Functional properties of the purified cholinergic receptor protein fromelectrophorus electricus

Brain Research, 62 (1973) 307-315 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

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F U N C T I O N A L PROPERTIES OF T H E P U R I F I E D C H O L I N E R G I C RECEPTOR PROTEIN FROM ELECTROPHORUS ELECTRICUS

JEAN-CLAUDE MEUNIER, HIROYUKI SUGIYAMA, JEAN CARTAUD*, ROBERT SEALOCK Argo JEAN-PIERRE CHANGEUX

D~partement de Biologie Mol~culaire, Institut Pasteur, Paris 75015 (France)

Since the discovery that the cholinergic (nicotinic) receptor protein can be selectively labeled by snake venom a-toxinsT, 25,85 and solubilized by mild detergents without loss of its ability to bind cholinergic agonists and antagonists 6-8,85, an increasing number of studies have been recently undertaken14,16,2a,27,a2,aa,a6, a9 (see also ref. in 18) on this important regulatory 3,1°,11,21 protein. In this brief report, we shall summarize the results obtained in our laboratory with the receptor protein from the electric organ of Electrophorus electricus. (1) Isolation and purification. The major requirements for the isolation and purification of a receptor protein are (1) a tissue particularly rich in receptor, (2) a set of specific ligands, (3) the possibility to correlate physiological response and in vitro properties of the isolated molecule. The electric organ of Electrophorus satisfies these three requirements. The electric organ of an average sized eel (1 m long) weighs up to 1 kg and contains about 101° identical synaptic contacts. These synapses are cholinergic and sensitive to typical nicotinic agents. The most effective agonists are acetylcholine, carbamylcholine or decamethonium. Antagonists like D-tubocurarine, Flaxedil or hexamethonium block the effect of these agonists in a 'competitive' manner; local anesthetics like tetracaine or procaine in a 'non-competitive' manner. In addition, snake venom a-toxins like that from Bungarus multicinctus (74 amino acids, 5 disulfide bridges, mol. wt. = 8,000) 25 or Naja nigricollis (61 amino acids, 4 disulfide bridges, mol.wt. ---- 6,800)~2, 25 behave in this system7, 8 as powerful and slowly reversible curarizing agents. The a-toxin from Naja nigricollis was tritiated by the method of Fromageot, that is, iodination followed by catalytic dehalogenation in the presence of tritium gas 30. The tritiated a-toxin, with a specific radioactivity of 10--14 Ci/mmole, presents the same structure as the native a-toxin except that one hydrogen atom is replaced by a tritium atom, and possesses all its pharmacological properties3L It has been used to label and assay the receptor protein. The physiological response to cholinergic agents can be recorded by electro* Laboratoire de Microscopic Electronique, Institut de Biologic Mol6culaire, Universit6 Paris VII, Paris 75005, France.

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physiological techniques applied to single electroplax dissected from the electric organ~0, 41. Decrease in membrane potential as a function of concentration of bath applied agonists gives dose-response curves and 'apparent' dissociation constants. A more reliable method consists of measuring steady-state slope conductances by voltage clamp 26. With carbamylcholine the apparent dissociation constant is 2.5 ::~:_ 0.5 × 10-2 M b y the first method and 3 ~ 0.9 × 10-4 M b y the second 2°,26. With decamethonium, the values obtained by both techniques fall between 10-6 M and 10-2 M. As shown by Kasai and Changeux z4, the pharmacological and ionic properties of the response are preserved, in vitro, in excitable membrane fragments or microsacs purified from crude homogenates of electric tissue. The passive permeability of these vesicles to Na ÷, K ÷ and Ca 2+ increases in the presence of agonist. The same vesicles bind [3H]decamethonium and [3H]a-toxin24,zL 'Reduction' of the system from the cellular to the subcellular level, therefore, does not result in a dramatic alteration of the physiological function. Mild detergents like sodium deoxycholate or cholate, Triton X-100, Emulphogen etc. completely solubilize excitable microsacs without loss of the ability to bind cholinergic ligands and snake a-toxins 7,~5. With the solubilized material, as well as with the membrane fragments, the amount of bound [aH]decamethonium displaced by unlabeled a-toxin and conversely of bound [all]a-toxin displaced by an excess of unlabeled decamethonium was considered as specifically associated with the cholinergic receptor protein 6-s. Several assays for the solubilized toxin binding material have been used: ammonium sulfate precipitation 15,32, adsorption on DEAE cellulose p a p e d 7,4° etc. The most reliable and fast one was, in our hands, precipitation in the absence of detergents followed by Millipore filtration an. Massive Triton X-100 extraction of a crude membrane preparation from E l e c 0.3

