Identification of the Acetylcholine Receptor Subunit in the Lipid Bilayer of Torpedo Electric Organ Excitable Membranes*

Transcription

1 ~ ~~ THE JOURNAI. OF BIOLOGICAL CHEMISTRY VnI No 3, Issue of February 10, pp , 1980 Prrnfedrn 1l.S.A. Identification of the Acetylcholine Receptor Subunit in the Lipid Bilayer of Torpedo Electric Organ Excitable Membranes* Rebeca Tarrab-Hazdai,$ Tuvia Bercovici,g Valentina Goldfarb,$ and Carlos Gitlers (Received for publication, July 20, 1979) From the $ Department of Chemical Immunology and the 9 Department of Membrane Research, The Weizmann Institute of Science, Rehouot, Israel The polypeptides of acetylcholine receptor-rich membrane fragments (from the electric organ of Torpedo californica), which are embedded or span the lipid bilayer, have been identified by their photolabeling with the lipid-soluble reagent, 5-[ 251]iodonaphthyl-l-azide. This reagent has been shown previously (Bercovici, T., and Gitler, C. (1978) Biochemistry 17, ) to label only those proteins in contact with the membrane lipids. Of the many polypeptide chains observed after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the acetylcholine receptor-rich membrane fragments, only two components, of M, = 90 X lo3 and 40 X lo3, were found to be strongly labeled. Minor incorporation was also consistently observed in the polypeptide region of 55 to 60 X lo3 daltons. This pattern of incorporation of the label was unaffected by the presence of high levels of a-bungarotoxin, carbamylcholine, and/or of the aqueous nitrene-scavenger glutathione. Purification by affinity chromatography of the ace- chromatography. It is essential for the dissection of the mechanism of channel formation to identify those polypeptides of the AChR-rich membrane vesicles and of the purified AChR that are embedded in or span the lipid bilayer. To accomplish this, advantage tylcholine receptor from membranes prelabeled with 5- has been taken of the findings that 5-[1 IJiodonaphthyl-1- [ 251]iodonaphthyl-l-azide, indicated thathe M, = azide photolabels only those domains of membrane proteins 40,000 subunit was the nearly exclusive site of label incorporation. Trypsinization of this purified acetylcholine receptor indicated that the M, = 40,000 subunit that are embedded in the lipid bilayer. EXPERIMENTAL PROCEDURES is split into a nonlabeled segment of 27,000 and a la- Preparation of AChR-rich Membranes from Torpedo califorbeled lipid-embedded domain of 13,000. Photolabeling nica-membranes were prepared from electric organs of Torpedo of the purified acetylcholine receptor in a Triton X-100- californica quick-frozen in liquid nitrogen (Pacific Bio-marine, Vensoluble form resulted in labeling of the four subunits in ice, CA). The frst step, which included homogenization, sonication, marked contrast to the results obtained when it was labeled in situ. Upon agonist binding to the acetylcholine receptor, and ultracentrifugation through a sucrose gradient, was performed changes in transmembrane ion permeability ensue. Those proteins, herein identified as embedded within the membrane bilayer, are those likely to be involved in the ionophoric functions. Current evidence indicates that agonist binding to acetylcholine receptors (AChR) leads to the transitory formation of transmembrane channels, which result in the diffusion of ions down their concentration gradients (1-5). This phenomenon which underlies neurotransmission has been reproduced in vesicles formed from the membranes of the electric organ of different fishes (6-9). These membranes are a rich source of the acetylcholine receptor which can be purified after solubi- lization with nonionic detergents by affinity chromatography using immobilized Naja naja siamensis neurotoxin. Poly- * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact