The structure of the skeletal muscle Calcium Channel. · School, Cranmcr Terrace, looting, London...

Ion Transport

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DAVID KEELING Smith Kline & French Research Limited Welwyn, Hertfordshire, UK

CHRIS BENHAM Smith Kline & French Research Limited Welwyn, Hertfordshire, UK

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Contributors

P . I . Aaronson Department o f Pharmacology, St George's Hospi ta l M e d i cal School, Cranmcr Terrace, Too t ing , London SW17 O R E , U K

M . H . Akabas Departments of Medicine and Physiology, Columbia Universi ty , College of Physicians and Surgeons, 630 W 168th Street, New-York , N . Y . , USA

Q. Al -Awqat i Departments of Medicine and Physiology, Columbia Univers i ty , College of Physicians and Surgeons, 630 W 168th Street, New York , N . Y . , USA

R . W . A l d r i c h Department of Neurobiology, Stanford Univers i ty School of Medicine, Stanford, C a l i f 94305-5401, USA

D . Anderson C U R E , Wadswor th V A and U C L A , Los Angeles, C a l i f , U S A

D . A u r e s - F i s c h e r C U R E , Wadswor th V A and U C L A , Los Angeles, Calif. , USA

E . A . B a r n a r d M R C Molecular Neurobiology U n i t , M R C Centre, Hi l l s Road, Cambridge CB2 2 Q H , U K

L . B i a n c h i n i Universi ty Laboratory of Physiology, Parks Road, Oxford Ο Χ Ι 3PT, U K

G . Belagi Allergan, I rv ine , C a l i f , USA

C D . Benham Smith Kl ine & French Research L i m i t e d , The Frythe, We lwyn , Herts A L 5 9AR, U K

M . B ie l Ins t i tu t fur Physiologische Chemie, Medizinische Faku l t ä t , Un ive r s i t ä t des Saarlandes, D-6650 Homburg/Saar , West Germany

T . B . Bolton Department of Pharmacology, St George's Hospital Medical School, Cranmcr Terrace, Toot ing , London SW17 O R E , U K

ν

vi Contributors

M.S. B r a i n a r d Department of Neurobiology, Stanford Universi ty School of Medicine, Stanford, C a l i f 94305-5401, USA

M . G . Dar l i son M R C Molecular Neurobiology U n i t , M R C Centre, Hi l ls Road, Cambridge CB2 2 Q H , U K

A . C . D o l p h i n Department of Pharmacology, St George's Hospital Medical School, Cranmcr Terrace, l o o t i n g , London SWT7 O R E , U K

A . E d e l m a n Departments o f Medicine and Physiology, Columbia University, College of Physicians and Surgeons, 630 VV 168th Street, New York . N . Y . , USA

D . T . E d m o n d s The Clarendon Laboratory, Parks Road, Oxford O X 1 3PU, U K

J . C . E l l o r y Universi ty Laboratory of Physiology, Parks Road, Oxford Ο Χ Ι 3PT, U K

V . F lockerz i Ins t i tu t fur Physiologische Chemie, Medizinische F a k u l t ä t , Un ive r s i t ä t des Haarlandes, D-6650 Homburg/Saar, West Germany

L . G e r l a c h Physiologisches Inst i tu t , Alber t Ludwigs Unive r s i t ä t Freiburg, Hermann Herder Strasse 3, D-7800 Frei burg, West Germany

R . Greger Physiologische Inst i tu t , Alber t Ludwigs U n i v e r s i t ä t Freiburg, Hermann Herder Strasse 3, D-7800 Freiburg, West Germany

A . C . H a l l Universi ty Laboratory of Physiology, Parks Road, Oxford O X 1 3PT, U K

K . H a l l C U R E , Wadsworth Y A and U C L A , Los Angeles, Calif. , USA

S. H e r i n g Department of Pharmacology, St George's Hospital Medica l School, Cranmcr Terrace, Footing, London SW1 7 O R E , U K

S.J . Hersey Emory Universi ty, At lanta , Georgia, USA

P. H e s s Department of Cellular and Molecular Physiology, Harva rd Medical School, 25 Shattuck Street, Boston, Mass. 02115. USA

B . H i l l e Department of Physiology and Biophysics, SJ-40, Washington Universi ty , Seattle, Wash. 98195, USA