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PROPERTIES OF RECEPTOR PROTEIN FROM E. electricus

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Fig. 2. Negative staining of a highly purified fraction of receptor protein (from Cartaud et al.~).

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trophorus electric organ gives a convenient starting material for purificationZ% Successful purification of the toxin binding material was achieved by affinity chromatography on a column of Sepharose derivative with cholinergic arms resembling Flaxedil followed by chromatography on DEAE cellulose and sucrose gradient centrifugation3a,36. The material purified in a satisfactory yield (30 ~o from crude extract) gives one major band by sucrose gradient centrifugation and gel electrophoresis in the presence of Na cholate and Emulphogen. In both cases (Fig. 1) a reasonable superimposition of protein and toxin binding was achieved. The specific activity was 5.9 ~ 0.5 #moles/g protein (assayed by Lowry method) or 6.8 :~ 0.5 #moles/g protein (determined from the amino acid composition). The purified preparation contained less than one catalytic site of acetylcholinesterase (assuming 65,000 g/site) per 100 [aH]a-toxin binding sites. The amino acid composition does not deviate from that of standard globular protein. The 'polarity'1 of the protein is close to 0.47. In addition, the purified protein is precipitated by concanavaline A and thus carries carbohydrate residues. Electron microscopy on the purified fraction2 shows, after negative staining by uranyl acetate, a homogenous population of particles with an average diameter of 8-9 nm, a characteristic electron dense center and a subunit pattern of 5-6 subunits with a diameter of 3-4 nm (Fig. 2). (2) The receptor protein as an integral membrane protein. The receptor protein is isolated in our laboratory from membrane fragments of Electrophorus electric organ which have been homogenized in water and subsequently washed in high (0.8 M) and low (0.02 M) ionic strength buffers, all without significant solubilization of the receptor. Only after extraction with Triton X-100 (1 ~) or Na-cholate solutions is the receptor found in soluble form. The receptor molecule is strongly membrane bound. By contrast, acetylcholinesterase, whose binding properties (and hence possibly its origin in evolution) present some analogies with that of the receptor, is released from the membrane during the wash with 0.8 M NaC1. Following S. J. Singer 42 acetylcholinesterase would be a 'peripheral' protein, the receptor protein an 'integral' one.

In the presence of detergent the receptor protein shows unusual hydrodynamic properties. On Sepharose 6B columns equilibrated with buffers containing Triton X-100(l ~) the receptor has an apparent Stokes radius of 7.3 nm(slightly larger than that of fl-galactosidase, mol.wt. 550,000). On the other hand, upon sedimentation in sucrose gradients in the presence of Triton the receptor has an apparent sedimentation constant of 9.5 S, considerably less than the 16 S found for fl-galactosidase 3~,~3,39. Sedimentation in D20 instead of H20 al shows that the receptor has an unusually low density or high partial specific volume (re = 0.78), while ~¢calculated from the amino acid composition (0.73-0.74) is typical of proteins in general 34. This low density, which most likely arises from the binding of considerable quantities of Triton (~ --~ 0.99) accounts to a large degree for the low observed sedimentation constant. A possible contribution from asymmetry of the receptor protein cannot be eliminated, however, on the basis of these data.