peptide analyses of the purified AChR indicate the main component to have an apparent molecular weight of 40,000 (8, 10-12). Three other subunits are also generally reported to be present but some variability in their stoichiometry and molecular weight has been reported (13-15). The M, = 40,000 subunit of the AChR was found to bind a-bungarotoxin and to be the main component reacting with affinity labels mim- icking agonist molecules (14, 16, 17). Attempts to reconstitute the agonist-mediated channel formation, using the purified AChR (18, 19) have been less successful than those based on the use of the AChR-rich membrane fragments (20, 21). Thus, it is as yet unclear whether both the agonist binding and the channel-forming functions reside in the AChR that can be purified by affinity according to Cohen et al. (22). The receptor-rich fractions were pooled, diluted 10-fold with phosphate-buffered saline (0.9% NaCl solution) plus M phenylmethylsulfonylfluoride (Pi/NaCI-PMSF), and sedimented at 16,700 X g (4 C. 10 min). The membranes derived from 50 g of original tissue were finally suspended in 5 ml of Pi/NaCI- PMSF. The a-bungarotoxin binding activity of the resulting receptorrich membranes had an average value of 2500 pmol of toxin bound/ mg of protein. Preparation of the Purified AChR-The detailed procedure employed for the purification of the receptor has been described previously (15). Essentially, it involved the solubilization of the membranes in Triton X-100 and the subsequent purification by affinity chromatography on a Sepharose-N. naja siamensis neurotoxin resin. Elution was performed with carbamylcholine. AChR activity was assayed by measuring the amount of radioactive I-a-bungarotoxin which COprecipitated with the receptor in a 35% saturated ammonium sulfate solution (15), and the specific activity was determined as described by Olsen et al. (23). The purified AChR had an average specific activity of 10,OOO pmol of toxin bound/mg of protein. It showed a single 9 S peak on sucrose gradient centrifugation and by analytical -~.. ~ The abbreviations used are: AChR, acetylcholine receptor; INA, 5-[1 I]iodonaphthyl-l-azide; Pi/NaCl-PMSF, phosphate-buffered saline plus lo- M of phenylmethylsulfonylfluoride; albumin, bovine serum albumin; a-butx, a-bungarotoxin; SDS, sodium dodecyl sulfate.

2 Lipid-embedded Domain of the ACh Receptor 1205 ultracentrifugation; it gave one precipitation line with anti-achr serum in both immunodiffusion and immunoelectrophoresis. Labeling Experiment~-5-['~~I]iodonaphthyl-l-azide was prepared according to Bercovici and Gitler (24). The membranes or protein to be labeled were suspended at a concentration of 1 to 2 mg of membrane protein/ml of the desired buffer in a cylindrical glass cuvette. Usually 2 ml of solution was irradiated at a time. The cuvette was placed in a glass holder through which water could be circulated to keep the membrane suspension at 37 C. The membrane suspension was stirred with a magnetic stirrer during the period of irradiation. Light from a Wild microscope UV source, containing a 200 watt mercury arc lamp (Osram HBO), was focused to 1 cm2 in the center of the cuvette. The light source was 16 cm from the cuvette. Corning 7-60 and 0-54 filters were used to remove light below 305 and above 400 nm. The INA in alcohol was added to the membrane suspension (I%, final alcohol concentration) under red fdtered light and the suspension was stirred for 5 min and then exposed to the UV light for the desired time. Noncovalently attached ['251]INA products were removed after irradiation by washing with Pi/NaCl containing albumin (12 mg/ml) by sedimentation (24). The membranes were finally resuspended in Pi/NaCl-PMSF. Electrophoresis-Sodium dodecyl sulfate-gel electrophoresis