S .B . H l a d k y Department of Pharmacology, Universi ty of Cambridge, Cambridge CB2 2 Q D , U K

F . Hof fman Ins t i tu t für Physiologische Chemie, Medizinische F a k u l t ä t , Un ive r s i t ä t des Saarlandes, D-6650 Homburg/Saar , West Germany

Contributors vii

S.J .D. K a r l i s h Department of Biochemistry, Weizmann Insti tute of Science, Rehovoth, Israel

K . K u n z e l m a n n Physiologisches Ins t i tu t , Alber t Ludwigs Unive r s i t ä t Freiburg, Hermann Herder Strasse 3, D-7800 Freiburg, West Germany

D . W . L a n d r y Departments of Medicine and Physiology, Columbia Univers i ty , College of Physicians and Surgeons, 630 W 168th Street, New-York . N . Y . , USA

D . E . Logothetis Department o f Cardiology at Children's Hospi ta l , Harvard Medica l School, Boston, Mass. 02115, US A

M . L . M a y e r U n i t of Neurophysiology and Biophysics, Bui ld ing 36, Room 2 Λ 2 1 , L D N . N I C H D , N I H , Bethesda, M d . 20892, U S A

J . M a r s h a l l M R C Molecular Neurobiology U n i t , M R C Centre, Hi l l s Road, Cambridge CB2 2 Q H , U K

J . E . Merrit t Smith K l ine & French Research L i m i t e d , The Fry the, We lwyn , Herts A L 5 9AR, U K

K . M u n s o n C U R E , Wads wor th Y A and U C L A , Los Angeles, C a l i f , USA

R . K . Nakamoto Department o f H u m a n Genetics & Cellular and Molecular Physiology, Yale Universi ty School of Medicine, New Haven, Conn. 06510, USA

M . R . P l u m m e r Department of Cellular and Molecular Physiology, Harva rd Medical School, 25 Shattuck Street, Boston, Mass. 02115, USA

H . Porz ig Department of Pharmacology, Universi ty of Berne, CH-3010, Β e r η e, S w i t ζ e r 1 a η d

R . R a o Department of H u m a n Genetics & Cellular and Molecular Physiology, Yale Universi ty School of Medicine, New Haven, Conn. 06510, USA

C . R e d h e a d Departments of Medicine and Physiology, Columbia University, College of Physicians and Surgeons, 630 W 168th Street, New York , N . Y . , U S A

H . Reuter Department of Pharmacology, Universi ty of Berne, CH-3010 Berne, Switzerland

T . J . R i n k Smith K l ine & French Research L i m i t e d , The Frythe, Welwyn , Herts A L 5 9AR, U K

viii Contributors

P. R u t h Ins t i tu t für Physiologisches Chemie, Medizinische F a k u l t ä t , U n i v e r s i t ä t des Saarlandes, D-6650 Homburg/Saar , West Germany

G. Sachs C U R E , Wadsworth V A and U C L A , Los Angeles, Calif. , U S A

D . B . Sattelle A F R C U n i t of Insect Neurophysiology and Pharmacology, Department o f Zoology, Universi ty of Cambridge, Cambridge CB2 3BJ, U K

R . H . Scott Department of Pharmacology, St George's Hospital Medica l School, Cranmer Terrace, Toot ing , London SW17 O R E , U K

C . W . S layman Department o f H u m a n Genetics & Cellular and Molecular Physiology, Yale Universi ty School of Medicine, New Haven, Conn. 06510, U S A

C . K . Sole Department of Neurobiology, Stanford Universi ty School of Medicine, Stanford, Calif. 9 4 3 0 5 - 5 4 0 1 U S A

W . N . Zagotta Department of Neurobiology, Stanford Univers i ty School of Medicine, Stanford, Calif. 94305-5401, USA

Contents

Contr ibutors ν Foix4 word ix In t roduct ion xvi i

P A R T 1: P - t y p e C a t i o n P u m p s

1 Extracytoso l ic Funct ional Domains of the Η , Κ ' - A T P a s e C o m p l e x 3 G. Sachs, K. Alunson, K. HalL D. Aures-Fischer, I). Anderson. V. Belagi and S.J. Hersey

