Proc. Natl. Acad. Sci. USA Vol. 76, No. 2, pp. 690-694, February 1979

Biochemistry

Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides (acetylcholine receptor/ion channel/polypeptide composition)

RICHARD R. NEUBIG, ELIZABETH K. KRODEL, NORMAN D. BOYD, AND JONATHAN B. COHEN* Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Communicated by Arthur B. Pardee, November 16, 1978

ABSTRACT After alkaline extraction, purified subsynaptic fragments isolated from Torpedo electric tissue exhibit on sodium dodecyl sulfate/polyacrylamide gel electrophoresis predominant peptides apparent Mr 41,000, 50,000, and 65,000 (i.e., the peptides characteristic of the nicotinic receptor purified and isolated in detergent solutions). The peptide of Mr 43,000 that is also found in the isolated postsynaptic membranes is recovered in the supernatant after alkaline extraction. The alkaline-extracted membranes were functionally 'intact, as demonstrated by the following criteria. The kinetics of binding of 13H]acetylcholine in the presence and absence of dimethisoquin were quantitatively unaltered. In the presence of 30 ;&M carbamoylcholine to'occupy acetylcholine binding sites, "14Cmeproadifen [2e(diethylmethylaminoethyl.,2,diphenylvalerate iodide] was bound with a dissociation constant; KD, of 0.3 ± 0.1 pM to 0.3 ± 0.1 site per 13H]a-toxin site. This binding was displaced by perhydrohistrionicotoxin. The carbamoylcholinestimulated efflux of 22Na+ from the Torpedo vesicles were preserved after alkaline extraction. It is concluded that not only the acetylcholine binding site, but also the local anesthetic binding site, must be associated with the peptides of the cholinergic receptor itself and not that of Mr 43,000. Those peptides remaining after alkaline extraction are also sufficient for permeability control.

Permeability control by nicotinic cholinergic receptors involves the binding of acetylcholine (AcCho) or other cholinergic agonists by the nicotinic cholinergic receptor and the utilization of the energetics of 'ligand binding to change the structure of the plasma membrane in such a manner as to increase its permeability to alkali cations. The AcCho receptor has now been purified to apparent homogeneity from detergent extracts of fish electric tissue and skeletal muscle, and considerable progress has been made in its characterization (for a review, see ref. 1). This protein, characterized by a Mr of about 250,000 under nondenaturing conditions, is made up of peptides of Mr 41,000, 50,000, 60,000, and 65,000. The peptide of Mr 41,000 is associated with the AcCho binding site, since that peptide is labeled by a covalent affinity label directed at that site (2), but no clear function has been associated with the others (3-6). A major question concerning the mechanism of permeability control is whether the AcCho binding protein itself contains the structure of the channel or whether there exists a distinct structure, termed the ionophore (7) or ion conductance modulator (8). Identification of a ligand interacting with the ion channel with high affinity and specificity would provide a method of identifying that structure. One promising class of ligands includes the aromatic amines such as dimethisoquin and proadifen as well as the piperidine alkaloid, histrionicotoxin (8, 9). These drugs act as potent noncompetitive antagonists of the cholinergic response of the isolated Electrophorus electroplax