was carried out according to Laemmli (25), as modified by Wyckoft et al. (26). After the electrophoretic run, the gels were first stained with Coomassie Brilliant Blue to visualize protein banding patterns and dried; then, autoradiography was performed. The autoradiograms were scanned at 560 nm in a Gilford spectrophotometer. In some cases, the dried gels were then cut into 2.0-mm slices and the radioactivity was quantified in a Packard y-counter. Tvptic Digestion of the AChR-rich Membrane-The AChR-rich membranes were incubated with varying amounts of trypsin for different periods of time (1 to 120 min) at 23 C. In some experiments, the reaction was terminated by the addition of soybean trypsin inhibitor (2 mg/mg of trypsin) and the digested membranes were then sedimented at 100,OOO X g (Beckman Airfuge) for 45 min. The pellet was resuspended in SDS sample buffer and analyzed by electrophoresis. In some cases, the trypsin digestion was stopped by direct addition of SDS sample buffer. RESULTS Incorporation of ['25ZJZNA Photolysis Products into the Components of AChR-rich Membrane Fragments-Rapid and efficient incorporation of label into the membrane proteins and lipids was observed. Fig. 1 shows that maximum incorporation (approximately 49%) was attained after 4 min of irradiation. Thereafter, a slight decrease in the level of incorporation was observed. For this reason, a 2-min period of exposure was generally used. The electrophoretic pattern of distribution of the label incorporated into AChR-rich membrane fragments (Fig. 2) consistently showed highest incorporation into polypeptides of apparent M, = 90 X lo3 and 40 X 10". The 90,000-dalton radioactive peptide does not coincide with the main Coomassie-stained band which is of slightly lower molecular weight. The 40,000-dalton radioactive peptide does coincide with an intense Coomassie-stained band. A minor broad radioactive band was consistently observed in the region of M, = 55,000 to 64,000. In gradient gels, after exhaustive washing, a 12,000 to 14,000 distinct minor radioactive band was sometimes observed. The radioactivity associated with the front was variable. It is probably due to labeled lipids in the membrane fragments and minor photolysis products which are not re- 7 0 min FIG. 1. Covalent incorporation of ["'IIINA as a function of time of exposure to light (314 mm). The curve represents the photolabeling by ['*'II]INA (4 X IO' cpm) of AChR-enriched membrane fragments (1 X 10"' M of toxin binding sites; 1.6 mg of protein) in 1.5 ml of Pi/NaCl-PMSF. At each point, 100 p1 of the suspension was added to 0.9 ml of acetone at -20 C. The precipitate was washed twice with acetone/lo% water, and once with acetone, and dried. '"I content: determined in the precipitate (0) and in the supernatant (0). FIG. 2. Incorporation of the ['251]INA into the enriched AChR membrane fragments. Membranes prepared from T. californica (2 mg/ml) with specific activity of 1340 pmol of toxin binding sites/ mg of protein were labeled with 6 X 10' cpm of ["'IIINA. A, electrophoretic pattern; scan of the Coomassie Brilliant Blue-stained gel. B, scan of the autoradiogram of the same gel. The sample buffer contained 1% SDS, 0.02 M Tris-HC1, ph 6.8, 10% glycerol, 1% 2-mercaptoethanol, and 5 to 30 pg of proteins. The samples were incubated 5 min at 100 C and layered into 7.5 to 15% polyacrylamide gradient gels. The gels were fixed in 25% isopropanol, 10% acetic acid. Absorbance 1.0 in the scan corresponds to about 10' cpm. The arrou~s indicate the positions of the polypeptides used as molecular weight standards. These are in order of decreasing molecular weight bovine serum albumin, immunoglobulin heavy chain, ovalbumin, immunoglobulin light chain, and ribonuclease. M, = 67, 55, 43, 25, and 13 x IO', respectively.