1 Introduction 3 2 Results 6 3 Discussion 13

Acknowledgeinen ts 16 References 17

2 T h e Mechanism of Cation Transport by the N a 1 , K - A T P a s e 19 S.J.I). Karl is h

1 Introduction 19 2 The transport mechanism 19 3 Cation occlusion 21 4 Cation selectivity 22 5 Trans effects of Na ! 23 6 Cation slippage fluxes 24 7 Electrogenie potentials 25 8 Effects of voltage on the pump 26 9 The structure of the cation-binding sites 29 10 Future directions 30

Acknowledgements 31 References : 3i

xi

xii Contents

3 T h e Nucleot ide-binding Site of the Plasma-membrane H + - A T P a s e of Neurospora crassa: A Compar i son with other P-type A T P a s e s 35 Rajini Rao, Roberl K. Nakamolo and Carolyn W. Slayman

1 Introduction 35 2 Nucleotide binding 37 3 Sequence comparisons 39 4 Structure of the nucleotide-binding site 47

Acknowledgements 50 References 50

P A R T 2: I o n C h a n n e l s a n d t h e i r M o d u l a t i o n

4 Voltage-gated Sodium Channe l s s ince 1952 57 Berlil Hille

1 Introduction 57 2 Distribution 57 3 Molecular Structure 59 4 Gating 60 5 Selectivity filter and pore 63 6 Modulated receptors 64 7 Conclusion 68


5 Single Potass ium C h a n n e l s in Drosophila Nerve and Musc le 73 Richard W. Aldrich, Charles K. Sole, William Λ'. Zagotla and Michael S. Brainard

1 Introduction 73 2 Advantages of Drosophila as a system for the study of ion channels 73 3 Tissue culture systems 75 4 Α ι channels 77 5 Λ.> channels 78 6 K| ) channels 79 7 K , channels 80 8 K 0 channels 81 9 Ks,- channel 81 10 Shaker differential splicing does not explain the diversity of

channel types 82 Acknowledgement 83 References 84

Contents x i "

6 C a l c i u m Channe l s : Properties and Modulat ion 87 II. Reuler and H. Porzig

1 Introduction 87 2 Ca 2 f channel selectivity 88 3 C a 2 + channel gating 88 4 C a 2 + channel modulation 90


7 C a l c i u m C h a n n e l s in M a m m a l i a n Sympathetic Neurons and P C 12 Ce l l s 97 Mark R. Plummer, Peter Hess and Diomedes E. Logothetis

1 Introduction 97 2 Results and discussion 98


8 Voltage-dependent C a l c i u m C h a n n e l s of Smooth Musc le C e l l s 117 T.B. Bolton, S. Hering and P.I. Aaronson

1 Introduction 117 2 Inward current 118 3 Conclusions 123

A c k η ο w 1 edge t u e η t s 124 References 124

9 Modulat ion of C a l c i u m and other Channe l s by G Proteins: Impl icat ions for the Contro l of Synaptic T r a n s m i s s i o n 127 Annette C. Dolphin and Roderick PI. Scott

1 Introduction 127 2 Modulation of C a 2 f channels by G protein activation 128 3 Evidence for G proteins coupling to K 1 channels 138 4 Role of G protein-coupled ion channels in the modulation of

synaptic transmission 140 5 Conclusion 141

References 142

10 T h e Structure of the Skeletal Musc le C a l c i u m C h a n n e l 147 Peter Ruth, Veit Flockerzi, Marlin Biel and Franz Hoffman

1 Introduction 147 2 Structural composition of the purified skeletal muscle Ca 2 '

channel 148

xiv Contents

3 Phosphorylation of the purified CaCB-receptor 148 4 Structure of the a,- and jS-subunits of the skeletal muscle Ca 2 r

channel 150 5 Identification of L-type C a 2 + channel proteins in other tissues . . 152 6 Reconstitution of an L-type C a 2 + channel from the skeletal

muscle CaCB-receptor 153 7 Conclusions 154


11 Structural Characterist ics of Cat ion and A n i o n C h a n n e l s Direct ly Operated by Agonists 159 Ε.Λ. Barnard, M.G. Darlison.J. Marshall and D.B. Sattelle