(10, 11), and they are bound by a specific site in the isolated Torpedo postsynaptic membranes that is distinct from the site of binding of cholinergic agonists and competitive antagonists (12-16). Although it has not yet been possible to ascertain whether the anesthetic binding site is actually a part of the ion channel itself or a distinct regulatory site, electrophysiological studies of the action of related ligands such as procaine and lidocaine yield results consistent with the notion that these ligands may interact directly with the site of ion translocation (17, 18). A comparison of the peptide composition of Torpedo nicotinic postsynaptic membranes with that of the AcCho receptor purified in detergent solutions revealed only one major difference, the presence of a peptide of Mr 43,000 that was not present in the purified AcCho receptor (19). Upon detergent dissolution of the membranes, [3H]histrionicotoxin may be interacting with a site distinct from the AcCho receptor protein itself (13), and spectroscopic evidence indicated that the local anesthetics and histrionicotoxin may interact with the isolated 43,000 Mr protein (19). We report here an analysis of the structure and function of postsynaptic membranes altered by alkaline extraction, a procedure used previously to solubilize peripheral plasma membrane proteins (20, 21). MATERIALS AND METHODS Isolation of Nicotinic Postsynaptic Membranes. AcCho receptor-enriched membranes were prepared from fresh Torpedo nobiliana and T. californica electric organ (1.5-2.5 kg/fish) as described by Sobel et al. (3). The specific activities obtained were: 2-3 ,imol of Naja nigricollis [3H]a-toxin sites per g of protein for T. nobiliana (three fish) and 1-2 gimol of a-toxin sites per g for T. californica (three fish). Removal of Peptides from AcCho Receptor-Rich Membranes by Alkalinization. AcCho receptor-rich membranes in 37% (wt/wt) sucrose/0.02% NaN3 were adjusted to about 1 g of protein per liter with 37% sucrose, then diluted with an equal volume of distilled water. Aliquots of 1 M NaOH were added with rapid mixing until the pH was 11.0; this mixture was incubated for 60 min at 23°C and then centrifuged at 40C for 30 min at 194,000 X g. The supernatant was removed and the pellet was resuspended in Torpedo physiological saline (250 mM NaCI/5 mM KCI/3 mM CaCl2/2 mM MgCl2/5 mM NaPi, pH 7/0.02% NaN3) at 0.3-0.8 g of protein per liter. The pH of these resuspended membranes was 7.0 + 0.2, and no further adjustment of pH was carried out. Polyacrylamide Gel Electrophoresis. Electrophoresis in the presence of sodium dodecyl sulfate (NaDodSO4) was performed Abbreviations: AcCho, acetylcholine; (yBgTx, av-bungarotoxin; CarCho, carbamoylcholine; NaDodSO4, sodium dodecyl sulfate; Torpedo physiological saline, 250 mM NaCI/5 mM KCI/3 mM CaCl2/2 mM MgCl2/5 mM NaP,, pH 7/0.02% NaN3. * To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement " in accordance with 18 U. S. C. §1734 solely to indicate this fact. 690

Biochemistry: Neubig et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

by the method of Laemmli (22) with 8% acrylamide (Sigma) in the separating gel. Gels were stained with Coomassle blue or periodic acid-Schiff reagent according to Steck and Yu (20). Binding of [3HjAcCho and [14C]Meproadifen to Torpedo Membranes. The equilibrium binding of [3H]AcCho (250 Ci/mol, 9250 GBq/mol; Amersham) and a slow (>10 see) component of the ligand-association kinetics were determined by an ultrafiltration assay (14, 23, 24). A detailed report of the experimental techniques and the results obtained will be presented elsewhere, but relevant experimental protocol is summarized. AcCho binding studies were carried out in the pres*ence of 0.1 mM diisopropylphosphofluoridate. To measure the kinetics of association of [3H]AcCho with the membrane-bound cholinergic receptor, we started the binding reaction by mixing rapidly in a "T" connector the contents of two syringes, one containing the membrane suspension and the other containing the appropriate concentration of [3H]AcCho in Torpedo physiological saline supplemented with 0.1 mM diisopropylphosphofluoridate. One-milliliter samples were filtered every 10 sec on Whatman glass fiber (GF/F) filters. Equilibrium binding was determined after incubation of the mixture for 15-20 min. Specific binding of [3H]AcCho to the membranebound AcCho receptor at each time (B') was calculated from the difference between the total radioactivity retained on the filter and that radioactivity retained on a filter when a suspension was filtered containing the same free [3H]AcCho in equilibrium with Torpedo membranes pretreated with abungarotoxin (aBgTx) to block all AcCho binding sites. The equilibrium binding of [14C]meproadifen to Torpedo membranes in physiological saline in the presence of 30,M carbamoylcholine (CarCho) was measured as described (16) by ultracentrifugation in a Beckman Airfuge at 4VC. Measurement of 22Na+ Efflux. To measure the efflux of 22Na+ (New England Nuclear) from native or alkaline-extracted Torpedo membranes, we used a filtration assay that is a modification of published procedures (25-27). Native or alkaline-extracted membranes (extraction carried out at 40C) were incubated overnight at 4 g of protein per liter with 20 MCi of 22Na+ per ml in a buffered salt solution (100 mM NaCI/5 mM NaPi, pH 7.0) at 4VC. A 0.5-ml sample of this suspension was passed over a Dowex 50-W column to remove readily exchangeable 22Na+ and was then diluted to 12 ml with salt solution (at 40C). CarCho was added to 6 ml of the diluted susTable 1. Comparison of protein, toxin sites, and AcCho binding in native and alkaline-extracted Torpedo postsynaptic membranes