3 1206 Lipid-embedded Domain of the Receptor ACh moved by the albumin washing nor by the destaining procedures. The pattern of INA incorporation was not modified when 15 mm glutathione, an aqueous nib-ene-scavenger, was present in the suspension medium before and after irradiation. Furthermore, in results not shown, the presence of excess (Ybungarotoxin (3 X M) carbamylcholine or (1 X M) before and during the INA labeling were found not to modify the pattern or the extent of incorporation. The Incorporation of ("'I]INA into the AChR Purified from Prelabeled Membranes-The INA-labeled AChR could be efficiently purified by affinity chromatography using Sepharose-bound N. naja siamensis toxin. This indicates, as discussed below, that the labeling does not affect toxin binding capacity. The pattern of radioactivity and the polypeptides obtained in the purified receptor are shown in Fig. 3. The 40,000-dalton peptide was the nearly exclusive site of INA incorporation. Minor radioactivity was sometimes associated with the M, = 48,000 subunit. The patterns of Coomassie staining bands were found to be somewhat variable in different batches of the purified AChR. Consistently, the main bands found were (in order of decreasing intensity) 40, 48, and 64 x 10:'-dalton peptides. In addition, some preparations showed lower concentrations of 43, 55, 38, 37, and 80 X 10'-dalton peptides. Consistently, however, the 40,000-dalton polypeptide was the only labeled band. Pattern of Incorporation of ["'IIINA into Purified Detergent-soluble AChR-The affinity chromatography-purified receptor was labeled with INA while dissolved in Triton X Under these conditions, all the polypeptides were labeled (Fig. 4). In all cases, the major incorporation of label was into the 40,000-dalton polypeptide followed by the 48,000-dalton band. Effect of (12RI]INA Labeling on the a-butx Binding by AChR-enriched Membranes and Purified AChR-When AChR membranes were labeled with INA, no inhibitory effects were observed in the a-butx binding capacity when compared with unlabeled membranes (Table I). When the a- BuTx binding was determined as the fraction not inhibited by IO-,' M nicotine, a marked enhancement was observed after INA labeling. In comparison, the capacity to bind a-butx was decreased when Triton X-100-soluble purified AChR was labeled with INA. Trypsin Digestion of INA-labeled AChR-enriched Membranes and Purified Detergent-soluble AChR-Trypsin TABLE: I Effect of INA labeling on the binding of wbutx hy AChR-enriched membranes Sample Specific activity" pmol ofn HuTx hound/mg protein Membrane 2,800 Membrane ["'I]INA 4,700 AChR AChR ['"IlINA The amount of bound toxin uersus receptor concentration was plotted and the receptor activity was calculated, as described by Olsen et al. (23). Average of three different experiments. The background subtraction used was the amount of ['251]a-BuTx bound in the pres- ence of lo-"' M nicotine. This includes the nonspecific binding of the toxin and the fractions of counts due to the ["'I]INA labeling, The blank values were less than 1076of the total counts due to toxin binding in the absence of nicotine. I I OB " - Ulslonce h i FIG. 3 (left). Incorporation of the ['251]LNA into the purified AChR prelabeled in situ. ['251]TNA-labeled membranes (6 mg of protein/3 ml (prepared as in the legend to Fig. 1)) were solubilized with 1%) Triton X-I00 followed by affinity chromatography of the AChK in 3 g of Sepharose containing bound N. naja siamensis toxin; elution was with 4 ml of 0.7 M carbamylcholine. The eluted material was concentrated with Amicon membranes. SDS-polyacrylamide gel electrophoresis was performed as indicated in the legend to Fig. 2. A, electrophoretic pattern; scan of the Coomassie Brilliant Blue-stained gel. B, scan of the autoradiogram of the same gel. Absorbance 1.0 corresponds to 3 X IO' cpm. Polypeptides used as a molecular weight standards are indicated in the legend to Fig. 2. FIG. 4 (rzght). Incorporation of ['"IIINA into the purified AChR while dissolved in detergent solution. Purified AChR (1 mg/ml) with specific activity of 10,500 pmol of toxin binding site/mg of protein was labeled with 6 X IO' cpm of ["'I]INA while dissolved in a 0.1% Triton X-100 solution. A, electrophoretic pattern; scan analysis of the Coomassie Brilliant Blue-stained gel. B, scan of the autoradiogram of the same material. SDS-polyacrylamide gel electrophoresis was performed as indicated in the legend to Fig. 2, but using 10% polyacrylamide in the gel. Absorbance 1.0 corresponds to about lo4 cpm. Polypeptides used as molecular weight standards are indicated in the legend to Fig. 2.

