channel gating behavior. In these studies, we have taken aim at the mechanisms which involve inactivation gating. We presume, as a working hypothesis, that the closed channels in the liposomes are in inactivated or slow-inactivated(i.e., desensitized) states. Because inactivation gating mechanisms can be removed in native membrane preparations (Armstrong et al., 1973; Oxford et al., 1978; Wang, 1984), we may hope to produce nontoxin-modified channel activity in proteins which have had inactivation removed. We then intend to identify the sites of modification leading to inactivation removal, and to characterize the remaining activation gating behavior in the presence and absence of BTX. The following describes studies which concern inactivation removal. IV. CHEMICAL MODIFICATIONS THAT ALTER REGULATION OF ION CONDUCTANCE A.

Inactivation Gating

In classical studies of Na channels in the squid giant axon it proved possible to selectively modify Na channel gating by treatment with proteolytic enzymes (Armstrong et d.,1973). While external proteases produced no effects, internal perfusion with Pronase, a mixture of bacterial enzymes, resulted in Na currents which failed to inactivate. Tested under optimal incubation conditions, voltage-clamped Na currents activated normally, but were markedly prolonged. Normally declining after 3-5 msec, after proteolysis these currents remain high for hundreds of milli-

17. VOLTAGE-SENSITIVE CHANNELS

35 1

seconds. In such preparations the voltage-dependent kinetics of activation were not affected. Reversal potentials, reflecting permeation selectivity, were not altered. The currents were still blocked by TTX and STX and by both membrane-permeant and -impermeant local anesthetics. [Interestingly, the use-dependence of the impermeant anesthetics, extensively documented in later studies, was altered in a manner consistent with the removal of the inactivation gating mechanism (Cahalan, 1978).] In these and later studies it was observed that peak Na currents gradually declined with extended exposure to the proteases, ultimately followed by a general loss in membrane resistance due to the development of an unselective leak. It was subsequently found that the active component of Pronase was alkaline proteinase b, a serine protease specific for arginine-containing sites (Rojas and Rudy, 1976). Other enzymes such as chymotrypsin and trypsin had similar effects (Sevcik and Narahashi, 1975). The reagents Nbromoacetamide (NBA) and N-bromosuccinimide (NBS) similarly eliminated the inactivation phenomenon (Oxford et al., 1978). The latter reagents are capable of oxidizing cysteines, methionines, and aromatic amino acids such as histidine, tyrosine, and tryptophan. Under optimal conditions of pH, in many proteins, aromatic amino acid oxidation is followed by rearrangement and cleavage of the peptide backbone (Spande et al., 1966). It was thus suggested that these various reagents cleaved at the cytoplasmic surface of the protein at one or more sites involved in inactivation. In these preparations all processes not related to inactivation remained intact. Later, experiments characterizing asymmetric displacement currents connected with the movement of charge associated with the voltage sensors of the channel further supported the selectiveness of these effects (Armstrong and Bezanilla, 1977). These experiments suggested that any of several modifications of a cytoplasmic domain of the sodium channel molecule could lead to loss of inactivation gating. Thus, it might be possible to use such modifications to identify a domain on the reconstituted protein that is associated with this important function. The utility of this approach is evident in results of the following studies carried out by Cooper in our laboratory (Cooper et al., 1987). 6. Biochemical Modification of the Reconstituted Protein

The rationale for these experiments is straightforward. Modification of the cytoplasmic surface of reconstituted proteins in vesicles with membrane-impermeant reagents should remove the inactivation mechanism. In the absence of an imposed membrane potential this should open the

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conductance pathway. We recognized that if the proteins were in a desensitized ("slow-inactivated") state insensitive to these reagents (Oxford et al., 1978; Rudy, 1978; Quandt, 1987), a change in the permeability state of the protein might not be observed. 1. EFFECTSOF NBA

AND

NBS

In these experiments, a vesicle suspension was treated with NBA or NBS at a particular concentration. At intervals after adding the reagent, an aliquot was removed and tested in 1-min radiotracer flux assays. Duallabeled isotopes were used, specifically "Na+ and 86Rb+. Because the activated Na channel selects strongly for Na" over R b + , a selective influx can help distinguish between induction of nonspecific membrane leak and a Na channel-mediated flux. In the experiment illustrated in Fig. 10, membranes were exposed to 1.1 mM NBA and the flux rates in 1-min uptake assays determined. In the first 2 min there was a marked stimulation of 22Na+uptake, but little change in the 86Rb+permeability. After this initial rise, the permeability to Na' declined to control levels by 8 min, after which developed a very slow leak which was nonselective to Na+ over Rb'. In several control experiments, parallel incubations with NBA and with BTX were carried out with the following conclusions. We observed that

0

2

4 6 8 10 12 14 Minutes of NBA incubation

16

18

FIG.10. Time course of flux activation by 1. I mM NBA. FTS vesicles were diluted 20fold into Tris-sulfate buffer and incubated at room temperature for 10 min. Controls, not shown here, indicated that tracer uptake with or without 2.5 pM BTX stabilized in 5 min and remained high for at least I hr. "Na ( 0 ) and *6Rb( 0 )uptake was determined in I-min assays, before or after the addition of NBA to 1.1 mM. (From Cooper ef d.,1987.)

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2.5 FM BTX, in the presence of I p M external TTX, produced fluxes approximately 2- to 2.5-fold greater than the peak of NBA stimulation. Further, the addition of 2.5 p M BTX to maximally NBA-stimulated vesicles did not yield signals exceeding those measured with BTX alone. Thus, the size of the NBA-elicited signal did not exceed about half that which could result from activation of the inside-out channel population. As indicated in Fig. 1 I , the NBA time course was concentration dependent. However, the peak of influx was not markedly increased or decreased by varying NBA. Rather the time for nse and fall was progressively shortened at higher reagent concentration. Thus, it seems likely that there are competing reactions, one leading to flux activation and one to deactivation, and they may exhibit comparable concentration dependencies. Failure to activate more than half of the outside-out oriented proteins may result from the substantial competing deactivation. In experiments of slightly different design, the sensitivity of the NBAelicted fluxes to TTX and QX-314 was examined. Samples of vesicles were treated with 500 p M NBA in the presence o r absence of the channel blockers, and the time courses of uptake for "Na' and S6Rb+were measured. As seen in Fig. 12A, sodium uptake, but not rubidium uptake was enhanced by the NBA. The enhanced sodium uptake was completely insensitive to I p M external TTX (Fig. 12B), but was completely blocked

n N

75

0

2

4

6

0

Incubation time, minutes

FIG.I I . Dependence of "Na uptake on NBA and NBS concentration. "Rb uptake, determined in dual-label assays. was low and omitted for clarity. Vesicles were diluted 20fold into Tris-sulfate buffer and incubated at room temperature for 10-20 min. Tracer uptake was determined before and after the addition of NBA at S mM (A),I . I mM ( 0 ) .or 0.S mM ( 0 ) .or of NBS at 5 pM (A).(From Cooper cf id.. 1987.)

g

354

WILLIAM S. AGNEW ET AL.

A

4:-

0

60 120 180

0

60 120 180

0

60 120

Time. seconds FIG.12. Rapid activation of sodium-selective uptake by SO0 FM NBA. ( A ) "Na uptake in the presence ( 0 )or absence (A)of SO0 pM NBA and "Rb uptake in the presence (m) or absence (IJ) of NBA. (B) Insensitivity of NBA-activated "Na uptake to external I pM TTX. 0 , NBA alone: 0.NBA in the presence of TTX; (A), TTX alone. (C) Complete block of "Na uptake by 3 m M external QX-314. 0 , NBA alone; 0.NBA in the presence of QX-314; and A , QX-314 alone. (From Cooper et d.,1987)

by the impermeant anesthetic QX-314 (3 mM) (Fig. 12C). In other experiments, similar to examples illustrated in protease studies below (Section IV,B,2), when internal plus external TTX was preequilibrated with the vesicles before addition of NBA, the stimulated flux was never observed. Thus, the pharmacological sensitivity suggests that sodium channels are rendered permeant by the NBA treatment, but only those proteins with their local anesthetic-binding sites exposed externally, and their 'ITX sites internally, are activated. In other experiments, NBS was tested for its ability to make the Na channel permeable. NBS has a similar specificity, but is generally more reactive than NBA. We found that NBS similarly produced a triphasic stimulation of flux, but at approximately 100-fold lower concentrations. Indeed, at fairly low concentrations it was apparently possible to titrate the reaction to different extents. In Fig. 11 it is seen that 5 p M NBS results in flux activation comparable to that seen with 500 pLM NBA, but the activated permeability fails to decline over many minutes. At these concentrations, NBS is approximately 1000 molar excess over the Na channel protein, suggesting that competing reactions may serve to quickly deplete the reagent. (Further additions would cause the decline evident in the NBA example.) It was observed that the NBS effect did not result in higher peak levels of flux activation than did NBA.

355

17. VOLTAGE-SENSITIVE CHANNELS

2. EFFECTS OF

PRONASE A N D

TRYPSIN

We proceeded, with similar experimental design, to test the ability of Pronase and the purified enzyme trypsin to convert the Na channel population to a permeable state. In an extensive series of experiments we found that Pronase stimulated **Na+influx, but not *'Rb+ influx, in a time course with a similar pattern to that seen with NBA and NBS. In Fig. 13A are shown the **Na+portions of three time courses carried out with 25, 100, and 500 pg Pronasdml. At the highest enzyme level, only the

300

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7 1 0

2

4

6

8

B'

I

I

I

b

y.

I

0

I

I

I

2 4 6 Minutes of proteolysis

I

8

FIG. 13. Dependence of "Na uptake on Pronase E and TPCK-treated trypsin concentration. *Rb uptake was low and i s not shown. ( A ) Vesicles were diluted 20-fold into Trissulfate and incubated at room temperature for 10-20 min. One-minute uptake of "Na was determined before or after the addition of Pronase E at 500 pg/ml (A),I 0 0 &ml ( 0 ) .or 25 pg/ml (0). ( B ) "Na uptake into 10-fold diluted vesicles before and after the addition of TPCK-treated trypsin at 7.5 p g h l (A)*2.S pg/ml ( o ) , or 1.25 &nl ( 0 ) .(From Cooper el id.. 1987.)

356

WILLIAM S. AGNEW ET AL.

declining phase of the activation is observed, while at 100 pg/ml, the rise and fall are seen, and at 25 pg/ml a more sustained flux activation is observed. It was consistently observed that the peak fluxes stimulated by Pronase were about 2-fold higher than those with NBA and NBS, and in control experiments were equal to that seen with 2.5 p M BTX in the presence of 1 pM external TTX, i.e., equal to that expected for complete activation of the inside-out oriented channel population. The reason for this seems to be that the declining phase of the reaction is slower, relative to flux activation, than that seen with NBA and NBS. Thus, more nearly complete activation may be achieved before deactivation becomes significant. Use of carefully limited proteolysis offers the possibility of assessing the extent and sites of modification associated with flux activation. Thus we further tested the utility of trypsin, a pure enzyme specific for sites involving lysine and arginine, and which can be stopped abruptly by addition of pancreatic trypsin inhibitor. [The active ingredient in Pronase, at least in physiological experiments,' was alkaline proteinase b, an enzyme specific for sites involving arginine side chains (Rojas and Rudy, 1976).] As seen in the example of Fig. 13B, trypsin was fully as active as Pronase in producing opened Na channels. In the illustrated experiments TPCKtreated trypsin at 1.25, 2.5, or 7.5 pg/ml, stimulated inftux. At progressively higher concentrations it was observed that flux activation was followed by a declining phase. Although not shown, "Rb' influx was not enhanced by this treatment: all experiments involved dual-label incubations. In all cases, trypsin activation was comparable to the Pronase signal, about 2to 2.5-fold that seen with NBA and NBS. In Fig. 14A are illustrated the pharmacological sensitivities of the trypsin-stimulated fluxes. The stimulated flux was minimally affected by addition of 1 p M TTX (external), but was completely inhibited by 3 mM QX-314, and failed to develop when TTX was preequilibrated with the vesicles before addition of the enzyme. Thus, the signal observed is TTX sensitive, but the TTX-binding sites are accessible only to the vesicle interior. In contrast, the local anesthetic-binding sites are accessible to the external solution, consistent with activation only of inside-out oriented proteins. In other experiments, the permeation selectivity of the Pronase- and trypsin-activated Na channels was compared to BTX-activated molecules. In the histograms of Fig. 14B, it is observed that the selectivity sequence expected for Na channels (Na' > K' > Rb' > Cs') was observed for the enzymatically activated fluxes. Indeed, the enzymatically activated proteins seemed to be rather more selective for Na' than the BTX-activated samples. This is expected from physiological studies in which BTX

[:

17. VOLTAGE-SENSITIVE CHANNELS A

250-

-# 0

200-

8 (c

0

#

150-

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3 100-

0

2

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0

BTX

PRON TRYP

Fic;. 14. (A) Sensitivity of trypsin-stimulated flux to external TTX. external QX-314. and external plus internal 'ITX. Aliquots of vesicles were subjected to freeze-thaw-sonication (FTS). In some samples. I pLM T T X was added before FTS. The vesicles were then diluted 20-fold into Tris-sulfate buffer containing no blockers (control). I pM TTX, or 3 mM QX314. After 15 min at room temperature. I-min "Na uptake assays were conducted either before or at intervals after the addition of trypsin to 2.5 pg/ml. 0.control: A,external TTX; a, external plus internal 'ITX: 0. external QX-314. ( B ) Cation selectivity of reconstituted sodium channels modified with BTX. with Pronase. or trypsin. Vesicles were incubated for 45 min at 30°C with or without 2.5 pmM BTX. diluted 10-fold into Tris-sulfate buffer. and incubated at room temperature for 10-20 min. For BTX-treated samples and controls, duplicate I-min uptake assays were performed with pairs of radiotracers. Net BTX-activated uptake was determined and is expressed as a percentage of "Na uptake. For protease-treated samples, I-min dual-label radiotracer assays were performed before or 1.5, 2.0. 3.5. or 4.0 min after the addition of Pronase E at SO pg/ml or TPCK-treated trypsin at 7.5 pg/ml. UPtake before enzyme addition was subtracted from protease-stimulated uptake to determine net flux. Data for each tracer (normalized to "Na uptake) are the average (with SD) of the four time points. (From Cooper er d..1987.)

has been found to decrease permeation selectivity significantly (Khodorov, 1978). The observations made here are consistent with the original hypothesis, namely that modification of the cytoplasmic surface of the protein could produce chronically activated channels. These channels would retain sensitivity to TTX and to local anesthetics, and would be ion selective. This is consistent with damage to or removal of the inactivation gating mechanism. Because local anesthetics such as QX-314appear not to bind to channels which are in the resting or inactivated state, we propose that local anesthetic-binding sites have been exposed by the modification re-

358

WILLIAM S. AGNEW ET AL.

action (Courtney, 1975; Hille, 1977; Cahalan, 1978; Yeh, 1978). This sensitivity is strongly suggestive, therefore, that the inactivation process has been removed. Further, we were encouraged that reagents with different specificities produced qualitatively similar results. Thus, most probably, damage to any of several sites in a general domain of the molecule results in inactivation removal. This could prove extremely useful in further studies, as mentioned below. We noted that physiological studies indicate that Pronase-modified or NBA-modified Na channels still undergo slow-inactivation or desensitization (Oxford et a / . , 1978; Rudy, 1978; Quandt, 1987). Thus, we might have expected the reactions observed not to be the formal equivalent of the physiological results. However, it is unclear whether the desensitization seen in physiological studies after modification is exactly equivalent to that seen before modification, although recent studies by Quandt (1987) have examined this question in some detail. It may well be that in our studies, the form of the protein observed in the declining phase of the chemical modification time cdurse represents an infrequently opening (i.e., desensitized) form of the channel. The true state of affairs, obviously, will be discovered in patch-clamp studies on the modified forms of the protein. These experiments are presently underway. 3. EXTENTOF PEPTIDE MODIFICATION CORRELATED WITH FLUXACTIVATION

Because all of the reagents tested here are capable of peptide cleavage, it seemed possible to examine the peptide fragmentation pattern to see the extent of the damage to the protein structure associated with flux activation. Further, if fragments could be closely correlated with flux activation, they could be isolated and sequenced. Given the known peptide sequence, with the exception of a region containing many consecutive glutamates, any four consecutive amino acids uniquely identify the location in the protein, permitting one to relate the cleavage site to models of the protein folding pattern. NBA and NBS do not always produce peptide cleavage (Oxford er al., 1978). Indeed, in preliminary experiments we found that the sodium channel peptide was not cleaved by NBS after maximal flux activation. Thus, the effects of these reagents appear to result from oxidation reactions alone. These are more difficult to localize than the cleavages produced by the enzymes. In experiments with trypsin, the time course of peptide cleavage was correlated with the time course of flux activation. In the experiments illustrated in Fig. 15A, 5 kg/ml trypsin was incubated for various periods

359

17. VOLTAGE-SENSITIVE CHANNELS

IB

1

1. 3

4

k t

t

B

T I

2

4

0

t

t

B

T

i T

3

0

0

100

200

750

Proteolysis time, s

t

t

T

B 4

t

T

t

B

FIG.15. Correlation of trypsin-stimulated radiotracer uptake with peptide cleavage. ( A ) Vesicles were slowly (5-10 min) passed over a small column of Tris-Dowex (ratio of column volume to vesicle suspension volume of I : 6 ) to exchange external Na' for Tris and create a sodium concentration gradient. Before, or at the indicated intervals (numbered arrows) after the addition of trypsin ( 5 )Lglml), samples were diluted 10-fold into Tris-sulfate buffer containing pancreatic trypsin inhibitor and radiotracers [for I-min uptake assays of "Na ( 0 ) and &Rb ( O ) ] or trypsin inhibitor alone (for SDS-PAGE). Samples were run on a 6-20%, linear gradient gel, stained with silver, and subjected to scanning densitometry ( B ) . Tracing numbers correspond t o the intervals on the uptake plot. Peaks indicated on densitometry tracings in B are as follows: I , sodium channel glycopeptide, -260 kDa; *, 100-kDa contaminant, presumably the a-subunit of Na,K-ATPase: 2. -130-kDa; fragment; 3, 70-kDd fragment; 4. 38-kDa fragment; 5 . 45-kDa fragment; and 6, 24-kDa fragment. T and B are top and bottom. (From Cooper P I at., 1987.)

-

360

WILLIAM S. AGNEW ET AL.

of time with a vesicle suspension. At the indicated times, the reaction was stopped by addition of pancreatic trypsin inhibitor. Aliquots were removed for 1-min dual-label ("Na' and *'Rb+) uptake assays. The remainder of each sample was denatured in SDS and analyzed by SDSPAGE. In the illustrated experiment, it was observed that the trypsin exposure rapidly induced a flux that was selective for **Na+over 86Rb+.Because low levels of trypsin were used, the declining phase of the reaction was avoided. For the samples taken at the indicated time points, SDS gels and their densitometer tracings are indicated in Fig. 15B. We observed that, within the first 40 sec, about 60% of the maximum flux was stimulated. The channel preparation used for these studies was about 90% homogeneous, contaminated only by a small amount of a peptide of 100 kDa, most likely the a-subunit of the Na,K-ATPase. After the 40-sec time point, the Na channel peptide decreased somewhat as new peptides appeared. A diffuse peptide of 135-150 kDa was observed: the fuzzy appearance of the band indicates extensive glycosylation. Other peptides of 70 kDa and, prominently, of 38 kDa were observed. These results suggest that the protein was not extensively damaged at this early stage of flux activation, and that as few as two cleavages might be responsible for the altered protein function. At 160 sec, the flux was about 90% of maximum. At this point, the large Na channel peptide was reduced in intensity by about 50%. Still observed were fragments of I50 and 38 kDa. The 70-kDa peptide was present, though somewhat reduced in intensity. In addition, by this stage of the incubation, peptides of 56 and 24 kDa began to emerge, becoming more prominent later on. Thus, the 70-kDa fragment might be the precursor to these smaller fragments. At the latest stages (280 sec) of the incubation, the initial Na channel peptide was still present, though it was clearly at a lower level than seen at the 160-sec point. It appears that part of the protein is rather resistant to proteolysis, and this may be that fraction of the molecules facing outsideout. (This may be due to steric protection by carbohydrate or, as predicted by models discussed earlier, to the exposure of only a small percentage of the protein mass to the extracellular surface.) The peptide fragmentation pattern at this later stage of the reaction is more complex and diffuse, indicating more extensive degradation had occurred. We conclude from these initial experiments that rather limited peptide cleavage can result in the functional modifications detected in the biochemical flux studies. Indeed, perhaps as few as two cleavages result in chronic opening of the channel conductance pathway. It should prove possible to carry out more precise correlation studies and to elute and

17. VOLTAGE-SENSITIVE CHANNELS

361

sequence the fragments involved. In patch-clamp recording studies, we hope to characterize the gating properties of each of the identified forms of the protein, to begin to identify the relationship between structural modification and functional behavior. In addition, because reagents of different chemical specificity produce similar functional consequences, it seems likely that a domain, rather than a very restricted site, is involved in occluding the channel pathway. Thus, proteases with widely different specifcities may result in similar functional states. Comparing the similar and dissimilar locations of protease attack could thus help in identifying domains involved in inactivation regulation. V.

CONCLUSION

Clearly, we have advanced considerably in our knowledge of the structure of Na channel proteins, and in our ability to reconstruct and investigate their functional behavior. The improvement of techniques for correlating selective chemical modifications of the protein structure with functional behavior should provide an important avenue for dissecting the mechanisms of gating, conductance, selectivity, and sensitivity to neurotoxins and drugs. These methods are likely to be an important complement to site-directed mutagenesis studies. REFERENCES Agnew. W. S. ( 1984). Voltage-regulated sodium channel molecules. Annrr. R ~ YPlrysiol. . 46, 5 17-530. Agnew. W. S.. and James, W. (1988). Polysialic acid substituents of the electroplax Na channel crossreact with antibodies raised against bacterial polysialic acid. Biophvs. J . 53. 536a. Agnew. W . S . . and Raftery. M. A. (1979). Soluhilized tetrodotoxin binding component from the electroplax of Elec/rop/iorrrs e/i~c/riiw.s;stability as a function of mixed lipid-detergent micelle composition. Bioc~liunris/rv10, 1912-1919. Agnew. W. S.. Levinson. S. R.. Brabson. J. S.. and Raftery, M. A. (1978). Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Elcc~/rop/iorrrsa1ecrricw.s membranes. Proi,. Ntrrl. A w d . Sci. U.S.A. 75, 2606261 I . Agnew. W. S.. Moore. A. C.. Levinson. S. R.. and Kaftery. M. A. (1980). Identification of a large peptide associated with the tetrodotoxin binding protein from EIec~irop/iorrrs decrricrrs. B i d i e n i . Bioip/rys. Rcs. Comrnrin. 92. XhO-866. Agnew. W. S.. Miller. J. A.. Ellisman. M . H.. Kosenberg. R. L.. Tomiko. S. A.. and Levinson. S. R. ( 1983). The voltage-regulated sodium channel from the electroplax of Elrc~ropliorrrse1ecrric~ri.s.Cold Spring Hcrrhor Syrrip. Q r i r r n r . Biol. 48, 165- 179. Agnew. W. S.. Rosenberg. R. L.. and Tomiko. S. A. (1986). Reconstitution of the sodium eluc.tric,ri.s. In “Ion Channel Reconstitution” (C. Miller. channel from E1ec~r~~pliortr.s ed.). pp. 307-322. Plenum. New York._

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Agnew. W. S.. Rudnick. H. F.. and Emerick. M. C. (1988). (Submitted.) Aldrich. R. W.. Corey. D. P., and Stevens. C. F. (1983). A reinterpretation of mammalian sodium channel gating based on single channel recording. Nriirrrc~(London) 306, 4 3 6 441. Armstrong. C. M. (1981). Sodium channels and gating currents. Physiol. R w . 61, 644-683. Armstrong. C. M.. and Bezanilla. F. (1977). Inactivation of the sodium channel 11. Gating current experiments. J. Gen. Physiol. 70, 562-590. Armstrong. C. M.. Bezanilla, F.. and Rojas. E. (1973). Destruction of sodium conductance inactivation in squid axons perfused with pronase. 1.Gen. P/rysic~l.62, 375-391. Bezanilla. F. (1985). Gating of sodium and potassium channels. J. Mcvnhr. Biol. 88, 97-111. Cahalan. M. D. (1978). Local anesthetic block of sodium channels in normal and pronasetreated squid giant axons. Biotphys. J . 23, 285-33 I . Cahalan. M. (1981). Molecular properties of sodium channels in excitable membranes. I n “The Cell Surface in Neuronal Function” (C. W. Cotman, G. Poste. and G. L. Nicolson. eds.). pp. 1-47. Elsevier. Amsterdam. Cahalan. M. D.. and Begenisich. T. (1976). Sodium channel selectivity. Dependence on internal permeant ion concentrations. J . Gen. Physiol. 68, I 11-125. Cooper. E. C.. Tomiko. S. A.. and Agnew. W. S. (1987). Reconstituted voltage-sensitive sodium channel from Elec~troplzor~rs c~lecirir~ns: Chemical modifications that alter regulation of ion permeability. Proc. Nail. Acrid. Sci. U . S . A . 84, 6282-6286. Correa. A. M.. Zhou. J.. and Agnew. W. S. (1988a). Optimized fusion of native or reconstituted membranes to artificial liposomes for single channel recording. B i ~ t p l i yJ~..Ahsir. (in press). Correa. A. M.. and Agnew. W. S. (1988b). Biophvs. J . (in press). Courtney. K. R. ( 1975). Mechanism of frequency-dependent inhibition of sodium currents rog myelinated nerve by the lidocaine derivative GEA 968. J . Plicirincic,ol. E x p . Tlier. 195, 225-236. Ebert. G . A.. and Goldman. L. (1976). The permeability of the sodium channel in Mv.ricdcr to the alkali cations. J. Gen. Physiol. 68, 327-340. Edelman. G. ( 1983). Cell adhesion molecules. Science 219, 450-457. Emerick. M. C.. and Agnew. W. S. (1988a). Biophys. J. Ahstr. (in press). Emerick. M. C.. and Agnew. W. S. (1988b). (Submitted). Finne. J. ( 1985). Polysialic acid-a glycoprotein carbohydrate involved in neural adhesion and bacterial meningitis. Trends Biochetn. Sci. 10, 129-132. Green. W. N.. Weiss. L. B.. and Andersen, 0. S. (1987a). Batrachotoxin-modified sodium channels in planar lipid bilayers. Ion permeation and block. J . Crn. Physiol. 89, 841872. Green, W. N.. Weiss. L. B.. and Andersen. 0. S. (1987b). Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures. J. Geiz. f h y s i o l . 89, 873-904. Guy. R. H.. and Seetharamulu. P. (1986). Molecular model of the action potential sodium channel. Proc. Nail. A w d . S1.i. U.S.A. 83, 508-512. Henderson. R.. and Wang. J . H. (1972). Solubilization of a specific tetrodotoxin binding component from garfish olfactory nerve membranes. Bioc~hc~mistry 11, 4565-4569. Hille. B. (1971). The permeability of the sodium channel to organic cations in myelinated nerve. J. Gen. P/i.v.siol. 58, 599-619. Hille. B. (1972). The permeability of the sodium channel to metal cations in myelinated nerve. J. GiJn.Physiol. 59, 637-658.