Protein (M native) Toxin sites (% native) [1HjAcCho binding Total sites (% native)

K1)(nM) Hill coefficient 3 (% totalsites)

Membhranes AlkalineNative extracted 100 43 i 6

Supernatant n 37 ± 10 4

100

78 ± 13

3i1

7

100 13+7 1.37 + 0.11 29 ± 6 53+5

84 ± 7 9+3 1.35 ± 0.16 30+ 9

ND ND ND ND ND

4 4 4 4

4 42±4 tl/9(sec) BF and t 1/2 refer to the kinetics of [3H]AcCho binding and are, re-

spectively, the amount of rapid (10 sec) phase of binding (see text). Values are mean ± SD; n is the number of each type of measurement; ND means not

determined.

691

pension (final concentration 0.1 mM), and l-ml aliquots of the membrane suspension with or without CarCho were filtered alternately on Millipore HA filters and washed with 10 ml of salt solution. RESULTS Membrane Extraction. The AcCho receptor-enriched membranes were subjected to alkaline protein extraction at various conditions of pH and time. After exposure of the membranes to pH 11 at low ionic strength for 1 hr, the [3H1a-toxin-binding activity was preserved, but half the membrane proteins were extracted (Table 1). Although this treatment did not significantly alter the [3H]a-toxin binding, two plasma membrane enzymes were inactivated. The total activity of acetylcholinesterase recovered was only 3.3% of the initial activity, while the Na+,K+-ATPase activity was less than 2%. Analysis of the polypeptide composition of the base-extracted membranes by NaDodSO4/polyacrylamide gel electrophoresis and Coomassie blue staining revealed that the 2-fold increase in the specific activity of a-toxin sites in the alkaline-extracted membranes was due to a selective extraction of polypeptides that are not part of the AcCho receptor (Fig. 1). In particular, the peptide of Mr 43,000 was recovered in the supernatant, while the three dominant peptides remaining in the membranes (Mr 41,000,50,000, and 65,000) are those characteristic of the AcCho receptor purified in detergent solution.t The lengthy exposure to pH 11 was necessary to extract quantitatively the 43,000 Mr peptide. After alkaline extraction, all peptides were recovered without significant alteration except that of Mr 92,000. We do not understand the reason for the loss of that peptide which is probably the major subunit of Na+,K+-ATPase. When the gels were stained for carbohydrate by the-use of the periodic acid-Schiff stain (20), the three peptides of the AcCho receptor in the alkaline-extracted membranes were stained, while none of the extracted peptides were. Binding of [3H]AcCho to Alkaline-Extracted Membranes. The equilibrium binding of [3H]AcCho to the alkaline-extracted membranes resuspended in Torpedo physiological saline at pH 7 was unaltered from that of the "native" AcCho receptorenriched membrane (Table 1). To characterize the conformational equilibria of the membrane-bound cholinergic receptor, the kinetics of ligand binding must be analyzed. Results obtained by various techniques (14, 23, 28-30) provide evidence that in the absence of cholinergic ligands the Torpedo receptor exists in two conformations in equilibrium with each other, and the effect of a cholinergic ligand is to shift the conformational equilibria from one that binds AcCho weakly to another that binds AcCho tightly. t The postsynaptic membranes used in these studies are not as highly purified as those isolated from T. marmorata electric tissue (3). Analysis of the peptide compositions of different membrane fractions isolated from a continuous sucrose gradient established that the peptides of Mr 41,000, 43,000, 50,000, and 65,000 were concentrated in fractions containing the highest amount of t3Hla-toxin and 4CImeproadifen (16) sites (38% wt/wt sucrose). Those of Mr 52,000, 55,000, and 92,000 were enriched in adjacent fractions (41, 41, and 34% sucrose, respectively). Their presence probably represents contamination of the isolated postsynaptic membrane, and for this reason we emphasize that the alkaline-extracted membranes no longer contain the 43,000 Mr peptide. The peptide of Mr 55,000 extracted from these membranes is not the y chain reported for purified Torpedo receptor (2, 4-6). In one preparation, an additional peptide of M, 56,000 was observed that remained in the alkalineextracted membranes. On the basis of the integrated scans of the densitometer tracing of Fig. 1, the AcCho receptor peptides constitute 50% of the protein on the alkaline-extracted membranes, a value consistent with the observed specific activity of a-toxin sites (3.3 Amol/g of protein). Clearly it remains important to extract membranes of higher purity (higher specific activity of a-toxin sites).