PROPERTIES OF RECEPTOR PROTEIN FROM

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One simple interpretation of the solubility properties and of the high affinity for detergents is that after extraction from the membrane the receptor protein possesses hydrophobic areas exposed to the solvent. In its membrane bound state these areas would anchor the receptor protein to the apolar phase of the membrane. An interesting problem is then raised by the fact that the amino acid composition of the receptor protein, like that of several other integral proteins, does not differ markedly from that of water soluble proteins. As already discussed, one explanation would be that part of the protein is folded in such a way that hydrophilic groups face the inside of the protein, hydrophobic ones its outside4. The tertiary structure would be 'inverted' as compared to that of water soluble protein. An alternative is that, as in cytochrome b543 or erythrocyte major glycoprotein28, the protein possesses 'segments' or 'tails' particularly rich in hydrophobic amino acids which would preferentially interact with the aliphatic chains of membrane lipids. In any case, since the protein acts as a receptor, at least part of it must be exposed to the solvent. This is, indeed, confirmed by freeze-etching pictures of receptor-rich membrane fragments from TorpedoL An attractive but still highly hypothetical structure of the molecule would be that subunits or globules are exposed to the solvent with the usual globular structure and carry the receptor site and that others, with an 'inverted' structure, are more directly involved in ionic translocation. (3) The cholinergic receptor protein as a regulatory protein. As already extensively discussed ~8, the cholinergic receptor protein can be viewed as a regulatory protein which controls the selective translocation of small cations through the membrane. To account for the coupling between the receptor site for agonists and the site for ion translocation, the receptor-ionophore complex was postulated to exist under at least two conformational states: the 'resting' state, with a low permeability for cations but a high affinityfor antagonists, and the 'active' state with a high permeability and a high affinity for agonists. Although these ideas are still hypothetical, several experimental results make them plausible. The yield of the purification procedure is high enough to study binding of cholinergic agonists and antagonists to the receptor protein by equilibrium dialysis. As shown in Fig. 3, decamethonium bound to the purified protein is completely displaced by N. nigricollis a-toxin and by a cholinergic antagonist Flaxedil. Interestingly, the affinity of the receptor protein for the three agonists tested is one to two orders of magnitude larger than the apparent affinity of the same agonists with either the isolated electroplax or the excitable microsacs29. On the other hand, no significant difference is found with the antagonists~9. Among the several interpretations which can be proposed for this phenomenon, one is that solubilisation by detergents and purification release a membrane 'constraint' created by either membrane lipids or proteins 29 or by both and stabilize the molecule in an 'active' conformation exhibiting high affinity for agonists. The limited changes of affinities for the antagonists would be caused by their non-exclusive binding to both the 'active' and 'resting' conformations. A sigmoid shape of the dose-response curve of the electroplax to cholinergic agonists was already noticed in the early work of Higman et al. ~° and subsequently

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confirmed for a variety of agonists with the same systemg,is or excitable microsacs 24. Isolation and purification of membrane fragments from Torpedo electric tissue particularly rich in receptor protein lz favored accurate analysis of the binding curve of acetylcholine to the cholinergic receptor site46. Clearcut deviation from the Langmuir isotherm has been observed: the Hill coefficient of the binding curve ranges from 1.3 to 1.5. Cooperative binding of acetylcholine, therefore, occurs 46. It is not known yet whether these cooperative effects are associated with the oligomeric structure of the receptor protein (interaction between subunits) or are relevant to its organization into a lattice structure (interaction between oligomers). It should be mentioned however that a lattice organization of the cholinergic receptor protein has been demonstrated by freeze-etching with the same membrane fragments 2. Finally, recent studies with a dansylated cholinergic ligand introduced by Weber et al. 45 provide direct evidence for a structural transition of the receptor protein upon binding of agonists lz. These still fragmentary results are consistent with the hypothesis that the receptor protein is a regulatory protein, although its final demonstration requires a more extensive documentation on the structural properties of the pure protein. (4) Immunological studies with the receptor protein. Among the several hypotheses which have to be mentioned concerning the changes of affinity observed after purification, one is that the purified protein is not the macromolecule which accounts for the electrogenic action of acetylcholine. Although the arguments based on the binding specificity of the isolated macromolecule for both cholinergic ligands and snake a-toxins are rather convincing, the immunological studies with the purified protein brought additional support that this protein carries the physiological receptor site for acetylcholine44. Antibodies directed against the purified protein were raised in rabbits by injecting 0.5 mg of protein in Freund's adjuvant per rabbit. After 3 weeks a booster injection of the same amount of purified protein was given. Four days later the rabbits