4 Lipid-embedded Domain of the ACh Receptor 1207 branes, was deduced from the following findings (24, and present results). First, INA is highly hydrophobic since it partitions into the membrane to an extent of >99% even at concentrations of INA of <lo-" M and levels of membrane lipids of 1 mg/ml. Second, the label attaches covalently to membrane proteins and lipids to the extent of 20 to more than 60% (present results, 48%). This suggests that only a minor fraction, if any, of the nitrene exits from the lipid into the aqueous phase, since there it would preferentially react with the buffer components and with water. The presence of glutathione (15 mm) in the aqueous medium during INA photo-or the patternof incorporation labeling did not alter the extent of the label into the polypeptide chains (Ref. 30 and present results). Glutathione is an effective nitrene-scavenger (31). Therefore, these findings strongly suggest that the photoderived products of INA are hydrophobic and do not exit into the aqueous layers surrounding the membrane. Third, red blood cell membrane glycophorin is labeled almost exclusively in the trypsin-insoluble peptide (29). The majority of the available evidenceindicates thatglycophorin spans themembrane by means of a segment of some 20 mainly apolar amino acid residues contained in the trypsin-insoluble peptide (32). Fourth, in membranes suchas those of the human erythrocyte and of rabbit muscle sarcoplasmic reticulum, where a clear distinction is available between intrinsic and extrinsic proteins, INAlabelsonlyintrinsic proteins (24, 30). Finally, lhlonce (cm) exhaustive proteolytic digestion with different enzymes refig.5 (left).effect of trypsin on thephotolabel distribution leased negligible amounts of the INA-labeled peptides while of AChR-enriched membrane fragments. Membranes were labeled (asindicated in the legend to Fig. 1) and treated with trypsin removing a major fraction of the membrane protein. A, electrophoretic pattern and labeling Of the many polypeptides found in the AChR-rich mem("experimental Procedures"). in the absence of trypsin; B, with trypsin (5 min) in a ratio of 1:1000 brane fragments, two main polypeptide chains were found to ((w/w) receptor/trypsin); C, a 1:20 ratio ((w/w) receptor/trypsin) be labeled by INA and therefore to be in contact with or (120 min). Sodium dodecyl sulfate-polyacrylamide gradient gel elecspanning the lipid bilayer. The f i t labeled polypeptide of trophoresis slab gels were then run (asindicated in the legend to Fig. 2), fixed, stained, and sliced into 1.5-mm slices and the '"'I-content apparent M, = 90,000 has not been identified although it is likely that itcorresponds to the heavy chain of the (Na', K+)was determined. purified AChR isolated ATPase. Torpedo electricorgan membranes are known to FIG.6 (right). Effect of trypsin on the from membranes prelabeled with ['251]INA. Membraneswere contain a high concentration of this enzyme (33)presumably labeled (as indicatedin the legend to Fig. 1) and the AChRwas to repolarize the membranefollowing acetylcholine-mediated isolated and purified.a, electrophoretic pattern without trypsin; scan of the autoradiogram. B, with added trypsin in a ratio of 1:1000 ((w/ depolarization. This suggestion is supported by the fact that w) receptor/trypsin). C, trypsin ratio of 1:20 (w/w). Absorbance 1.0 the purified (Na', K')-ATPase fromkidney is extensively corresponds to about 3 X IO' cpm. Unlabeled bands correspond to labeled by INA; exhaustive trypsin digestion of this enzyme is molecular weights of 36,000 and 32,000 in B and 30,000 and 27,000 in known to lead to theformation of a single INA-labeled peptide C. For details, see "Experimental Procedures." Polypeptides usedas of M,= 13,000 to 14,000 (28). Results shown in Fig. 5 indicate molecular weight standards are indicated inthe legend to Fig. 2. that a similarpeptide arises fromthe proteolysis of the 90,000dalton peptide band (see below). digestion of INA-labeled AChR-enriched membranes