692

Proc. Nati. Acad. Sci. USA 76 (1979)

Biochemistry: Neubig et al. V:

A.

I

t

',

\

504341 TD i~~i

65

Ij:

I

I/

!,-

.1.

I;

,

'%

I-r,.

R

By

V.

92

s

j

u

r,

.IJi

in i" "

11

2

0?

"-..,

04 08 06 Relative mobility

1

0

FI(;. 1. Selective extraction of proteins from AcCho receptor-rich membranes of T. nobiliana by treatment with pH 11 at low ionic strength. (Lower) Scans of Coomassie blue-stained NaDodSO4/ polyacrylamide gels. I, AcCho receptor-rich membranes from 7'. nobiliana (27 pg of protein, 1.8 Mmol of (V-toxin sites per g). II, Pellet after incubating AcCho receptor-enriched membranes at pH 11 for I hr at room temperature and centrifuging for 30 min at 194,000 X g (14 pg of protein, 3.3 pmol/g). III, Supernatant from alkaline extraction. Pellet and supernatant samples contain the material obtained from 27 pg of I. Numbers indicate apparent Mr in thousands. (Upper) Photograph of gel from which scans in Lower were made. Samples l-IIl numbered as in Lower.

The kinetics of association of [3H]AcCho with the native and alkaline-extracted Torpedo membranes were determined by ultrafiltration (Fig. 2). At each time the amount of [3H]AcCho bound specifically to the receptor (B;) did not differ significantly between native and extracted membranes, and in both cases equilibrium was attained only slowly. The time constant (t 1/2) for the observed slow component of the association process, and the amount of binding associated with that slow process (B'), was determined from a plot of the logarithm of B'. -B against time. The amount of binding occurring rapidly

(B1) is equal to Be. - B8. The particular concentration of [3H]AcCho used in the-experiments in Fig. 2 was chosen, on the

basis of a knowledge of the kinetic and equilibrium parameters, as one at which the extent of the rapid component of binding (B1) and t 12 of the slow component are expected to be quite sensitive to changes in the intrinsic rates and equilibria. For experiments with four different membrane preparations, the value of B1 was unaltered by alkaline extraction, while tI/2 was decreased by only 20% (Table 1). Hence, neither the equilibrium binding nor the more fundamental conformational equilibria of the membrane-bound Torpedo receptor were significantly altered by removal of the 43,000 Mr peptide from the membranes. Effect of Dimethisoquin on 13HJAcCho Binding. We examined the effect of the local anesthetic dimethisoquin on the kinetics of binding of [3H]AcCho (30 nM) to suspensions of base-extracted or native Torpedo membranes (25 nM [3H]atoxin sites). In the absence of local anesthetic, 60% of the [3H]AcCho binding occurred with a half-time of 50 sec, whereas in the presence of 10 ,uM dimethisoquin, the ligand binding was at equilibrium within 10 sec of mixing (Fig. 2). Dimethisoquin had the same effect on the base-extracted as on the native membranes. Equilibrium Binding of ["4C]Meproadifen. Direct measurement of the equilibrium binding of local anesthetics to the alkaline-extracted membranes was determined by the use of ['4C]meproadifen. Because ['4Cjmeproadifen also has the capacity to interact weakly with the AcCho binding site in the Torpedo membranes (16), all binding studies were carried out in the presence of 30 ,uM CarCho, a concentration sufficient to occupy all AcCho binding sites. In Fig. 3 are shown the total binding of [14C]meproadifen to suspensions of native or alkaline-extracted membranes and the nonspecific partitioning of ["4CImeproadifen into those membranes in the presence of 30 1AM meproadifen. Both the total binding and nonspecific partitioning were not significantly altered by alkaline extraction. The specifically bound meproadifen is characterized by a KD of 0.3 + 0.1 M, and and the number of sites is equal to 0.33

60-

m

0.