1 Classes of receptor-operated channels 159 2 Ligand-operated ion channels 161 3 Vertebrate nicotinic acetylcholine receptors 162 4 Homo-oligomeric forms of nicotinic receptors in insects 166 5 G A B A A and glycine receptors 171 6 The ion channel in the structure 174 7 Conclusions 175

References 176

12 Act ivat ion and Desensitization of Glutamate Receptors i n M a m m a l i a n C N S 183 Mark L . Mayer

1 Introduction 183 2 Techniques used for rapid perfusion to limit desensitization. . . . 185 3 Responses to fast applications of excitatory amino acids 186 4 Glycine modulates desensitization at N M D A receptors 188 5 Dose-response analysis for activation of N M D A and quisqualate

receptors 189 () Implications lor synaptic transmission 191


13 Receptor-mediated C a l c i u m E n t r y 197 CD. Benimm, J.Ε. Merrill and T.J. Rink

1 Introduction 197 2 Electrophysiological approaches 199 3 Studies with fluorescent indicators of [Ca 2 f J, 203 4 Conclusion 210


Contents xv

P A R T 3: I o n s a n d F l u i d T r a n s p o r t

14 Pathways for C e l l V o l u m e Regulat ion v ia Potass ium and C h l o r i d e L o s s 217 A.C. Hall, L Bianchini and J.C. Ellory

1 Introduction: transport processes invoked in RYD 217 2 Coupled KCl co-transport in hepatocytes 222 3 Anion dependence and kinetic properties of KCl co-transport in

red cells 224 1 Specific inhibitors of KCl co-transport 226 5 Loss of KCl co-transport on "young" red cell maturation 228 6 Discussion 229


15 Epi the l ia l C h l o r i d e Channe l s : Properties and Regulat ion 237 R. Greger, L . Gerlach and K. Kunzelmann

1 Introduction 237 2 Properties of epithelial Cl~ channels 238 3 Regulation of epithelial CI"' channels 241 4 Conclusion 244


16 Purif ication and Reconsti tut ion of the Epi the l ia l C h l o r i d e C h a n n e l 249 Donald W. Landry, Μ vies H. Akabas, Christopher Redhead, AI ek sander Kdelman and (la is Al-Awqali

1 Introduction 249 2 Solubilization and affinity chromatography 250 3 Reconstitution 253 4 Discussion 257


P A R T 4: M o d e l s o f I o n P e r m e a t i o n a c r o s s M e m b r a n e s

17 Models of I o n Permeation through Membranes 263 S.B. Hladkv

1 introduction 263 2 Molecular and Brownian dynamics 264

xvi Contents

3 Interpretation of experimental data 265 References 275

18 T o p i c s Relat ing to the Model l ing of I o n C h a n n e l Funct ion 279 D.T. Edmonds

1 A threshold model of N a + channel kinetics 279 2 A kinetic role for ionizable residues in channel proteins 286

References 291

A p p e n d i x : Abstracts of Posters 293

I n d e x 375

C o l o u r Plate Between pages 8 and 9

10

The Structure of the Skeletal Muscle Calcium Channel

PETER RUTH, VEIT FLOCKERZI, MARTIN BIEL and FRANZ HOFFMAN Institut für Physiologische Chemie, Medizinische Fakultät, Universität des Saarlandes, Homburg/Saar, West Germany

1 Introduction

Voltage-activated C a 2 + channels are classified into three types ( Τ , Ν and L ) , which differ in their pharmacological behaviour and functional significance (Nowycky et αι., 1985). L-type channels are sensitive to organic drugs the C a 2 + channel blockers (CaCB) which include the dihydropyridines (1 ,4-DHPs) , the phenylalkylamines (PAAs) and the benzothiazepines (BTZs) (Hofmann et αι., I n cardiac muscle, ß - a d r e n o e e p t o r agonists increase the open state probabi l i ty of this channel type either by phosphorylation of the channel or a channel associated protein via the catalytic subunit of cAMP-dependent protein kinase (Trau twein et αι., 1986), or by stabilizing the open state via the α - s u b u n i t of the G T P b ind ing protein G s (Yatani et al., 1987). L-type channels occur in peripheral and central neurons, smooth muscle, invertebrate skeletal muscle and heart. The i r biological function in vertebrate skeletal muscle has not been established unequivocally, but probably the channel has the same function as in cardiac muscle, i.e. par t ic ipat ing in the maintenance of an adequate amount of intracellular C a 2 _ h for muscle contraction (Ildefonse et αι., 1985). However, the channel protein may participate in skeletal muscle excitation contraction-coupling not only as Ca2"1" channel but also as a voltage sensor (Berwc et aL, 1987; L a m b and Walsh , 1987; Rios and B r u m , 1987). L-type C a 2 + channels of different tissues probably are not identical and w i l l be divided up further