PROPERTIES OF RECEPTOR PROTEIN FROM E. electricus

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developed a flaccid paralysis and died rapidly. A similar effect was previously observed by Patrick and LindstrOm a7 with a preparation of protein prepared with a different affinity column and interpreted by them as an autoimmune response of the rabbit to the receptor protein from Electrophorus. The antibody precipitates the toxin binding protein from both a crude extract and a purified preparation, and cross-reacts, but with a different ratio of antigen to serum at equivalence, with the receptor protein f r o m Torpedo and chick embryo. The serum gives a single precipitation band by Ouchterlony's double diffusion technique against the purified protein. Interestingly, a 10-fold dilution of the serum applied to the electroplax for 20 min blocks in an irreversible manner the response to bath applied carbamylcholine. Therefore, the protein, which in vitro binds cholinergic agonists with a very high affinity, is that which mediates the electrical response of the electroplax to the same agonists but with a 10-40 times smaller 'apparent' affinity. (5) Epilogue. Concerning the molecular mechanism of membrane excitation, several questions remain without answer: is the ionophore part of the purified protein or does it constitute a separate entity ? What is the exact mechanism of ionic translocation and of its control by the cholinergic receptor site? Reconstitution of an excitable system with pure lipid and protein components is in progress and should bring answers to these essential questions 5,19. Dr. P. Boquet deserves special thanks for supplying the a-toxin of N a j a nigricollis, and Drs. A. Menez, J. L. Morgat and P. Fromageot for its tritiation. This work was supported by funds from the Centre National de la Recherche Scientifique, the D616gation G6n6rale A la Recherche Scientifique et Technique, the Coll6ge de France, the Commissariat A l'Energie Atomique, and the National Institutes of Health. 1 CAPALDI, R. A., AND VANDERKOOI, G., The low polarity of many membrane proteins, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 930-932. 2 CARTAUD,J., BENEDETTI,L., COHEN, J. B., MEUNIER,J. C., AND CHANGEUX,J. P., Presence of a lattice structure in membrane fragments rich in nicotinic receptor protein from the electric organ of Torpedo marmorata, FEBS Letters, 33 (1973) 109-113. 3 CHANGEUX,J. P., Responses of acetylcholinesterase from Torpedo marmorata to salts and curarizing drugs, Molec. PharmacoL, 2 (1966) 369-392. 4 CHANGEUX,J. P., General discussion. In Polymerisation in Biological Syswms, Ciba Foundation Symposium 7, Excerpta Medica, Amsterdam, 1972, pp. 289-290. 5 CHANGEUX,J. P., HUCHET, i . , ET CARTAUD,J., Reconstitution partielle d'une membrane excitable apr6s dissolution par le deoxycholat¢ de sodium, C.R. Acad. Sci. (Paris), 274D (1972) 122125. 6 CHANGEUX, J. P., KASAI,M., HUCHET,M., ET MEUNIER, J. C., Extraction ~t partir du tissu 61ec-