015 20

/BY IC I/F 3

E

?A0o 0

005

0

9Y£

02

04

06

['4C]meproadifen, jiM FIlG. 3. Equilibrium binding of l'4C]meproadifen to alkalineextracted and native T. nobiliana membranes in the presence of CarCho. Total binding of 1'4C]meproadifen (0, 0) and binding in the presence of 30MM meproadifen (A, *) was determined by ultracentrifugation for a control membrane supsension (0, &; 0.7 MM [:Hla-toxin sites, 0.45 g of protein per liter) and for alkaline-extracted membranes (0, *) resuspended to yield a suspension 0.7 MM in VH]a-toxin sites if the extraction occurred without loss of sites. Each Free

Time,

sec

Ft(c. 2. Effect of dimethisoquin on the kinetics of binding of I3H]AcCho to native and alkaline-extracted T. nobiliana membranes at 230C. Equal volumes of I:3H]AcCho (60 nM) and native (0, 0; 50 nM I:'H](Y-toxin sites) or alkaline-extracted (0, *) membranes were mixed, aliquots were filtered at the indicated times, and specific binding was calculated. Solutions contained either 0 (0, 0) or 10 (0, *) MM dimethisoquin.

data point is the mean of duplicate samples. (Inset) Double-reciprocal plot [1/B (pM-') against 1/F (MM')] of the specific binding of ['4C]meproadifen to alkaline-extracted (-) and native (3) membranes.

Biochemistry: Neubig et al.

c80

Proc. Natl. Acad. Sci. USA 76 (1979)

A

-

0

C, W

* 60

o0

MO

A

20

0r

7

6 5 -log [local anesthetic]

4

FIC. 4. Effect of local anesthetics and perhydrohistrionicotoxin on the equilibrium binding of ['4Clmeproadifen to alkaline-extracted and native T. nobiliana membranes. Membrane suspensions (0.7 PM I3Hla-toxin sites) in Torpedo physiological saline were equilibrated with 30 pM CarCho before addition of [14Clmeproadifen (0.4 MM). Open symbols, binding to native membranes: closed symbols, alkaline-extracted membranes. Specific binding in the presence of dimethisoquin (3, *) and perhydrohistrionicotoxin (A, A) is expressed as a percent of the [14C]meproadifen bound specifically in the absence of other anesthetics. Each point is the mean of duplicate samples.

0.1 per [3H1a-toxin site. The binding of [14Cjmeproadifen to T. nobiliana membranes is the same as that observed to T. marmorata membranes (16). The pharmacological specificity of the ['4Cjmeproadifen binding to the alkaline-extracted AcCho receptor-enriched membranes was the same as that of the native membranes (Fig. 4). The ligands perhydrohistrionicotoxin and proadifen displaced [14C]meproadifen when present at micromolar concentrations, and dimethisoquin displaced [14C]meproadifen only when present at concentrations an order of magnitude higher. The affinity of these ligands for the anesthetic binding site in the T. nobiliana membranes is similar to that reported (16) for the anesthetic binding site in T. marmorata mem-

branes. Permeability Response of Alkaline-Extracted Postsynaptic Membranes. In order to assess the permeability properties of

693

the alkaline-extracted membranes, we measured the efflux of 22Na+ from Torpedo membranes in the presence and absence of CarCho by ultrafiltration (Fig. 5). Qualitatively, the permeability response of the alkaline-extracted vesicles was identical to that of the native vesicles. In the absence of CarCho, the efflux was characterized by a half-time of about 600 sec. Upon addition of 100 ,M CarCho, 70% of the 22Na+ was released before the first time point (20 sec) and the remaining 22Na+ was released only very slowly from the vesicles (half-time t600 sec). Preincubation of either the alkaline-extracted or native vesicles with aBgTx prevented the CarCho-stimulated release of 22Na+. Quantitatively, the difference between the native and alkaline-extracted vesicles resided not in the efflux rates, but in the apparent volume of the vesicles. In the absence of CarCho, the amount of 22Na+ retained in the vesicles was equivalent to 0.3-0.6 ,l/mg of protein (three experiments); for the alkaline-extracted membranes the value was only 15-30% of that (two experiments). DISCUSSION We report here a procedure to selectively extract peptides from AcCho receptor-enriched membranes of T. nobiliana and T. californica with minimal alteration of the membrane structure. It was reported (19) that in highly purified preparations of postsynaptic membranes from T. marmorata, the only peptide of Mr greater than 20,000 other than the peptides characteristic of the purified nicotinic receptor was a peptide of Mr 43,000. Upon alkaline extraction, we observed that the 43,000 Mr peptide is quantitatively solubilized (i.e., greater than 85%) and the peptides of 41,000, 50,000, and 65,000 Mr remained in the extracted membranes (Fig. 1). The fact that the 43,000 Mr peptide was extracted indicates that it should be considered a peripheral rather than an integral membrane protein (31). The 43,000 Mr protein solubilized by base does spontaneously aggregate at neutral pH in the presence of salt, and it-is this property that was used previously (19) to purify the peptide from detergent extracts of the Torpedo postsynaptic membranes. Even though the alkaline extraction procedure inactivated plasma membrane enzymes such as acetylcholinesterase and Na+,K+-ATPase, the functional properties of the membranebound cholinergic receptor remained unaltered. Not only the equilibrium binding, but also the kinetics of association, of [3H]AcCho were the same after alkaline extraction as before

(Fig. 2).