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Hille. B. (1977). Local anesthetics: hydrophilic and hydrophobic pathways for the drugreceptor reaction. J . Gen. Physiol. 69, 497-5 15. Hodgkin. A. C.. and Huxley. A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. J . P/rysio/. ( L o m h r ) 117, 500-544. Hoffman. J. F.. and Laris. P. C. (1974). Determination of membrane potentials in human and A/tip/ii/rni(r red blood cells by means of a fluorescence probe. J. Plrysiol. (London) 239. 519-552. James. W. M.. and Agnew, W. S. (1987a). Evidence for alpha-2.8-linked polysialic acid in the glycopeptide of the voltage-sensitive Na channel from Elac~rroplror/rsc 4 c ~ r r i c w . SOC. Nrrrrosci. Ahs/r. 13, 29.3. James. W. M.. and Agnew. W. S. (1987b). Multiple oligosaccharide chains in the voltagesensitive Na channel from E/eclrop/rorrr.s C / P L ~ / ~ I ‘ C I I S : evidence for alpha-2.8-linked polysialic acid. B i o c k w ~ Biophys. . Res. C ‘ o ~ ~ i n r / r148. ~ r . 817-826. James. W. M.. and Agnew. W. S. (1988a). Sialic acid-specific lectin affinity resin for purification and reconstitution of the electroplax Na channel. Biophys. J. Ah.str. (in press). James. W. M.. and Agnew. W. S. (I988b). Polysialic acid substituents of the eel electroplax Na channel cross-react with antibodies raised against bacterial polysialic acid. Bic~plry.s. J . A h w . (in press). James. W. M.. and Agnew. W. S. (1988~).I n preparation. Khodorov. B. I.( 1978). Chemicals as tools to study nerve fiber sodium channels: Effects of batrachotoxin and some local anesthetics. / / I “Membrane Transport Processes’’ (D. C. Tosteson. Y. A. Ovchinnikov. and R. Latorre. eds.). pp. 153-174. Raven. New York. Koshland. D. E.. Jr. (1970). The molecular basis for enzyme regulation. //I “The Enzymes” (P. D. Boyer. ed.). Vol. I . pp. 342-397. Academic Press. New York. Levinson. S. R. (1975). Studies on excitable membrane proteins. Ph.D. Thesis, Cambridge Univ.. Cambridge. England. Levinson. S. R.. Duch. 0. S.. Urban. B. W.. and Recio-Pinto. E. (1986). The sodium channel from Elccvroplrorrts l k w i c x s . A m . N. Y . Actrcl. S ( , i . 479, 162- 178. Lyles. J. M.. Linnemann. D.. and Bock. E. (19x4). Biosynthesis of the 02-cell adhesion molecule: Post-translational modifications. intracellular transport. and developmental changes. J . Cell Biol. 99, 2082-2091. Miller. J. A , . Agnew. W. S.. and Levinson. S. R. (19x3). Principal glycopeptide of the tetrodotoxin/saxitoxin binding protein from E / c ~ / r i ~ p / r o r ri~/ec/ric.r/s: is isolation and partial chemical and physical characterization. ~ h J c / l < ’ / t l ~ . Y /22, / . y 462-470. Monod. J.. Wyman. J.. and Changeux. J.-P. (1965). On the nature of allosteric transitions: a plausible model. J . Mol. B i ~ l 12. . 88-1 I X . Nakamura. Y.. Nakagima. S.. and Grundfest. H. (1965). Analysis of spike electrogenesis and depolarizing K inactivation in electroplaques of E / ~ T / ~ o ~ / I c(4Je c~~ / r/k./ rs. c .J . Gcn. P / I ~ s ~ 49. o / . 32 1-350. Noda. M.. Shimizu. S.. Tmabe, T.. Takai. T.. Kayano. T.. Ikeda. T.. Takahashi. H.. Nakayama. H.. Kanaoka. y . . Minamino, N., Kangawa. K.. Matsuo. H.. Raftery. M. A,. Hirose, T.. Inayama. S . . Hayashida. H.. Miyata. T.. and Numa. S. (1984). Primary structure of Elc~/rop/rorr/sc4cc.tric.ri.v sodium channel deduced from c 0 N A sequence. N ( / / / / fLO/ld(J/l) V~ 312, 121-127. Noda. M.. Ikeda. T.. Kayano. T.. Suzuki. H., Takeshima. H.. Kurasaki. M.. Takahashi, H.. and Numa. S. (1986). Existence of distinct sodium channel messenger RNAs in rat brain. Nrrtrrrc (Lo/rdori)320, 188-192.

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Nonner. W. (1980). Relations between the inactivation of sodium channels and the immobilization of gating charge in frog myelinated nerve. J . Physiol. (London) 299,573-603. Oxford. G. S.. Wu. C. H.. and Narahashi. T. (1978). Removal of sodium channel inactivation in squid giant axons by N-bromoacetamide. J. Gen. Pliysiol. 71, 227-247. Popot. J.-L.. and Changeux. J.-P. (1984). Nicotinic receptor of acetylcholine: structure of an oligomeric integral membrane protein. Plivsiol. Rev. 64, 1162-1239. Quandt. F. N. (1987). Burst kinetics of sodium channels which lack fast inactivation in mouse neuroblastoma cells. J . Pliysiol. (London) 392, 563-585. Regier. F.. Grumet. M.. and Edelman. G. M. (1985). N-CAM at the vertebrate neuromuscular junction. J . Cell Biol. 101, 285-293. Ritchie. J. M..and Rogart. R. B. (1977). The binding of saxitoxin and tetrodotoxin to excitable tissue. Reit. Plivsiol. Biocliein. Plitrrmrrcol. 79, 1-50. Rojas. E.. and Rudy. B. (1976). Destruction of the sodium channel inactivation by a specific protease in perfused nerve fiber from Loligo. J . Pliysiol. (London) 262, 501-531. Rosenberg. R. L.. Tomiko, S. A.. and Agnew. W. S. (1984a). Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of E1eciropliorrr.s e1ecvricrr.s. Proc. Ntiil. Acad. Sci. U . S . A . 81, 1239- 1243. Rosenberg. R. L.. Tomiko. S. A.. and Agnew. W. S. (1984b). Single-channel properties of the reconstituted voltage-regulated Na channel isolated from the electroplax of Eleciropliorirs elec.iricrrs. f r o c . Nrril. Actrd. Sci. U . S . A . 81, 5594-5598. Rudy. B. (1978). Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance. J. Pliysiol. (London) 283, 1-21. Rutishauser. U.. Watanabe. M.. Silver. J.. Troy. F. A.. and Vimr. E. R. (1985). Specific alteration of N-CAM-mediated cell adhesion by an endoneuraminidase. J . Cell Bid. 101, 1842-1849. Salkoff, L.. Butler, A., Aguan, W.. Scavarda. N., Giffen, K., Ifune. C.. Goodman, R., and Mandel. G. (1987). Genomic organization and deduced amino acid sequence of a putative sodium channel gene in Drosopliilu. Science 237, 744-749. Salkoff, L., Butler, A.. Scavarda, N.. and Wei, A. (1987). Nucleotide sequence of the putative sodium channel gene from Drosopliilu: the four homologous domains. N i d e i c Acids Res. 15, 8569-8572. Schwarz, T. L.. Tempel, B. L.. Papazian, D. M.,Jan, Y. N., and Jan, L. Y. ( 1987). Multiple potassium-channel components are produced by alternative splicing at the Shciker locus in Drosophilu. Nuiirre (London). (in press). Sevcik, K. C.. and Narahashi, T. (1975). Effects of proteolytic enzymes on the ionic conductance of the squid axon. J. Memhr. Bid. 24, 329-339. Sims. P. J . , Waggoner, A. S.. Wang, C.-H., and Hoffman. J. F. (1974). Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biocliemisiry 13, 33 15-3330. Spande. T. F., Green. N. M..and Witkop, B. (1966). The reactivity toward N-bromosuccinimide of tryptophan in enzymes, zymogens, and inhibited enzymes. Bioc+ieinisiry 5, 1926-1933. Tanabe. T., Takeshima, H., Mikami, A.. Flockerzi, V., Takahashi. H.. Kangawa. K., Kojima. M., Matsuo, H., Hirose, T., and Numa, S. (1987). Primary structure of the receptor for calcium channel blockers from skeletal muscle. Ntiirrre (London) 328, 313-318. Ternpel, B. L.. Papazian, D. M.. Schwartz. T. L.. Jan. Y. N.. and Jan. L. Y. (1987).Sequence of a probable potassium channel component encoded at Shuker locus of Dro.sop/iiltr. Science 237, 770-775.

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Tomiko. S. A.. and Agnew. W. S. (1987). Selectivity sequence of metal and organic cafions for batrachotoxin-modified reconstituted Na channels purified from E/w/rophorirs P ~ C tric'its. SOC. Nerrrosci. Ahstr. 12, 410.5. Tomiko. S. A , , and Agnew, W. S. (1988). (Submitted). Tomiko. S. A,. Rosenberg. R. L.. Emerick. M.C., and Agnew. W. S . (1986). Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ. Biochemisti? 25, 2 162-2 174. Vandenberg, C. A., and Horn, R. (1984). lnactivation viewed through single sodium channels. J. Gen. Pliysiol. 84, 535-564. Wang. G. K. (1984). Modification of sodium channel inactivation in single myelinated nerve fibers by methionine-reactive chemicals. Biopliys. J. 46, 121-124. Yeh. J. 2. (1978).Sodium inactivation mechanism modulates QX314 block of sodium channels in squid axons. Bioplrvs. J. 24, 569-574.

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Part IV

Calcium Channels

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

Chapter 18

Molecular Properties of Voltage-Sensitive Calcium Channels WILLIAM A . CATTERALL, MICHAEL J . SEAGAR,' MASAMI TAKAHASHI,' AND BENSON M . CURTIS" Department of Pharmacology Universiiy of Washington Seattle, Washington 98195

I. II.

111.

IV.

lntroduct ion Identification and Purification of Calcium Channels from Skeletal Muscle A. Organic Calcium Antagonists as Probes of Calcium Channel Structure and Function B. Molecular Size of the Calcium Antagonist Receptor of the Calcium Channel C. Identification of Protein Components of the Calcium Antagonist Receptor by Covalent Labeling D. Characterization of the Detergent-Solubilized Calcium Antagonist Receptor E. Skeletal Muscle Transverse Tubules as a Model System for Examination of the Biochemical Properties of Calcium Channels F. Purification and Characterization of the Calcium Antagonist Receptor from Skeletal Muscle Transverse Tubules G. Phosphorylation and CAMP-Dependent Regulation of Calcium Channels Functional Properties of the Purified Calcium Antagonist Receptor in Phospholipid Vesicles Subunit Structure of Dihydropyridine-SensitiveCalcium Channel A. Polypeptide Components B. Subunit Glycosylation C. Covalent Labeling of Calcium Channel Subunits D. Analysis of Noncovalent Subunit Interactions E. An Oligomeric Model for the Dihydropyridine-SensitiveCalcium Channel

'Permanent address: CNRS-INSERM. Laboratoire de Biochimie, Fac. Med. Nord. Marseille. France. 'Permanent address: Mitsubishi-Kasei Institute of Life Sciences, Machida-Shi. Tokyo. Japan. 'Present address: Immunex Corp.. Seattle. WA.

369 Copyright (1 ' I Y X X hy Academic Pre5s. Inc. All rights of repniduction in any form reserved.

WILLIAM A. CATTERALL ET AL.

370 V. VI.

lmmunospecific Identification of Calcium Channel Components in Other Tissues Conclusion References

1.

INTRODUCTION

In muscle tissues, voltage-sensitive calcium channels mediate calcium influx during cellular depolarization and play an important role in excitation-contraction coupling (reviewed in Hagiwara and Byerly, I98 1 ; Reuter, 1979). In neurons, they produce action potentials in dendrites (Llinas et al., 1981; Schwartzkroin and Slawsky, 1977) and couple changes in membrane potential at nerve terminals to the release of neurotransmitter (Katz and Miledi, 1969). Multiple classes of calcium channels have been distinguished in neurons (Armstrong and Matteson, 1985; Carbone and Lux, 1984; Nowycky et al., 1985) and in cardiac muscle cells (Nilius et al., 1985; Bean, 1985). This article focuses on molecular properties of calcium channels that are blocked by dihydropyridine calcium antagonists. These are the most prominent calcium channels in smooth, cardiac, and skeletal muscle and they are also present in neurons and neurosecretory cells. II. IDENTIFICATION AND PURIFICATION OF CALCIUM CHANNELS FROM SKELETAL MUSCLE A. Organic Calcium Antagonists as Probes of Calcium Channel Structure and Function

Calcium channels are inhibited by three different classes of organic antagonists: the dihydropyridines including nifedipine and nitrendipine, the phenylalkylamines and diphenylalkylamines including verapamil, and the thiobenzazepine diltiazem (Triggle, 1982). 3H-Labeled derivatives of nitrendipine and other dihydropyridines have been prepared and their binding to high-affinity receptor sites in homogenates and membrane preparations from excitable tissues has been extensively examined (Janis and Scriabine, 1983). A single class of high-affinity binding sites is observed with a K D for nitrendipine in the range of 0.1 to 1 .O nM. The structure-activity relationships for occupancy of these high-affinity binding sites correlates closely with the concentrations of various dihydropyridines required to block contraction of smooth muscle over a wide range of K D values (Bolger et al., 1982). Similar relative structure-activity relationships are observed

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in other tissues, but the absolute concentration required to block calcium currents is often substantially greater than that required to occupy the high-affinity nitrendipine binding sites (Almers et al., 1981; Lee and Tsien, 1983). This apparent discrepancy has now been resolved by the finding that dihydropyridine binding and block of calcium channels is highly voltage sensitive (Bean, 1985; Schwartz et al., 1985; Uehara and Hume, 1985). In cardiac cells, half-maximal block of calcium currents at the resting membrane potential requires 700 nM nitrendipine. In contrast, when cells are depolarized to - 20 mV to inactivate 70% of the calcium current, only 0.8 nM nitrendipine is required for half-maximal block. This value is in close agreement with K , values for high-affinity binding sites in cardiac membrane preparations which are depolarized by tissue homogenization and cell disruption (Janis and Scriabine, 1983). These results resolve an important discrepancy between previous biochemical and physiological measurements and provide additional strong support for the view that measurements of specific binding of nitrendipine and other dihydropyridines represent drug interaction with calcium channels or a closely associated regulatory component. The binding of nitrendipine to its receptor site is modulated via an allosteric mechanism by diltiazem and various mono- and diphenylalkylamines such as verapamil (Murphy et af., 1983) which interact with two separate receptor sites (Garcia et al., 1986). Verapamil decreases nitrendipine binding and enhances the rate of dissociation of the nitrendipine-receptor complex, whereas diltiazem increases binding and slows the rate of dissociation of the nitrendipine-receptor complex. Thus, the calcium antagonist receptor of the calcium channel contains at least three drug binding sites, one specific for dihydropyridines and one each for diltiazem and phenylalkylamines, which interact allosterically in modulating calcium channel function and have opposite effects on binding of dihydropyridine calcium channel blockers. The discovery that the binding of dihydropyridine catcium antagonists is state dependent and favors the inactivated state of the channel suggests that they may block channel function by altering normal channel gating rather than by physically occluding the ion-conducting pore. This conclusion is illustrated more clearly by the discovery of dihydropyridine calcium channel activators, exemplified by BAY K 8644 (Schramm et al., 1983). These agents bind at the same receptor site as the dihydropyridine channel blockers, but cause smooth muscle contraction and stimulation of the heart by favoring activation of calcium channels at more negative membrane potentials (Hess et al., 1984; Kokubun and Reuter, 1984). Thus occupancy of the same receptor site by agents of closely related structure

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can cause either channel block or channel activation. These results provide firm evidence that the dihydropyridine receptor modulates the gating of the calcium channel as its principal mode of action. The development of radioactively labeled derivatives of high affinity and specificity now allows the use of these drugs as probes to identify and isolate the protein components of calcium channels and understand their molecular properties. B. Molecular Size of the Calcium Antagonist Receptor of the Calcium Channel

The first estimates of the molecular size of the component(s) of the calcium channel that bind dihydropyridines have been derived from radiation inactivation measurements. Estimates of the target size for inactivation of [3H]nitrendipinebinding to receptor sites are 180,000 or 210,000 for skeletal muscle (Ferry et al., 1983; Norman et al., 1983), 210,000 for brain (Norman et al., 1983), and 280,000 for guinea pig ileum (Venter er al., 1983). The apparent target size derived from radiation inactivation studies depends upon the radiolabeled calcium antagonist used in the receptor binding assay and on the assay and irradiation conditions, suggesting that the calcium antagonist receptor is a complex oligomeric structure (Ferry et al., 1983). C. Identification of Protein Components of the Calcium Antagonist Receptor by Covalent Labeling

The dihydropyridines are amenable to structural modification at several positions and therefore are potentially valuable tools for affinity chromatography and covalent labeling. Nitrendipine itself is photolabile and can be covalently incorporated into a polypeptide with apparent molecular weight of 32,000 in cardiac sarcolemma by high intensity irradiation (Campbell er al., 1984). A photoreactive azidobenzoyl analog of nitrendipine has been prepared by Ferry el al. (1984). This compound labels a polypeptide with an apparent molecular weight of 145,000 in skeletal muscle transverse tubule membranes. In each case, the covalent labeling reactions described were prevented by incubation with excess unlabeled calcium antagonist, suggesting that the polypeptides labeled are components of the calcium antagonist receptor in these different tissues. Either the calcium antagonist receptor from these different tissues has different polypeptide components or, as argued below, the receptor is an oligomer of subunits of different molecular weight which are differentially labeled by the probes used.

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D. Characterization of the Detergent-Solubilized Calcium Antagonist Receptor

Since it is likely to be an intrinisic membrane protein, the first essential step in purification and biochemical characterization of the calcium antagonist receptor is solubilization from an appropriate membrane source and characterization of the solubilized protein. After a survey of several detergents, we concluded that digitonin is the most effective detergent for solubilization of a specific ['Hlnitrendipine-receptor complex from brain and skeletal muscle transverse tubule membranes (Curtis and Catterall. 1983, 1984). Up to 40% of the receptor-ligand complex is solubilized. The dissociation of bound [3H]nitrendipine from the complex is accelerated by verapamil and slowed by diltiazem through allosteric interactions between the nitrendipine binding site and the binding sites for those ligands as previously observed in intact membranes. These results show that the three different binding sites for calcium antagonist drugs remain associated as a complex after detergent solubilization of the calcium antagonist receptor and provide further support for the conclusion that a specific receptor complex has been solubilized under conditions which allow retention of the functional allosteric regulation of dihydropyridine binding. Sedimentation of the solubilized ['Hlnitrendipine-receptor-digitonin complex through sucrose gradients gives a single peak of specifically bound nitrendipine with a sedimentation coefficient of 19 to 20 S. Comparison of the sedimentation behavior of the solubilized complex from brain, heart, and skeletal muscle indicates that they have identical size. These results provide support for the view that the calcium antagonist receptor in different tissues is quite similar. Many plasma membrane proteins are glycosylated during their synthesis and transport to the cell surface. The solubilized calcium antagonist receptor from brain or skeletal muscle is specifically adsorbed to and eluted from affinity columns with immobilized wheat germ agglutinin or other lectins (Borsotto et ul., 1984; Curtis and Catterall, 1983. 1984; Glossmann and Ferry, 1983). Evidently, one or more of the subunits of the calcium antagonist receptor are glycoproteins.

E. Skeletal Muscle Transverse Tubules as a Model System for Examination of the Biochemical Properties of Calcium Channels Skeletal muscle fibers have substantial voltage-activated calcium currents that have been measured under voltage clamp (Sanchez and Stefani,

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1978). These currents originate almost entirely in the transverse (T) tubular system and are blocked by dihydropyridine calcium antagonists (Almers er al., 1981). They have been presumed to play a role in excitation-contraction coupling, although direct evidence for such a role has not been obtained. Transverse tubule membranes can be extensively purified from skeletal muscle by a combination of differential and density gradient centrifugation (Rosemblatt et al., 1981). Antibodies prepared against the most highly purified fractions of T-tubule membranes stain only T-tubules and not sarcolemma or sarcoplasmic reticulum of intact muscle, indicating a high degree of purity of this preparation. Analysis of [3H]nitrendipine binding to membrane fractions from skeletal muscle reveals a specific localization of the calcium antagonist receptor in the T-tubule fraction (Fosset et al., 1983; Glossmann and Ferry, 1983). These membranes contain a 10-fold greater concentration of calcium antagonist receptor than any other membrane preparation described to date (Janis and Scriabine, 1983). Since T-tubule membranes are the most enriched source of calcium antagonist receptors and they have a substantial voltage-activated calcium current that is blocked by dihydropyridines, they provide a favorable experimental preparation for examination of the molecular properties of the calcium antagonist receptor and its relationship to voltage-sensitive calcium channels. It is anticipated that the T-tubule calcium channel will resemble those of other tissues which are blocked by dihydropyridines, and therefore that information on the molecular properties of this calcium channel will give insight into others. F. Purification and Characterlzation of the Calcium Antagonist Receptor from Skeletal Muscle Transverse Tubules

We have purified the calcium antagonist receptor solubilized from Ttubule membranes by digitonin 330-fold by affinity chromatography on wheat germ agglutinin-Sepharose, ion-exchange chromatography on DEAE-Sephadex, and velocity sedimentation through sucrose gradients (Curtis and Catterall, 1984). The purified preparation contained 1950 pmol calcium antagonist receptor per milligram of protein, 81% of the value expected for a protein of 416,000 Da (see below) which binds one calcium antagonist per molecule. Analysis of the purified preparation by polyacrylamide gel electrophoresis (PAGE) after denaturation in sodium dodecyl sulfate (SDS) without reduction of disulfide bonds revealed three major protein bands, as illustrated in Fig. I . Their molecular weights are a,167,000; p, 50,000; and y, 33,000. All three of these polypeptides quantitatively comigrate with the [3H]nitrendipine-receptor complex during

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FIG.I. Noncovalently associated components of the purified calcium channel. The dihydropyridine-sensitive calcium channel from skeletal muscle T-tubules was purified as described by Curtis and Catterall (1984). Lane I. purified calcium channels were analyzed by SDS-PAGE without reduction of disulfide bonds and proteins were visualized by silverstaining. Three size classes of polypeptide components are recognized under these conditions: a. 167.000; p, 50,000; and y, 30.000. Lane 2, the purified calcium channel was phosphorylated by reaction with CAMP-dependent protein kinase and [y-”P]ATP as described by Curtis and Catterall (1985). analyzed by SDS-PAGE as for lane I. and visualized by autoradiography. The a and p protein bands are labeled by 12P.

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velocity sedimentation in sucrose gradients and therefore are likely to be components of the calcium antagonist receptor. G. Phosphorylation and CAMP-Dependent Regulation of Calcium Channels

The classic work of Reuter, Tsien, and their colleagues (Reuter, 1974, 1983; Tsien et ul., 1972) established that the positive inotropic effect of epinephrine and norepinephrine on the heart is mediated by a cyclic AMPdependent increase in inward calcium current. This effect is due to an increase in the number of calcium channels that are active during the cardiac action potential (Cachelin et d., 1983; Tsien et ul., 1983). These effects are mimicked by intracellular injection of the catalytic subunit of CAMPdependent protein kinase, suggesting that protein phosphorylation mediates the effects of cAMP (Brum et al., 1983). l t is uncertain whether the substrate for CAMP-dependent phosphorylation is the calcium channel itself or another regulatory component. In addition to these studies on mammalian heart, calcium channels in mammalian dorsal root ganglion neurons and both calcium channels and calcium-activated K' channels in molluscan neurons are regulated by CAMP-dependent protein phosphorylation (reviewed in Rossie and Catterall, 1987). Recent results indicate that calcium channels in cultured skeletal muscle cells are also regulated in this manner (Schmid et ul., 1986). Thus, modulation of calcium channel properties by cellular regulatory processes may be a general phenomenon. Although regulation of calcium channels via a pathway involving cAMP and protein phosphorylation is now well established, it is not known with certainty whether the site of phosphorylation is the calcium channel itself or another intracellular protein which in turn regulates channel function. As a step toward resolving this question, we have investigated whether the subunits of the purified calcium antagonist receptor can serve as substrates for CAMP-dependent protein kinase in the purified state and in intact T-tubule membranes (Curtis and Catterall, 1985). When incubated with physiological concentrations of CAMP-dependent protein kinase, proteins in the a- and P-subunit bands are phosphorylated as illustrated in Fig. I . Up to 0.85-0.9 mol "P per calcium antagonist receptor can be incorporated into each subunit band at rates that are consistent with a physiologically significant phosphorylation reaction. In contrast, the ysubunit is not a substrate for CAMP-dependent protein kinase, even under forcing reaction conditions (Curtis and Catterall, 1985). These results identify proteins in the a- and p-subunit bands of the calcium antagonist

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receptor as potential sites of regulation of the voltage-sensitive calcium channel by CAMP-dependent phosphorylation. 111.

FUNCTIONAL PROPERTIES OF THE PURIFIED CALCIUM ANTAGONIST RECEPTOR IN PHOSPHOLIPID VESICLES

We purify the calcium antagonist receptor as a preformed complex with the calcium antagonists [3H]nitrendipine or [3H]PN 200-1 10. After solubilization, it retains allosteric interactions among the separate binding sites for verapamil. diltiazem, and dihydropyridines (Curtis and Catterall, 1983). However, other aspects of calcium channel function cannot be assessed in detergent solution. It is important, therefore, to return the purified calcium antagonist receptor to a membrane environment and to determine whether the purified protein is capable of mediating voltage-dependent calcium flux. In order to maximize the probability of purification of the calcium channel in an active state, the calcium antagonist receptors in T-tubule membranes were incubated with sufficient (3H]PN 200-1 10 to label approximately 1% of the binding sites. This label was used to identify the calcium antagonist receptor during purification as in previous studies. The T-tubule membranes were then incubated in an excess of the specific calcium channel activator BAY K 8644 so that the remaining 99% of dihydropyridine sites would be occupied this agent. The calcium antagonist receptors were then solubilized in digitonin and purified in the continued presence of BAY K 8644 using previously described procedures (Curtis and Catterall, 1986). Phosphatidylcholine for reconstitution was solubilized in the zwitterionic detergent CHAPS because it is poorly soluble in digitonin. Purified calcium antagonist receptor dispersed in digitonin was then mixed with phosphatidylcholine dispersed in CHAPS and single-walled phospholipid vesicles were formed by removal of the detergents by molecular sieve chromatography. Analysis of the resulting vesicle preparations by sucrose density gradient sedimentation show that the purified calcium antagonist receptors are quantitatively incorporated into phosphatidylcholine vesicles and I5 to 25% of vesicles contain at least one calcium antagonist receptor. These preparations therefore provide a suitable system for analysis of the ion transport properties of this purified protein. Initial rates of influx of 4'Ca" or '33Ba2+into reconstituted phosphatidylcholine vesicles were measured under countertransport conditions in which the intravesicular compartment contains a high concentration of unlabeled Ca" or Ba" . These conditions greatly increase the amount of

WILLIAM A. C A T E R A L L ET AL.

