Proc. Nati. Acad. Sci. USAVol. 75, No. 1, pp. 510-514, January 1978Neurobiology

Distinct protein components from Torpedo marmorata membranescarry the acetylcholine receptor site and the binding site for localanesthetics and histrionicotoxin

(acetylcholine ionophore/quinacrine fluorescence/subunit interactions)

ANDRE SOBEL, THIERRY HEIDMANN, JOHN HOFLER, AND JEAN-PIERRE CHANGEUXNeurobiologie Moleculaire & Laboratoire Associe Centre National de la Recherche Scientifique, Interactions Moleculaires et Cellulaires, Institut Pasteur,75015 Paris, France

Communicated by Franqois Jacob, October 12, 1977

ABSTRACT Highly purified subsynaptic membrane frag-ments prepared from Torpedo marmorata electric organ (spe-cific activity, >4 imol of Na'a nigricoilis a-[3H toxin per mg ofprotein) exhibit, on sodium dodecyl sulfate/polyacrylamide gelelectrophoresis, two major protein bands of apparent molecularweight 40,000 and 43,000, respectively. Dissolution of thesemembranes by the. nondenaturing detergents Triton X-100 andBerol 043 followed by standard fractionation yielded (j)the 9Sacetycholine-receptor protein which still binds the cW4ltoxinand after further purification-yielded, in the presence of sodiumdodecyl sulfate, the 40,OOWdalton component, covalently labeledby the affinity reagent 4-(N-maleimido)phenyl[3H]trimethyl-ammonium; only serine was found as the NHrterminal aminoacid of this protein; and (iil) a high molecular weight aggregatenamed 43,000 protein which was resolved in denaturing gelsalmost exclusively as the 43,000-dalton band. In the absence ofdetergents, the 43,000 protein binds compounds known to in-teract with the acetylcholine ionophore: a fluorescent localanesthetic quinacrine and histrionicotoxin (apparent dissocia-tion constant, 7 i 1 x 10-7 M). The regulation of quinacrinefluorescence by carbamylcholine, observed in the intact mem-brane, no longer occurs with the isolated 43,000 component.

The elementary functional unit that accounts for the regulationby acetylcholine (ACh) of cation translocation through an ex-citable membrane comprises, a priori, two main categories ofsites: (i) the ACh receptor site, which binds cholinergic agonists,antagonists (1), and snake venom a-toxins (2); and (ii) a siteinvolved in the selective permeation of small cations. It wasfurther postulated that these two sites were topographicallydistinct, although coupled by "allosteric" interactions and mostlikely carried by different polypeptide entities referred to asthe ACh-receptor protein sensu stricto, and the ACh ionophore(3) [or ion conductance modulator (4)]. The ACh-receptorprotein has been isolated and purified from fish electric organand skeletal muscle in several laboratories (see ref. 5) on the basisof its ability to bind the snake. a-toxins.

Local anesthetics such as prilocaine, tetracaine, and Quotaneaffect in vwo (6) and in vitro (7) the permeability response tobath-applied ACh in a manner rather different from that of"competitive" antagonists like curare or Flaxedil. These localanesthetics block the response by decreasing the amplitudewithout changing the apparent dissociation constant for theagonists. When individual current pulses corresponding tosingle ionophore openings are recorded in the presence of oneof these anesthetics, the square contour of the pulse becomeschopped by multiple fast blocking events that are interpretedas representing the repetitive and reversible binding of the localanesthetic directly to the ion gate (E. Neher, personal com-

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked"advertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

munication). Local anesthetics may therefore be consideredas potential markers of the ACh ionophore (8).

Binding and spectroscopic studies--carried out in vitro withmembrane fragments from the electric organ of Torpedomarmorata highly enriched in ACh receptor (9) and containinga functional ACh ionophore (10, 11) have led to the demon-stration that in these membranes there are specific sites for localanesthetics which are distinct from-the ACh receptor site (12)but strongly coupled to it (13-15) through typical "allosteric"(16) interactions.