liberthe second highly labeled polypeptide present in AChRated about 30% of the membrane protein withoutsignificant rich membranefragments corresponds tothe a subunit release of radioactivity. Membranes subjected mild to trypsin- (40,000-dalton peptide) of the purified AChR. During the ization (Fig. 5) yielded intermediate bandsbetween 55,000 and AChRpurificationprocedure, the radioactivityassociated 66,000 and small fragments in the region of 10,000 to 13,000. with this band is quantitatively retainedin the Sepharose-N. Exhaustive trypsinization showed significant labeling only in naja siamensis toxin column and then eluted by carbamylthe 10,000- to 13,000-dalton region. choline. The a-butx binding to AChR-rich membranes or Equivalent treatment of the Triton X-100-soluble purified purified AChR measured directlyis unalteredby INA labeling AChR derived from INA-labeled membranes indicated that and thelabel incorporation is not affected by the presence of the labeled 40,000-dalton peptide band is converted into a a-butx or carbamylcholine. These results indicate that the labeled 10,000- to 13,000-dalton peptide band and unlabeled an site of INA labeling is distinct from that site which binds a27,000- to 30,000-dalton polypeptide (Fig. 6). The radioactive BuTx or carbamylcholine. This is in contrast to previous band stained weakly withcoomassiebrilliant Blue. This photolabeling experiments (14, 17,34). Digestion with trypsin could imply excess of negative charge or its hydrophobic of the purified AChR prelabeled with INA while in the memnature, or both. brane fragmentsleads to a fragmentation of the 40,000-dalton polypeptide into nearly unlabeled 27,000- to 30,000- and ladiscussion beled 10,000- to 13,000-dalton polypeptide bands, respectively. In this study, we have identified the lipid-embedded poly- It has recently been shown that after trypsin digestion of peptides of AChR-rich membrane fragments andof the puri- purified AChR, the a-butxbinding activity is unaffected and tied AChR by their photolabelingwith 5-iodonaphthyl-l- is associated withthe 27,000 to 30,000 band (35). This suggests two doazide. That the photolysis of INA resulted in a nitrene that that the 40,000-dalton polypeptidebandcontains labeled, while dissolved within the lipid phase of the mem- mains: a 27,000- to 30,000-dalton peptide segmentexposed to 1

5 1208 Lipid-embedded Domain of the ACh Receptor the aqueous phase containing the toxin and agonist binding sites and a 10,000- to 13,000-dalton peptide domain embedded in the membrane lipids. Intimate association between the two domains is suggested by the fact that sedimentation of membrane fragments after trypsin digestion shows the generated 27,000 to 30,000 segment to be retained with the membrane. Furthermore, after digestion and sedimentation, toxin binding activity is not decreased (when expressed as specific activity, an enhancement of 2-fold is observed due to release of other proteins). Of the other subunits co-purified with the 40,000- dalton peptide main band of the receptor, only minor labeling was sometimes observed in the polypeptide of48,000. The 64,000- and 55,000-dalton peptide bands were never found to be labeled. It should be noted that variability was observed in the amounts of the different bands other than 40,000 present. Furthermore, in some instances, a 43,000 shoulder was seen. The membranes used were obtained from commercially frozen material and this might explain some of the variability. Our results consistently indicate the M, = 40,000 subunit to be the almost exclusive site of INA incorporation. The question can then be made why are the other subunits of the receptor not labeled. First, it could mean that the other subunits are peripheral and not in contact with the lipid bilayer. The recent findings that 0.1 N NaOH removes some of these bands would support this contention (36,37). Second, the quaternary struc- ture of the receptor within the bilayer might be such that the M, = 40,000 subunit shields