378

45Ca2' or '33Ba2+uptake required to achieve isotopic equilibrium and greatly slow the approach to isotopic equilibrium as described previously for reconstituted sodium channels (Talvenheimo et al., 1982). They therefore maximize any ion flux mediated by reconstituted channels. Under these conditions, calcium influx into phosphatidylcholine vesicles containing reconstituted calcium antagonist receptors was two- to threefold greater than influx into protein-free phosphatidylcholine vesicles in the presence of BAY K 8644 (Fig. 2). This increase is completely blocked by verapamil at a concentraion of 100 pM. If the calcium channel activator BAY K 8644 is removed from the vesicle preparation by molecular sieve chromatography, 45Ca2' influx is markedly reduced. These results show that at least a fraction of the purified calcium antagonist receptors can function as calcium channels when incorporated into phosphatidylcholine vesicles. The inhibition of the reconstituted calcium channels by different conM was centrations of organic calcium channel blockers from lo-' to

800

I-

/

600

400

200

I-/

/

-

I

4

10

Time(minutes) FIG.2. Calcium flux mediated by the purified calcium channel. The calcium antagonist receptor from skeletal muscle transverse tubules was purified and incorporated into phosphatidylcholine vesicles as described by Curtis and Catterall (1986). Initial rates of "Ca" influx into reconstituted vesicles were measured in the presence of I pbf BAY K 8644 (0). I pM BAY K 8644 and 100 pM verapamil (A). or no added drugs (0).

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examined. Half-maximal inhibition was observed with approximately 1.5 p M verapamil, 1.0 p M D600, or 0.2 p M PN200-110. These concentrations are similar to those that give half-maximal inhibition of voltage-activated calcium currents in intact skeletal muscle fibers consistent with the conclusion that the calcium flux in reconstituted vesicles is mediated by functional purified calcium channels. To determine whether the calcium influx stimulated by BAY K 8644 and blocked by PN200-I 10 and verapamil required the presence of the subunits of the calcium antagonist receptor and not other detectable proteins, purified preparations were sedimented through sucrose gradients and each fraction was examined for bound [3H]PN200-l10, polypeptide composition, and ability to mediate ‘33Ba’+influx when incorporated into phosphatidylcholine vesicles (Curtis and Catterall, 1986). A close quantitative correlation was observed between the presence of the a-,(3-, and y-subunit bands of the calcium antagonist receptor, as analyzed by SDSPAGE without reduction of disulfide bonds, and the ability to mediate ‘33Ba” flux. No other polypeptides were rsted whose presence correlated with ion flux activity. Thus, these results are also consistent with the conclusion that the purified calcium antagonist receptor is capable of mediating ion flux with the pharmacological characteristics expected of voltage-sensitive calcium channels. IV. SUBUNIT STRUCTURE OF DIHYDROPYRIDINE-SENSITIVE CALCIUM CHANNEL

A.

Polypeptide Components

As shown above (Fig. I). analysis of the purified protein by SDS-PAGE under alkylating conditions and silver staining revealed three classes of polypeptides that we have designated (Y (167 kDa). p (54 kDa), and y (30 kDa) (Fig. 3. lane I ) . When disulfide bonds were cleaved with dithiothreitol. the a band split into two clearly resolved protein populations of 175 and 143 kDa (Fig. 3, lane 2). In the initial studies from this laboratory, the anomalous behavior of t h e a polypeptide was ascribed to partial cleavage and/or reformation of intrachain disulfide bonds, resulting in a variable fraction of the protein with smaller apparent size (Curtis and Catterall, 1984).The more recent use of a battery of specific labeling methods has now shown that the 175- and 143-kDa polypeptides are two distinct calcium channel subunits, a,and a:, which have similar size but clearly different properties (Takahashi rt a / ., 1987).

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FIG.3. Differential labeling ofcalcium channel subunits. Lanes I and 2. purified calcium channels were analyzed by SDS-PAGE and silver staining with or without reduction of disulfide bonds as indicated beneath each lane. Lane 3. polypeptides. separated by SDSPAGE with reduction of disulfide bonds, were electrophoretically transferred to nitrocellulose strips and immunolabeled by incubation with PAC-10. or followed by incubation with '"1labeled protein A. washing, and autoradiography. Lane 4, calcium channel subunits were transferred to a nitrocellulose sheet and labeled with ["'IjConA. Lane 5 . calcium channel subunits were labeled directly in the polyacrylamide gel with ["'IIWGA. Lane 6. p/ioior!fliniiy Ittheling.: T-Tubule membranes (0.4 mg/ml) in 25 mM HEPES. I mM CaCI,, adjusted to pH 7.5 with Tris base. were incubated with 6 nM ['Hlazidopine and irradiated for 15 min at 4°C with a 30 watt U V source (A",.," 356 nm). The membranes were solubilized in I%, digitonin. 10 mM HEPES. 185 mM NaCI. 0.5 mM CaCI,. 0. I mM PMSF. I pM pepstatin A adjusted to pH 7.5 Tris base, and calcium channels were partially purified by chromatography on WGA-Sepharose and analyzed by SDS-PAGE and fluorography. Lane 7. /ivdrop/iohic /tiheling: ['251]TlD(15 Ci/mmol) was prepared. and purified calcium channel was labeled with 100 pCilml ["'I]TID in a buffer containing 0. I% digitonin as previously described (Reber and Catterall. 1987). Lane 8. pliosphoivlttrion: Purified calcium channel was incubated with 0.3 pM CAMP-dependent kinase catalytic subunit and 0.12 pM carrier free ly-"P]ATP for 15 min at 37°C as previously described (Curtis and Catterall. 1985).

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A polyclonal antibody (PAC-101, obtained from the ascites fluids of a SJL/J mouse immunized with purified calcium channel, selectively labeled only the 175-kDa polypeptide after reduction (Fig. 3 , lane 3). No immunolabeling was observed with preimmune serum or with PAC-I0 which had been preadsorbed with purified calcium channel. These observations indicate that the 175-kDa and 143-kDa components are distinct polypeptides. 6. Subunit Glycosylation

Solubilized ['Hldihydropyridine receptors specifically bind to various immobilized lectins and affinity chromatography on wheat germ agglutininSepharose is the most efficient purification step (Curtis and Catterall, 1983, 1984; Glossmann and Ferry, 1983; Borsotto et al., 1984). These results imply that at least one subunit is glycosylated. The oligosaccharide chains of the purified calcium channel were detected by separating subunits by SDS-PAGE and probing the resolved polypeptides with "'I-labeled concanavalin A (["-'I]ConA) or "'1-labeled wheat germ agglutinin ([ "'I]WGA). After disulfide reduction ['"I]ConA labeled only the a,-subunit (Fig. 3, lane 4). ["'IIWGA bound to both the a,-and y-subunits after dithiothreitol treatment (Fig. 3, lane 5). In addition, disulfide reduction lead to the appearance of two new ["'IIWGA labeled components at 24-27 kDa that are clearly distinct from the y-subunit. These polypeptides were also detected, but much less distinctly, by silver staining (Fig. 3, lane 2). They appear to be disulfide linked to the a,-subunit under nonreducing conditions. Previous immunochemical evidence (Schmid et ul., 1986) suggests that the smaller polypeptide may be proteolytically derived from the larger, so we refer to them collectively as the &subunit. No labeling of a ,or p was detected with either lectin. {"'l]ConA and ["'IIJWGA binding to calcium channel subunits was blocked in the presence of 100 mM a-methylmannoside or N-acetylglucosamine. respectively (not shown). To determine the extent of glycosylation and the core polypeptide size of the calcium channel subunits, purified channel preparations were labeled with '"1, incubated with glycosidases to remove oligosaccharide chains, and analyzed by SDS-PAGE and autoradiography . Sequential deglycosylation with neuraminidase and endoglycosidase F caused a reduction in the apparent sizes of the az- and y-subunits, reaching core polypeptide sizes of 105 kDa and 20 kDa, respectively. Poor iodination of the 8-subunit prevented estimation of its carbohydrate content by this method. No shift in the mobility of the at-and P-subunits was noted, confirming the absence of N-linked carbohydrate in these two subunits.

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C. Covalent Labeling of Calcium Channel Subunits

['H]PN2OO-I 10 and [-'H]azidopine have been shown to covalently label a 145- to 170-kDa polypeptide in T-tubule membranes (Ferry el al., 1984, 1985; Galizzi et al., 1986) and purified calcium channels (Striessnig el al., 1986) that presumably corresponds to one of the two a-subunits. In our preparations, ['Hlazidopine was incorporated by UV photolysis into a polypeptide which migrated as a band of 175 kDa after dithiothreitol treatment (Fig. 3, lane 6). The electrophoretic behavior of this polypeptide identifies it as the a,-subunit. The a,-subunit is not labeled and no labeling of a,was observed in the presence of 2 pit4 PN200-I 10. Ion-channel-forming polypeptides should contain transmembrane segments which may be detected using the hydrophobic probe ["'1]3-(trifluoromethyl)-3-(m-iodophenyl)diazirine(TID). This photoreactive compound partitions into free detergent micelles and detergent associated with the major hydrophobic domains of integral membrane proteins and is specifically incorporated into these regions by photolysis (Brunner and Semenza, 1981). The a,-and y-subunits were prominently labeled by TID, with a much lower level of incorporation into a, and 6 (Fig. 3, lane 7). The P-subunit was not detectably labeled. Quantitation of ["'I]TID in excised protein bands showed that the aI-and y-subunits incorporated I0-fold more TID per unit mass than the a2-or b-subunits, even though, as shown below, nearly all aI-and y-subunits are associated with an a,subunit. These results indicate that the a,-and y-subunits are the principal transmembrane components of the purified calcium channel complex. Previous work showed that protein components of the a-and @-subunit bands were good substrates for CAMP-dependent protein kinase and were therefore likely to be the sites of CAMP-dependent regulation of the calcium channel (Curtis and Catterall, 1985) (Fig. 1). Comparison of the electrophoretic mobility of the phosphorylated bands after reduction of disulfide bonds (Fig. 3, lane 8) showed that the only a,-subunit is a good substrate for this enzyme, while the a, and b polypeptides are not labeled. The psubunit was more weakly labeled at the low ATP concentration used in this experiment (see legend). These results identify the a,-and @-subunits of the calcium channel as the probable sites of regulation of the voltagesensitive calcium channel by CAMP-dependent phosphorylation. D. Analysis of Noncovalent Subunit Interactions

By the use of several labeling techniques, we have established that a, has the properties expected of the calcium channel, including a binding site for dihydropyridine calcium antagonists, at least one CAMP-dependent

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phosphorylation site, and extensive hydrophobic domains. It is important to determine whether other polypeptides present in the purified preparation are persistent impurities or specifically associated components of the oligomeric calcium channel complex. Experiments with "'I-labeled lectins established ConA as a specific probe for the a,-subunit (Fig. 3 . lane 4). Lentil lectin has the same specificity as ConA, but its lower affinity facilitates elution of bound glycoproteins with a-methylmannoside. The calcium channel was labeled with '"1 by the Bolton-Hunter procedure (Fig. 4, lane I ) . The incorporation of ','I into a2 was anomalously low, presumably indicating a low content of accessible primary amines. When the calcium channel protein was denatured by treatment with I % SDS and then exchanged into 0.5% Triton X-100 by gel filtration, only a, bound specifically to lentil lectin-agarose (Fig. 4, lane 4). while a,.p, and y remained in the flow through. This result confirms the conclusion that only a, binds to lectins with the specificity of lentil lectin and ConA. In contrast to these results after denaturation of the channel subunits, in detergent conditions (0. I%digitonin or 0. I% CHAPS) known to retain dihydropyridine binding, allosteric coupling of the three calcium antagonist receptor sites, and ion conductance activity (Curtis and Catterall, 1984; Flockerzi et a / . , 1986a.b; Striessnig et a / . , 1986; Borsotto et a / . , 1985; Curtis and Catterall, 19861, an a,a,py complex was specifically bound to lentil lectin (Fig. 4, lanes I and 2), indicating that all four polypeptides behave as a complex under native conditions. Although a I and a,are associated in 0. I% digitonin or CHAPS, in 0.5% Triton X- 100, which causes complete loss of dihydropyridine binding activity (Curtis, 1986; Curtis and Catterall, 19831, the a,-subunit alone binds specifically to lentil lectin-agarose (Fig. 4. lane 3). while the a I - ,p-, and y-subunits dissociate and appear in the column flow through. Considered together, these results indicate a correlation between retention of the dihydropyridine binding activity of the solubilized calcium channel in various detergents and association of the el-, a?-,p-. and y-subunits. Complementary data supporting these observations have also been obtained using specific antibodies against the a,-subunit. These antibodies immunoprecipitate all five subunits as a complex under native conditions, but only the a,-subunit after denaturation in SDS (Takahashi e t u l . , 1987).

E. An Oligomeric Model for the Dihydropyridine-Sensitive Calcium Channel On the basis of present knowledge of the structure of the dihydropyridine-sensitive calcium channel, and in analogy with current models of

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FIG.4. Analysis of noncovalent subunit interactions by lentil lectin affinity chromatography. One hundred nanograms of '"I-labeled calcium channel in I50 p,I of the detergents indicated below and 25 mM HEPES. 150 mM NaCI, I m M CaCI:. I mM MnCI?. 0.1 mM PMSF. and 1 mM pepstatin A (adjusted to pH 7.5 with Tris base) were incubated for 90 min at 4°C with 50 p,I of lentil lectin-agarose. equilibrated in the same buffer. under agitation. The resin was spun down and the flow-through collected. The resin was washed three times with I ml buffer and resuspended in I50 pl containing 0.2 M a-methylmannoside. The eluate was collected after a 90-min batch incubation. Specifically bound samples were analyzed by SDS-PAGE and autoradiography. Lane I , 0. I% digitonin: lane 2, 0.1% CHAPS: lane 3. 0.5% Triton X-100: lane 4, samples treated with 1% SDS were exchanged into 03% Triton X-100by filtration over a 2-ml Sephadex (3-50column and analyzed in 0.5% Triton X-100.

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the structure of voltage-sensitive sodium channels (Catterall, 1986), we propose a model (Fig. 5) based on a central ion-channel-forming element interacting with three other noncovalently associated subunits. The a,subunit, which contains the calcium antagonist binding sites, CAMP-dependent phosphorylation sites, and the largest hydrophobic domains, is proposed to be the central ion-channel-forming component of the complex. Its apparent molecular weight of 175,000 from SDS-PAGE is likely to be a reasonable approximation of the true polypeptide molecular weight since no N-glycosylation was detected. This calcium channel subunit is therefore large enough to contain four homologous transmembrane domains analogous to those of the rat brain sodium channel a-subunit, whose mRNA alone encodes a functional ion channel (Noda et al., 1986; Goldin et al., 1986). Like a,,the sodium channel a-subunit also contains CAMP-dependent phosphorylation sites (Catterall, 1986) and extensive hydrophobic domains that are efficiently labeled by TID (Reber and Catterall, 1987). The p-subunit is also a substrate for CAMP-dependent kinase (Curtis and Catterall, 1985). but hydrophobic labeling indicates that it does not interact with the membrane phase and it is not a glycoprotein. Since it remains associated with a ,in Triton X-100 while a2 is dissociated, it is probably therefore tightly associated with an intracellular domain of a , (Takahashi et al., 1987). The y-subunit of 30 kDa interacts independently with a,,contains at least one transmembrane segment, and consists of approximately 30% carbohydrate. All these properties are similar to those of the P,-subunit of the rat brain and skeletal muscle Na4 channels (Catterall, 1986). A polypeptide of similar size appears to be associated with the apamin-sensitive calcium-activated potassium channel (Seagar et al., 1986). and it is interesting to speculate that this subunit may be a conserved constituent of voltage- or calcium-dependent ion channels.

FIG.5 . Proposed model for calcium channel structure. Sites of CAMP-dependent phosphorylation (PI,glycosylation, and interaction with the membrane are illustrated.

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The a,6 dimer appears to interact more weakly with a],although the conditions necessary to achieve dissociation result in a loss of dihydropyridine binding activity. The 105-kDa core polypeptide of a? contains a heavily glycosylated extracellular domain but displays weak hydrophobic labeling, indicating a limited intramembrane domain. For this reason, it seems unlikely that the ion channel is formed jointly by a ,and a2at their zone of interaction. The proposed model assumes a complex containing one of each subunit type. Our present results and previous data showing quantitative binding of solubilized calcium channels to ConA (Glossmann and Ferry, 1983) suggest that each complex contains at least one a I - and one a,-subunit, but do not specify the stoichiometry of any subunits. a I and a2appear to be present in approximately equal amounts on silver-stained gels, and the aI-and P-subunits incorporate approximately 1 mol of 32Pper mole of complex. A complete hydrodynamic analysis of the skeletal muscle calcium channel has not been reported. However, a size of 370 kDa, determined for the rat ventricular muscle dihydropyridine receptor (Horne et a / . , 1986), is within reasonable range of the predicted size of the complex represented in Fig. 4 (416 kDa). Thus, an assumption of I mol of each subunit in the complex is plausible, but requires direct experimental verification.

V. IMMUNOSPECIFIC IDENTIFICATION OF CALCIUM CHANNEL COMPONENTS IN OTHER TISSUES In order to identify the molecular components of calcium channels in tissues other than skeletal muscle, the purified calcium channel from rabbit T tubules was injected into mice and polyclonal antisera were obtained after multiple injections. To determine the immunological cross-reactivity of this antiserum with calcium channels from heart and brain, radioimmune assays were performed. In each tissue, calcium channels were labeled with [3H]PN200-I10, solubilized with digitonin, and purified by chromatography on WGA-Sepharose. PAC-I0 antiserum, which we have used to characterize the a,-subunit of the calcium channel (see Fig. 3), showed poor cross-reactivity with other tissues. However, another antiserum, designated PAC-2, recognized [3H]PN200-I 10-labeled calcium channel in skeletal muscle, heart, and brain. The concentration dependence of immunoprecipitation showed that each channel was precipitated to a similar extent by maximum concentrations of antiserum and the ratios of antiserum concentrations for half-maximal immunoprecipitation were

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1.0 : I .8 : 7.9 for skeletal muscle, heart, and brain. These results indicate that antigenic determinants present on the skeletal muscle calcium channel are also present on calcium channels in heart and brain. Since these antibodies recognize calcium channels in heart and brain, it is possible to use them to identify and characterize the related proteins in these tissues. For this purpose, membrane preparations from heart and brain were solubilized with digitonin, and glycoproteins were purified by chromatography on WGA-Sepharose. These purified glycoproteins were labeled with I2'I, and the components recognized by antibodies against the skeletal muscle calcium channel were isolated by immunoprecipitation with specific antiserum and a protein A-Sepharose immunoadsorbent. In each tissue, a polypeptide with the characteristics of the calcium channel a,-subunit was identified (Takahashi and Catterall, 1987a,b). The imrnunoprecipitated component had a molecular weight of 170,000 in heart and 169,000 in brain in nonreducing conditions and 141,000 and 140,000, respectively, after reduction. In each case, immunoprecipitation was blocked by prior incubation of the antiserum with purified calcium channel. Thus, we conclude that dihydropyridine-sensitive calcium channels in skeletal muscle, heart, and brain are all associated with an a,-subunit that is homologous, but not identical in the three tissues. Polypeptides homologous to other subunits of the skeletal muscle calcium channel have not yet been detected using these techniques.

VI. CONCLUSION

The molecular properties of dihydropyndine-sensitive calcium channels are now being elucidated by following a general strategy that was used previously in studies of voltage-sensitive sodium channels in this laboratory (reviewed in Catterall, 1986) and in other laboratories whose work is presented in this volume (Chapters 12 and 17). This strategy involves identification by specific ligand binding and covalent labeling, solubilization and isolation by conventional protein purification methods, reconstitution of channel function in v i m , and analysis of the properties of channel components by specific antibodies and labeling methods. The work reviewed here illustrates the substantial progress achieved with this approach to date. Future directions include further definition of the functional properties of the purified calcium channel complex in reconstituted phospholipid vesicles, analysis of the mechanism of regulation of the channel by protein phosphorylation, determination of the primary structure of the channel subunits, and comparison of these molecular properties of calcium

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chan-nels with those of voltage-sensitive sodium channels in order to define common structural themes underlying the function of voltage-sensitive ion channels in general. REFERENCES Almers, W., Fink, R., and Palade, P. T. (1981). Calcium depletion in frog muscle tubules: The decline of calcium current under maintained depolarization. J . Physiol. (London) 312, 177-207. Armstrong, C. M., and Matteson, D. R. (1985). Two distinct populations of calcium channels in a clonal line of pituitary cells. Science 227, 65-67. Bean, B. P. (1985). Two kinds of calcium channels in canine atrial cells. Differences in kinetics, selectivity, and pharmacology. J. Gen. Physiol. 86, 1-30. Bolger, G. T.. Gengo, P. J., Luchowki, E. M., Seigel, H., Triggle, D. J., and Janis, R. A. (1982). High affinity binding of a calcium channel antagonist to smooth and cardiac muscle. Eiochem. Eiophys. Res. Commun. 104, 1604-1609. Borsotto, M., Barhanin, J., Norman, R. I . , and Lazdunski. M. (1984). Purification of the dihydropyridine receptor of the voltage-dependent Ca’+ channel from skeletal muscle transverse tubules using ( + ) [.‘H]PNZOO-I 10. Eiochem. Eiophys. Res. Commun. 122, 1357-1365, Borsotto, M., Barhanin, J., Fosset, M., and Lazdunski, M. (1985). The 1.4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca’ . J . Eiol. Chem. 260, 14255-14263. Brum, G., Flockerzi, V., Hofmann, F., Ostenieder, W., and Trautwein, W. (1983). Injection of catalytic subunit of CAMP-dependent protein kinase into isolated cardiac myocytes. Pj7uegers Arch. 398, 147-154. Brunner. J., and Semenza. G. (1981). Selective labeling of the hydrophobic core of membranes a carbene generating reagent. with 3-(trifluoromethyl~-3-(m-[”’I]iodophenyl~diazirine, Eiochemisfiy 20, 7174-7182. Cachelin, A. B., de Peyer, J. E., Kukubun, S., and Reuter, H. (1983). Ca” channel modulation by 8-bromocyclic AMP in cultured cells. Nature (London) 304, 462-464. Campbell, K. P., Lipschutz, G. M., and Denney, G. H. (1984). Direct photaffinity labeling of the high affinity nitrendipine-binding site in subcellular membrane fractions isolated from canine myocardium. J. Eiol. Chem. 259, 5384-5387. Carbone, E., and Lux, H. D. (1984). A low voltage-activated fully inactivating Ca channel in vertebrate sensory neurones. Nature (London) 310, 501-502. Catterall, W. A. (1986). Molecular properties of voltage-sensitive sodium channels. Annrr. Rev. Eiochem. 55, 953-985. Curtis, B. M. (1986). Purification and reconstitution of the calcium antagonist receptor of the voltage-sensitive calcium channel. Ph.D. Thesis, Univ. of Washington, Seattle. Curtis, B. M., and Catterall. W. A. (1983). Solubilization of the calcium antagonist receptor from rat brain. J. Biol. Chem. 258, 7280-7283. Curtis, 9. M., and Catterall, W. A. (1984). Purification of the calcium antagonist receptor of the voltage-sensitive calcium channel from skeletal muscle transverse tubules. Biochemistry 23, 21 13-21 18. Curtis, B. M., and Catterall, W. A. (1985). Phosphorylation of the calcium antagonist receptor of the voltage-sensitive calcium channel by CAMP-dependent protein kinase. Proc. Ntrtl. A c ~ dSci. . U.S.A. 82, 2528-2532. Curtis, B. M . , and Catterall, W. A. (1986). Reconstitution of the voltage-sensitive calcium channel purified from skeletal muscle transverse tubules. Biochemistry 25, 3077-3083. +

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Ferry, D. R., Goll. A.. and Glossmann, H. (1983). Putative calcium channel molecular weight determination by target size analysis. Nuiinyn-SclimiedeherX'sArch. f l i u r m c r c d . 323, 292-297. Ferry, D. R., Rombusch, M., Goll, A., and Glossman, H. (1984). Photoaffinity labelling of Ca' ' channels with ['Hlazidopine. FEBS L e f t . 169, 112-167. Ferry. D. R., Kampf, K., Goll. A + . and Glossmann, H. (1985). Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the I ,4-dihydropyridine photoaffinity probe ['HI-azidopine. EMBO J. 4, 1933-1940. Flockerzi, V.. Oeken, H.-J.. and Hoffman, F. (1986a). Purification of a functional receptor for calcium channel blockers from rabbit skeletal muscle microsomes. Ew. J . Biocliem. 161, 217-224. Flockerzi. V.. Oeken, H.-J., Hofmann, F., Pelzer. D., Cavalie, A.. and Trautwein, W. (1986b). Purified dihydropyridine binding site from skeletal muscle T-tubules is a functional calcium channel. Nature (London) 323, 66-68. Fosset. M., Jaimovich, E., Delpont, E., and Lazdunski, M. (1983). ['Hlnitrendipine receptors in skeletal muscle. J. B i d . Chem. 258, 6086-6092. Galizzi. J. P.. Borsotto. M., Barhanin, J., Fosset, M., and Lazdunski. M. (1986). Characterization and photoaffinity labeling of receptor sites for the Ca" channel inhibitors dcis-diltiazem, (-t)-bepridil. dismethoxyverapamil and ( + )PN200-110 in skeletal muscle transverse tubule membranes. J . B i d . Chern. 261, 1393-1397. Garcia, M. L., King, V. F., Siegl, P. K. S.. Reuben, J. P., and Kaczorowski, G. J. (1986). Binding of calcium entry blockers to cardiac sarcolemmal membrane vesicles. Characterization of diltiazem-binding sites and their interaction with dihydropyridine and aralkylamine receptors. J. Biol. Chem. 261, 8146-8157. Glossmann. H.. and Ferry, D. R. (1983). Solubilization and partial purification of putative calcium channels labelled with ('HI-nimodipine. Nurtnyn-Sc/rmiedeher~'.sArch. Pkurmarol. 323, 279-291. Goldin. A. L.. Snutch. T., Lubbert, H.,Dowsett, A,. Marshall, J . , Auld, V.. Downey, W.. Fritz, L. C., Lester, H. A., Dunn, R., Catterall. W. A., and Davidson, N . (1986). Messenger R N A coding for only the a subunit of the rat brain Na channel is sufficient for expression of functional channels in Xenopus ooryfes. f ro c. N o / / . Acud. Sci. U.S.A. 83, 7503-7509. Hagiwara, S., and Byerly. L. (1981). Calcium channel. Annu. Re\!. Nerirosci. 4, 69-125. Hess. P.. Lansman, J. B., and Tsien. R. W. (1984). Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. N u t w e (London) 311, 538-544. Horne, W. A,, Weiland. G. A., and Oswald, R. E. (1986). Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle. J. Biol. Cli
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technique for analysis of electrical activity of neuronal circuit function. Fed. Proc.. Fed. Am. Soc. Exp. Biol. 40, 2240-2245. Murphy, K. M. M., Could, R. J., Largent, B. L., and Snyder, S. H. (1983).A unitary mechanism of calcium antagonist drug action. Proc. Narl. Arad. Sci. U.S.A. 80, 860864. Nilius, B., Hess, P., Lansman, J. B., and Tsien, R. W. (1985).A novel type of cardiac calcium channel in ventricular cells. Nature (London) 316, 443-446. Noda. M., Ikeda, T., Suzuki, H., Takeshima, H.. Takahashi, T.. Kuno, M., and Numa, S. (1986).Expression of functional sodium channels from cloned cDNA. Nuture (London) 322, 826-828. Norman, R. I., Borsotto, M., Fosset, M., Lazdunski, M., and Ellory, J. C. (1983).Determination of the molecular size of the nitrendipine-sensitive Ca” channel by radiation inactivation. Biocliem. Biophys. Res. Commun. 111, 878-883. Nowycky, M. C., Fox, A. P., and Tsien, R. W. (1985).Three types of neuronal calcium channel with different calcium agonist sensitivity. Nutrue (London) 316, 440-443. Reber, B. F. X.,and Catterall. W. A. (1987).Hydrophobic properlies of the pl and (32 subunits of the rat brain sodium channel. J. B i d . Chem. 262, 11369-1 1374. Reuter, H. (1974).Localization of p adrenergic receptors, and effects of noradrenaline and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. J. Plivsiol. 242, 429-451. Reuter. H. (1979).Properties of two inward membrane currents in the heart. Annrr. Rev. Plivsiol. 41, 413424. Reuter, H. (1983).Calcium channel modulation by neurotransmitters, enzymes and drugs. Nutiire (London) 301, 569-574. Rosemblatt. M., Hildao. C.. Vergara. C.. and Ikemoto, N. (1981).Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle. J. Biol. Chem. 256, 8140-8148. Rossie, S.. and Catterall. W. A. (1987).Regulation of ionic channels. In “The Enzymes.” vol. 18,pp. 335-358. Academic Press, New York. Sanchez. J. A.. and Stefani, E. (1978).Inward calcium current in twitch muscle fibers of the frog. J. Physiol. (London) 283, 197-209. Schmid. A.. Barhanin. J., Coppola. T., Borsotto, M.. and Lazdunski, M. (1986).Immunochemical analysis of subunit structures of 1.4-dihydropyridine receptors associated with voltage dependent Ca” channels in skeletal. cardiac and smooth muscle. Biochemistry 25, 3492-3495. Schramm, M., Thomas, G.. Towart. R., and Franckowiak, G. (1983).Novel dihydropyridines with a positive inotropic action through activation of Ca” channels. Nutiire (London) 303,535-537. Schwartz, L. M..McCleskey, E. W.. and Almers. W. (1985).Dihydropyridine receptors in muscle are voltage-dependent but most are not functional calcium channels. Nufirre (London) 314, 747-75 1. Schwartzkroin, P. A.. and Slawsky, M. (1977).Probable calcium spikes in hippocampdl neurons. Brain Res. 135, 157-161. Seagar. M. J.. Labbe-Julle, C.. Granier. C., Goll, A., Glossmann. H., Van Reitschoten. J., and Couraud. F. (1986).Molecular structure of the rat brain apamin receptor: Differential photoaffinity labeling of putative K’ channel subunits and target size analysis. Biochemistry 25, 4051-4057. Striessnig. J.. Moosburger, K., Goll. A,. Ferry, D. R.. and Glossmann. H. (1986).Stereoselective photoaffinity labeling of the purified 1.4-dihydropyridine receptor of the voltage dependent calcium channel. Eur. J. Biocliem. 161, 603-609.