Histrionicotoxin (HTX) is a potent toxic compound isolatedfrom the skin of a Columbian frog (17). It behaves in vivo (4,18, 19) and in vitro on ACh-receptor-rich membranes (20) ina manner similar, if not identical, to that of a local anesthetic.This analogy is further supported by the recent observation thatlocal anesthetics displace a radioactive derivative of HTX boundto membrane fragments from T. ocellata (21). Most likely, HTXand local anesthetics label the same class of membrane sites andit was suggested, from experiments done in the presence ofdetergents, that these sites belong to a protein unit distinct fromthe ACh receptor (21).

In this communication, we report on experiments performedwith highly purified receptor-rich membranes isolated fromthe electric organ of T. marmorata (22, 23). Neutral detergentextraction of these membranes yields two distinct proteincomponents which are separated and purified by conventionalmethods: one is labeled by Naja nigricollis a-[3H]toxin (24) andtherefore is the ACh-receptor protein sensu stricto; the other,"the 43,000 protein" binds the fluorescent local anestheticquinacrine and HTX in the absence of detergents.

MATERIALS AND METHODS

Purification of ACh-Receptor-Rich Membranes, ACh-Receptor Protein, and 43,000 Protein. ACh-receptor-richmembranes were purified as described (22, 23) from fresh T.marmorata electric organ of small animals (20-25 cm in di-ameter). Only the fractions having a specific activity higherthan 4 ,mol of a-[3H]toxin binding sites per g of protein wereused. The membranes were dissolved in 2.5% Triton X-100/2.5% Berol 043 in buffer I (100mM NaCl/10mM Tris-HCI, pH7.4/15mM 2-mercaptoethanol/0.1 mM phenylmethylsulfonylfluoride/0.02% NaN3), and the ACh-receptor protein waspurified as described (22, 23). The purified fraction was freedfrom detergent by centrifugation on a 6-ml 12% (wt/wt) sucrosecushion in buffer II [100 mM NaCl/10 mM Na phosphate

Abbreviations: ACh, acetylcholine; HTX, histrionicotoxin; MPTA,4-(N-maleimido)phenyltrimethylammonium; IAcOH, iodoacetic acid;NaDodSO4, sodium dodecyl sulfate.

510

Proc. Natl. Acad. Sci. USA 75 (1978) 511

buffer, pH 7.4, in solution I (0.1 mM phenylmethylsulfonylfluoride/0.02% NaN3)] for 6 hr at 60,000 rpm in a Beckmann75 Ti rotor. The pellet was resuspended and dialyzed againstbuffer II.To purify the 43,000 protein, 4 ml of dissolved membranes

(10-20 mg of protein) was layered on the following discontin-uous gradient: 3 ml of 43% (wt/wt) sucrose in solution I, 3 mlof 5% (wt/wt) sucrose, and 0.05% Berol 043 in buffer I. After4 hr of centrifugation at 60,000 rpm in a Beckmann 75 Ti rotor,fractions were collected. The fractions corresponding to theinterface between the 5 and 43% sucrose layers contained theACh-receptor protein. The pellet was resuspended, homoge-nized with an all-glass homogenizer, and dialyzed against so-lution I; during dialysis, a white precipitate, referred to as the43,000 protein, was formed; the suspension was homogenizedagain before use.

Labeling. The ACh-receptor-rich membrane fragmentswere partially labeled with 4-(N-maleimido)phenyl[3H]tri-methylammonium ([3H]MPTA) (less than 10% of the a-[3H]-toxin binding sites) by the procedure of Karlin and Cowburn.(25).

Iodo[14C]acetic acid ([14C]IAcOH) labeling of the [3H]-MPTA-labeled membranes was done as follows: (i) 20 mg ofmembrane protein was incubated with 5 mM dithiothreitol inbuffer III (100 mM NaCl/10 mM Tris-HC1, pH 8/1 mMEDTA) for 30 min at room temperature; (ii) 20 mM [14C]-IAcOH (1 nuCi/mmol) was added for 3 hr at room temperature;and (iii) the excess unreacted reagent was separated from themembranes by centrifugation for 30 min at 100,000 X g.