ΪΟΝ T R A N S P O R T Copyright © 1989 Academic Press Limited ISBN 0.12.403985.5 Ml rights of reproduction in any form reserved

147

148 P. Ruth et al.

as our knowledge of their electrophysiological and molecular properties, their functional significance and their modula t ion by hormones, neurotransmitters and drugs expands.

2 Structural composition of the purified skeletal muscle C a 2 + channel

Most o f the informat ion on the structural composition o f the L-type C a 2 1

channel originates from studies w i t h rabbi t skeletal muscle. SDS-gcl analysis of the purified skeletal CaCB-receptor yields several stained bands wi th apparent molecular weights o f 165 kDa (ctj), 55 kDa (β) and 32 kDa (γ) in a constant ratio o f 1:1.7:1.4 (Sieber el al., 1987). A further protein containing a 130-(a 2 ) and 28-kDa (8) disulfide-linked peptide co-purifies in variable amounts w i t h the α,- , β- and γ - s u b u n i t s o f the CaCB-receptor. The a 2 - and the γ - p e p t i d e s are heavily glycosylated, whereas the a,- and /3-subunit contain none or a low amount of carbohydrates (Takahashi et αι., 1987). The purified receptor binds al l three major classes o f C a 2 +

channel blockers (i.e. DHPs , PAAs and B T Z s ) in a stereospecific manner. The photo-affinity analogues of the 1,4-DHPs and PAAs, azidopinc and L U 49888, label only the 165-kDa (a , ) subunit , indicat ing that this protein carries the d rug receptor sites for 1,4-DHPs and PAAs (Galizzi et al., 1986; Sieber et αι., 1987; Tanabe et αι., 1987; Striessnig et al, 1986, 1987). The constant stoichiometry o f the 165-, 55- and 32-kDa proteins suggests that these proteins are constituents of the C a 2 + channel. Further evidence for the existence o f a functional complex w i t h this composition comes from studies w i t h antibodies against the a,-subunit which precipitate the a r , β- and γ - s u b u n i t s (Takahashi et al., 1987). Antibodies specific against the a,- or ß - s u b u n i t s immunoprecipitates the a r or /3-subunits (Leung el al., 1988). Furthermore, antibodies specific against the a r , β- and γ - s u b u n i t s modulate the C a 2 + current in vivo (Campbell el al., 1988; M o r t o n et al., 1988; V i l v e n el al., 1988). Attempts to isolate only the a,-subunit under non-denaturing conditions have not been successful so far, suggesting that the β- and γ - s u b u n i t s stabilize the channel in a high-affinity CaCB binding-conformation. A t present, i t is not known whether or not the 130/28-kDa protein belongs also to this structure ('Leung el al., 1988) or is only a contaminant.

3 Phosphorylation of the purified CaCB-receptor

The 165- and 55-kDa subunits o f the purified CaCB-receptor are phosphorylated readily by cAMP-dependent protein kinase (Curtis and

Skeletal Muscle Calcium Channel 149

Table 1 Kinase-specific phosphopeptides of the skeletal muscle CaCB-receptor

Phosphopeptide number

Protein kinase 165 kDa 55 kDa

cAMP-kinase cGMP-kinase Protein kinase C Casein kinase I I

none 7,11

1,10 1,2

7 1,3 7/8 9,10

T h e pu r i f i ed receptor was phosphory la t cd by each kinase as described by J a h n et al., (1988) . T h e phospho ry l a t ed subuni t s were separated by SDS-gcl electrophoresis. I n d i v i d u a l gel pieces c o n t a i n i n g the phosphory la t ed a,- or ß - s u b u n i t were digested by t ryps in over n igh t . T h e phosphopept ides were then separated by 2 -D th in - layer ch roma tog raphy . T h e n u m b e r o f the kinase-specific phosphopept ide(s ) is shown . See also J a h n et al. (1988) .