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Takahashi. M.. and Catterall. W. A. (1987a). Identification of an a subunit of dihydropyndinesensitive brain calcium channels. Scitncr 236, 88-92. Takahashi, M.. and Catterall, W. A. ( 1987b). Dihydropyridine-sensitive calcium channels in cardiac and skeletal muscle membranes: Studies with antibodies against the a subunits. B i o c k e t n i s r ~26, ~ 55 18-5526. Takahashi. M.. Seagar. M. J.. Jones, J . F.. Reber. B. F. X., and Catterall. W. A. (1987). Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle. Proc. N d . Acrid. Sci. U.S.A. 84, 5478-5482. Tdlvenheimo. J. A.. Tamkun. M. M.. and Catterall, W. A. (1982). Reconstitution of neurotoxin-stimulated sodium transport by the voltage-sensitive sodium channel purified from rat brain. J. B i d . C l w n . 257, 11868-1 1871. Tnggle. D. J. (1982). Biochemical pharmacology of calcium blockers. I n “Calcium Blockers: Mechanism of Actions and Clinical Applications” (S. F. Flaim and R. Zelis, eds.). pp. 121-134. Urban & Schwarzenberg, Munich. Tsien. R. W., Giles. W.. and Greengard, P. (1972). Cyclic-AMP mediates the action of epinephrine on the action potential plateau of cardiac Purkinje fibers. Nutiire (London). New B i d . 240, 181-183. Tsien. R. W.. Bean, €3. P., Hess. P.. and Nowycky. M. (1983). Calcium channels: Mechanisms of p-adrenergic modulation and ion permeation. Cold Spring Harbor Symp. Quant. Biol. 48, 201-212. Uehara. A,, and Hume. J . R. (1985). Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells. J. Gen. Phvsiol. 85, 621-647. Venter. J. C.. Fraser. C. M.. Schaber. J. S., Jung, C . Y., Bolger, G.. and Triggle. D. J . (1983). Molecular properties of the slow inward calcium channel. J. B i d . C l w n . 258, 9344-9448.

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CUKKENT TOPICS IN MEMBRANES A N D T R A N S W K T . V O L U M E 11

Chapter 19

Cardiac Calcium Channels: Pore Size and Symmetry of Energy Profile R . L. ROSENBERG,' E. W . McCLESKEY,' P . HESS,' AND R . W . TSIEN Department of Cellular and Molecular Physiology Yale School of Medicine New Haven. Connecticut M510

I. Introduction 11. A Model for the Ca Channel: A Single-File Pore with Two Binding Sites 111. Recordings in Intact Cells to Estimate Pore Size A. Permeation by Organic Cations B. Block by Diamino Compounds IV. Recordings in Planar Bilayers A. Comparison with Cell-Attached Patch Recordings B. Tests of Pore Symmetry V. Discussion A. Pore Size B. Pore Symmetry C. Pore Structure References

1.

INTRODUCTION

Calcium channels are among the most interesting examples of the intrinsic membrane proteins that control transmembrane ion flow and cellular function (Hagiwara and Byerly, 1981; Reuter, 1983; Tsien et al., 1987a,b). Present in the surface membranes of all known excitable cells, they open 'Present address: Department of Pharmacology. The University of North Carolina-X'hapel Hill. Chapel Hill, North Carolina 27599. 'Present address: Department of Cell Biology and Physiology. Washington University. St. Louis. Missouri 631 10. 'Present address: Department of Physiology and Biophysics. Harvard Medical School, Boston. Massachusetts 021 15.

393 Copyright 111 1988 by Acildsmic Prehh. Inc. All rishts of r e p r d u c l w n in any form rersrved.

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in response to membrane depolarization and allow Ca” to move down a steep electrochemical gradient into the cell. The flow of depolarizing charge may help generate action potentials, pacemaker activity, or bursting patterns. The Ca” influx may also act as a chemical signal when decoded by calcium-receptor proteins inside the cell. Rises in cytosolic Ca2+can initiate diverse cellular responses such as contraction, secretion, activation and inactivation of ion channels, and changes in metabolism and gene expression. Calcium channels work under some rather stringent conditions. In a typical extracellular solution, Na’ ions vastly outnumber Ca” ions. Yet Ca channels are highly effective in transporting Ca” to the exclusion of Na’, K’, Mg”, and other physiological ions, while permitting large open channel fluxes (- 10“ sec- ’ under experimental conditions). There is great interest, therefore, in understanding the mechanism of ion permeation in molecular terms. At present, structural studies of Ca channels have advanced to the stage of biochemical analysis of the subunits necessary for function (reviewed in Chapter 18, this volume) and the cloning and sequencing of a putative Ca channel, the dihydropyridine-binding protein from skeletal muscle (Tanabe et ul., 1987). No direct information is available yet about the structure of the pore itself. Present understanding of the Ca channel pore comes largely from electrical recordings (for reviews see Hagiwara and Byerly, 1981; Almers et al., 1986; Tsien et al., 1987b). Studies of ion selectivity, permeation, and block have been camed out in cardiac myocytes (Reuter and Scholz, 1977; Lee and Tsien, 1984; Hess and Tsien, 1984; Matsuda and Noma, 1984; Hess et al., 1986; Lansman et al., 1986), skeletal muscle cells (Almers et ul., 1984; Almers and McCleskey, 1984; Cota and Stefani, 19841, neurons (Akaike et al., 1978; Kostyuk and Krishtal, 1977; Kostyuk et at., 1983; Byerly et al., 1985; Chesnoy-Marchais, 1985; Lux, 1986), and other cells (see, e.g., Hagiwara and Ohmori, 1983; Fox and Krasne, 1984; Fukushima and Hagiwara, 1985).All of the work seems consistent with the hypothesis that the Ca channel pore contains multiple high-affinity binding sites for Ca” arranged in single file (Hess and Tsien, 1984;Almers and McCleskey, 1984; Tsien et d.,1987b). This model provides a starting point for experiments aimed at gaining a clearer picture of the physical structure of the pore. In this article, we describe studies on L-type Ca channels in heart cells, probably the most extensively characterized of the many voltage-dependent Ca channels that have been distinguished in recent years (see reviews in Tsien et al., 1987a; Miller, 1986). The two major aims were ( I ) estimating the radial dimensions of the pore and (2) looking for possible asymmetry of the energy profile along the length of the pore.

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

39s

II. A MODEL FOR THE Ca CHANNEL: A SINGLE-FILE PORE WITH TWO BINDING SITES

Figure I illustrates a two-binding site model for the Ca channel pore as developed by Hess and Tsien (1984) for cardiac myocytes and Almers and McCleskey (1984) for skeletal muscle, and patterned after other multisite models (Hille and Schwarz, 1978; Urban et al., 1980). The basic postulates of the model may be listed as follows: (1) ions move through the Ca channel in single file, (2) the pore contains two binding sites with high affinity for Ca” ions (dissociation constant 1 p M ) and low affinity for Na’ and other monovalent cations, (3) cations at the outer and inner sites repel each other (for review see Tsien et d., 1987b). Figure IA shows a plot of the potential energies of a Ca” ion (solid curve) and a Na’ ion (dashed curve) against position in the electric field of an empty pore. The inherent difficulty in a model with high-affinity ion binding ( K , = M) is that, given a diffusion-limited on-rate (IO’M-’ sec-’), the maximum off-rate would be only lo3 see-', or roughly three orders of magnitude less than observed flux rates (Almers and McCleskey, 1984; Lansman et al., 1986). However, two sequential high-affinity sites can solve the problems associated with only one: the difficulty of the slow off-rate from an isolated high-affinity Ca”-binding site is eliminated when a second Ca” ion occupies the pore because the two ions are expected to repel each other, forcing a more rapid exit. Double occupancy of the model pore is essential for high Ca” fluxes. Figure IB depicts all possible states of pore occupancy for two ions, where 0 indicates an empty site and A and B indicate different ions. With physiological concentrations of Ca”, the pore is almost always occupied by one or more Ca” ions and cycles among the four states depicted in Fig. IC. When the outer site is empty, a Na+ ion has a chance to enter the pore, but it binds weakly and is quickly repelled from the channel. The situation is different when a second Ca” ion enters the pore. If. for example, the membrane potential is zero, there is a 50% chance that the inner Ca” will enter the cytoplasm, resulting in the net transfer of charge. Since Ca’+ entry into the cytoplasm is essentially irreversible (the cytoplasmic concentration being so low that Ca” efflux is extremely unlikely), there will be net clockwise traffic around the upper triangle of states. The energy is provided by the electrochemical gradient for Ca’+ . This model can account for block of monovalent currents by micromolar Ca”, even though millimolar Ca” is required for detectible Ca” flux. Also, it can account for the anomalous mole fraction effect seen in mixtures

-

FIG. I . Theoretical description of a two-site Ca channel pore (Tsien ef d.,1987b).(A) Cartoon of the model (above)and the corresponding energy profile (below) where potential energies of a Ca2' ion (solid line) and a Na' ion (dashed line) in an otherwise empty pore in the absence of an electric field are plotted against the fraction of the transmembrane potential at any given point in the pore (adapted from Almers and McClesky. 1984). (€3) State diagram of the possible states of occupancy of two permeant ions, A and 6.in both internal and external solutions, with 0 indicating an empty site (from Hess and Tsien. 1984). (C) Predominant states of occupancy under physiological conditions.

19.

FORE SIZE AND SYMMETRY OF Ca CHANNELS

397

of divalent and monovalent ions (Hess and Tsien, 1984; Almers and McCleskey, 1984; Tsien et a / . , 1987b). In addition, the model has many experimental implications. It suggests that the selectivity of Ca channels occurs by selective ion binding rather than by exclusion of ions at a molecular sieve. This can be tested by probing the pore with large organic cations. Also, the model places the ion-binding sites symmetrically within the electric field in the membrane. This can be tested by observing the ion fluxes under symmetrical ionic conditions.

111. A.

RECORDINGS IN INTACT CELLS TO ESTIMATE PORE SIZE Permeation by Organic Cations

Since Ca channels are highly permeable to monovalent cations in the absence of Ca'+ (Kostyuk and Krishtal, 1977; Hess and Tsien, 1984; Almers et al., 1984; Hess et al., 1986), the relative permeability of large organic cations can be used to approximate the pore diameter. This strategy has been used to describe the pore diameter of a wide variety of ion channels (Hille, 1971, 1973; Dwyer et al.. 1980; Coronado and Miller, 1982; Bormann et al., 1987). including Ca channels from skeletal muscle (McCleskey and Almers, 1985; Coronado and Smith, 1987). Figure 2 shows currents recorded from a heart cell in four different external solutions containing the methylated ammonium ion indicated. Clear inward currents with a time course characteristic of cardiac L-type Smaller inward currents Ca channels were recorded with Me,". were recorded in Me,". and in Me3" inward "tail currents" were recorded when the cell was repolarized to the holding potential, where the driving force for inward current was the greatest. In this cell, no inward currents at any potential were recorded with Me," in the external solution. In other cells, Me," currents were just large enough to be detectable. Figure 3A shows an example of Me," currents, compared to those recorded with Et,N+ as the extracellular cation. Clearly there was more current carried by Me,". Figure 3B shows the current-voltage relations of Me," and Et,N+ tail currents recorded after a depolarizing pulse large enough to activate most of the Ca channels in the cell. Me," carried more current and had a more positive reversal potential. To determine that the Me," currents were carried by Ca channels, we tested for the block of the currents by micromolar external Ca". Figure 3C shows that the Me," currents were almost completely blocked by 3.3 Ca". The dramatic effect of micromolar Ca" provided clear evidence that these

398

R. L. ROSENBERG ET AL.

A

Me, N+

2n

A

l

40 ms

FIG.2. Currents carried by methylated ammonium ions. (A-D) Membrane currents in isolated cardiac myocytes during voltage pulses to -50. -30, - 10, + 10, and +20 mV from a holding potential of -90 mV. Extracellular solutions contained (in m M ) 140 XCI (where X is the indicated methylated ammonium cation), 10 EDTA. 0.01 TTX. and 10 MOPS titrated to pH 7.2 with Et,NOH. lntracellular solution contained I10 CsF. 10 CsCI, 15 EGTA, 40 HEPES-Et,NOH. pH 7.2. Cell E04A.

currents were indeed through Ca channels, since only Ca channels display such high-affinity Ca2* block. The relative permeability of the organic cations can be determined from the reversal potentials (the voltages at which inward and outward currents balance to give zero net current) measured with each of the external solutions, according to the Goldman equation for monovalent cations: P x / P y = ([Y]/[X]) exp{(E,

-

Ey)*F/RT}

where P x and Py are the permeabilities of cations X and Y, [XI and [Y] are the concentration of the ions in the extracellular medium, Ex and EY are the measured reversal potentials, and F, R , and T have their usual meanings. Figure 4 shows a plot of the permeabilities of the methylated ammonium compounds relative to Me,". The data show that the permeability decreased as the diameter of the ion increased for this series of chemically related ions. Since Me," was the largest ion to permeate the Ca channel, we can take the molecular dimensions of this ion as an indication of the minimum diameter of the pore, at least in the absence of Ca'+. Thus, the pore diameter can be no less than 5.5 A. This is equivalent to the pore diameter of dihydropyridine-sensitive Ca channels from skeletal muscle (McCleskey and Almers, 1985; Coronado and Smith, 1987).

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

399

2n A L

2 n A L

10 rns

10 rns

0

0

Me4N+ 0

FIG.3. Me,N + permeability. (A) Currents evoked by a depolarization to +30 mV from the holding potential of -90 mV. Extracellular solutions were as described in Fig. 2 with the indicated organic cation. lntracellular solution contained (mM) I10 Et,NF, 10 CsCI. 15 ECTA. 40 HEPES titrated to pH 7.2 with Et,NOH. Cell E06B. (B) Plot of the peak amplitude of the tail currents carried by external Me,N ' (open circles) and Et,N (tilled circles) as a function of the repolarization potential following a voltage pulse to +30 mV. (C) Me,N + currents in the absence and presence of 3.3 pM Ca'+. Voltage pulses were to + 10 mV from a holding potential of -90 mV. Ca-free extracellular solution as described for Fig. I. Cacontaining solution included (in mM) 140 Me,NCI. 5 CaCI,. 10 HEDTA. 10 MOPS adjusted to pH 7.2 with Et,NOH. lntracellular solution was the same as in (A). Cell E06D. +

B. Block by Diamino Compounds

While the pore diameters and Ca"-binding sites are common to both cardiac and skeletal Ca channels, other aspects of ion permeation are different. For example, in skeletal muscle, butylammonium ion was found to be impermeant whereas diaminobutane, a larger molecule, could carry substantial inward current (McCleskey and Almers, 1985). This is probably because diaminobutane has two charged nitrogens separated by a chain of four carbon atoms, and electrostatic repulsion causes the molecule to

400

R. L. ROSENBERG ET AL.

1.5

I

:a: 1.0

0

Me,N+

UI

L I-

2

Me2N+

0.5

U

Me3N+ 0.0 L ' 1

I

1

I

I

1

2

3

4

5

6

ION SIZE (A)

FIG.4. Permeability of Na' and organic cations through cardiac Ca channels. PermeMe,", and Me," relative to Me," are plotted against the abilities of Na', Me,", diameter of the ion as measured from Corey-Pauling-Koltum (CPK) models. Relative permeability was calculated from the reversal potential of currents measured during the pulse (Na. Me,N, Me,N) or after the end of the pulse (Me,N). Each point is the average of at least three cells; the mean error is indicated.

assume a linear form which can fit lengthwise through a 5.5 A pore. Removing one of the nitrogens to form butylammonium allows the four carbon atoms to assume a globular shape that is larger than the pore. Different results were obtained with cardiac Ca channels. Neither diaminobutane nor butylammonium were found to be measurably permeant. However, diaminobutane was found to block monovalent ion currents (Fig. 5A) whereas butylammonium had no effect (Fig. 5B). Interestingly, the nature of the current carrier was an important factor in the efficacy of block by diaminobutane; Fig. 5C shows that Ba2+currents were blocked less than Na' currents at the same concentration of diaminobutane. Single-channel recordings (Fig. 5D)show that the block of monovalent currents appeared as a decrease in the single-channel current without any increase in the noise of the open channel current, indicating that the blocking reaction is fast compared to the filtering frequency of 1000 Hz. These recordings also show that the block by diaminobutane was voltage dependent; there was significantly more block at - 50 mV (upper traces) than at - 10 mV (lower traces). The current-voltage relationships (Fig. 5E) in the absence (open circles) and presence (filled circles) of diaminobutane confirm that above - 10 mV there was very little block, but as

PORE SIZE AND SYMMETRY OF Ca CHANNELS

19.

4 7 m M Na+ *

401 C

I

10 m M

~2'+

DIAMINOBUTANE

10 ms

D

140 mM Li+

,

140 mM Li+ DIAMINOBUTANE

E

2P A L 20 ms

Fici. 5 . Block of cardiac Ca channels by diaminobutane. (A-C) Whole-cell currents camed by Na' (A. B) or Ba" (C) before and after the addition of 10 m M diarninobutane (A, C ) or butylarnmonium (6).Currents were evoked by a depolarization to - 20 mV (A. B) or 0 mV (C) from a holding potential of - 70 mV (A, B) or - ho mV (C). The intracellular solutions were as described for Fig. I . The extracellular solutions in A and B were as described for Fig. I with 47 m M NaCl and 93 rnM Et,NCI. Extracellular solution in C contained (mM) 10 BaCI,. 135 Et,NCI, 10 HEPES-E1,NOH. pH 7.2. A. B: cell E06E: C: cell E06G. (D) Recordings of L i t currents through single Ca channels in the absence (left) and presence (right) of 0. I rnM diaminobutane. Test potentials were -50 rnV (upper traces) or - 10 mV (lower traces). Pipette solufion (mM): 140 LiCI. 10 EDTA. 0.01 TTX. 10 MOPS-E1,NOH. pH 7.2. Extracellular solution: 140 potassium aspartate. 10 ECTA. 0.005 BAY K 8 W . 10 HEPES-KOH, pH 7.2. ( E ) Single channel current is plotted as a function of the test potential in the absence (open circles) or presence (filled circle) of 0. I mM diaminobutane. The lines represent I-V relations obtained from membrane potential ramps (Hess et c d . , 1986).

the potential is made more negative, diaminobutane block is more and more potent. This voltage dependence is consistent with the idea that the diaminobutane must enter the pore to gain access to the blocking site, since a negative membrane potential would tend to drive the cationic blocker into the channel. The block of monovalent but not divalent currents by an organic molecule that has two separated charges provides independent evidence for multiple intrapore ion-binding sites. This result also suggested that changing the length of the carbon chain separating the two charges might have an effect on the efficacy of the block and provide information on the physical distance separating the two sites (Miller, 1982). However, we found that the potency of block did not depend on the chain length for the eight compounds tested from C3 (diaminotriane) to C 10 (diaminodecane). Each

402

R. L. ROSENBERG ET AL.

of these organic ions was half-maximally effective at a concentration of about 3 mM. This lack of effect of chain length may reflect the flexibility of the carbon chain; we have not yet tested any rigid, unsaturated molecules. IV. RECORDINGS IN PLANAR BILAYERS

Patch-clamp recordings have yielded much information about the permeation of ions through L-type Ca channels, but they are limited to cellattached recording configuration, where the intracellular medium cannot be experimentally modified. Ca channels can be reconstituted in planar lipid bilayers where their functional properties can be studied under ionic conditions and membrane voltages that are difficult to achieve with intact cells. A. Comparison with Cell-Attached Patch Recordings

Unitary Ca channel activity was observed after the incorporation of bovine cardiac sarcolemmal membrane vesicles into lipid bilayers (Rosenberg et al., 1986),as illustrated in Fig. 6A. The dihydropyridine agonist BAY K 8644 was included to promote long openings of L-type Ca channels (Hess and Tsien, 1984; Kokubun and Reuter, 1984; Ochi et af., 1984). Membrane depolarization to + 10 mV evoked voltage-dependent Ca channel activity that was seen as inward current pulses of 1.2 pA carried by external Ba”. The L-type Ca channel activity recorded in the planar lipid bilayers was virtually identical to that seen in intact heart cells (Rosenberg et al., 1986, 1988).With external Ba“, the conductance at 0 mV was 23 pS, and with 100 mM external Ca*+,the slope conductance was -7 pS (Reuter er al., 1982;Cavalie er at., 1983;Nilius et af., 1985).The mean current (Fig. 6A, bottom trace), activated rapidly following depolarization, inactivated slowly during a maintained depolarization, and deactivated very rapidly following repolarization, giving a time course that is typical for Ba’+ currents through cardiac L-type Ca channels in the presence of BAY K 8644 (Hess et al., 1984;Kokubun and Reuter, 1984;Ochi et af., 1984). In the bilayers, as in cell-attached patch recordings of single Ca channels, there was considerable variability in the inactivation rate from channel to channel (Cavalie er al., 1986). The single-channel kinetics were also very similar to that expected for long-lasting channel openings promoted by BAY K 8644 (Rosenberg et al., 1986, 1988);in the experiment illustrated in Fig. 6B,the mean open

-

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

A

403

B

+I0mV

80 m

-

(3

$

60-

w

n

-

0 L

0 K

40

-

w

-

5z

-

m 20

-

0~~

OPEN TIME (ms)

P

-50

0

50

100

150

200

V (mV)

FIG.6. Properties of cardiac Ca channels incorporated in planar lipid bilayers. The extracellular solution contained ( m M ) 100 BaCI,. SO NaCI. 1 EDTA, 10 HEPES-NaOH. pH 7.4. The intracellular solution contained (mM) SO NaCI, I EDTA, 10 HEPES-NaOH. pH 7.4. (A) Unitary currents recorded during voltage pulses to + 10 mV from a holding potential of -70 mV; openings appear as downward transitions in the records. ( B ) Channel open time distribution at 0 mV. The smooth curve is the least-squares fit of the data with a single exponential (time constant, 20 msec). (Ct Open probability plotted versus test potential. Open probability was determined for each sweep containing channel activity; null sweeps were excluded. The average probability and standard errors are plotted at each potential. The smooth curve was drawn according to the equation Pupen = 0.82S/{ I + exp[(VI, - V ) / K ] ) with VI, = 18 mV and K = 16 mV. (From Rosenberg ef a / . , 1986.)

time was -20 msec at 0 mV and in a total of six experiments at this potential the mean open time averaged 16.5 & 3.9 msec. Closed-time distributions were usually fitted with two exponential components with time constants of 6.2 ? 1.4 and 28.5 8.0 msec ( n = 5 ) at 0 mV. Open- and closed-time duration were voltage dependent. Open times increased with increasing depolarization from -5 msec at - 20 mV to a limiting value of 20-50 msec above + 20 mV. The slower time constant of the closed-time distribution decreased as the membrane was depolarized, from -100 msec

*

404

R. L. ROSENBERG ET AL.

at weak depolarizations to -5 msec above + 20 mV. This voltage dependence of channel open and closed times leads to the characteristic voltage dependence of open probability, as illustrated in Fig. 6C. We directly compared the current-voltage (I-V) relations of Ca channels in planar bilayers and intact cells under closely matched ionic conditions (Fig. 7). There was a near-perfect superposition of the I-V relations in the intermediate voltage range where currents could be measured with both experimental designs (23 pS at 0 mV). Because the artificial membranes were more stable at very strong depolarizations than cell-attached patches, we were able to record unitary outward currents carried by internal K' (Fig. 7) or internal Na+ (filled symbols, Fig. 8C). In both cases, the slope conductance for the outward currents increased steeply with voltage, producing an asymmetric, strongly inflected I-V relation. The steeply increasing conductance for outward monovalent current most likely reflects a voltage-dependent decrease of channel occupancy by Ba2* (Hess and Tsien, 1984; Almers and McCleskey, 1984; Hess et al., 1986). As Ba2+is driven out of the pore by the large positive transmembrane potential, the internal monovalent ions can enter and permeate rapidly, as suggested by the model. Thus, when the membrane is depolarized sufficiently, rapid permeation by monovalent cations can occur even in the presence of divalent cations, indicating that monovalent permeability is a basic property of the channel pore, and does not result from a large-scale modification of the channel by the lack of divalent ions. B. Tests of Pore Symmetry

To determine whether the nonlinearity in I-V relationships was a consequence of the asymmetric distribution of ions or whether the channel itself was functionally asymmetric, we measured the open channel I-V relationship with symmetric ionic conditions (open symbols, Fig. 8C). As expected, the reversal potential is 0 mV. On either side of the reversal potential, inward and outward currents were equal and opposite. Thus, the open channel behaves like a functionally symmetric pore under these symmetric ionic conditions. The linearity of the I-V relation with symmetric Ba" stands in sharp contrast to the nonlinearity seen with external Ba2+and internal monovalent ions (filled symbols, Fig. 8C). In the symmetric 100 mM Ba'+, we measured a conductance of 23 pS over a wide range of voltages from -60 to + 150 mV (Fig. 8A). Under identical recording conditions, Ca channels from skeletal muscle transverse (T) tubules also show a linear, symmetric I-V relationship, but with a conductance of I 1-12 pS (Rosenbergel al., 1986;Ma and Coronado, 1988).