the other subunits from access to the INA. That such quaternary structure exists is suggested by the fact that exhaustive proteolysis does not alter, for example, the anion channel of the human erythrocyte (38). Third, INA has recently been shown not to insert readily into C-H bonds (30). Thus, labeling of unsaturated phospholipid liposomes was to the extent of 44% while that containing saturated phospholipids was less than 2% This implies that the iodonaphthylnitrene is not energetic enough to react randomly and that there exist different susceptibilities of the various amino acid side chains to be labeled. This could mean that a protein in contact with the lipid but devoid of susceptible side chains would be labeled to a lesser extent than one containing such reactive moieties. Therefore, unlabeled peptides might not necessarily be those excluded from the bilayer. It should be noted, however, that in human erythrocyte membranes, all the known intrinsic proteins were labeled (24, 30). Different nitrene- and carbene-generating molecules might have different reactivities and might preferentially be located in a different region of the lipid bilayer (30). This might explain the difference in the results reported here from those published at the end of our study by Sator et a1 (39). These authors reported that [: H]pyrenesulfonyl azide labeled preferentially the M, = 48,000 and 55,000 subunits. Attempts to explain the differences in these reports must await further characterization of the labeling properties of the pyrenesulfonyl azide. The labeling of the AChR in situ gave results which showed selective labeling of the M, = 40,000 subunit. Equivalent labeling of the purified AChR while in Triton X-100 solution led to all subunits being labeled. These results indicate the existence of susceptible residues in all the subunits. They further emphasize that the interaction of the AChR with the membrane lipids is different from that with the Triton X-100 micelles. Clearly, this difference is of interest in reconstitution experiments since the purified AChR reincorporated into phospholipid liposomes and then INA-labeled should show, if properly reconstituted, labeling only in the M, = 40,000 subunit. There is indirect evidence that the AChR is a transmem- brane protein. Thus, agonist binding results in the transloca- FEBS Lett. 26,43-47 tion of ions across the membrane (1-6). Ultrastructural studies utilizing freeze-fracture or negative staining of receptor-rich membrane fragments, reveal an organized hexagonal array of closely packed particles in a significant portion of the membrane vesicles (40-42). The dimensions of these particles closely resemble the values obtained for pure AChR. X-ray diffraction results further suggest that the AChR molecules traverse the end plate membrane, extending some 15 A on one side of the bilayer and 55 A on the other (43). Furthermore, studies on the localization of AChR in the enriched membranes, using a specific (44) immunoferritin-labeled anti- AChR (Fab),, suggest that the antigenic sites of the receptor molecule are exposed on both sides of the excitable membrane (45). There is no agreement in the literature whether channel formation after agonist binding is a function exclusively of those polypeptides present in the purified AChR (46,47). The present results indicate that the 90,000- and 55,000- to 64,000- dalton polypeptide regions contain other components, independent of those purified by toxin-bound Sepharose, which are embedded or span the lipid bilayer. Their identification relates them to transmembrane function. It is evident, furthermore, that the presence of a membrane-embedded domain in the 40,000-dalton polypeptide makes it a prime candidate for the ionophoric function of the AChR. Acknowledgment-We would like to express our gratitude to Prof. Sara Fuchs for providing R. T. H. with the means to carry out these experiments and for her interested discussion of our results. REFERENCES 1. Nachmansohn, D. (1953; 1954) Harvey Lect. 49, Nachmansohn, D., ed (1975) Chemical and Molecular Basis of Nerve Actiuity, Academic Press, New York 3. Katz, B., and Miledi, R. (1972) J. Physiol. (Lond.) 224, Changeux, J.