Analytical Methods. Electrophoresis in the presence of 0.1%sodium dodecyl sulfate (NaDodSO4) was carried out in 10%polyacrylamide gels by the procedure of Laemmli (26) asmodified by Anderson and Gesteland (27). [3H]MPTA and[14C]IAcOH were measured by drying the gel on a Whatman3 MM paper, cutting it into 2- to 4-mm pieces that were incu-bated for 24 hr in a 10% tissue solubilizer containing scintillationmedium, and then assaying for 3H and 14C in an IntertechniqueSL 30 liquid scintillation counter. Amino acid analysis of theACh-receptor and the 43,000 proteins was done on duplicatesamples. The proteins were hydrolyzed in vacuo for 24 hr in6 M HCI at 1100. The amino acid content was determined ona Joel JLC-5AH amino acid analyzer with the two-columnmethod described by Spackman et al. (28). The NH2-terminalamino acid was determined by the NaDodSO4/dansyl chlorideprocedure of Pett et al. (29).

Fluorescence Measurements. All fluorescence measure-ments were carried out at 200 with a FICA spectrofluorimeter.Quartz fluorescence cells (Hellma) with a 10 X 10 mm crosssection were filled with 2.0 ml of sample containing approxi-mately 0.4 mg of membrane proteins or 0.2 mg of the purifiedACh-receptor or 43,000 protein in buffer II supplemented with1 mM dithiothreitol and 5 ,uM quinacrine. All measurementswere done under conditions of energy transfer from proteins:wavelength of excitation, 290 nm; recording, 517 nm. Duringrecording the suspension was shaken every 5 min to avoidsedimentation and to accelerate the stabilization of the fluo-rescence signal.

Histrionicotoxin. HTX was purified by G. Kato (20) fromskin extracts of Dendrobates histrionicus by the method of Dalyet al. (17).

RESULTSPolypeptide Composition of ACh-Receptor-Rich Mem-

brane Fragments Isolated from T. marmorata ElectricOrgan. Recent improvements (22, 23) of the original method

>. B

Ia

.0

LO TD.

.0

.0

< ~

7-

064x5- ~~~~~~~~X

I3 '| | =~~~~~3E

3 2

'2 I

0 10 20 30 40 50 60 70Distance, mm

FIG. 1. NaDodSO4 polyacrylamide gel electrophoresis of highlypurified ACh-receptor-rich membranes (A), purified ACh-receptor(B), and 43,000 proteins (C), shown as densitometric scans-of the gelsafter staining with Coomassie brilliant blue. (D) Distribution of theradioactivity along the gel obtained from membranes labeled with[3H]MPTA and [14C]IAcOH. Preincubation of the membrane frag-ments with a-toxin completely blocked [3H]MPTA labeling. T.D.,tracking dye.

of Cohen et al. (9) have led to the separation of membranefragments in significantly higher amounts (more than 150mgof protein per kg of fresh electric organ) and with a specificactivity higher than 4 ,umol of a-[3H]toxin sites per mg of pro-tein, which is about half that reported with the detergent-ex-tracted purified receptor protein (5). Fig. 1A shows the proteinpattern obtained from a membrane fraction with a particularlyhigh specific activity after NaDodSO4/polyacrylamide gelelectrophoresis and Coomassie blue staining. Two main proteinbands with apparent molecular weights (determined withwater-soluble globular proteins as standards) of 40,000 ± 1000and 43,000 + 1000 were observed. Components of highermolecular weight (50,000, 66,000, and 100,000), often reportedin preparation of receptor-rich membrane fragments (30,31),were present but only in trace quantities, The relatively smallamounts of these bands most likely does not result from pro-teolysis because the fractionation was carried out in the presence

Neurobiology: Sobel et al.

I S

Proc. Natl. Acad. Sci. USA 75 (1978)

of a protease inhibitor and proteins of identical apparent mo-lecular weight were present in other membrane fractions withlow specific activity. Densitometric scanning of the stained gelsindicated approximately equivalent amounts of the 40,000 and43,000 peaks, within +50%.The affinity labeling reagent [3H]MPTA binds covalently

and with a high selectivity to the ACh-receptor site aftertreatment with the disulfide bond reducing agent, dithiothreitol(25, 31). In agreement with previous studies (25, 31), [3H]MPTAonly labeled the 40,000 component (Fig. 1D) which, therefore,belongs to the ACh-receptor protein. The 43,000 band was notlabeled by [3H]MPTA but covalently bound [14C]IAcOH whenthe membrane suspension was incubated with this reagent afterreduction (Fig. 1D); under these conditions [14C]IAcOH actsas a marker of the 43,000 component.