A

B

CELL-ATTACHED PATCH

-?---

PLANAR LIPID BILAYER

Vtest

-80mV

-80mV

-30mV

-20mV

+ I O

U

1p A L 20 ms

1p A L 100 ms

C

3

I (PA)

[

FIG. 7. Comparison of Ca channels in cell-attached patches and planar lipid bilayers. (A) Single-channel recordings from a cell-attached patch of a guinea pig myocyte. The pipette solution contained (in m M ) I10 BaCI,. HEPES-Et,NOH, pH 7.4. The external solution contained ( m M ) 140 potassium aspartate. 10 EGTA. I MgCI?. 10 HEPES-Et,NOH, 0.001 BAY K 8644.The patch was held at -80 mV and was depolarized to the test potentials indicated. ( B ) Single Ca channels recorded in planar lipid bilayers after the incorporation of cardiac membrane vesicles. The external solution contained ( m M ) I10 BaCI,, 10 HEPES-NaOH. pH 7.4. 0.001 BAY K 8644.The internal solution contained I50 KCI, I EDTA. 10 HEPESNaOH. pH 7.4,0.001 BAY K 8644. The membrane was held at -80 mV and was depolarized to the test potentials shown. (C) Open channel I-V relationships from the cell-attached patch (line) and planar lipid bilayer (filled circles). The I-V from the cell-attached patch was obtained from a ramp depolarization as described by Hess et t r l . ( 1986).

-

R. L. ROSENBERG ET A t .

406

A

100Ba,50Na//100Ba,50Na

Vtest

B

100Ba,50N a//50N a

Vtest

I

1

-80rnV

I

-80mV

= -30mV

v I

-10

C

I

I

I

100

-100

P -3

Y

200

V (mVf

-

FIG.8. Comparison of Ca channel currents with symmetric and assymmetric solutions. (A) Current recordings obtained with symmetric solutions (in mM) of 50 NaCI. 100 BaCI,. I EDTA, 10 HEPES-NaOH. pH 7.4,O.OOl BAY K 8644. Membranes were depolarized from a holding potential of -80 m V to the indicated test potentials. (6)Current recording with asymmetric solutions. External solution was the same as in A, internal solution contained (mM)50 NaCI, 1 EDTA, 10 HEPES-NaOH, pH 7.4. (C) I-V relationships under asymmetric (open circle) and symmetric (filled circles) ionic conditions.

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

407

Recordings of purified, reconstituted dihydropyridine receptors from skeletal muscle T-tubules include the activity of a functionally symmetric channel with a conductance of 20 pS (Flockerzi er al., 1986), in addition to one with a conductance of 12 pS (Smith er al., 1987; Talvenheimo et al., 1987). Thus, Ca channels from different tissues appear to be functionally symmetric, despite differences in channel conductance. Another way to examine the symmetry of the pore is to study-Ca’+ block of Ba” currents when it is added from either side of the membrane. From the outside, in the presence of 50 mM Ba”. the K, for Ca” block is approximately 10 mM (Lansman et a / . , 1986). From the inside, with only 50 mM Na’ present, the K i is approximately 4 mM (not shown). The difference is consistent with the different occupancy of the sites, and probably indicates that that block is equally effective from the inside and outside.

-

V.

DISCUSSION

These experiments provide a glimpse of the structure of the cardiac Ltype Ca channel from two distinct vantage points: an end-on view of its minimal radial dimensions, and a lateral view of its energetic symmetry along the pathway of conduction. Inferences from electrophysiological measurements may be complementary to emerging evidence from other approaches.

A.

Pore Size

In showing significant permeability to organic cations as large as tetramethylammonium, the L-type Ca channel in heart resembles its counterpart in skeletal muscle (McCleskey and Almers, 1985). As summarized in Fig. 9, the large apparent pore diameter (5.5-6.0 A) distinguishes Ca channels from other voltage-sensitive channels such as K or Na channels, which exclude all methylated ammonium ions (Hille, 1971, 1973). The Ca channel pore approaches the apparent size of the nicotinic acetylcholine receptor channel (Dwyer et al., 1980). Whereas the acetylcholine receptor channel shows little selectivity among monovalent cations, Ca channels show considerable selectivity among monovalent cations and great selectivity (- lo3) for divalents over monovalents. In this light, the apparent size of the Ca channel is particularly noteworthy. The large diameter of the pore provides a simple basis for arguing against a sieving mechanism for selectivity which accepts or

408

R. L. ROSENBERG ET AL. K

Na

END-PLATE

Ca

3.1 * 5.1 A

6.5 6.5 A

6.0 * 6.0 A

@ 3.3 3.3 A

FIG.9. Deduced pore sizes of some physiological ion channels and a representation to scale of a Ca ion with a water molecule. The cartoon of the pores of K . Na, and end-plate channels is taken from Dwyer e/ d.(1980); that of the Ca channel is from McCIeskey and Almers (1985).

rejects ions on the basis of their unhydrated size. On the other hand, the pore size is compatible with other lines of evidence suggesting that the very high selectivity of Ca channels for Ca2+and other divalent ions depends upon high-affinity binding of divalent ions rather than selective exclusion at a narrowing in the pore (see Tsien ef al., 1987b, for review). How can the Ca channel be so effective in binding Caz+ ions whose diameter is only 2 A, if the minimal diameter of the pore itself appears to be 5.5 A? A likely possibility is that peptide side chains within the pore can move inward to complex the Ca” ion. That is, the presence of Ca” in the pore may induce a local conformational change of the protein, causing a constriction of the pore to -2 A and permitting effective ion binding. Alternatively, the pore may remain 5.5 A in diameter, and a water molecule may accompany the Ca ion into the pore, providing for indirect coordination in the binding “pocket,” as proposed for Ca” binding to troponin C (Herzberg and James, 1985). It is interesting to note that a water-Ca’+ pair has a molecular size that would allow a close fit into the pore of the Ca channel (McCleskey and Almers, 1985). B. Pore Symmetry

Bilayer recordings with symmetric solutions provide information about the energetic profile seen by ions as they pass through the channel. Finding that inward and outward currents are equal in magnitude for a given driving force places strong constraints on how asymmetric the energy profile can be. For example, the ion-binding sites (“wells”) must be positioned at roughly equal electrical distances from the bulk solutions; also, the height and steepness of the peripheral barriers must be approximately equal as viewed from either end of the pore.

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

409

C. Pore Structure

Inferences about pore structure from electrophysiogical approaches will need to be combined with more direct evidence from biochemistry or molecular biology. Such a combination of techniques may not be long in coming, now that Tanabe PZ cil. (1987) have reported the primary sequence

TABLE I COMPARISON OF CARDIAC A N D SKELETAL MUSCLECALCIUM CHANNELS Cardiac Permeation Single channel conductance (100 m M Ba") Single channel conductance (I50mMLi') High-affinity Ca" site, Kd Anomalous mole fraction effect Pore diameter Cd'+ block IC,,, Mg" permeability Diamino compounds Kinetics Activation Inactivation Drug sensitivity Dihydropyridines Verapamil Diltiazem P-Adrenergic modulation

Skeletal

23 ps"

11-12 and 20 pS"

45 pS'

18 pS"

1.0 p M

0.7 pM1 Yes'

Yes" >5.5 A'a 10 pM'

Not measurable' Block''

>5.5

At

>I00 pMh Yes' Permeate'

2 msec' V. Cadependent'

200 msec'

Yes'" Yes Yes Yes

Yes' Yes Weak Yes

~

"Reuter et id., 1982: Cavalie e / d..1983. 1986; Hesb e / d.,1986: Rosenberg e / ( I / . , 1986. "Rosenberg ei d . . 1986; Flockerzi rt d..1986: Coronado and Smith, 1987; Smith PI ( I / . , 1987: Talvenheimo e / . d . , 1987. 'Hess et d..1986. "Coronado and Smith. 1987. "Hess and Tsien. 1984, and references therein. 'Almers e / d..1984. and references therein. 'Almers and McCleskey. 1984. "This paper. 'McCleskey and Almers. 1985: Coronado and Smith. 1987. 'Lansman e / d . . 1986. and references therein. 'Almers et id., 1985. and references therein. 'Lee e / id., 1985. "'Reuter. 1983.

41 0

R. L. ROSENBERG ET AL.

of the skeletal muscle dihydropyridine receptor. This protein may prove to be the Ca channel itself, or a closely related protein that acts as a voltage sensor. In either case, it may soon be possible to compare the structures and functions of various Ca channels from various tissues. At present, it may be useful to summarize some similarities and differences between Ca channels in cardiac and skeletal muscle (Table I). The channels are very similar in their high affinity for Ca” and their large pore size (Almers and McCleskey, 1984; McCleskey and Almers, 1985; Hess and Tsien, 1984; see also this chapter, Section IILA). They also share sensitivity to organic Ca antagonists, with the exception of diltiazem, which blocks cardiac Ca channels much more potently than skeletal muscle Ca channels (Lee and Tsien, 1983; Almers and McCleskey, 1984). Cardiac and skeletal channels differ greatly in the speed of activation (Almerset al., 1985) and in certain aspects of ion permeation. Cd2+is a highly potent blocker of the cardiac L-type Ca channel but distinctly weaker on skeletal muscle Ca” currents (Lee and Tsien, 1984; Lansman et al., 1986; Almers et al., 1985). Mg’+ and diamino compounds are permeant in skeletal muscle (McCleskey and Almen, 1985), but block the L-type cardiac Ca channel (Hew et al., 1986; see also this chapter, Section 111,B).A dihydropyridine-sensitive Ca channel in skeletal muscle has a Ba” conductance of 10-12 pS (Affolter and Coronado, 1985; Rosenberg et al., 1986; Ma and Coronado, 1988; Smith et al., 1987; Talvenheimo e f al., 1987), whereas the conductance of the L-type Ca channel from heart is 23-25 pS (Reuter et a / . , 1982). The differences in permeation and blocking properties could reflect detailed differences in the intrapore binding sites, but there seems to be little doubt that the overall mechanism of selection by affinity is the same for channels in both tissues. REFERENCES Affolter. H.. and Coronado, R. (1985). Agonists Bay K 8644 and CGP 28392 open calcium channels reconstituted from skeletal muscle transverse tubules. Biophys. J. 48, 341341. Akaike, N., Lee, K. S.. and Brown, A. M. (1978). The calcium current of Helix neuron. J. Gen. Phvsiol. 71, 509-531. Almers, W., and McCleskey, E. W. (1984). Non-selective conductance in calcium channels in frog muscle: calcium selectivity in a single-file pore. J. Physiol. (London) 353, 585608. Almers, W., McCleskey, E. W., and Palade, P. (1984). A non-selective cation conductance in frog muscle membrane blocked by micromolar external calcium ions. J. Physiol. (London) 353, 565-583. Almers, W., McCleskey, E. W., and Palade, P. (1985).Calcium channels in vertebrate skeletal muscle. In “Calcium in Biological Systems” (R. P. Rubin, G. B. Weiss, and J. W. Putney, eds.), pp. 321-330. Plenum, New York. Almers, W., McCleskey, E. W., and Palade, P. (1986). The mechanism of ion selectivity in calcium channels of skeletal muscle membrane. Forrschr. 2001. 33, 61-73. Bormann, J., Hammill, 0. P., and Sakmann, B. (1987). Mechanism of anion permeation

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

41 1

through channels gated by glycine and y-aminobutyric acid in mouse cultured spinal neurons. J . Physio/. (London) 385, 243-286. Byerly, L.. Chase, P. B., and Stimers, J. R. (1985). Permeation and interaction of divalent cations in calcium channels of snail neurons. J . Gen. Physiol. 85, 491-518. Cavalie, A,, Ochi, R., Pelzer, D., and Trautwein. W. (1983). Elementary currents through Ca” channels in guinea pig myocytes. Pj7uegers Arch. 398, 284-297. Cavalie, A., Pelzer, D., and Trautwein, W. (1986). Fast and slow gating behaviour of single calcium channels in cardiac cells. Relation to activation and inactivation of calciumchannel current. Pguegers Arch. 406,241-258. Chesnoy-Marchais. D. (1985). Kinetic properties and selectivity of calcium-permeable single channels in Ap/vsiu neurons. J. Physiol. (London) 367, 457-488. Coronado, R.. and Miller, C. (1982). Conduction and block by organic cations in a K-selective channel from sarcoplasmic reticulum incorporated into planar phospholipid bilayers. J . Gen. Pliysiol. 79, 529-547. Coronado, R., and Smith, J. S. (1987). Monovalent ion current through single calcium channels of skeletal muscle transverse tubules. Biophys. J . 51, 497-502. Cota. G., and Stefani. E. (1984). Saturation of calcium channels and surface charge effects in skeletal muscle fibres of the frog. J . Pliysiol. (London) 351, 135-154. Dwyer. T. M., Adams. D. J., and Hille, B. (1980). The permeability of endplate channels to organic cations in frog muscle. J . Gen. Phvsiol. 75, 469-492. Flockerzi, V., Oeken. H.-J., Hofmann, F.. Pelzer, D., Cavalie. A.. and Trautwein. W. ( 1986). Purified dihydropyridine-binding site from skeletal muscle t-tubules is a functional calcium channel. Nutiire (London) 323, 66-68. Fox, A. P.. and Krasne. S. (1984). Two calcium currents in N e m t h e s urrn~icoedenttrtirs egg cell membranes. J. Phvsiol. (London) 356, 491-505. Fukushima, Y . . and Hagiwara. S. (1985). Currents carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes. J. Physiol. (London) 358, 255284. Hagiwara, S.. and Byerly, L. (1981). Calcium channel. Annri. Rev. Neiirosci. 4, 69-125. Hagiwara. S .. and Ohmori. H. (1983). Studies of single calcium channel currents in rat clonal pituitary cells. J . Physiol. (London) 336, 649-661. Herzberg, 0.. and James. M.-N. G. (1985). Structure of the calcium regulatory muscle protein troponin-C at 2.8 A resolution. Nuritre (London) 313, 653-659. Hess. P., and Tsien. R. W. (1984). Mechanism of ion permeation through calcium channels. Nature (London) 309,453-456. Hess, P., Lansman. J. B.. -nd Tsien. R. W. (1984). Different modes of Ca channel gating behavior favoured by dihydropyridined Ca agonists and antagonists. Ntiture (London) 311, 538-544. Hess. P.. Lansman. J. B.. and Tsien. R. W . (1986). Calcium channel selectivity for divalent and monovalent cations. Voltage- and concentration-dependence of single channel current in guinea pig ventricular heart cells. J . Gen. Pliysiol. 88, 293-319. Hille, B. (1971). The permeability of the sodium channel to organic cations in myelinated nerve. J. Gen. Plrvsiol. 58, 599-619. Hille, B. (1973). Potassium channels in myelinated nerve. Selective permeability to small cations. J. G m . Plivsiol. 61, 669-686. Hille, B.. and Schwarz. W. (1978). Potassium channels as multi-ion, single-file pores. J. Gerr. Ptivsiol. 72, 4 0 9 4 2 . Kokubun, S.. and Reuter. H. (1984). Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells. /‘roc. N u t / . Acud. Sci. U.S.A. 81, 48244827. Kostyuk. P. G.. and Krishtal. 0. A. (1977). Effects of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones. J . Plivsiol. (London) 270, 569-580.

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Kostyuk, P. G., Mironov. S. L., and Shuba. Y. M. (1983). Two ion-selecting filters in the calcium channel of the somatic membrane of mollusc neurons. J . Memhr. Biol. 76, 8393. Lansman. J. B., Hess, P.. and Tsien. R. W. (1986). Blockade of current through single Ca channels by C d 2 + ,Mg” and Ca”. Voltage and concentration dependence of Ca entry into the pore. J . Gen. Physiol. 88, 321-347. Lee, K. S., and Tsien, R. W. (1983). Mechanism of calcium channel blockade by verapamil. D600, diltiazem and nitrendipine in single dialysed heart cells. Nature (London) 302, 790-794. Lee. K. S., and Tsien, R. W. (1984). High selectivity of calcium channels in single dialysed heart cells of the guinea pig. J . Physiol. (London) 354, 253-272. Lee. K. S., Marban, E., and Tsien, R. W. (1985). Inactivation of calcium channels in mammalian heart cells: Joint dependence on membrane potential and intracellular calcium. J. Physiol. 364, 395-4 I I . Lux. H. D. (1986). Na permeability of the low-voltage activated Ca channel. Proc. In,. Union Physiol. Sci. 16, 559. Ma, J., and Coronado, R. (1988). Heterogeneity of conductance states in calcium channels of skeletal muscle. Biophys. J . 53, 387-395. McCleskey, E. W., and Almers, W. (1985). The Ca channel in skeletal muscle is a large pore. Proc. Null. Acad. Sci. U.S.A. 82, 7149-7153. Matsuda, H., and Noma, A. (1984). Isolation of calcium current and its sensitivity to monovalent cations in dialysed ventricular cells of guinea pig. J . Physiol. (London) 357,553573. Miller, C. (1982). Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K’ channel. J . Gen. Physiol. 79, 869-891. Miller, R. J. (1987). Calcium channels in neurones. I n “Structure and Physiology of the Slow Inward Calcium Channel” (D.J. Triggle and J. C. Venter, eds.), Receptor Biochemistry and Methodology, Vol. 9, pp. 161-246. Alan R. Liss, New York. Nilius, B., Hess. P., Lansman, J. B., and Tsien, R. W. (1985). A novel type of cardiac calcium channel in ventricular cells. Nuture (London) 316, 443-446. Ochi, R., Hino, N., and Niimi, Y. (1984). Prolongation of calcium channel open time by the dihydropyridine derivative Bay K 8644 in cardiac myocytes. Proc. J p n . Acud., Ser. B 60, 153-156. Reuter, H. (1983). Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature (London) 301, 569-574. Reuter, H.. and Scholz, H. (1977). A study of the ion selectivity and the kinetic properties of the calcium dependent slow inward current in mammalian cardiac muscle. J. Physiol. (London) 264, 17-47. Reuter, H., Stevens, C.F., Tsien, R. W., and Yellen, G. (1982). Properties of single calcium channels in cultured cardiac cells. Nature (London) 297,501-504. Rosenberg. R. L., Hess, P.. Reeves, J., Smilowitz, H., and Tsien, R. W. (1986). Calcium channels in planar lipid bilayers: new insights into the mechanisms of permeation and gating. Science 231, 1564-1566. Rosenberg, R. L., Hess, P., and Tsien, R. W. (1988). Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative mernbrane potentials. J . Gen. Physiol. (in press). Smith, J. S., McKenna, E. J., Ma, J., Vilven. J., Vaghy, P. L., Schwartz, A., and Coronado, R. (1987). Calcium channel activity in a purified dihydropyridine receptor preparation of skeletal muscle. Biochemistry 26, 7182-7188.

19. PORE SIZE AND SYMMETRY OF Ca CHANNELS

41 3

Talvenheimo. J. A.. Worley. J . F.. and Nelson, M. T. (1987). Heterogeneity of calcium channels from a purified dihydropyridine receptor preparation. Biophvs. J. 52, 891899. Tanabe, T., Takeshima. H., Mikami, A., Flockerzi. V., Takahashi, H., Kangawa, K.. Kojima, M.. Matsuo, H.. Hirose, T., and Numa, S. (1987). Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nufure (London) 328, 313-3 18. Tsien. R. W., Fox, A. P., Hess, P.. McCleskey. E. W.. Nilius, B., Nowycky, M. C.. and Rosenberg, R. L. (1987a). Multiple types of calcium channel in excitable cells. Soc. Gen. Physiol. Ser. 41, 167-187. Tsien. R. W.. Hess. P., McCleskey, E. W., and Rosenberg. R. L. (t987b). Calcium channels: Mechanisms of selectivity, permeation. and block. Annu. Rev. Eiophys. Biophys. Chem. 16, 265-290. Urban, B. W., Hladky, S. B., and Haydon, D. A. (1980). Ion movements in gramicidin pores: an example of single-file transport. Biochim. Eiophys. Acra 602, 331-354.

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Part V

Conclusion

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CUKRENT TOPICS I N MEMBRANES AND TRANSPOKT. VOLUME 33

Chapter 20

o-Conotoxins and Voltage-Sensitive Calcium Channel Subtypes LOURDES J . CRUZ,*t DAVID S . JOHNSON,* JULITA S . IMPERIAL,* DAVID GRIFFIN,* GARTH W . LeCHEMINANT,* GEORGE P . MILJANICH, ** AND EALDOMERO M . OLIVERA* *Department of Biology University of Utah Salt Lake City, Utah 84112 AND ?Marine Science Institute University of the Philippines Metro-Manila, Philippines AND **Department of Biological Sciences University of Southern California Los Angeles, California 90089 I.

Introduction A . Ca Channels and w-Conotoxins B. Biological Background 11. The w-Conotoxins and Their Receptor Targets A. Biochemistry of w-Conotoxins B. Specificity of w-Conotoxin GVlA C. Cross-linking to Receptor Targets D. Receptor Targets in Mammalian versus Avian Systems E. w-Conotoxin MVIlA versus GVlA 111. Discussion References

1. A.

INTRODUCTION

Ca Channels and o-Conotoxins

The use of toxins has become a central approach for the characterization of receptors or ion channels in excitable cells. Recent advances in un41 7 Copyright @ I IYXX hy ALadrmir. Prr% InL All rights ot reprodwtion in m y form rererved

41 8

LOURDES J. CRUZ ET AL.

derstanding the nicotinic acetylcholine receptor and voltage-sensitive sodium channels have been achieved largely because of the availability of a-bungarotoxin (Karlin, 1980; Conti-Tronconi and Raftery, 1982) and saxitoxin/tetrodotoxin (Catterall, 1984; Agnew, 1984) as molecular probes for these large receptor-channel complexes. Voltage-sensitive Ca channels are key macromolecules in the function of the nervous system. Ultimately, electrical signals are decoded into biochemical events by the passage of Ca ions across a membrane. At the presynaptic terminus, neurotransmitter release is controlled by voltageactivated Ca channels. The molecular characterization of neuronal Ca channels would therefore be a major advance in understanding the coupling of electrical and biochemical events in the nervous system. The major probes that have been used to characterize voltage-sensitive Ca channels in excitable tissue are a group of drugs which have collectively been called the organic blockers (Hagiwara and Byerly, 1981; Tsien, 1983; Janis and Triggle, 1983; Murphy el af.,1983), to differentiate them from inhibitory cations (Co2+,La”). These include the phenylalkylamines such as verapamil, the benzothiazepines such as diltiazem, and, most important, the dihydropyridines such as nitrendipine or nimodipine. The use of these agents has permitted the purification of the first voltage-sensitive Ca channels, from the T-tubules of skeletal muscle (Glossmann et al., 1983; Curtis and Catterall, 1984; Borsotto et al., 1984). Recently, our laboratories have been studying the venoms of fish-hunting cone snails (Olivera et af., 1985). We isolated and characterized a group of toxins from these venoms, the o-conotoxins, which inhibit presynaptic Ca channels (Olivera et al., 1984). In addition to the Ca channel toxins, the venoms of these remarkable snails contain two additional paralytic toxins, the a-conotoxins which inhibit the nicotinic acetylcholine receptor (Gray et al., 19811, and the pconotoxins which selectively inhibit voltagesensitive sodium channels in muscle, but not in motor neuron axons (Cruz et al., 1985a). In addition, there are numerous biologically active components in the venoms of the fish-hunting cone snails, some of which have been biochemically characterized (McIntosh et at., 1984; Clark et al., 1981). In this article, however, we will focus only on the o-conotoxins and their receptor targets. Our studies have demonstrated that the o-conotoxins show remarkable tissue specificity, at least in comparison with other presently known Ca channel blockers, including the dihydropyridines. Thus, at the present time w-conotoxins are known to target only to voltage-sensitive Ca channels in neuronal tissue, but not to Ca channels in skeletal muscle, smooth muscle, or cardiac muscle (Cruz and Olivera, 1986; McCleskey et al., 1987). However, within vertebrate neuronal tissue, we will present evidence that the w-conotoxins have multiple receptor subtypes. The results

20. W-CONOTOXINS

41 9

of these studies suggest that, at presynaptic terminii, there may well be several different voltage-sensitive Ca channel subtypes. It should be noted that multiple types of voltage-sensitive Ca channels have been detected by physiological and pharmacological criteria. In one prominent classification scheme (Nowycky et at., 1985). three channel types have been proposed, the L, T, and N Ca channels. Our present understanding of the relationship between the Ca channel types previously defined, and the subtypes detected by the biochemicaVtoxicological criteria used here, is discussed in the last section of this article.