The interactions of local anesthetic noncompetitive antagonists with the Torpedo postsynaptic membranes were unaltered by alkaline extraction. This was demonstrated indirectly by the fact that dimethisoquin still had a profound effect on the

7oG clo 00

+

0 "R

o

g 80 II

A~~V

CD

- 60 IA If+

Z 40

E 20

CL C-

II-.

-//

-

200 '"5 iir Time, sec CarCho-stimulated 22Na+ efflux from native and alka0

Fic. 5.

I

~j

I~~~~~~~--tr-o n of T* -// l00

line-extracted T. nobiliana membrane vesicles. Native (open symbols) or alkaline-extracted (closed symbols) membranes were equilibrated with 22Na+ and prepared for filtration. Ordinate, percent 22Na+ cpm

retained, expressed relative to the first measurement in the absence

ranged from 2000 to 3000 cpm for native membranes (three experiments) and 500 to 550 cpm for alkalineextracted membranes (two experiments). Abscissa, time after addition of CarCho to half of the diluted membrane suspension. Membranes: without ligands (A, A); with aBgTx (v, v); with 100 MM CarCho (3, U); with (yBgTx and 100 MM CarCho (0, 0). of CarCho. That value

kinetics of binding of [3H]AcCho (Fig. 2). Direct binding studies established that in the alkaline-extracted and native membranes, [14C]meproadifen was bound with a KD of 0.3 gM to one site per four a-neurotoxin sites. That binding was displaced by the potent noncompetitive antagonists perhydrohistrionicotoxin, proadifen, and dimethisoquin. The binding site identified in the Torpedo membranes by the use of [14C]meproadifen has the same apparent affinity for perhydrohistrionicotoxin as that characterized by the binding of [3H]histrionicotoxin (12, 13) and is undoubtedly the same site. Because the equilibrium binding of [14C]meproadifen reported here was determined after preincubation of the membrane with high concentrations of CarCho, the observed high-affinity binding must reflect a structural feature of the Torpedo postsynaptic membrane in equilibrium with cholinergic agonists; i.e., the "desensitized" membrane (32). Further studies are necessary to determine the relationship between the anesthetic binding site and the site of ion translocation.

694

Biochemistry: Neubig et al.

The comparison of the ligand-binding properties of the alkaline-extracted membranes with those of the native membranes has been emphasized because they can be quantified with great precision. The characterization of the permeability response of the membranes is more qualitative. It is not possible to characterize the ion transport per a-toxin site. Despite this limitation, we have presented evidence that the agonist-stimulated permeability response of the Torpedo membranes is preserved after alkaline extraction (Fig. 5). The major conclusion of this report is that the site of binding of local anesthetics and that of AcCho remain in the structure of the alkaline-extracted postsynaptic membrane. It is most likely that the anesthetic binding site resides within the peptides of the AcCho receptor. Since greater than 80% of the anesthetic binding is retained in the alkaline-extracted membranes after removal of at least 85% of the 43,000 Mr peptide, that peptide cannot contain the binding site. Further studies are necessary to identify the particular peptide containing the binding site and also to rule out the possibility that there are important low molecular weight peptides in the native and alkaline-extracted membranes distinct from the AcCho receptor peptides. We thank Mr. A. Ku for expert technical assistance. We also thank Drs. P. Boquet, A. Menez, J. L. Morgat, and P. Fromageot for a gift of 13H la-toxin of N. nigrwollis, and Dr. Y. Kishi for a gift of perhydrohistrionicotoxin. This research was supported by U.S. Public Health Service Grant NS-12408 and by a grant from the Sloan Foundation. R.R.N. is supported by U.S. Public Health Service Predoctoral Training Grant GM-02220; E.K.K. and N.D.B. are supported by fellowships from the Muscular Dystrophy Association of America; and J.B.C. is a recipient of U.S. Public Health Service Research Scientist Career Development Award NS-00155. 1. Heidmann, T. & Changeux, J. P. (1978) Annu. Rev. Biochem.