Separation and Purification of the ACh-Receptor and43,000 Proteins. Treatment of high specific activity membranefractions with a mixture of nondenaturing detergents (2.5%Triton X-100/2.5% Berol 043) in the presence of 15 mM 2-mercaptoethanol clarified the suspension and released intosolution the 9S form of the ACh-receptor protein, in a state thatstill bound the a-[3H]toxin. High-speed centrifugation of themembrane extract on a discontinuous sucrose gradient (5-43%)in the presence of 0.05% Berol 043 yielded two major proteinfractions: the a-[3H]toxin-labeled ACh-receptor protein at theinterface between the 5 and 43% sucrose layers, and a pellet.The receptor protein was further purified (see Materials andMethods) and the pellet was resuspended, centrifuged againand dialyzed. Under these conditions, 20 mg of membraneproteins yields approximately 7 mg of purified ACh-receptorprotein and 5 mg of purified 43,000 protein. Fig. 1 B and Cshows the densitometric scans of the purified receptor proteinand of the resuspended pellet after NaDodSO4/polyacrylamidegel electrophoresis and Coomassie blue staining. The first scanwas characterized by one major band of apparent molecularweight 40,000, labeled by [3H]MPTA. Bands of higher molec-ular weights were still present but in small amounts. The pelletalso gave a single band, of apparent molecular weight43,000.

Table 1 shows the average amino acid compositions of thepurified ACh-receptor and 43,000 proteins. The values foundfor the purified receptor superimpose well with those reportedpreviously after purification by affinity chromatography fromthe same (32) or different Torpedo species (see ref. 5). The43,000 protein differs in composition from the ACh-receptorprotein for at least three amino acids: glycine, alanine, andvaline (possibly also for isoleucine and phenylalanine). Otherdifferences between the two proteins include the following: the43,000 protein makes high molecular weight aggregates underconditions where the 9S receptor protein remains in solution,and it does not bind to concanavalin A covalently bound toSepharose beads, under conditions where the receptor proteinattaches to them (33). NH2-terminus determination with thepurified receptor protein gave only one amino acid, serine.

Fluorescence Studies with Quinacrine and HTX. Previousspectroscopic studies with the fluorescent local anestheticquinacrine have shown that, in vitro, this compound binds toa set of saturable sites present on ACh-receptor-rich membranesas well as to nonspecific sites (14). The signal given by these sitesis enhanced by cholinergic agonists such as carbamylcholineand is abolished by the nonfluorescent local anesthetics Quotaneand prilocaine (14). Binding studies (20) done in vitro withreceptor-rich membranes show that HTX enhances [3H]AChbinding in a manner similar to that of a potent local anesthetic.

Table 1. Amino acid composition of detergent-purified ACh-receptor protein and 43,000 protein from T. marmorata

mol/100 mol of amino acidAmino acid ACh-receptor protein 43,000 protein

Lys 5.9 6.1His 2.4 2.3Arg 3.9 4.6Asp 11.2 9.2Thr 6.5 5.4Ser 7.2 7.8Glx 10.5 12.6Pro 5.2 6.7Gly 6.1 10.2Ala 5.9 8.2Val 7.2 5.3Met 2.6 2.5Ile 7.3 4.2Leu 10.1 9.5Tyr 3.7 3.0Phe 4.2 3.2Trp* + +Cys NDt NDt

* The presence of tryptophan was determined by spectral analysis.t Not determined.

or interfere with those of quinacrine, it was used as a competingligand to ascertain the origin of the fluorescence signal givenby quinacrine.HTX caused a significant reduction (15%) of the total fluo-

rescence signal given by ACh-receptor-rich membranes equi-librated with quinacrine, under conditions of energy transferfrom proteins (Fig. 2A). The decrease of fluorescence intensityin the presence of HTX followed a slow time course (t1/2 = 15min) and tended to a plateau. From the amplitude of the signalrecorded as a function of HTX added to the suspension, anapparent KD of 0.7 i 0.1 juM was found. Preequilibration with10 mM prilocaine or 0.1 mM Quotane decreased the amplitudeof the HTX-sensitive signal by 89 or 81%, respectively. Thedetergent Triton X-100, which has been shown (34) to act onthe electroplaque system like a local anesthetic both in vivo andin vitro, also decreased the fluorescence signal sensitive to HTX(17% at 20 ,uM and 77% at 200 ,uM). Therefore, the quinacrinesignal sensitive to HTX is related to the local anesthetic bindingsite.