B. Biological Background

Most of the fish-hunting cones such as Conus striutus, Conus magus, and Conus purpurascens capture fish using a hook-and-line method; a feeding sequence of this type has recently been published (Olivera et al., 1985). However, Conus geogruphus (the species of Conus from which wconotoxin GVIA was isolated and characterized) uses a trapping technique. The animal can detect the presence of a fish by a chemoreceptor mechanism. It responds to this by opening its distensible rostrum, often to an extent where this is the widest point of the whole animal. When an unsuspecting fish enters what looks like a white crevice in the coral reef, it is engulfed by the snail closing the rostrum. Once engulfed, the fish is then stung with a disposable, harpoon-like tooth, thereby introducing a paralyzing venom into the prey. The paralyzed fish is then digested in the distensible stomach of the snail and a few hours later the fish scales and bones, as well as the Conus harpoonlike tooth that was used, are regurgitated. The venom apparatus of cone snails is shown in Fig. 1. The harpoonlike teeth are continuously produced, specimens of C. geographits typically carrying several dozen at any time. The venom is actually made in a long tubelike venom duct. As outlined above, there are three basic toxins which are produced in the venom duct of C . geogruphus, the w, a , and pconotoxins. Preliminary data suggest that these toxins are processed from a larger precursor, which is probably contained within the ellipsoidal granules that are prominently found in the venom. II. THE w-CONOTOXINS AND THEIR RECEPTOR TARGETS A.

Biochemistry of o-Conotoxins

The o-conotoxins were originally discovered because they cause a characteristic tremor when injected intracerebrally (ic) into mice. To date,

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LOURDES J. CRUZ ET AL.

FIG.I . The venom apparatus of Conr/s. The long thin tube is the venom duct: at its end is the venom bulb. The radular apparatus, where the disposable harpoon-like teeth are made, is also indicated. In this reconstruction, one harpoon has been moved to the tip of the proboscis (drawing courtesy of Kerry Matz).

nine o-conotoxins have been isolated and characterized from three different piscivorous species, the geography cone, C. geographus (Olivera et al., 1984, 1985), C. magus (Olivera et al., 1987), and C. striatus (G. Zafaralla and L. J. Cruz, unpublished observations). The two best characterized of the o-conotoxins are o-conotoxin GVIA from C. geographus, and w-conotoxin MVIIA from C. magus (for a note regarding the nomenclature of conotoxins, see Cruz et al., 1985b). These two peptide toxins have been chemically synthesized (Olivera et ul., 1985,

42 1

20. W-CONOTOXINS

1987; Nishiuchi et al., 1986; Rivier et al., 1987), radiolabeled, and their binding to receptor targets extensively characterized (Cruz and Olivera, 1986; Abe et al., 1986; Cruz et al., 1987; Olivera et al., 1987). Most electrophysiological and biochemical studies carried out so far have employed w-conotoxin GVIA, which at present is the most widely available of the w-conotoxins. A comparison of the sequences of w-conotoxin GVIA and MVIIA is shown in Fig. 2.

B. Specificity of o-Conotoxin GVlA A brief review of the specificity of the w-conotoxin which has been studied in most detail, w-conotoxin GVIA, is given here. Both electrophysiological data, as well as binding data indicate that wconotoxin GVlA is highly tissue specific. The toxin does not have biological effects on, nor does it have high-affinity binding sites for, voltagesensitive Ca channels in skeletal muscle T-tubules or cardiac muscle (Cruz and Olivera, 1986; McCleskey et al., 1987). Recent data suggest that even neuronlike cell lines (such as those derived from adrenal chromaffh cells) are not sensitive to w-conotoxin (E. Neher, personal communication; R. Miller, personal communication). In addition, in a developing system such as chick retinal neuron, axon outgrowth is not inhibited by w-conotoxin (B. Olivera and F. Bonhoeffer, unpublished results). Furthermore, in Rohan Beard neurons in the developing amphibian embryo, sensitivity to wconotoxin was not detected (D. Johnson and N . Spitzer, unpublished observations). The present data therefore lead to the tentative conclusion that w-conotoxin receptor targets are largely restricted to differentiated neuronal tissue. There appears to be a phylogenetic gradient of sensitivity to wconotoxin GVIA. Thus, a combination of in vivo experiments, as well as binding experiments, indicate that presynaptic termini at the neuromuscular junction are sensitive to w-conotoxin GVlA in fish, frog, and avian neuromuscularjunctions (Ken and Yoshikami, 1984; Olivera et al., 1985; Z. Bagabaldo and D. Yoshikami, unpublished observations). In all these animals, when w-conotoxin GVIA is injected intraperitoneally (ip), paralysis and death results. In contrast, mice are insensitive to an ip injection

b.

GVI A MWA

CK

S H i 6 S 8 C S HYT S 1 I

3&

C K 6 I6 A K F S R 1 II Y D

FIG. 2. Sequences of w-conoloxins GVlA and MVIIA. The two sequences have been aligned at their cysteine residues. The one-letter code is used: Hy. hydroxyproline. The asterisk indicates that the C-terminus i s blocked with an amide group.

422

LOURDES J. CRUZ ET AL.

of w-conotoxin GVIA, suggesting that mammalian neuromuscular junction presynaptic Ca channels are not a high-affinity target for this toxin. Binding experiments have been done on the electric organ of Torpedo;a low-affinity site is present at the presynaptic terminus (Yeager er af., 1987). Data with synaptosomes would indicate that instead of affinities in the subnanomolar range, in the Torpedo electric organ, voltage-sensitive Ca channels are inhibited at micromolar concentrations of the toxin. Thus, from teleosts to the electric ray, there is a > lo3decrease in w-conotoxin affinity for the presynaptic receptors. All experiments that have been done on invertebrate preparations indicate that w-conotoxin is inactive; the toxin has specifically been tested on the Drosophila neuromuscular junction, and no biological effects were found (D. Yoshikami, unpublished observations). C. Cross-Linking to Receptor Targets

A number of experiments have been carried out to identify w-conotoxin targets by using affinity cross-linking. The basic strategy is to bind radiolabeled w-conotoxin GVIA to its receptor target, and to analyze what proteins become specifically radiolabeled. As has been previously published (Cruz et af., 1987). using chick membranes, a polypeptide with a denatured molecular weight of approximately 135,000 is the primary target radiolabeled by cross-linking using the bivalent cross-linker DSS (disuccinimidyl suberate). Recent studies using a more extensive variety of protocols have revealed that in chick brain membranes, up to seven different polypeptide targets can be cross-linked to radiolabeled w-conotoxin GVIA (J. Imperial and L. J. Cmz, unpublished observations). Some of the cross-linked species have a much higher apparent MW than 135,000 (i.e., 240-300 K). These results raise the possibility that the different receptor targets radiolabeled in cross-linking experiments may represent multiple voltage sensitive Ca channel subtypes. D. Receptor Targets in Mammalian versus Avian Systems

The in vivo effects of w-conotoxin GVIA are strikingly different in mammalian and avian systems. In chicks, w-conotoxin GVIA causes paralysis and lethality whether it is injected by the ip or ic routes. In contrast, in mice, injection of w-conotoxin GVIA ip causes no obvious biological effects; however, when injected by the ic route, the toxin is not lethal but a characteristic shaker syndrome is induced. We hypothesize that the results above can be explained by postulating a minimum of two types of neuronal voltage-sensitive Ca channels in avian and mammalian systems; we will tentatively call these A and B Ca channel

423

20. w-CONOTOXINS

subtypes. One of these (type A) controls neurotransmitter release at the neuromuscularjunction (and perhaps other excitatory synapses), and the second (type B) is primarily involved in the central nervous system (at inhibitory synapses?). Our results suggest that w-conotoxin GVIA can inhibit the postulated type A voltage-sensitive Ca channel in avian systems, but not in mammalian systems; in contrast, the toxin inhibits type B channels in both avian and mammalian systems. Inhibition of type A channels causes paralysis and lethality ip, and lethality ic. Inhibition of the second type of channel causes shaker symptoms in mammals; we observed that injection of chicks with sublethal doses of o-conotoxin GVIA ic will also elicit shaker symptoms. Consistent with the hypothesis above are measurements of 4sCauptake using brain synaptosomal preparations. Although in chick o-conotoxin GVIA can inhibit >90% of 4’Ca uptake in the most favorable experiments (Rivier et a / . , 1987), inhibition of 4’Ca uptake in bovine brain synaptosomes is only about 50%, even at the highest toxin concentrations (Reynolds et ul., 1986; J. Wagner, personal communication). This is consistent with a significant fraction of 45Cauptake in bovine synaptosomes being mediated by a Ca channel which is not o-conotoxin sensitive. We postulate that this o-conotoxin-insensitive Ca mammalian channel is the type A channel. A summary of this hypothesis is given in Table I.

TABLE I COMPARISON OF U-CONOTOXIN EFFECTSI N AVIANAND MAMMALIAN SYSTEMS System Avian Treat men t w-Conotoxin, ip injection w-Conotoxin, ic injection w-Conotoxin. synaptosomal 4’Ca uptake Nitrendipine. ic injection Nitrendipine. synaptosomal T a uptake Hypothesis A channels inhibited by w-conotoxin? B channels inhibited by w-conotoxin? A or B channels inhibited by nitrendipine?

Lethal Lethal

Mammalian

>90% inhibition

N o effect Nonlethal, “shaker symptoms” -50% inhibition

No effect No inhibition

No effect N o inhibition

Yes

No

Yes

Yes

No

No

424

LOURDES J. CRUZ ET AL.

It should be noted that, under the experimental conditions where wconotoxin elicits marked biological effects, injection of nitrendipine does not elicit either lethality or shaker symptoms, and, in most experiments with synaptosomes, does not inhibit 4JCauptake in either the avian or the mammalian system. E. w-Conotoxin MVllA versus GVlA

The hypothesis above proposing two types of voltage-sensitive Ca channels is also supported by the in vivo results, as well as binding competition data, using two different w-conotoxins, GVlA and MVIIA. In chick and mice, the in vivo effects of w-conotoxin MVllA are indistinguishable from the effects of GVlA described above. In contrast, we discovered that, in amphibians, the two w-conotoxins had different effects. A comparison of the in vivo effects of two toxins, w-conotoxin GVIA isolated from C. geogruphus and w-conotoxin MVlIA isolated from C. mugris, is shown in Table 11. Since o-conotoxin MVllA is not lethal upon ip injection into frogs, using the hypothesis above we would interpret this to mean that, in the amphibian system, w-conotoxin GVlA inhibits type A channels while w-conotoxin MVllA does not. We have tested whether there is a correlation between binding and in vivo activity. As we have previously shown (Fig. 3, left), w-conotoxin MVIIA does not compete for all of the sites that are bound by w-conotoxin GVIA (Olivera et d.,1987). In contrast, when chick synaptosomes are examined, there is competition for all w-conotoxin GVlA sites (Fig. 3, right). Thus, we interpret the differential binding data, and the different in vivo effects, in the following way. In amphibians, w-conotoxin GVlA binds and inhibits both type A and type B channels; in contrast, we postulate that w-conotoxin MVllA has a high affinity only for type B channels, and

In

ViVO

TABLE I1 EFFECTS OF w-CONOTOXINS AND NITRENDIPINE Lethality, ip injection

Fish Amphibian GVIA MVllA Nitrendipine

+ + -

+

-

Avian

Mammalian

+

-

-

-

+

-

Shaker syndrome, ic injection, mammals

+ + -

425

20. W-CONOTOXINS

I

.2=g C

1 .o m \\ TI Q) 0.8 -\ \ \\ .E

5 0.6 -\\\ \ \ Q

.-o .-

0.4

c

2O .EU

.

\

- \\

m-m

\

0.2 - \

LLD

0

-

A+ B -\\

\ \

\ t-A

A

0.1

0.2

-

A

A-A

0.1

0.2

FIG.3 . Binding competition between unlabeled w-conotoxin MVllA and '"I-labeled wconotoxin G V l A in frog brain membrane preparations (a) and chick brain membrane preparations (b). The methods used are described in Olivera c't t i / . ( 1987). The membrane preparations were preincubated with either unlabeled MVllA ).( or unlabeled G V l A (A)before '"I-GVIA was added. The fraction of binding represented by the postulated type A and type B channels are indicated (see text).

not for type A channels. Finally, as suggested above, since neither toxin causes lethality and paralysis in mammals, but both toxins cause shaker symptoms, we suggest that neither o-conotoxin G V l A nor M V l l A binds type A channels in mammalian systems, but both toxins bind type B channels. Experiments measuring 4'Ca uptake by mammalian synaptosomes indicate that 50% of 45Cauptake is not inhibited by o-conotoxin GVIA. However, within the class of Ca channels which are w-conotoxin inhibitable, there are two distinguishable affinity classes (Reynolds et d.,1986). Thus, approximately 30% of the total 4cCauptake is inhibited at 50 pM toxin, while an additional 20% is inhibited at o-conotoxin concentrations >5 nM. These experiments suggest that the type B channels which we have proposed in the hypothesis above may perhaps be further subdivided into BI and B11 subclasses. We explain the profile of inhibition of %I uptake in mammalian brain synaptosomes by postulating a BI subclass of Ca channels inhibited by w-conotoxin at 50 pM levels. a BII subclass inhibited by o-conotoxin at nanomolar levels, and the A subclass, which is not inhibited by w-conotoxin in the mammalian system. In contrast, all of the major subclasses of Ca channels are inhibited by w-conotoxin GVlA in avian brain synaptosomes. It should be noted that, since nitrendipine does not inhibit 45Cauptake under these conditions, none of the 4'Ca uptake seen in such synaptosomal

426

LOURDES J. CRUZ ET AL.

preparations is mediated by Ca channels which belong to the “L class” as defined by the presently proposed pharmacological criteria.

111.

DISCUSSION

Our data clearly indicate that w-conotoxins are much more tissue specific with respect to binding voltage-sensitive Ca channels than are the wellcharacterized organic blockers of Ca channels that have been developed in the last decade. This specificity for neuronal Ca channels may be a consequence of the biological function of w-conotoxins in the venom of piscivorous Conus spp. It is clearly desirable for these snails to concentrate the toxins where they will be most effective in paralyzing prey. Since there are voltage-sensitive Ca channels in a large number of peripheral tissues, a voltage-sensitive Ca channel antagonist would potentially have a large number of binding sites if it did not discriminate between different voltage-sensitive Ca channel subtypes. There is thus very strong selective pressure for such channel subtype discrimination to have evolved. It would appear that the presynaptic terminus is the most critical Ca channel target in the prey animal. We suggest that these toxins not only have structural features to bind with high affinity to their presynaptic neuronal Ca channel targets, but that the w-conotoxins have specific structural features to prevent them from binding to muscle and cardiac Ca channels. Despite the high tissue specificity of o-conotoxins, the evidence summarized here strongly indicates that there are multiple receptor subtypes which are targets of w-conotoxins in neuronal tissue. Partly, this evidence comes from the comparative toxicological approach using both voltagesensitive neuronal Ca channels in different vertebrate systems, and different w-conotoxins (GVIA and MVIIA). We suggest that there are probably three neuronal Ca channel subtypes which are targets for w-conotoxins. Curiously, by pharmacological criteria, none of the subtypes are inhibited by the dihydropyridines and would therefore be putative N-type Ca channels (i.e., dihydropyridine-insensitive and, at least potentially, wconotoxin-sensitive). Of course, a critical examination of these different subtypes by physiological criteria has not yet been carried out at the present time, but their pharmacology is consistent with being N subtypes. The results are consistent with the suggestion that N Ca channels can be further subdivided into distinguishable subtypes. The N voltage-sensitive Ca channel is believed to be the primary neuronal Ca channel type which controls neurotransmitter release (see, e.g., Miller, 1987). The possibility of different Ca channel subtypes being in-

427

20. o-CONOTOXINS

volved in the control of neurotransmitter release is attractive since Ca channels would be the focal control point for the kinetics of neurotransmitter release. Kinetic differences in the release of excitatory versus inhibitory neurotransmitters would be expected. In addition, presynaptic terminii apposed to neurotransmitter receptors which directly control ion channels may have quite different transmitter release kinetics as compared to termini across neurotransmitter receptors which operate through second messenger systems. We suggest that these different types of nerve endings would require different Ca channel subtypes, depending on the neurotransmitter to be released and the receptor to which the neurotransmitter targets. One model for how different Ca channel subtypes may be distributed is given in Fig. 4.

T channels

i;;

Neurotransmitter Vesicles

0-0 0-0 cN channels

FIG.4. A working model illustrating one scheme for the distribution of different Ca channel subtypes. ( A ) One possible disposition of the three physiological types of Ca channels proposed by Nowycky C I t i / . (1985). The L and T channels may be primarily located on the cell body. while N channels may be at nerve endings. Different subtypes of N channels could be distributed as shown in ( B ) ; presynaptic termini which are primarily excitatory might have different C a channels from those which are inhibitory. In 8 . three types of presynaptic termini are shown. one excitatory and two inhibitory, mediating presynaptic and postsynaptic inhibition. The different N Ca channel subtypes might be found in different types of presynaptic termini. a s represented by the different square symbols.

428

LOURDES J. CRUZ ET AL.

ACKNOWLEDGMENTS This work was supported by the National Institutes of General Medical Sciences (GM22737) and the International Foundation for Science, Stockholm. Sweden. REFERENCES Abe, T., Kogano, K., Saisu, H., Nishiuchi, Y., and Sakakibara, S. (1986). Binding of wconotoxin to receptor sites associated with the voltage-sensitive calcium channel. Neurosci. Lett. 71, 203-208. Agnew, W. S. (1984). Voltage-regulated sodium-channel molecules. Annu. Rev. Physiol. 46,517-530. Borsotto, M., Barhanin, J., Norman, R. I., and Lazdunski, M. (1984). Purification of the dihydropyridine receptor of the voltage dependent Ca” channel from skeletal muscle transverse tubules using ( + ) - ’H-PN 2001 10. Biochem. Biophys. Res. Commun. 122, 1357-1 366. Catterall, W. (1984). The molecular basis of neuronal excitability. Science 233, 653-661. Clark, C., Olivera, B. M., and Cruz, L. J. (1981). A toxin from the venom of the marine snail Conus geogruphus which acts on the vertebrate central nervous system. Toxicon 19, 691-699. Conti-Tronconi, B. M., and Raftery, M. A. (1982). The nicotinic cholinergic receptor: Correlation of molecular structure with functional properties. Annu. Rev. Biochem. 51, 49 1-530. Cruz, L. J., and Olivera, B. M. (1986). Calcium channel antagonists. w-Conotoxin defines a new high affinity site. J. Biol. Chem. 261, 6230-6233. Cruz, L. J., Gray, W. R., Olivera, B. M., Zeikus, R., Kerr, L., Yoshikami, D., and Moczydlowski, E. (l985a). Conus geogruphus toxins that discriminate between neuronal and muscle sodium channels. J. B i d . Chem. 260, 9280-9288. Cruz, L. J., Gray, W. R., Yoshikami, D., and Olivera, B. M. (1985b). Conus venoms: A rich source of neuroactive peptides. J. Toxicol.--Toxin Rev. 4, 107-132. Cruz, L. J., Johnson. D. S., and Olivera, B. M. (1987). Characterization of the w-conotoxin target. Evidence for tissue-specific heterogeneity in calcium channel types. Biochemistry 26, 820-824. Curtis, B. M., and Catterall, W. A. (1984). Purification of the calcium channel antagonist receptor of the voltage sensitive calcium channel from skeletal muscle transverse tubule. Biochemistry 23, 21 13-21 18. Glossmann, H., Ferry, D. R., and Boschek, C. B. (1983). Purification of the putative calcium channel from skeletal muscle with the aid of [’HI-nimodipine binding. Nuunyn-Schmiedeberg’s Arch. Phurmacol. 323, 1-1 I . Gray, W. R.. Luque, A., Olivera, B. M., Barrett, J., and Cruz, L. J. (1981). Peptide toxins from Conus geogruphus venom. J. Biol. Chem. 256,4734-4740. Hagiwara, S . , and Byerly, L. (1981). Calcium channel. Annu. Rev. Neurosci. 4, 69-125. Janis. R. A.. and Triggle, D. J. (1983). New developments in Ca” channel antagonists. J . Med. Chem. 26, 775-785. Karlin, A. (1980). Molecular properties of nicotinic acetylcholine receptors. I n “The Cell Surface and Neuronal Function” (C. W. Cofman, G. Poste, and G. C. Nicolson. eds.). pp. 191-260. Elsevier, Amsterdam. Kerr, L. M., and Yoshikami, D. (1984). A venom peptide with a novel presynaptic blocking action. Noture (London) 308, 282-284. McCleskey, E. W., Fox, A. P., Feldman, D., Cruz, L. J., Olivera, B. M., Tsien. R. W.,

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429

and Yoshikami. D. ( 1987). Calcium channel blockade by a peptide from Conits: Specificity and mechanism. Proc. Null. Acrid. Sci. U . S . A . 84, 4327-4331. Mclntosh. J . M.. Olivera. B. M., Cruz, L. J., and Gray, W. R. (1984). y-Carboxyglutamate in a neuroactive toxin. J. Eiol. Chem. 259, 14343-14346. Miller, R. J . (1987). Multiple calcium channels and neuronal function. Science 235, 46-52. Murphy, K . M. M.. Gould. R. J., Sargent, B. L., and Snyder, S. H. (1983). A unitary mechanism of calcium antagonist drug action. Proc. Nut/. Acud. Sci. U . S . A . 80, 860864. Nishiuchi, Y ., Kumagaye, K., Noda, Y ., Watanabe, T . X., and Sakakibara, S. (1986). Synthesis and secondary-structure determination of w-conotoxin GVIA: A 27-peptide with three intramolecular disulfide bonds. Eiopolvmers 25, 561-568. Nowycky, M. C.. Fox, A. P., and Tsien, R. W. (1985). Three types of neuronal calcium channel with different calcium agonist sensitivity. Nurure (London) 316, 440-443. Olivera. B. M.. Mclntosh, J. M., Cruz, L. J.. Luque, F. A., and Gray, W. R. (1984). Purification and sequence of a presynaptic peptide toxin from Conus geogrriphus venom. Eiocliemislry 23, 5087-5090. Olivera. B. M.. Gray, W. R., Zeikus. R., McIntosh. J . M.. Varga, J., Rivier, J., de Santos. V., and Cruz. L. J. (1985). Peptide neurotoxins from fish-hunting cone snails. Science 230, 1338-1343. Olivera. B. M., Cruz. L. J.. de Santos, V.. LeCheminant, G., Griffin, D., Zeikus, R., Mclntosh. J. M., Galyean, R.. Varga, J., Gray, W. R.. and Rivier, J . (1987). Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using w-conotoxin from Conus mugus venom. Biochemistry 26, 2086-2090. Reynolds. I . J.. Wagner. J. A,. Snyder, S. H.. Thayer. S. A.. Olivera. B. M.. and Miller, R. J . ( 1986). Brain voltage-sensitive calcium channel subtypes differentiated by w-conotoxin fraction GVIA. Proc. Ntitl. Aciril. S c i . U . S . A . 83, 8804-8807. Rivier, J., Galyean, R., Gray, W. R.. Azimi-Zonooz, A.. Mclntosh. J. M., Cruz. L. J., and Olivera, B. M. (1987). Neuronal calcium channel inhibitors. Synthesis of w-conotoxin GVIA and effects on "Ca uptake by synaptosomes. J. Eiol. Chem. 262, 1194-1 198. Tsien. R. W. (1983). Calcium channels in excitable cell membranes. Annu. Rev. Physiol. 45, 341-358. Yeager, R. E.. Yoshikami, D., Rivier, J.. Cruz,L. J., and Miljanich, G. P. (1987). Transmitter release from presynaptic terminals of electric organ: lnhibition by the calcium channel antagonists, omega Coniis toxin. J. Neurosci. 7 , 2390-2396.