47,317-357.

2. Weill, C. L., McNamee, M. G. & Karlin, A. (1974) Biochem. Biophys. Res. Commun. 61,997-1003. 3. Sobel, A., Weber, M. & Changeux, J. P. (1977) Eur. J. Biochem.

80,215-224.

4. Chang, H. W. & Bok, E. (1977) Biochemistry 16,4513-4519. 5. Witzemann, V. & Raftery, M. A. (1977) Biochemistry 16,

5862-5868. 6. Hamilton, S. L., McLaughlin, M. & Karlin, A. (1977) Biochem. Biophys. Res. Commun. 79,692-699. 7. Changeux, J. P., Podleski, T. R. & Meunier, J. C. (1969) J. Gen.

Physiol. 54, 225S-244S.

Proc. Natl. Acad. Sci. USA 76 (1979) 8. 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. 9. Daly, J. W., Karle, I., Myers, C. W., Tokuyama, T., Waters, J. A. & Witkop, B. (1971) Proc. Natl. Acad. Sci. USA 68, 18701875. 10. Cohen, J. B., Weber, M. & Changeux, J. P. (1974) Mol. Pharmacol. 10, 904-932. 11. Kato, G. & Changeux, J. P. (1976) Mol. Pharmacol. 12, 92100. 12. Elliott, J. & Raftery, M. A. (1977) Biochem. Biophys. Res. Commun. 77, 1347-1353. 13. Eldefrawi, A. T., Eldefrawi, M. E., Albuquerque, E. X., Oliveira, A. C., Mansour, N., Adler, M., Daly, J. W., Brown, G. B., Burgermeister, W. & Witkop, B. (1977) Proc. Natl. Acad. Sci. USA

74,2172-2176. 14. Cohen, J. B. (1978) in Molecular Specialization and Symmetry in Membrane Function, eds. Solomon, A. K. & Karnovsky, M. (Harvard Univ. Press, Cambridge, MA), pp. 99-128. 15. Krodel, E. K. & Cohen, J. B. (1978) Fed. Proc. Fed. Am. Soc. Exp.

Biol. 37,578 (abstr.). 16. Krodel, E. K., Beckman, R. A. & Cohen, J. B. (1978) Mol. Pharmacol., in press. 17. Adams, P. R. (1977) J. Physiol. 268, 291-318. 18. Neher, E. & Steinbach, J. H. (1978) J. Physiol. 277, 153-176. 19. Sobel, A., Heidmann, T., Hofler, J. & Changeux, J. P. (1978) Proc. Nati. Acad. Sci. USA 75,510-514. 20. Steck, T. L. & Yu, J. (1973) J. Supramol. Struct. 1, 220-232, 233-248. 21. Shanahan, M. F. & Czech, M. P. (1977) J. Biol. Chem. 252, 6554-6561. 22. Laemmli, U. K. (1970) Nature (London) 227,680-685. 23. Cohen, J. B. & Boyd, N. D. (1977) Biophys. J. 17, 123a. 24. Boyd, N. D. & Cohen, J. B. (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37, 650 (abstr.). 25. Kasai, M. & Changeux, J. P. (1971) J. Membr. Biol. 6, 1-80. 26. Hess, G. P., Andrews, J. P., Struve, G. E. & Coombs, S. E. (1975) Proc. Natl. Acad. Sci. USA 72,4371-4375. 27. Gasko, 0. D., Knowles, A. F., Shertzer, H. G., Suolinna, E. M. & Racker, E. (1976) Anal. Biochem. 72,57-65. 28. Weiland, G., Georgia, B., Lappi, S., Chignell, C. F. & Taylor, P. (1977) J. Biol. Chem. 252,7648-7656. 29. Heidmann, T., Iwatsubo, M. & Changeux, J. P. (1977) C. R. Hebd. Seances Acad. Sci. Ser. D 284, 771-774. 30. Quast, U., Schimerlik, M., Lee, T., Witzemann, V., Blanchard, S. & Raftery, M. A. (1978) Biochemistry 17,2405-2414. 31. Singer, S. J. (1971) in Structure and Function of Biological Membranes, ed. Rothfield, L. J. (Academic, New York), pp. 145-222. 32. Sugiyama, H., Popot, J. L. & Changeux, J. P. (1976) J. Mol. Biol. 106,485-496.