In agreement with previous results (14), carbamylcholinecaused a marked increase of fluorescence intensity of thequinacrine-labeled membranes, with an apparent KD of 2 ±1 ,uM. HTX added in the presence of a saturating level of car-bamylcholine caused a large decrease of fluorescence intensity.Under these conditions, the plateau reached at high concen-trations of HTX decreased slightly steeper than in the absenceof carbamylcholine, but the apparent KD for HTX still was 0.7+ 0.1 ,uM. Preincubation of the membrane fragments witha-toxin blocked the fluorescence response to carbamylcholine,which is therefore triggered by carbamylcholine binding to theACh-receptor site. Interestingly, a-toxin also blocked theHTX-sensitive signal observed in the presence of carbamyl-choline and significantly decreased that observed in the absenceof agonist.When the same experiment was carried out with the purified

receptor protein, carbamylcholine still caused a slight a-toxin-sensitive increase of fluorescence intensity (14) but HTXhad no significant effect (it caused a small linear decrease) onthis response (Fig. 2B). The intensity of the fluorescence signal

512 Neurobiology: Sobel et al.

"Because BTX has spectroscopic properties that do not overlap

Proc. Natl. Acad. Sci. USA 75 (1978) 513

aeoD l,.,I ...I.1 I, , I, ...

LL BLL

105a-toxin

10095-

100 -Ap 11A ~~~~~~a-toxin

90

85

,, I .I0 25 50

Carb, pMM I.-.aII..0 5 10 5

HTX,,4MFIG. 2. Fluorescence studies with quinacrine. F/Fo is the ratio

of the fluorescence intensity measured in the presence of effector tothe initial fluorescence intensity (Fo) recorded after equilibration ofthe membrane fragments with quinacrine. HTX was added before(A) or after (0) addition of carbamylcholine or after 1 hr of prein-cubation with 5 ,uM a-toxin (0) and subsequent addition of car-bamylcholine (0). *, Detergent-free purified ACh receptor in theabsence of carbamylcholine and a-toxin. (A) ACh receptor-richmembranes. (B) Purified ACh-receptor protein. (C) Purified 43,000protein.

recorded with the quinacrine-labeled 43,000 protein (Fig. 2C),on the contrary, decreased markedly in the presence of HTX,by approximately 8% (at a protein concentration half that ofthe suspension of membrane fragments in Fig. 2A). The ap-parent KD for HTX was the same as with the native membranesbut neither a-toxin nor carbamylcholine influenced this re-sponse (carbamylcholine caused only a slight linear decreaseof fluorescence under resting conditions).

In the course of these experiments, it was noticed that theamplitude of the fluoresence response to HTX given by the43,000 protein was maximal in the absence of divalent cations,in a range of quinacrine concentration of 5-10,uM and wasenhanced by 1 mM dithiothreitol. The response to HTX wasnot observed in the presence of low concentrations of TritonX-100. Finally, the amplitude of the HTX-sensitive signalvaried from one preparation to another and disappeared aftera few days of storage at 40.To ascertain that the absence of HTX-sensitive fluorescence

signal with the purified receptor was not due to contaminationby detergent, the following control experiment was done. Thepurified receptor protein was precipitated by centrifugationthrough a 16% sucrose cushion in the absence of detergent,dialyzed, and resuspended by the same procedure as for the

43,000 protein; again, no effect of HTX on the fluorescencesignal was found (Fig. 2B).