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Index

A

Ab initio methods, gramicidin A computational models, 98-100 ion permeation through channels, 95-96 quantitative predictions, 100-102 site-to-site transition rates, 102-104 StNCtUR, 97-98 valence selectivity, 104-107 Acetylcholine, single channel currents in BC3H-I cells, 135 closed-duration histograms at different concentrations, 138-139 at intermediate concentrations, 141-142 concentration dependence closed-duration histograms, 139 effective channel opening rate, 142-143 limiting low concentrations, 139-140 open-duration histograms at different concentrations, 135-138 saturating concentrations, 140-141 Acetylcholine receptors chemical kinetic measurement, 198-201 inhibitory site, 199-200 molecular genetics techniques for subunits, 201 rapid desensitization, 201 regulatory mechanisms, 200 slow inactivation (desensitization), 201 stable expression system, advantages, 221 subunit clones (I: azl~ornica) acetylcholine-induced"Na uptake, 225 acetylcholine-inducedsingle channel currents, 225 a-bungarotoxin binding, 225 cDNA introduction into cultured cells DNA-mediated gene transfer, 225-227

a-protein expression in fibroblasts and muscle cells, 236-238 viral infection, 227-228, 236 cDNA library construction, 222-223 cell surface, expression in oocytes, 229-230 cotransformation efficiency in fibroblast cells, 232-233 full-length, identification, 229 functional expression of transfected DNA in murine fibroblasts, 233-235 in muscle cells, 235 labeling and immunoprecipitations, 228 mRNA microinjection into oocytes. 223-224 mRNA transcripts, preparation. 223-224 oocyte preparation, 224 screening libraries for, 223 a-subunit expression in fibroblasts and muscle cells, 238-241 subunits, expression in S.cemisiae, 202-214 aimmunofluorescence detection, 205 orientation, 209-211 6-, immunofluorescence detection in plasma membrane, 206 immune blotting, 204 insertion into plasma membrane, 204-205, 207 ligand-binding properties, 207-209 plasmid construction, 202 posttranslation modifications, 204 'I: caliJomiaz tubular crystals, threedimensional image analysis, 198-199 N-Acetyl-gramicidin channels, gaps, 118, 120 431

432 Aconitine, effects on sodium channel kinetics and selectivity, 305 Action potentials, Itrmmecium, 2 Affinity chromatography, calcium antagonist receptor purification, 374 Alamethicin, X-ray diffraction, 290 Allosteric model, guanidinium toxin inhibition of sodium channels, 312, 315-316 a-helices ridges into grooves packing theory, 300-301 sodium channel transmembrane segments, 296-297 transmembrane segments, 291 eprotein, Torpedo, expression in fibroblasts and muscle cells, 236-238 a-toxin. hydrophobic side chains, 291-293 Amino acid analysis, sodium channel subunits in skeletal muscle, 253 Amino acid composition, eel electroplax sodium channel, 335 Amino acid sequences channel protein structure modeling by, 290 gramicidin, 35 sodium channel eel electroplax, 336-339 skeletal muscle, 259-260 Ammonium ions. methylated. currents carried by cardiac Ltype calcium channels, 3W-398 Anesthetics, local, inside blocking of sodium channels by, 304 Antibodies anti-peptide, acetylcholine receptor ligand-binding site probes, 174-175 a-subunit main immunogenic region localization, 172-174 subunit proteolytic fragments, identificationand mapping, 171-172 nicotinic acetylcholinereceptor, generation with synthetic peptides, 167 Antisense RNA probes, for sodium channel type 11, 281-282 Antisera, synthetic oligopeptide, for sodium channel topography, 261-263 Arginine side chains, sodium channel gating and, 301 Autocovariance function, power spectra of gramicidin A channel currents, 116-122 Azidopine, 'H-labeled, covalent labeling of calcium channel subunits, 382

INDEX

B & c i h bratis, gramicidin from, 15 Backbone structures, transmembrane segments, 291 Batrachotoxin activation of cation flux in reconstituted sodium channels, 255-257 effects on sodium channel kinetics and selectivity, 305 single sodium channel activity in lipid bilayers, 314-315 BAY K 8644, 371, 377-379 Binding constants cesium ion in gramicidin A channel, NMR estimation, 67 ions in gramicidin pores, 21 current-voltage relations, 27 equilibrium dialysis. 28 flw ratio exponents, 28 permeability ratios, 28 spectral changes, 28-29 water permeability, 28 Brain calcium channel components, immunospecific identification, 386-387 sodium channel type I1 mRNA, RNase protection analysis, 282-284, 286 N-Bromoacetamide, effect on reconstituted protein, 352-353 N-Bromosuccinimide,effect on reconstituted protein, 352-353 Brownian dynamics, ion permeation through channel proteins, 93 a-Bungarotoxin binding by acetylcholine receptor a-subunit in yeast membrane, 207, 209 by nicotinic acetylcholine receptor a-subunit synthetic peptide, 177-184 map of. 180 by Xenopus luevis cell-surface acetylcholine receptors, 225

C Calcium-43, interactions with gramicidin A transmembrane channel, 62-63 Calcium antagonist receptor detergent-solubilized,characterization, 373

INDEX

molecular size, 372 protein components, 372 purification and characterization from skeletal muscle transverse tubules, 374-376 purified, functional properties in phospholipid vesicles, 377-379 Calcium/calmodulin-dependent protein kinases, 148 Calcium channels brain tissue, immunospecific identification, 386-387 calcium antagonist receptor detergent-solubilized,characterization, 373 molecular size, 372 protein components, 372 purification and characterization from skeletal muscle transverse tubules, 374-376 purified, functional properties in phospholipid vesicles, 377-379 cardiac and skeletal, comparison, 409 o-conotoxin GVIA wconotoxin MVIIA and, 424-426 receptor targets cross-linking to, 422 specificity, 421-422 subtypes A and B, 422-423 distribution, model, 427 w-conotoxins, 417-419 dihydropyridine calcium antagonists, 370-372 heart tissue, immunospecific identification, 386-387 models single-file pore with two binding sites, 395-397 structure, 385 organic calcium antagonists as probes, 370-372 Fbramecium, action potential and, 2 phosphorylation and CAMP-dependent regulation, 376-377 pore size, 407-408 pore size estimation block by diaminobutane, 399-402 permeation by organic cations, 397-399 pore structure, 409410 pore symmetry, 408 tests of, 404-407

433 purified calcium flux mediated by, 378 noncovalently associated components, 375 recordings in intact cells, 397-402 in planar bilayers, 402-407 skeletal muscle transverse tubules, model for biochemical properties, 373-374 subunits, dihydropyridine-sensitive covalent labeling, 382 differential labeling, 380 glycosylation, 381 noncovalent subunit interactions, 382-383 oligomeric model for, 383-386 polypeptide components, 379-381 Calcium phosphate precipitation, DNAmediated gene transfer with, 225-227 Calcium/phospholipid-dependentprotein kinases, 148 Calmodulin, Fbmmecium, wild-type and pntA mutants, 3-4 CAMP-dependent protein kinases, 148 phosphorylation of nicotinic acetylcholine receptor y- and &subunits, 152 phosphorylation site protein sequencing, 154 CAMP-dependent regulation, calcium channels, 376-377 Carbohydrate analysis, sodium channel subunits in skeletal muscle, 253 Carbohydrate composition. large peptide from eel electroplax sodium channel, 335 Carbonyl carbon chemical shifts, ioninduced, gramicidin A, 58-61 Carbonyl group, gramicidin A, reorientation, 103 Cardiac muscle, sodium channels comparison with skeletal muscle, 333 type I1 mRNA, RNase protection analysis, 282-284, 286 Cations monovalent, Goldman equation, 398 organic permeation of intact cardiac cells, 397-399 relative permeability, 398 Cesium ion in gramicidin A channels interactions, 62-63

434

INDEX

low-energy permeation pathways, 105-106

single channel currents calculation with NMR-derived constants, 66, 74 as function of ion activity, 63-66 solvation energy, 105 valence selectivity, 140-107 in gramicidin channel, solvation energy profile, 101 leaving gramicidin A channel ion correlation time, 69-70 off-rate constants, 70-74 transverse relaxation times, 68 rate of transfer in gramicidin pores, 29 cGMFdependent protein kinases, 148 Channel proteins, membrane a-toxin, 291 channel lining identification, 293 hydrophobicity plots, 291-292 model development, 291-292 modeling, 290-294 surface charge, 317-318 transmembrane segments, see 'Ransmembrane segments Chlorine ion in gramicidin A channels low-energy permeation pathways, 105-106

solvation energy, 105 valence selectivity, 140-107 in gramicidin channels, solvation energy profile, 101 Circular dicbroism, gramicidin channel and pore forms, effect of binding ions, 40 conformations, 39-40 modeling, 36 Complementary DNA derived mRNA encoding for rat brain sodium channel, 271-272

T carifmica library construction, 223 library screening for acetylcholine receptor subunit clones, 233 Computational models, gramicidin A,

Conduction, ion, see Ion conduction Conformational analysis, ion permeation through channel proteins, 93 Conformations, gramicidin pores, ion interactions and, 24-26 a-Conotoxins, 418 p-Conotoxins, 418 guanidinium toxin binding site, lipid bilayers, 314-315 w-Conotoxins biochemistry, 419-421 GVIA calcium channel subtype distribution model, 427 subtypes A and B, 422-424 in vivo effects, 424-426 receptor targets in avian and mammalian systems, 422-424

cross-linking to, 422 specificity, 421-422 sequence, 421 MVIIA in vivo effects, 424-426 sequence, 421 Continuum electrostatic model, ion permeation through channel proteins, 93

Conus geogmphus, C magus, C. purpumscens, and C. striam, 419-420 Cotransformation efficiency, four T cal&%nica acetylcholine receptor cDNAs into same cell, 232-233 Covalent labeling calcium channel subunits, 382 protein components of calcium antagonist receptor, 372 Crystallography, gramicidin structure, 42-46 C-terminus, eel sodium ion channel, 261-263

Curaremimetic neurotoxin, synthetic peptides, acetylcholine receptor binding, 184-185 Current-voltage relations, ion binding in gramicidin pores, 27

98-100

Conductance-concentration relation, ions in gramicidin pores, 26-27 Conductance ratio, dependence on potential and ionic strength for gramicidin and NaCI, 25

D Dansyl-gramicidinC, 18 Depolarization, low, noninactivating sodium channel activity, 274

INDEX

435

Desensitization, nicotinic acetylcholine receptors, MP-induced phosphorylation and, 156-159 Detergent-bound gramicidin, 41 Detergent solubilization calcium antagonist receptor by digitonin, 373 sodium channel in skeletal muscle, 252 Diaminobutane block, of cardiac calcium channels. 399-402 Diffusion limitations, ion interactions in gramicidin pores and, 23-24 Digitonin, solubilization of calcium antagonist receptor, 373 Dihydropyridine organic antagonists, 370-372 Dihydropyridine-sensitive calcium channel subunits covalent labeling, 382 differential labeling, 380 glycosylation, 381 noncovalent subunit interactions, 382-383 oligomeric model for, 383-386 polypeptide components, 379-381 Diltiazem, organic antagonist, 370 Dimerization, gramicidin A, 97 Dimers gramicidin, 36 gramicidin A, 98 gtamicidin pores, 17-18 Diphenylalkylamines,organic antagonists, 370 Diphytanoylphosphatidylcholine/n-decane membranes, gramicidin A single channel conductance histograms, 65 DNA-mediated gene transfer, acetylcholine receptor subunit cDNAs into cultured cells with calcium phosphate precipitation, 225-227 by viral infection, 227-228

purified, function reconstitution, 342-346 single channel recordings, 346-350 structure activation gating and, 340-341 amino acid sequence, 336-339 inactivation gating and, 341-342 Electrophysiology,guanidinium toxin inhibition of sodium channels, 311-313 End plates, concentration of sodium channels in, 265 Equilibrium dialysis, ion binding in gramicidin pores, 28 Escherichia coli, ion channels, 6-7

F Fibroblasts Ltk-aprt- cells cotransformation efficiency, four acetylcholine receptor cDNAs into same cell, 232-233 functional expression of transfected DNA, 233-235 NIH3T3 cells acetylcholine receptor cDNA introduction by viral infection, 236 expression of I: calijornica a-protein in, 236 partial characterization of I: cafijomica a-subunits, 238-241 Fluorescence imaging, gramicidin structure in lipid bilayers, 37 F l u ratio exponents ion binding in gramicidin pores, 28 ion conduction in gramicidin pores, 23, 26 Folding motifs, gramicidin channel and pore forms, 36 Forskolin, effect on desensitization of nicotinic acetylcholinereceptors, 157-159 Free-energy profile, gramicidin A, 95-96

E Electrical recordings, see Patch-clamp recordings; Voltage-clamp recordings Electron density maps gramicidin, 43-44 gramicidin/cesium complexes, 45 Electrphorus electricus electroplax, sodium channel protein isolation and characterization, 331-336

G

Gaps, in gramicidin A channel currents kinetic significance, 120-122 properties, 116-118 Gating activation eel electroplax sodium channel, 340-341

436

INDEX

sodium channel, 300-302 gramicidin A channel currents, gaps in, 116-122

inactivation eel electroplax sodium channel, 341-342 effect on ion conductance, 350-351 sodium channel activation, 300-302 eel electroplax, 340-341 helical screw mechanism, 301 Gene expression, tissue-specific sodium channel type 11, 279-281 Gene transfer, DNA-mediated, see DNAmediated gene transfer Glycosylation, dihydropyridine-sensitive calcium channel subunits, 381 Goldman equation, for monovalent cations, 398

Gramicidin amino acid sequence, 35 channel form cation binding sites, 41 effect of binding ions, 40 effects of altered lipid structures, 41 folding motif, 36 conformations, 39-40 crystal forms, 42-43 crystallographic studies, 43-46 C-terminus, 36 detergent- and lipid-bound forms, 41 dimer, 36 double helices, parallel and antiparallel intertwined, 37 electron density maps, 43-44 helical conformation, 36-37 ion binding mechanism, 40 ion binding sites, 40 membrane-bound form. 47-48 modeling with circular dichroism and NMR, 36-37 multiple isomorphic replacement phasing methods, 42 NMR modeling, 36 structure in lipid bilayers and SDS micelles, 37 N-terminus, 35-36 Patterson analysis, 43 pores, see Gramicidin pores single-file conductance, 41

single isomorphic replacement phasing methods, 43 structure in phospholipid bilayers, 37 X-ray diffraction, 42-46, 290 Gramicidin A cesium ion transport, single channel currents calculation with NMR-derived constants, 74 ion activity and, 63-66 "'CS-NMR binding constants, estimation, 67 interactions, 62-63 ion correlation time, 69-70 off-rate constants, 70-74 relaxation methods, 61-62 single channel currents, calculation, 74 transverse relaxation times, 68 gaps in channel currents kinetic significance, 120-122 methods, 114-116 N-acetyl-gramicidinchannels, 118, 120 properties, 116-118 heat incorporation with L-a-lysolecithin, 56-58

ion channels calcium-43 interactions in, 62-63 cesium433 interactions in, 62-63 ion-induced carbonyl carbon chemical shifts, 58-61 lipid bilayer state, 56-58 minimum energy conformation, stereo plots of, 35 structure, 52-56 ion permeation. ab fnftfo methods, 95-96 cation-binding sites, 102 computational models, 98-100 cooperative translocation, 103 free-energy profile, 95-% ion-binding sites, 102 ion-water-gramicidin interaction model, 99-100 monomer dimerization, 97 quantitative predictions, 100-102 site-to-site transition rates, 102-104 solvation energy profile, 96-97, 100-101 valence selectivity, 104-107 ion transport through channel, mechanism, 56 open-channel noise, 122-123 autocovariance function. 124-125

INDU

437

methods. 114-116 origin, 126-128 power spectra of channel currents, 123 spectra prediction from rate constants, 123-126

single channel conductance histograms in diphytanoylphosphatidylcholine/ n-decane membranes, 65 Gramicidin pores ' . and T1' rates of transfer, 29 CS', K form effect of binding ions, 40 folding motif, 36 three-dimensional structure, 43 formation, 16, 18 ion conduction, 19 binding constants, 21 current-voltage relations, 27 equilibrium dialysis, 28 flux ratio exponents, 28 permeability ratios, 28 spectral changes, 28-29 water permeability, 28 complex ion interaction, 22-23 conductance-concentration relation,

H Heart calcium channels comparison with skeletal channels, 409 components, immunospecific identification, 386-387 intact cells, recordings for calcium channel pore size, 397-402 Helical conformations, gramicidin, parallel and antiparallel intertwined, 37 Helical screw gating mechanism, sodium channel transmembrane segments, 301 Hydrophobicity, side chains. a-toxin and porins, 291-293 Hyperpolarization effect on skeletal muscle sodium channels, 259 Xenopus oocyte sodium channels, 272 I Immune blotting, expression of acetylcholine receptor subunits in yeast.

26-27

diffusion limitations and, 23-24 double-layer polarization and, 24 double occupancy, 26-28 flux ratio exponent. 23 mean residence time, 21 mole fraction effect, 23 pore conformations and, 24-26 rate of entry, 21 regulatory sites, 24 reversal potential, 21-22 second ion entry, 26-28 simple competition. 19-22 nature of, 18-19 one-ion two-conformation, 26 structure, kinetic data, 18-19 Grayanotoxin. effects on sodium channel kinetics and selectivity, 305 Guanidinium toxin, sodium channel binding interaction, 311-316 location, 324 in model lipid bilayers, 313-316 at selectivity filter, 312 sialic acid and, 324-326 site location, local fiied charges and, 322-323

204

Immunocytochemistry, C-terminus, sodium channel, 261-262 Immunofluorescence, acetylcholine receptor a-subunit in yeast, 205 &subunit in yeast, 206 Ion channel proteins, see Channel proteins, membrane Ion channels, see a h specific ion conduction, effect of negative surface charge, 318 E. mIi, 6-7 location, 7 pressure-gated, 6-7 voltage-gated, 6-7 ion permeation through, 94-95 hmeciurn, action potentials, 2 S

C W W ~ ~4-6 M,

voltage-clamp measurements, 289-290 Ion conduction channel-mediated, negative surface charge effects, 318 gramicidin pores complex interactions, 22-23 conformation effects, 24-26 diffusion limitations, 23-24

438

INDEX

double-layer polarization, 24 regulatory sites, 24 simple competition, 19-22 regulation biochemical modification of reconstituted protein N-bromoacetamide effects, 352-353 N-bromosuccinimide effects, 352-353 peptide modification, flux activation and, 358-361 Pronase effects, 355-358 trypsin effects, 355-358 inactivation gating and, 350-351 Ion-exchange chromatography, purification of calcium antagonist receptor, 374 Ion permeation, through channel proteins ub initio methods, 94-85 Brownian dynamics, 93 conformational analysis, 93 continuum electrostatic model, 93 Ion transport, nicotinic acetylcholine receptor, pre- and postphosphorylation, 156

L Ligand-binding sites, nicotinic acetylcholine receptor, anti-peptide antibody probes, 174-175 Lipid bilayers, planar, see uLro Phospholipid bilayers reconstituted calcium channels cell-attached patch recordings and, 402-404 pore symmetry, 404-407,408 sodium channel inhibition by guanidinium toxin, 313-316 modification by trimethyloxonium, 315-316 surface charge effects, 316-323 sodium channel reconstitution in, 257-259, 310-311 Lipid-bound gramicidin, 41 Lipid structures, altered, effect on gramicidin channel, 41 L-a-Lysolecithin, gramicidin A heat incorporation, 56-58 Lubrol-PX, sodium channel solubilization, 252

M Macro patches, 272 Mean residence time, ions in gramicidin pores, 21 Messenger RNA cDNA-derived, encoding for rat brain sodium channel, 271-272 sodium channel Northern blot analysis, 278-279 type I, oligonucleotide probes specific for, 278 type I1 oligonucleotide probes specific for, 278 RNase protection analysis, 282-284, 286 transcripts, T callfomica subunit clones, 223-224 Molecular size, calcium channel calcium antagonist receptor, 372 Mole fraction effects, in gramicidin pores, 23 Monoclonal antibodies map of binding domains on rat skeletal muscle, 264 structural probes of sodium channels, 263-264 Multiple isomorphic replacement phasing method, 42 Muscle cells BC3H-1 cells, single channel currents acetylcholine-induced. 135 closed-duration histograms acetylcholine concentration dependence, 139 at different acetylcholine concentrations, 138-139 at intermediate acetylcholine concentration, 141-142 effective channel opening rate, acetylcholine concentration dependence, 142-143 limiting low acetylcholine concentrations, 139-140 linear four-state activation scheme, 138-139, 144 open-duration histograms at different acetylcholine concentrations, 135-138

INDEX

saturating acetylcholine concentrations, 140-141 L6 cells acetylcholine receptor cDNA introduction by viral infection, 235 expression of T caiifornica a-protein in, 236-238 partial characterization of T wlijornica a-subunits, 238-241 L6 and BC,H-1 cells T calfornica acetylcholine receptor expression in, 235 Mutations Rmmecium cnrC, 4 ion channels, 2-3 pntA, 3-4 theoretical models, sodium channel functioning and, 275 Myotubes, nicotinic acetylcholine receptor desensitization, forskolin effect, 158-159

N Neurotoxins, effects on eel electroplax sodium channel, single channel recordings, 346-350 Neutron diffraction, gramicidin, 43 Nicotinic acetylcholine receptor, hydrophobic segments, 293 Nicotinic acetylcholine receptors antibodies, generation with synthetic peptides, 167 BC3H-1 cells, single channel currents acetylcholine-induced, 135 closed-duration histograms, 143-144 acetylcholine concentration dependence, 139 at different acetylcholine concentrations, 138-139 at intermediate acetylcholine concentrations, 141-142 effective channel opening rate, acetylcholine concentration dependence, 142 limiting low acetylcholine concentrations, 139-140 linear four-state activation scheme, 138-139, 144

open-duration histograms at different acetylcholine concentrations, 135-138 saturating acetylcholine concentrations, 139-140 ligand-binding site, anti-peptide antibody probes, 174-175 phosphorylation biochemical characterization, 150-155 by cAMP-dependent protein kinase, 156-159 forskolin effects, 157-159 ion transport properties, 156 modulation of ion channel properties by, 155 physiological significance, 155-159 rapid desensitization, 156-158 regulation, 150, 152 regulation of receptor localization and stabilization, 155-156 site conservation between species, 155 sites on subunits, 152-155 postsynaptic membranes preparations, 149 solubilization, 149 subunits arrangement, 1500151 C- and N-termini, membrane sidedness, 167-170 immunogenic region of a-subunit, 172-174 phosphorylation sites, 152-155 proteolytic fragements, identification and mapping, 171-172 synthetic peptide binding to abungarotoxin, 177-184 transmembrane orientation, 175-177 transmembrane structure, 150-151 types, 149-150 topological mapping with domaindirected antibodies, 167-177 Nitrendipine binding to receptor site, 371 3H-labeled, purification of calcium antagonist receptor, 377 in vivo effects, 424 photoreactive azidobenzoyl analog, 372 Noise, open-channel, see Open-channel noise Nonidet-P40, sodium channel solubilization, 252

440

INDEX

Northern blot analysis, sodium channel mRNA, 278-279 Nuclear magnetic resonance 133Cs -, gramicidin A channels binding constants, estimation, 67 interactions, 62-63 ion correlation time, 69-70 off-rate constants, 70-74 relaxation methods, 61-62 single channel currents, calculation, 74 transverse relaxation times. 68 gramicidin modeling, 36 structure in lipid bilayers and SDS micelles, 37 0

Oligomeric model, for dihydropyridinesensitive calcium channel, 383-386 Oligonucleotide probes, for sodium channel a-subunit mRNAs, 278 Oligopeptides, synthetic, for sodium channel topography, 261-263 Oocytes, Xenopus loevis acetylcholine-induced"Na uptake, 225 acetylcholine-induced single channel currents, 225 a-bungarotoxin binding, 225 cell surface acetylcholine receptor expression, 229-230 cloned sodium channels expressed in inactivation in potential-dependent manner, 272-273 macro patches, 272 mutations, theoretical models for, 275 noninactivating channel activity at low depolarizations, 274 step polarizations, 272 microinjection with mRNA from acetylcholine receptor subunit clones, 224-225

preparation, 224 Open-channel noise, gramicidin A channels, 122-123 origin, 126-128

power spectra of channel currents, 123 spectra prediction from rate constants, 123-126

Organic calcium antagonists. probes for calcium channel, 370-372 Organic cations permeation of intact cardiac cells. 397-399 relative permeability, 398

P PABS-gramicidin, 18 Pancuronium, inside blocking of sodium channels, 304 Pantophobiac mutants, hmmeciurn, 2-4 hmmeciurn. ion channels action potentials, 2 Ca2+-activatedcyclic nucleotide phosphodiesterase and myosin lightchain k i n e 4 cnrC mutants, 4 complementation groups, 2-3 mutations, 2-3 pantophobiac mutants, 2-4 wild-type calmodulin, 3-4 Patch-clamp recordings cloned sodium channels in Xenopus oocytes, 272-275 E. coli spheroplasts, 6 gramicidin A channels, open-channel noise, 122-123 Ltype calcium channels ion permeation, 397-399 recordings in planar bilayers and, 402-404

single channels in lower organisms, table, 9

Patterson analysis, gramicidin, 43 Peptides modification, flux activation of ion conductance and, 358-361 synthetic, see Synthetic peptides Permeability ratios ion binding in gramicidin pores, 28 ion conduction in gramicidin pores, 23 Phenylalkylamines, organic antagonists, 370 Phospholipid bilayers, see a h Lipid bilayers, planar gramicidin structure in, 37 Phospholipid vesicles purified calcium antagonist receptor in, 377-379

INDEX

441

sodium channel reconstitution in, 255-257

P hosphorylation calcium channels, 376-377 nicotinic aceytlcholine receptors biochemical characterization, 150-155 by CAMP-dependent protein kinase, 156-159

forskolin effects, 157-159 ion transport properties, 156 modulation of ion channel properties by, 155 physiological significance, 155-159 rapid desensitization, 156-158 regulation, 150, 152 regulation of receptor localization and stabilization. 155-156 site conservation between species, 155 sites on subunits, 152-155 Photosynthetic reaction center, 290-291 structure, 292 Planar lipid bilayers, see Lipid bilayers, planar Plasma membrane, insertion of acetylcholine receptor subunits in yeast, 204-205. 207

Plasmida, pRII-1, 272 Plugging model. guanidinium toxin inhibition of sodium channels, 312, 316 PN 200-110, lH-labeled covalent labeling of calcium channel subunits, 382 purification of calcium antagonist receptor, 377 Polarization, double-layer, ion interactions in gramicidin pores and, 23-24 Polypeptides, dihydropyridine-sensitive calcium channel subunits, 379-381 Pores calcium channel size, 407-408 size estimation, 397-402 structure, 409-410 symmetry, 408 symmetry, tests of. 404-407 gramicidin Cs', K ', and T1' rates of transfer, 29 form effect of binding ions. 40 folding motif, 36

three-dimensional structure, 43 formation, 16, 18 ion conduction, 19 binding constants, 21 current-voltage relations, 27 equilibrium dialysis, 28 flux ratio exponents, 28 permeability ratios, 28 spectral changes, 28-29 water permeability, 28 complex ion interaction, 22-23 conductance-concentration relation, 26-27

diffusion limitations and, 23-24 double-layer polarization and, 24 double occupancy, 26-28 flux ratio exponent, 23 mean residence time, 21 mole fraction effect, 23 pore conformations and, 24-26 rate of entry, 21 regulatory sites, 24 reversal potential, 21-22 second ion entry, 26-28 simple competition, 19-22 nature of, 18-19 one-ion two-conformation, 26 structure, kinetic data, 18-19 Porins E. coli, 7 hydrophobic side chains, 291-293 Posttranslational modifications, acetylcholine receptor subunits in yeast, 204

Potassium channels

E coli, 7 ion transfer rates in gramkidin pores, 29

Rmmeciurn, action potential and, 2

s cen?visiae,4-6

Potential-dependent inactivation, Xenopus oocyte sodium channels, 272 Probes oligonucleotide, for sodium channel a-subunit mRNAs, 278 organic calcium antagonists. 370-372 RNA antisense, for sodium channel type 11, 281-282

Pronase, effects on reconstituted protein ion conductance, 355-358

442

INDEX

Protein kinase C, phosphorylation of nicotinic acetylcholine receptor a-and &subunits, 152 Protein kinases, classes, 148 Proteins, channel, see Channel proteins, membrane Proteolytic fragments, nicotinic acetylcholine receptor subunits, identification and mapping, 171-172 Pyromellityl gramicidin, 18 0

R Rabies virus glycoprotein sequences, derived synthetic peptides, acetylcholine receptor binding, 184-185 Random coils, membrane protein structure modeling and, 291 Rate of entry, ions in gramicidin pores, 21 Regulatory sites, ion interactions in gramicidin pores and, 23-24 Reversal potential, ion conduction in gramicidin pores, 21-23 RNA antisense probes, for sodium channel type 11, 281-282 RNase protection analysis, sodium channel type I1 mRNA in brain and muscle, 282-284, 286

Sea anemone toxin, sodium channel inactivation, 305 Selectivity, gramicidin pores, 1617 Selectivity filter, sodium channel, 298-300 guanidinium toxin binding at, 312 Sialic acid, guanidinium toxin binding and, 324-326

Side chains a-toxin and porins, hydrophobicity, 291-293

arginine, sodium channel gating and, 301 Simple competition model, ion conduction in gramicidin pores, 19-22 Single channel recordings, skeletal muscle sodium channels incorporated into planar lipid bilayers, 258 Single isomorphic replacement phasing method, 43 Site-to-site transition rates, ions in gramicidin channels, 102-104 Skeletal muscle calcium channels, comparison with cardiac channels, 409 sodium channels activation of monovalent cation flux, 255-257

comparison with cardiac muscle, 333 concentration in end plate regions, 265 C-terminus, 261-263 inhibition of monovalent cation flux, 255-257

S

Sacchamrnyces cenwkiae

expression of acetylcholine receptor subunits detection by immune blotting. 204 immunofluorescencedetection of &subunit in plasma membrane, 206

insertion into plasma membrane, 204-205, 207 ligand-binding properties, 207-209 orientation, 209-211 plasmid construction, 202 posttranslational modification, 204 ion channels, 4-6 Saxitoxin, sodium channel inhibition, 255-257, 304, 311 a Scorpion toxin, sodium channel inactivation, 305

monoclonal antibodies as structural probes, 263-264 primary sequence, 259-260 purification, 252-253 reconstitution in phosopholipid vesicles, 255-257 in planar bilayers, 257-259 RNase protection analysis, 282-284, 286