trique de Gymnote d'une prot6ine pr6sentant plusieurs propri6t6s caract6ristiques du r6cepteur physiologique de l'ac.~tylcholine, C.R. Acad. Sci. (Paris), 270D (1970) 2864-3867. 7 CHANGEUX,J. P., KASAI,M., AND LEE, C. Y., Use of a snake venom toxin to characterize the cholinergic receptor protein, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1241-1247. 8 CHANGEUX,J. P., MEUNIER, J. C., AND HUCHET, M., Studies on the cholinergic receptor protein of Electrophorus electricus. I. An assay in vitro for the cholinergic receptor site and solubilization of the receptor protein from electric tissue, Molec. Pharmacol., 7 (1971) 538-553. 9 CHANGEUX, J. P., AND PODLESKI, T. R., On the excitability and cooperativity of the electroplax membrane, Proc. nat. Acad. Sci. (Wash.), 59 (1968) 944-950.

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choline 6tudi6es -/t l'aide d'une neurotoxine radioactive, C.R. Acad. Sci. (Paris), 273D (1971) 595-598. MEUNIER,J. C., OLSEN, R., SEALOCK,R., AND CHANGEUX,J. P., (1973) in preparation. MILDEI, R., MOLINOFF, P., AND POTTER, L. T., Isolation of the cholinergic receptor protein from Torpedo electric tissue, Nature (Lond.), 229 (1971) 554--557. OLSEN, R. W., MEUNIER,J. C., AND CHANGEUX,J. P., Progress in the purification of the cholinergic receptor protein from Electrophorus electricus by affinity chromatography, FEBS Letters, 28 (1972) 96--100. PATRICK,J., AND LINDSTROM,J., Science, 180 (1973) 871-872. PODLESIO,T. R., AND CHANGEUX,J. P., On the excitability and cooperativity of the electroplax membrane. In J. F. DANIELLI, J. F. MORAN AND D. J. TRIGGLE (Eds.), Fundamental Concepts

of Drug Receptor Interactions, Proc. 3rd Ann. Buffalo Milan Symp. on Molec. PharmacoL 1968, Academic Press, New York, 1970, pp. 93-119. 39 RAFTERY,M. A., SCHMIDT,J., CLARK, D. G., AND WOLCOTT, R. G., Demonstration of a specific a-bungarotoxin binding component in Electrophorus electricus electroplax membranes, Biochem. biophys. Res. Commun., 45 (1971) 1622-1629. 40 SCHMIDT,J., AND RArTERY, M. A., Purification of acetylcholine receptors from Torpedo californica electroplax by affinity chromatography, Biochemistry, 12 (1973) 852-856. 41 SCHOFEENIELS,E., AND NACHMANSOHN,D., An isolated single electroplax preparation. I. A new data on the effect of acetylcholine and related compounds, Biochim. biophys. Acta (Amst.), 26 (1957) 1-15. 42 SINGER,R. J., AND NICOLSON, G. L., The fluid mosaic model of the structure of cell membranes, Science, 175 (1972) 720-731. 43 SPATZ, L., AND STRITTMATTER,P., A form of cytochrome b5 that contains an additional hydrophobic sequence of 40 amino acid residues, Proc. nat. Acad. Sci. (Wash.), 68 (1971) 1042-1046. 44 SUGIYAMA,H., BENDA, P., MEUNIER,J. C., AND CHANGEUX,J. P., Immunological characterisation of the cholinergic receptor protein from Electrophorus electricus, FEBS Letters, (1973) in press. 45 WEBER,G., BORRIS,D., DE ROBERTIS,E., BaRRANTES,F., LA TORRE, J., AND DE CARLIN, The use of a cholinergic fluorescent probe for the study of the receptor proteolipid, Molec. Pharmacol., 7 (1971) 530. 46 WEBER, M., ANO CHAN~EUX,J. P., Binding of Na/a nigricollis 8H-a-toxin to membrane fragments from Electrophorus and Torpedo electric organs, Molec. Pharmacol., (1973) in press.