Cat tcra l l , 1985; Flockerzi et al., 1986a,b) and other kinases in vitro (Nastainczyk et al., 1987; O 'Cal lahan et al., 1988; Jahn et al., 1988). The α , - s u b u n i t is a good substrate for cAMP-kinase and casein kinase I I , whereas the 55-kDa subunit is preferentially phosphorylated by protein kinase C and cGMP-dependcnt protein kinase. Two-dimensional peptide maps yield 11 phosphopeptides from the 165-kDa subunit and 11 from the 55-kDa subunit using these kinases (Jahn et al., 1988). W i t h the exception o f protein kinase C, each kinase apparently phosphorylates one or two peptides specifically in each subunit (Tabic 1). Protein kinase C does not phosphorylatc specifically a peptide in the 165-kDa peptide, but modifies rapidly peptide 7/8 of the 55-kDa subunit. Neither the 32-kDa nor the 130/28-kDa peptides arc phosphorylated by the above-mentioned kinases. A t physiological concentrations c A Μ P-d c pend en t protein kinase incorporates 1 mole phosphate per mole 165-kDa subunit w i t h i n 10 m i n (Curt is and Cat tcra l l , 1985; Nastainczyk et al., 1987). This suggests that the phosphorylat ion of this site may be functionally important . A second site is phosphorylated dur ing prolonged incubation. The rapidly phosphorylated peptide was isolated and sequenced. The phosphorylated amino acid was identified as Ser 687 of the deduced amino acid sequence of the Of j - subun i t ( R ö h r k a s t e n et al., 1988). This serine is located in the cytoplasmic loop between transmembrane regions I I and I I I (see Fig. 1). I t is possible that in vivo cAMP-dependcnt phosphorylation of this serine increases the open state probabi l i ty of the C a 2 + channel.

150 P. Ruth et al.

J , , II , in , . !Y_

Fig. 1 Hydrophobicity profile and transmembrane topology of the skeletal muscle Ca~+ channel a}-subunit. The hydrophobicity profile of the α j-subunit according to Tanabe et al. (1987). Positive indices represent hydrophobic amino acid regions. The protein consists of four homologous domains ( I , I I , I I I , I V ) each composed of six membrane spanning helices ( l , 2, 3, 4, 5, 6). Transmembrane regions are based on their hydropathy value, polarity index and hydrophobic moment analysis according to Chou and Fasman (1978). The homologous regions ( I , I I , I I I , I V ) each containing six transmembrane spirals are shown linearly. They are supposed to form the ionic pore. The carboxy- and amino-termini are located at the cytoplasmic site of the plasma membrane. The phosphorylation site of cAMP-dependent protein kinase, serine residue 687, is indicated between domains I I and I I I .

4 Structure of the α Ί - and β-subunits of the skeletal muscle C a 2 +

channel

Identification and cloning of the a,- subunit of the C a 2 + channel from skeletal muscle was a major step in Ca 2 * channel research (Tanabe el al.,


1987). The cloned rabbit skeletal muscle a,-subunit has 29% homology wi th the voltage-dependent Na 1 channel. I t is assumed that four homologous regions, each consisting of five hydrophobic α-hel ices ( S I , S2, S3, S3 and S6) and one hydrophi l ic α-hclix (S4), span the membrane and form the C a 2 4 channel pore (Fig. 1). S4, which is present in each transmembrane region, is a positively charged helix that could act as a voltage sensor. A homologous helix is found in other voltage-activated ion channels, i.c the N a + channel o f eel, fly and rat and the Κ 1 channel of various tissues. The positive charges o f the S4 segment could respond to a change in the membrane potential by a transmembrane shift of its positive charges, and thereby affect the open/closed state of the channel.