Subtypes, 265-267 subunits amino acid and carbohydrate analysis, 253 chemistry, 253-255 stoichiometry, 253-255 topographical organization. 260-264 voltage dependence, 259 transverse tubules calcium antagonist receptor, purification and characterization, 374-376

INDEX

443

model for calcium channel biochemical properties, 373-374 Sodium channels activated over a range of Na+ concentrations, 321 channel lining, identification, 293 classical allosteric proteins and, 329-330 cloned, expression in Xenopus laevis oocytes, 271-272 hyperpolarization, 272 noninactivating channel activity at low depolarizations, 274 step polarizations, 272 theoretical models for mutations, 275

E. coli, 7 eel electroplax isolation and characterization, 331-336 protein function reconstitution, 342-346 single channel recordings, 346-350 protein structure activation gating, 340-341 amino acid sequence, 336-339 inactivation gating, 341-342 schematic, 332 guanidinium toxin binding site, local fixed charges and, 322-323

interaction, 311-316 hydrophobic segments, 293 ion entry into gramicidin pores, 29 in lipid bilayers guanidinium toxin binding. 324 guanidinium toxin interaction, 311-316 sialic acid role, 324-326 surface charge effects, 316-323 Rmntxiurn, action potential and, 2 reconstitution in lipid bilayers, 310-311

S cemisiae, 4-6 skeletal muscle activation of monovalent cation flux, 255-257

chemistry, 253-255 concentration in end plate regions, 265 C-terminus, 261-263 inhibition of monovalent cation flux, 255-257

monoclonal antibodies as structural probes, 263-264 primary sequence, 259-260 purification, 252-253

reconstitution in phosopholipid vesicles, 255-257 in planar bilayers, 257-259 stoichiometry, 253-255 Subtypes, 265-267 subunits, amino acid and carbohydrate analysis, 253 topographical organization, 260-264 voltage dependence, 259 surface charge effects, 316-323 tetrodotoxin binding competition, 313-314 cation flux inhibition in reconstituted channels, 255-257 in developing muscle, 267 inhibition, 311 subtypes sensitive to, 255-257 transmembrane segments activation gating, 300-302 a-helices, positions, 2%-297 arginine side chains, 301 coupling of inactivation to activation, 302-304

folding scheme, 295-2% helical screw gating mechanism, 301 identification, 293 inactivation, 302-304 location, 294-295 model, 294-2% pharmacology, 304-305 ridges into grooves a-helix packing theory, 300-301 secondary structun, 293-294 selectivity filter, 298-300 S4 segment, 295-2% transmembrane topology models, testing, 305

type I (brain) mRNAs, Northern blot analysis, 278-279

oligonucleotide probes specific for, 278 ontogeny, 278-279 type I1 (brain) generation of antisense RNA probe for, 281-282 mRNAs, Northern blot analysis, 278-279

oligonucleotide probes specific for, 278 ontogeny, 278-279 RNase protection analysis, 282-284. 286

444

INDEX

tissue-specific expression, 279-280 type I1 (muscle), RNase protection analysis, 282-284. 286 Soleus muscle, nicotinic acetylcholine receptor desensitization, forskolin effect, 158 Solubilization calcium antagonist receptor by digitonin, 373 sodium channel in skeletal muscle, 252 Solvation energy profile, ion permeation in gramicidin A channels, %-98, 100-101 Cs+and Cl-, 104-107 Southern blot analysis, bacteriophage with sodium channel gene I and gene I1 sequences, 283 Spectral changes, ion binding in gramicidin pores, 28-29 Step depolarization, Xenopus oocyte sodium channels, 272 Strychnine, inside blocking of sodium channels, 304 Surface charge effects, sodium channel, 316-323 Synthetic oligopeptides, and antisera for sodium channel topography, 261-263 Synthetic peptides from curaremimetic neurotoxin sequences, acetylcholinereceptor binding, 184-185 generation of antibodies to predetermined domains, 167 nicotinic acetylcholine receptor a-subunit, a-bungarotoxin binding, 177-184 from rabies virus glycoprotein sequences, acetylcholinereceptor binding, 184-185

T Tetrodotoxin, sodium channel binding competition, 313-314 in developing muscle, 267 inhibition, 304, 311 reconstituted, inhibition of cation flux, 255-257 subtypes, 265-267 Thallium ion, rate of transfer in gramicidin pores, 29 7brpedo calfornica acetylcholine receptor tubular crystals, three-dimensional image analysis, 198-199

a-protein expression in fibroblasts and muscle cells, 236-238 cDNA libraries construction, 222-223 screening for acetylcholine receptor clones, 223 nicotinic acetylcholine receptor ion transport, 156 a-subunit, phosphorylation site, 155 Wansition rates, site-to-site, ions in gramicidin channels, 102-104 Ifansloction, ions in gramicidin A channels, 103-104 Transmembrane segments a-helices, 291 backbone structures, 291 composition, 291 sodium channel activation gating, 300-302 a-helices. positions, 296-297 coupling of inactivation to activation, 302-304 folding scheme, 295-296 helical screw gating mechanism, 301 identification, 293 inactivation, 302-304 location. 294-295 model, 294-2% pharmacology, 304-305 ridges into grooves a-helix packing theory, 300-301 SWOndary StNctWe, 293-294 selectivity Fdter. 298-300 S4 segment, 295-296 topological models, testing, 305 Wsverse tubules, skeletal muscle calcium antagonist receptor purification and characterization, 374-376 model for calcium channel biochemical properties, 373-374 3-('Ififluoromethyl)-3-(m-iodophenyl)diazirine, labeling of calcium channel subunits, 382 l'timethyloxonium, treatment of sodium channels, 312 'Ityosine-specific protein kinase, phosphorylation of nicotinic acetylcholine receptor fl-, T-, and bsubunits, 152 nypsin, effects on reconstituted protein ion conductance, 355-358

445

INDEX

mosine-specific protein kinases, 148-149 phosphorylation site on nicotinic acetylcholine receptor B-, y , and 6subunits, 152 Qrothricin extract, IS

W Water permeability, ion binding in gramicidin pores, 28 X

Xenopus laevk

V Valence selectivity, ions in gramicidin A channels, 104-107 Valine-gramicidin A, 16 Velocity sedimentation, purification of calcium antagonist receptor, 374 Verapamil, organic antagonist, 370 Veratridine activation of cation flux in reconstituted sodium channels, 255-257 effects on sodium channel kinetics and selectivity, 305 Viral infection acetylcholine receptor cDNA introduction by, 236 DNA-mediated gene transfer with, 227-228 Voltage-clamp recordings ion channel measurements, 289-290 Rzmmecium, currents in plasma membrane, 2 Xenopus oocytes, cloned sodium channels, 272-275 Voltage dependence, sodium channels in skeletal muscle lipid bilayers, 259 Voltage-gated sodium channels, a-subunits, see Sodium channels, types I and I1 Voltage-sensitive channels, E. electricus, see Sodium channels, E. electricus

microinjection with T calvornica mRNA, 224 oocytes a-bungarotoxin binding, 225 cell surface acetylcholine receptor expression, 229-230 cloned sodium channels expressed in inactivation in potential-dependent manner, 272-273 macro patches, 272 mutations, theoretical models for, 275 noninactivating channel activity at low depolarizations, 274 step polarizations, 272 microinjection with mRNA from acetylcholine receptor subunit clones, 224-225 "Na uptake, acetylcholine-induced, 225 preparation, 224 single channel currents, acetylcholineinduced, 225 X-ray diffraction alamethicin, 290 gramicidin, 47-48, 290 gramicidinkiipalrnitoylphosphatidylcholine crystals, 48 gramicidin/cesium crystals, 44 structure, 42-46 membrane protein structure determination, 290-291

This Page Intentionally Left Blank

Contents of Recent Volumes Volume 22

The Squid Axon PART I. STRUCTURE Squid Axon Ultrastructure GLORIA M. VILLEGASAND RAIMUNDO VILLEGAS The Structure of Axoplasm RAYMOND J. LASEK PART 11. REGULATION OF THE AXOPLASMIC ENVIRONMENT Biochemistry and Metabolism of the Squid Giant Axon HAROLD GAINER, PAULE. GALLANT, ROBERT GOULD, AND HARISH c. PANT Ransport of Sugars and Amino Acids AND A. CARRUTHERS P. F. BAKER Sodium Pump in Squid Axons LUISBEAUGB Chloride in the Squid Giant Axon JOHNM. RUSSELL Axonal Calcium and Magnesium Homeostasis P. F. BAKERAND R. DIPOU) Regulation of Axonal pH WALTER F. BORON Hormone-Sensitive Cyclic Nucleotide Metabolism in Giant Axons of AND A. CARRUTHERS P. F. BAKER

Noise Analysis and Single-Channel Recordings FRANCOCONTI Membrane Surface Charge AND GERALD DANIEL L. GILBERT EHRENSTEIN Optical Signals: Changes in Membrane Structure, Recording of Membrane Potential, and Measurement of Calcium LAWRENCE B. COHEN,DAVID LANDOWNE, LESLIE M. Loew, AND BRIAN M. SALZEERG Effects of Anesthetics on the Squid Giant Axon D. A. HAYDON, J. R. ELLICIT, AND B. M. HENDRY Pharmacology of Nerve Membrane Sodium Channels TOSHIO NARAHASHI PART IV. INTERACTION BETWEEN GIANT AXON AND NEIGHBORING CELLS The Squid Giant Synapse RODOLFO R. L L I N ~ Axon-Schwann Cell Relationship JORGEVILLEGAS

Index Volume 23

PART 111. EXCITABILITY Hodgkin-Huxley: Thirty Years After H.Mev~s Sequential Models of Sodium Channel Gating AND CLAYM. ARMSTRONG DONALD R. MATIESON Multi-Ion Nature of Potassium Channels in Squid Axon TEDBEGENISICH AND CATHERINE SMITH

Genes and Membrsner: Mnspott Proteins and Receptors

PART I. RECEPrORS AND RECOGNITION PROTEINS Sensory "kansduction in Bacteria I. SIMON,ALEmDRA KRIKOS, MELVIN NORIHIRO MUTOH,AND ALANBOYD 447

CONTENTS OF RECENT VOLUMES

Structure and Function of the Signal Peptide K. GUYD. DUFFAUD, SUSAN LEHNHARDT, PAUL E. MARCH, AND MASAYORI INOWE The Use of Genetic Techniques to Analyze PART 11. CHANNELS Protein Export in Escherichia coli VYTAS A. BANKAITIS, J. PATRICK RYAN, Ca" Channels of fimmeclum: A AND BETHA. RASMUSSEN, Multidisciplinary Study CHINGKUNO AND YOSHIROSAIMI PHILIPJ. BASSFORD, JR. Structural and Thermodynamic Aspects of Studies of Shaker Mutations Affecting a K' the Transfer of Proteins into and across Channel in Dmsophila LILYYEH JAN,SANDRA BARBEL, LESLIE Membranes TIMPE,CHERYL LAFFER, LAWRENCE VON HEIJNE GUNNAR AND SALKOFF, PATRICK O'FARRELL, Mechanisms and Functional Role of YUH NUNGJAN Glycosylation in Membrane Protein Synthesis SHARON S. KM Sodium Channels in Neural Cells: Molecular Protein Sorting in the Secretory Pathway Properties and Analysis of Mutants ENRIQUE RODRIOUEZ-BOULAN, DAVID E. WILLIAM A. C A ~ R A LTOHRU L , GONOI, AND MARIA COSTA MIseK, DORAVEOADE SALAS, AND ENZO BARD PEDRO J. I. SALAS, PART 111. TRANSPORT SYSTEMS Transport of Proteins into Mitochondria GRAEME A. REID Assembly of the Sarcoplasmic Reticulum The Histidine Transport System of during Muscle Development Salmonella typhimurium DAVID H. MACLENNAN, ELIZAEZTH GIOVANNA FERRO-LUZZI AMES ZUBRZYCKA-GMRN, A Study of Mutants of the Lactose AND ANNELISE 0. JOROENSEN Transport System of Ercherichia coli T. HASTINGS WILSON, DONNA SETOReceptors as Models for the Mechanisms of Membrane Protein mrnover and Dynamics YOUNO, SYLVIE BEDU,RESHA M. PUTZRATH, H. STEVEN WILEY AND BENNO MULLER-HILL The Role of Endocytosis and Lysosomes in The Proton-ATPase of Ercherichia coli A. E. SENIOR Cell Physiology The Kdp System: A bacterial K+ Transport YVESJACQUES SCHNEIDER, JEAN-NOEL AND AND&TROUET ATPase OCTAVE, WOLFOANO EPSTEIN Regulation of Glucose Transporter and Hormone Receptor Cycling by Insulin in Molecular Cloning and Characterizaton of a Mouse Ouabain Resistance Gene: A the Rat Adipose Cell IANA. SIMPSON AND Genetic Approach to the Analysis of the SAMUEL W. CUSHMAN Na+,K*-ATPase ROBERT LEVENSON Index

Mutational Analysis of the Structure and Function of the Influenza Virus Hemagglutinin MARYJANE GETHINO, CAROLYN DOYLE, MICHAEL ROTH,AND Joe SAMBROOK

Index Volume 24

Volume 25

Membrane Protein Blosyntherlr and Turnover

Regulatlon of Calclum TMnrport across Murcle Membrane6

Application of the Signal Hypothesis to the Incorporation of Integral Membrane Proteins TOMA. RAPOPORT AND MARTIN WIEDMANN

Overall Regulation of Calcium Transport in Muscle ADILE. SHAMOO

449

CONTENTS OF RECENT VOLUMES

PART I. REGULATION OF CALCIUM TRANSPORT IN PLASMA MEMBRANES

PART I. GENERAL ASPECTS OF INTRACELLULAR pH REGULATION AND Na'-H+ EXCHANGE

Sarcolemmal Enzymes Mediating j3-Adrenergic Effects on the Heart LARRY R. JONES Properties of Myocardial Calcium Slow Channels and Mechanisms of Action of Calcium Antagonistic Drugs NICKSPERELAKIS, GORDON M. WAHLER, AND GHASSAN BKAILY The Sarcolemmal Sodium-Calcium Exchange System JOHNP. REEVES

Intracellular pH Regulation by LKch and Other Invertebrate Neurons AND W. R. SCHLUE R. C. THOMAS Approaches for Studying Intracellular pH Regulation in Mammalian Renal Cells WALTER F. BORON Aspects of pHi Regulation in Frog Skeletal Muscle RoseRT W. P ~ A AND M ALBERT Roos Molecular Properties and Physiological Roles of the Renal Na*-H+ Exchanger AND PETER IOARASHI PETERS. ARONSON

PART 11. REGULATION OF CALCIUM TRANSPORT IN SUBCELLULAR ORGANELLES

PART 11. Na+-H*EXCHANGE AND CELL VOLUME REGULATION

Volume-Sensitive Alkali Metal-H 'Itansport in Amphiuma Red Blood Cells Regulation of Calcium 'Pansport in Cardiac PETERM. CA~A Sarcoplasmic Reticulum Na-Proton Exchange in Dog Red Blood ADILE. SHAMOO, INDUS. AMBUDKAR, Cells AND JEANBIDLACK MARCS. JACOBSON, JOHNC. PARKER Role of Calmodulin in the Regulation of Activation of the Na+-H' Antiport by Muscle Contraction Changes in Cell Volume and by Phorbol ANNIEMOLLA, SIDNEY k k T Z , AND Esters; Possible Role of Protein Kinase JACQLES G. DEMAILLE S. GRINSTEIN, S. COHEN,J. D. GOETZ, Calcium Release from Sarcoplasmic AND A. ROTHSTEIN, A. MELWRS, Reticulum E. W. GELFAND M u m EN^ The Regulation of Mitochondrial Calcium PART 111. Na+-H+EXCHANGE AND Ttansport in Heart CONTROL OF CELL GROWTH MARTIN CROMFTON The Generation of Ionic Signals by Growth Factors PART 111. CELLULAR ION W. H.MOOLENMR, L. H. K. DEFIZE, REGULATION AND DISEASE P. T. VAN DER SM, AND S. W.DE LAAT Control of Mitogenic Activation of Na+-H' Cellular Ion Regulation and Disease: A Exchange Hypothesis P. ROTHENBERG, D. CASSEL, BENJAMIN F. TRUMP AND B. WHITELEY, D. Mmcuso, IRENE K. BEREZESKY P. SCHLESSINGER, L. REUSS, AND L. GLASER E. J. CRAGOE, Index Mechanisms of Growth Factor Stimulation of Na*-H+ Exchange in Cultured Volume 26 Fibroblasts MITCHELL. VILLEREAL, LESLIE L. MIXMULDOON, LUCIA M. VICENTINI, GORDON Na*-H' Exchange, Intmcellular pH, A. JAMIESON, JR., AND NANCY E. OWEN and Cell Function

CONTENTS OF RECENT VOLUMES

B Lymphocyte Differentiation: Role of Phosphoinositides, C Kinase, and Na+-H+ Exchange PHILIPM. Rosow AND LEWISC. CANTLEY Na+-H' Exchange and Growth Control in Fibroblasts: A Oenetic Approach JACQUES POUYSSlk3UR, ARLETTB FRANCHI, MICHIAKI KOHNO, GILLES I! ALLEMAIN, AND SONIA PARIS PART IV. ROLE OF Na+-H' EXCHANGE IN HORMONAL AND ADAPTIVE RESPONSES Hormonal Regulation of Renal Na+-H' Exchange Activity BERTRAM SACKTOR AND JAMES L. KINSELLA Adaptation of Na+-H' Exchange in the Proximal 'lbbule: Studies in Microvillus Membrane Vesicles AND JULIAN L. SEIFTER WMOND C. HARRIS The Role of Intracellular pH in Insulin Action and in Diabetes Mellitus RICHARD D. MOORE The Proton as an Integrating Effector in Metabolic Activation WILLIAM B. B u s Index

Volume 27 The Role of Membranes In Cell Growth and Dlfferentlstlon

PART I. DESCRIPTION OF ION TRANSPORT SYSTEM IN ACTIVATED CELLS Mitogens and Ion Fluxes LUIS R E U S , DANCASSEL, PAUL ROTHENBERG, BRIAN WHITELEY, AND Luis GLASER DAVID MANCUSO, Na*-H' and Na+-Ca" Exchange in Activated Cells MIKHELL. VILLEREAL

Chloride-Dependent Cation Cotransport and Cellular Differentiation: A Comparative Approach PETERK. LAUP PART 11. TRIGGERS FOR INCREASED TRANSPORT DURING ACTIVATION External 'Itiggers of Ion 'Itansport Systems: Fertilization, Growth, and Hormone Systems A SARAH JOAN BeLL, L O R E ~NIsLSEN, SARIBAN-SOHRABY,AND DALEBENOS Early Stimulation of Na+-H' Antiport, Na+-K+Pump Activity, and Ca*' Fluxes in Fibroblast Mitogenesis AND ENRIQUE ROZENOURT STANLEY A. MENDOW Volume-Sensitive Ion Fluxes in Amphiumu Red Blood Cells: General Principles Governing Na-H and K-H Exchange 'Itansport and CI-HCO, Exchange Coupling PETER M. CALA PART 111: CONSEQUENCES OF THE ALTERATIONS IN ION TRANSPORT OBSERVED DURING ACTIVATION Intracellular Ionic Changes and Cell Activation: Regulation of DNA, RNA, and Protein Synthesus U T H I GEERINO

Energy Metabolism of Cellular Activation, Growth, and Transformation LAZARO J. MANDEL Index Volume 28 Potasalum Transport: Physlology and Pathophydology

PART I. CELL MECHANISM OF POTASSIUM TRANSPORT Role of Potassium in Epithelial 'Ransport Illustrated by Experiments on Frog Skin Epithelium H. H. USSS~NG

451

CONTENTS OF RECENT VOLUMES Na', K+, and Rb' Movements in a Single lhrnover of the Na/K Pump 111 BLISSFORBUSH Properties of Epithelial Potassium Channels DAVID C. DAWN Role of Potassium in Cotransport Systems ROLFKr"e AND ERICHHEIN2 Functional Roles of Intracellular Potassium in Red Blood Cells JOSEPH F. HOFFMAN PART 11. RENAL AND EXTRARENAL CONTROL OF POTASSIUM PHYSIOLOGY Overview: Renal Potassium 'Ifansport along the Nephron FREDS. WRIGHT Potassium Recycling REXL. JAMISON AND ROLAND MULLER-SUUR Cell Models of Potassium 'Ifansport in the Renal 'hbule GERHARD H. GIEBISCH Adrenal Steroid Regulation of Potassium Transport RWERG. O'NEIL Potassium and Acid-Base Balance RICHARD L. TANNEN Renal Potassium Adaptation: Cellular Mechanisms and Morphology BRUCE A. STANTON Quantitative Analysis of Steady-State Potassium Regulation DAVID B. YOUNG PART 111: RENAL AND EXTRARENAL CONTROL OF PWASSIUM: DIURETICS AND PATHOPHYSIOMGY Regulation of Extrarenal Potassium Homeostasis by Insulin and Catecholamines RALPHA. DEFRONW Diuretics and Potassium S. WILCOX CHRISTOPHER Pathogenesis and Pathophysiological Role of Hypoaldosteronism in Syndromes of Renal Hyperkalemia MORRISSCHAMBELAN AND ANTHONYSEEASTIAN

Hypokalemia JORDAN J. COHEN Metabolism and Potassium JAMES P. KNWHEL PART IV. POTASSIUM TRANSPORT IN MUSCLE AND COLON Effects of Potassium Deficiency on Na.K Homeostasis and Na+,K+-ATPasein Muscle TORBEN CLAUSEN AND KELD KIELDSEN Relationship between Cell Potassium and Hydrogen Ion in Muscle SHELDON ADLER Electrophysiologyof Active Potassium Transport across the Mammalian Colon N. K. WILLS,C. CLAUSEN,AND W. C. CLAUSS Potassium Adaptation in Mammalian Colon JOHN P. HAYSLETT, HENRY J. BINDER, AND MICHAEL KASHGARIAN

Index Volume 29 Mernbmne Structum and Function Current Views of Membrane Structure ALANM. KLEINFELD Ultrastructural Studies of the Molecular Assembly in Biomembranes: Diversity and Similarity SEK-WEN HUI The Thermodynamics of Cell Adhesion AND GEOROE I. BELL MICAHDEMBO Rotational and Lateral Diffusion of Membrane Proteins and Lipids: Phenomena and Function MICHAEL EDIDIN Biosynthesis and Distribution of Lipids KENNETH J. LONOMUIR Lipid Exchange: Transmembrane Movement, Spontaneous Movement, and Protein-Mediated 'Ifansfer of Lipids and Cholesterol ELIEWRA. DAWIDOWICZ Membrane Fusion ROBERT BLUMENTHAL

CONTENTS OF RECENT VOLUMES The Control of Membrane Tkaffic on the Endocytic Pathway IRA MELLMAN, CHRISTINE How, AND ARIHELENIUS

Index

Volume 31 Molecular Neuroblology PART I. PEPTIDE ACTION

Volume 30 Cell Volume Control: Fundamental and Compamtlw AapocDI In Anlmrl Cdla

PART I. VOLUME CONTROL IN ISOSMOTIC CONDITIONS Volume Maintenance in Isosmotic Conditions ANTHONY D. C. MACKNIOHT Role of Cytoplasmic Vesicles in Volume Maintenance G. D.V. VAN ROSUht, M. A. Russo, AND J. C. SCHISSELEALIER The Cell Cytoskeleton: Possible Role in Volume Control JOHNW. MILLS

Peptides and Slow Synaptic Potentials STEPHEN W. JONES AND PAULR. ADAMS Molecular Mechanisms of Neurite Formation Stimulated by Insulin-Like Factors and Nerve Growth Factor DOUGLAS N. ISHIIAND JOHNE MILL Pituitary Regulation by GonadotropinReleasing Hormone (GnRH): Gonadotropin Secretion, Receptor Numbers, and Wget Cell Sensitivity WILLIAM C. GOROSPE AND P. MICHAEL CONN Thyrotropin-ReleasingHormone: Role of Polyphosphoinositides in Stimulation of Prolactin Secretion RICHARD N. KOLESNICK AND MARVIN C. GERSHENOORN PART 11. STEROID HORMONE ACTION

PART 11. VOLUME CONTROL IN ANISOSMOTIC CONDITIONS Volume Regulation in Epithelia MIKAEL LARSON AND KENNETHR. SPRINO Volume Regulation in Cultured Cells ELSEK. HOFFMAN Cell Volume Regulation in Lower Vertebrates LEONGOLDSTEIN AND ARNOST KLEINZELLER Volume Regulation in Cells of Euryhaline Invertebrates R. GILLES

Steroid Effects on Excitable Membranes S. D. ERULKARAND D. M. WETZEL Estradiol-Regulated Neuronal Plasticity V. MOEES AND CHARLES DONALD W. PFAFF Steroid Hormone Influences on Cyclic AMP-Generating Systems AND BRUCE MCEWEN ALLAN HARRELSON PART 111. MAGNOCELLULAR NEURONS

Non-Donnan Effects of Organic Osmolytes in Cell Volume Changes MARYE. CLARK

Expression of the Oxytocin and Vasopressin Genes AND DIETMAR RICHTER HARTWIG SCHMALE The Secretory Vesicle in Processing and Secretion of Neuropeptides JMes T. RUSSELL Mammalian Neurosecretory Cells: Electrical Properties in Vivo and in WDu D. A. POULAINAND J. D. VINCENT

Index

Index

PART 111. PHYSICOCHEMICAL PROSPECTIVES

453

CONTENTS OF RECENT VOLUMES Volume 32 Membrane Furlon In hrtlllzatlon, Cellular 'Ranrport, and Vlral lnhotlon PART 1. MEMBRANE FUSION IN FERTILIZATION AND DEVELOPMENT Sperm-Egg Fusion Rvuzo YANAGIMACHI Cortical Exocytosis in the Sea Urchin Egg ROBERT C. JACKSON AND JOSEPH H. CWB Myoblast Fusion-A Mechanistic Analysis MICHAEL J. 0. WAKELAM P A m 11. CELLULAR TRANSPORTEXOCYTOSIS AND ENDOCYTOSIS Exocytosis in Electropermeabilied Cells: Clues to Mechanism and Physiological Control PETERF. BAKER

Exocytosis and Membrane Recycling JACOPO MELDOLESI AND BRUNO CECCARELLI Exocytosis and Endocytosis: Membrane Fusion Events Captured in Rapidly Frozen Cells DOUGLAS E. CHANDLER Osmotic Effects in Membrane Fusion during Exocytosis KEITHW. BROCKLEHURSTAND HARV ~Y B. POLLARD Polyanionic Agents and Inhibition of Phagosome-Lysosome Fusion: Paradox Lost MAYERB. GOREN PART 111. VIRUS-CELL FUSION Fusion of Viral Envelopes with Cellular Membranes SHUN-ICHI OHNISHI Sendai Virus-Mediated Cell Fusion YOSHIOOWDA Fusion Activity of the Hemagglutinin of Influenza Virus MARYJAM GETHING, JEANHENNEBERRY, AND Joe SAMBROOK

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