DISCUSSIONAfter extraction by nondenaturing detergents and subsequentfractionation, highly purified ACh-receptor-rich membranesmost likely deriving from subsynaptic areas of Torpedo elec-troplaque plasma membrane yield two major protein compo-nents. When analyzed by NaDodSO4/gel electrophoresis, eachone yields only one major protein band of apparent molecularweight 40,000 and 43,000, respectively.The protein component that gives the 40,000-dalton band

is without ambiguity the ACh-receptor protein because it bindssnake a-toxin and cholinergic ligands and reacts with [3H]-MPTA. It only gives one NH2-terminal amino acid, andtherefore it consists of a single type of polypeptide chain. The40,000-dalton chain would thus be the repeating unit of theACh-receptor oligomer.The nature and the role of the 43,000 protein are more hy-

pothetical. Upon extraction of the membrane fragments bynondenaturing detergents, it forms large, less-soluble aggregatesthan does the ACh-receptor protein, has a different averageamino acid composition, and does not react with concanavalinA.

Fluorescence experiments done under conditions of energytransfer from proteins show that quinacrine and HTX bind toACh-receptor-rich membranes. A fluorescence signal is re-corded that is sensitive to HTX, to local anesthetics, and to lowconcentrations of detergents and most likely results from theinteraction of quinacrine, HTX, and these other ligands withsaturable sites referred to as local anesthetic binding sites. Theenhancement of fluorescence intensity caused by carbamyl-choline is consistent with previous results that demonstrate al-losteric interactions between the ACh-receptor site and the localanesthetic binding site (13-15). The large decrease of the flu-orescence signal caused by HTX in the presence of carbamyl-choline is consistent with the assumption that carbamylcholinestabilizes a high-affinity state for local anesthetics (14, 15, 35).The disappearance of the HTX-sensitive signal in the presenceof a-toxin would then result from a stabilization of the allostericequilibrium in favor of a resting state with a low affinity forlocal anesthetics. The existence of an HTX-sensitive signal inthe absence of carbamylcholine and its blocking by a-toxinwould then result from a stabilization of the high-affinity statefor local anesthetics by quinacrine itself.With the solubilized purified ACh-receptor protein, no ev-

idence has been found for an interaction of quinacrine withsaturable sites sensitive to HTX. However, in the presence ofquinacrine the 43,000 protein gives a fluorescence signal thatis sensitive to HTX and possesses several characteristic featuresof the signal found with the native membranes. The presentdata support the conclusion that the ACh-receptor site and thesite for local anesthetics and HTX are carried by differentprotein entities. In the native membrane these distinct proteinunits would be tightly associated in a manner similar to thatfound with regulatory enzymes (16). A conclusive demonstra-tion of this model should come from the functional reassociationof the purified components. Such reconstitution experimentsextended to a lipid membrane environment might, in addition,produce essential information on the function of the local an-esthetic binding site and on its role in the selective translocationof ions.

We thank Dr. G. Kato for a gift of pure histrionicotoxin, Dr. P. Bo-quet, A. Menez, J. L. Morgat, and P. Fromageot for supplying the la-

Neurobiology: Sobel et al.

Proc. Natl. Acad. Sci. USA 75 (1978)

beled a-toxin from N. nigricollis, Prof. G. Cohen for the amino acidanalyses of the purified proteins, and J. Davoust and Sylvie Schorgenfor technical assistance. This research was supported by grants fromthe Muscular Dystrophy Association of America, the College de France,the Delegation Generale a la Recherche Scientifique et Technique,the Centre National de la Recherche Scientifique, the Institut Nationalde la Sante et de la Recherche M6dicale, and the Commissariat al'Energie Atomique. J.H. was supported by a postdoctoral fellowshipfrom the Muscular Dystrophy Association of America.

1. Nachmansohn, D. & Neumann, E. (1975) in Chemical andMolecular Basis of Nerve Activity (Revised) (Academic Press,New York).

2. Lee, C. Y. (1970) Clin Toxicol. 3,457-472.3. Changeux, J. P., Podleski, T. R. & Meunier, J. C. (1969) J. Gen.

Physiol. 54, 225S-244S.4. Albuquerque, E. X.,- Barnard, E. A., Chiu, T. H., Lapa, A. J.,

Dolly, J. O., Jansson, S. E., Daly, J. & Witkop, B. (1973) Proc. Natl.Acad. Sci. USA 70,949-953.