The p r imary structure of the rabbit skeletal muscle j3-subunit has been deduced from the cloned c D N A . The c D N A has a length of 1.85 kilobasc. The deduced peptide consists of 524 amino acids w i t h a Λ/ λ of 58 kDa. The p r imary structure of the /3-subunit agrees wi th that of a peripheral membrane protein. I t contains four homologous α-hel ices (Fig. 2). Analysis of the pr imary structure reveals two further apparently specific protein kinase C phosphorylat ion sites. This is interesting since the /3-subunit is preferentially phosphorylated by protein kinase C in vitro. Furthermore,

Fig. 2 Predicted secondary structure of the /3-subunit of the skeletal muscle CaL > +

channel. The secondary structure of the deduced amino acid sequence was predicted by the method of Gamier et at. (1978). The /3-subunit contains four hydrophilic helical regions (shaded rods), each composed of 26-33 amino acids. The helices are joined by /3-sheets (arrows) and coils. These secondary structures are interrupted at three positions by helical structures with a length of 10-15 amino acids (open rods). The in vitro phosphorylation sites are indicated by P. The site closer to the amino terminus is the major in vitro phosphorylation site of cAMP-depenclent protein kinase.

152 P. Ruth et al.

there is ample evidence that protein kinase C alters L-type channel function in vivo (Kaczmarek, 1987). I n addi t ion, the pr imary sequence contains a site specific for cGMP-depcndent protein kinase. This was expected from the in vitro phosphorylation experiments (see Table 1).

The biological significance of these pr imary structures remains to be elucidated. Antibodies against the /3-subunit enhance C a 2 + currents through L-type channels and prevent the blocking action o f ni trendipine (V i lvcn el al., 1988), whereas antibodies against the γ - s u b u n i t inh ib i t C a 2 4 current (Campbell el αι., 1988). This suggests that the two smaller subunits of the skeletal muscle C a 2 + channel are necessary for proper channel function.

The pr imary structure of the γ - s u b u n i t is not known. However, the pr imary structure o f the 130-kDa (a2) protein has been deduced from cloned rabbit skeletal muscle c D N A (Ellis el al., 1988). The a2 protein has the sequence o f a hydrophi l ic protein and may contain up to three transmembrane helices and eight extracellular N-glycosylation sites. This predicted topography is in accordance w i t h the finding that the purified protein is heavily glycosylated. Hybr id iza t ion studies show that the m R N A for the a2 protein is expressed in many tissues, whereas the c D N A for the skeletal muscle a,-subunit hybridizes only weakly or not at all to the messenger R N A from other tissues (Ellis el al., 1988). Al though these data are not conclusive, they support the notion that the 130/28-kDa (<W8) protein may not be an essential part of the calcium channel.

5 Identification of L-type C a 2 + channel proteins in other tissues

A C a 2 ' channel a,-subunit o f a slightly larger size than that o f rabbit skeletal muscle has been identified in brain, smooth muscle and heart by northern blot analysis of the respective m R N A (Table 2). This difference in size is supported by photo-affinity labell ing of the high-affinity receptor for CaCBs. Azidopinc and L U 49888 identify a 195-kDa protein in a part ial ly purified preparation of the bovine cardiac muscle CaCB-receptor (Schneider and Hofmann, 1988). A n identically sized α - subun i t is labelled in hippocampus (Striessnig et al., 1988). These differences are not caused by species differences, since a monoclonal antibody to the a!-subunit o f rabbit skeletal muscle recognizes the same subunit i n the skeletal muscle of guinea pig, hamster, rat, cow and pig, but docs not b ind to the bovine heart subunit. This suggests that the differences arc real and reside in the pr imary sequence of the α - s u b u n i t s from different tissues.


Table 2 Size of the putative α-subunit of the C a 2 + channel in other tissues

Size of putative a{-subunit

Tissue Species Protein (kDa) mRNA (kB)

Skeletal muscle rabbit 165 195

6.2 8.3 8.2 8.1

Heart Lung Brain guinea pig

cow cow

195

M e m b r a n e s or a p a r t i a l l y pur i f i ed p repa ra t ion o f the CaCB-recep to r were af f in i ty- labe l led w i t h az idopine or L U 49888. T h e molecu la r we igh t was de te rmined by SDS-gel electrophoresis. T h e size o f the m R N A was de te rmined by h y b r i d i z a t i o n w i t h a probe der ived f rom the cloned α , - subuni t o f the r abb i t skeletal CaCB-recep to r . For fur ther de ta i l see Sicher et at. (1987) , Schneider and H o f m a n n (1988) and Striessnig et at. (1988) .