-P., Podleski, T. R., and Meunier, J. C. (1969) J. Gen. Physiol. 54, Albuquerque, E. X., Barnard, E. A,, Chiu, T. H., Lapa, A. J., Dolly, J. O., Jansson, S. E., Daly, J., and Witkop, B. (1973) Proc. Natl. Acad. Sci. U. S. A. 70, Kasai, M., and Changeux, J.-P. (1971) J. Membr. Biol. 6, Brisson, A,, Scandella, C. J., Bienvenue, A,, Devaux, P., Cohen, J. B., and Changeux, J.-P. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, Reed, K., Vandlen, R., Bode, J., Duguid, J., and Raftery, M. A. (1975) Arch. Biochem. Biophys. 167, Kato, G., and Changeux, J.-P. (1976) Mol. Pharmacol. 12, Karlin, A. (1974) Life Sci. 14, Changeux, J.-P., Benedetti, L., Bourgeois, J. P., Brisson, A., Cartaud, J., Devaux, P., Grunhagen, H., Moreau, M., Popot, J. L., Sobel, A., and Weber, M. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, Raftery, M. V., Vandlen, H. C., Reed, K. L., and Lee, T. (1975) Cold Spring Harbor Symp. Quant. Bid. 40, Karlin, A,, Weill, C. L., McNamee, M. G., and Valderrama, It. (1975) Cold Spring Harbor Symp. Quant. Biol. 40, Hucho, F., Layer, P., Kiefer, H. R., and Bandini, G. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, Aharonov, A., Tarrab-Hazdai, R., Silman, I., and Fuchs, s. (1977) Immunochemistry 14, Weill, C. L., McNamee, M. G., and Karlin, A. (1974) Biochem. Biophys. Res. Commun. 61, Witzemann, V., and Raftery, M. (1978) Biochemistry 17, Michaelson, D., Vandlen, R., Bode, J., Moody, T., Schmidt, J., and Raftery, M. A. (1974) Arch. Biochem. Biophys. 165, Shamoo, A. E., and Eldefrawi, M. E. (1975) J. Membr. Biol. 25, Schiebler, W., and Hucho, F. (1978) Eur. J. Biochem. 85, Epstein, M., and Racker, E. (1978) J. Biol. Chem. 253, Cohen, J. B., Weber, M., Huchet, M., and Changeux, J.-p. (1972)

6 Lipid-embedded Domain of the ACh Receptor Olsen, R. W., Meunier, J.-C., and Changeux. J.-P. (1972) FEBS Lett Bercovici, T., and Gitler, C. (1978) Biochemistry 17, Laemmli, U. K. (1970) Nature (Lond.) 227, Wyckoff, M., Rodbard, D., and Chrambach, A. (1977) Anal. Biochem. 78, Sigrist-Nelson, K., Sigrist, H., Bercovici, T., and Gitler, C. (1977) Biochim. Biophys. Acta 468, Karlish, S. J. D., Jorgensen, P. L., and Gitler, C. (1977) Nature 269, Kahane, I., and Gitler, C. (1978) Science 201, Gitler, C., and Bercovici, T. (1979) in Application of Photochemistry to Probe Biological Targets (Tometsko, A. M., and Richards, F. M., eds), New York Academy of Sciences, New York, in press 31. Bayley, H., and Knowles J. R. (1978) Biochemistry 17, Furthmayr, H., Galardy, R. E., Tomita, M., and Marchesi, V. T. (1978) Arch. Biochem. Biophys. 185, Jean, D. H., and Albers, W. R. (1977) J. Biol. Chem. 252, Witzemann, V., and Raftery, M. A. (1977) Biochemistry 16, Bartfeld, D., and Fuchs, S. (1979) Biochem. Biophys. Res. Commun 89, Steck, T. L., and Yu, J. (1973) J. Supramol. Struct. 1, , Neubig, R. R., Krodel, E. K., Boyd, N. D., and Cohen, J. B. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, Grinstein, S., Ship, S., and Rothstein, A. (1978) Biochim. Biophys. Acta 507, Sator, V., Gonzalez-Ros, J. M., Calvo-Fernandez, P., and Martinez-Carrion, M. (1979) Biochemistry 18, Cartaud, J. (1974) in Electron Microscopy (Sanders, J. V., and Goodchild, D. J., eds), Vol. 2, pp , Australian Academy of Science, Canberra, Australia 41. Cartaud, J., Benedetti, E. L., Cohen, J. B., Meunier, J.-C., and Changeux, J.-P. (1973) FEBSLett. 33, Nickel, E., and Potter, L. T. (1973) Brain Res. 57, Ross, M. J., Klymkowsky, M. W., Agard, D. A,, and Stroud, R. M. (1977) J. Mol. Biol. 116, Karlin, A., Holtzman, E., Valderrama, R., Damle, B., Itsu, K., and Reyes, F. (1978) J. Cell Biol. 76, Tarrab-Hazdai, R., Geiger, B., Fuchs, S., and Amsterdam, A. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, De Robertis, E. (1971) Science 171, Heidemann, T., and Changeux, J.-P. (1978) Annu. Reu. Biochem. 47,