5. Changeux, J. P. (1975) Handb. Psychopharmacol. 6,235-301.6. Podleski, T. R. & Bartels, E. (1963) Biochim. Biophys. Acta 75,

387-396.7. Kasai, M. & Changeux, J. P. (1971) J. Memb. Biol. 6, 1-23.8. Adams, P. R. (1977) J. Physiol. (London), 268,291-318.9. Cohen, J. B., Weber, M., Huchet, M. & Changeux, J. P. (1972)

FEBS Lett. 26, 43-47.10. Hazelbauer, G. L. & Changeux, J. P. (1974) Proc. Natl. Acad. Sci.

USA 71, 1497-1483.11. Popot, J. L., Sugiyama, H. & Changeux, J. P. (1976) J. Mol. Biol.

106,469-483.12. Weber, M. & Changeux, J. P. (1974) Mol. Pharmacol. 10, 35-

40.13. Cohen, J. B., Weber, M. & Changeux, J. P. (1974) Mol. Phar-

macol. 10, 904-932.14. Gruinhagen, H. & Changeux, J. P. (1976) J. Mol. Biol. 106,

497-535.-15. Cohen, J. B. (1976) Symposium on Molecular Specialization and

Symmetry on Membrane Function at Harvard Medical School,Boston, Mass. June 14-15.

16. Monod, J., Wyman, J. & Changeux, J. P. (1965) J. Mol. Biol. 12,88-118.

17. Daly, J. W., Karle, J., Myers, C. W., Tokuyama, T., Waters, J. A.

& Witkop, B. (1971) Proc. Natl. Acad. Sci. USA 68, 1870-1875.

18. Albuquerque, E. X., Kuba, K. & Daly, J. (1974) J. Pharmacol.Exp. Ther. 189, 513-524.

19. Dolly, J. O., Albuquerque, E. X., Sarvey, J. M., Mallick, B. &Barnard, E. A. (1977) Mol. Pharmacol. 13, 1-14.

20. Kato, G. & Changeux, J. P. (1976) Mol. Pharmacol. 12, 92-100.

21. Eldefrawi, A. T., Eldefrawi, M. E., Albuquerque, E. X., Oliveira,A. C., Mansour, N., Adler, M., Daly, J. W., Brown, G. B., Bur-germeister, W. B. & Witkop, B. (1977) Proc. Natl. Acad. Sci. USA74,2172-2176.

22. Sobel, A. & Changeux, J. P. (1977) Biochem. Soc. Trans. 5,511-514.

23. Sobel, A., Weber, M. & Changeux, J. P. (1977) Eur. J. Biochem.80,215-224.

24. Menez, A., Morgat, J. L., Fromageot, P., Ronseray, A. M., Boquet,P. & Changeux, J. P. (1971) FEBS Lett. 17,333-335.

25. Karlin, A. & Cowburn, D. (1973) Proc. Natl. Acad. Sci. USA 70,3636-3640.

26. Laemmli, U. K. (1970) Nature 227,680-685.27. Anderson, C. W. & Gesteland, R. F. (1972) J. Virol. -9, 758-

765.28. Spackman, D. H., Stein, W. M. & Moore, S. (1958) Anal. Chem.

30, 1190-1206.29. Pett, D. M., Vanaman, T. C. & Joklit, W. K. (1973) Virology 52,

174-186.30. Raftery, M. A., Vandlen, R. L., Reed, K. L. & Lee, T. (1976) "The

synapse," Cold Spring Harbor Symp. Quant. Biol. 40, 193-202.

31. Karlin, A., Weill, C., McNamee, M. & Valderrama, R. (1976)"The synapse," Cold Spring Harbor Symp. Quant. Biol. 40,203-210.

32. Eldefrawi, M. E. & Eldefrawi, A. T. (1973) Arch. Biochem.Biophys. 159, 362-373.

33. Meunier, J. C., Sealock, R., Olsen, R. & Changeux, J. P. (1974)Eur. J. Biochem. 45,371-394.

34. Brisson, A., Devaux, P. & Changeux, J. P. (1975) C. R. Hebd.Seances Acad. Sci. Ser. D 280, 2153-2156.

35. Changeux, J. P., Benedetti, L., Bourgeois, J. P., Brisson, A., Car-taud, J., Devaux, P., Grunhagen, H., Moreau, M., Popot, J. L.,Sobel, A. & Weber, M. (1976) "The synapse," Cold Spring HarborSymp. Quant. Biol. 40,203-210.

514 Neurobiology: Sobel et al.