6 Reconstitution of an L-type C a 2 + channel from the skeletal muscle CaCB-receptor

The purified d ihydropyr id ine receptor from skeletal muscle has been reconstituted into phospholipid vesicles which were fused w i t h a phospholipid bilayer (Flockerzi et al., 1986a; Hyme l el al., 1988; M a and Coronado, 1988; Pelzer el al., 1988; Talvcnheimo el ai, 1987). I n agreement wi th whole-cell recording data, the reconstituted protein channel has a single-channel conductance o f about 20 pS (Flockerzi el ai, 1986a; Pclzcr el al., 1988; Talvenheimo el ai, 1987). Its open state probabi l i ty (/>o) is reduced by the C a 2 + channel blockers gal lopamil and PN 200-110, and increases in the presence of the C a 2 + channel agonist Bay Κ 8644. The pa is also increased several fold by the addi t ion of A T P - M g and the catalytic subunit of cAMP-dependcnt protein kinase (Flockerzi el al., 1986a; Hyme l el al., 1988; Pelzer el ai, 1988). These results suggest that the reconstituted channel has some properties of the cardiac L-typc C a 2 1 channel. Further analysis o f the single-channel kinetics showed that open and closed times of the reconstituted channel are about 10 times longer than that of an in vivo cardiac muscle L-type channel (Pelzer el al., 1988), indicat ing that the reconstituted channel has properties which differ from that of the cardiac muscle channel. The slower channel kinetics of the purified receptor are not caused by proteolysis of the receptor dur ing purif icat ion. The same kinetics were obtained when solubilized microsomal membranes were reconstituted.

154 P. Ruth et al.

Both preparations, the solubilized membranes and the purified receptor, contain a second channel w i t h a conductance around 10 pS ( M a and Coronado, 1988; Pclzer el al., 1988; Talvcnheimo el al., 1987). The pa o f the channel showing the smaller conductance was not affected by phosphorylat ion, C a 2 + channel blockers or agonists (Pclzer el al., 1988). So far, an interconvcrsion of a small non-regulated into a large regulated conductance has not been observed (Pelzer el al., 1988). The two conductances could be distinguished further by the voltage-dependence of pa. The small conductance showed a regular voltage dependence of its open state probabi l i ty , whereas the large conductance yielded a bell-shaped dependency. pa was greatest at a membrane potential around 0 m V (Pelzer el al., 1988). These differences in electrophysiological parameters clearly dist inguish the two conductances.

7 Conclusions

T h e biochemical identi ty of these conductances is not clear at present. Recent reconstitution experiments of the isolated a}-subunit suggest that the 165-kDa subunit is the C a 2 4 - c o n d u c t i n g unit (Pelzer el al., 1988). This conclusion is supported by experiments in which the cloned c D N A of the « | - s u b u n i t was expressed in embryonic muscle cells from dysgenic mice (Tanabe el al., 1988). The dysgenic myotubes do not express the 6.5-kB m R N A of the a r s u b u n i t and are defective in EC-coupling. The intranuclear injection and the expression of the cloned aj-subunit m R N A in myotubes o f dysgenic mice restores EC-coupling and a slow L-type C a 2 + channel in these cells. Interestingly, the T- tubular voltage sensor and the C a 2 + channel is regained together w i t h the expression of the a r s u b u n i t , suggesting that both functions require the a,-subunit. These two functions may not depend alone on the presence of the 165-kDa (a , )-subunit, but correct functioning may require the presence of the other subunits. This conclusion is supported also by the modulatory effect of ß- (V i lvcn el al., 1988) and ^-(Campbel l el al., 1988) subunit-specific antibodies on C a 2 ' current (see above). Furthermore, the survival of a C a 2 + channel reconstituted from T- tubular membranes increases dramatically in the presence of activated G s (Yatani el al., 1988). I t is l ikely, therefore, that the 165-kDa subunit contains the C a 2 ' - c o n d u c t i n g part o f a L-type C a 2 + channel, which requires in vivo the presence of other smaller subunits and G proteins for proper function.

Acknowledgements

We thank M r s Siepmann for her work on the figures. This work was supported by Fonds der Chemischen Industrie and Deutsche Forschungsgemeinschaft.


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