Purification of Acetylcholine Receptors, Reconstitution ... · affinity chromatography using either Naja naja sia- mensis toxin I11 or concanavalin A coupled to agarose. Sucrose gradient

Purification of Acetylcholine Receptors, Reconstitution into Lipid Vesicles, and Study of Agonist-induced Cation Channel Regulation*

(Received for publication, November 13, 1979, and in revised form, March 13, 1980)

Jon Lindstrom,S§ Robert Anholt,$r[ Brett Einarson,S Andrew Engel,(l Mitsuhiro Osame,JI and Mauricio Montall From $The Salk Institute for Biological Studies, Sun Diego, California 92158, !The University of California, Sun Diego, Dewartments o f Biolom and Phvsics. La Jolla. California 92093, and IIThe Mayo Clinic, Department of Neurology, Ro'chester, Minnesota-~5901 "

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubilization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naja naja siamensis toxin I11 or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamylcholine-induced influx of "Na+ per mol of receptor. Carbamylcholine-induced "Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external "Na+ concentration, whereas in reconstituted vesicles "Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

* This work was supported by grants from the National Institutes of Health (J. L., A. E., M. M.), by grants from the Muscular Dystrophy Association (J. L., A. E.), by a grant from the Office of Naval Research ( J . L., M. M.), and by a grant from the Paul Stock Foundation. 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.

§ T o whom reprint requests should be addressed at: The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, CA 92158.

Binding of an agonist such as carbamylcholine to a ligand binding site on the AChR' initiates a conformational change in this protein which triggers the transient opening of a cation- specific channel across the postsynaptic membrane (reviewed in Refs. 1 and 2). AChR solubilized from membranes with detergent and purified by affinity chromatography retains ligand-binding activity, but until recently attempts to recon- stitute purified AChR into model membranes and demonstrate agonist-controlled cation flux have been unsuccessful or not readily reproducible (1, 2). Alkaline extraction (3) of highly purified membrane fragments from torpedo electric organ (4, 5 ) produces membrane fragments containing only the four polypeptide subunits characteristic of detergent-solubilized purified AChR (6). These purified membranes can be solubilized in cholate plus soybean lipid and then the AChR can be reconstituted into lipid vesicles by removing the cholate via dialysis ( 7 ) . The AChRs in these reconstituted vesicles retain agonist-controlled channel activity (6,8), showing that the cation channel is an integral component of the AChR macromolecule. Recently, AChR solubilized in cholate/lipid mixtures and then purified on an agonist affinity column has also been reconstituted into vesicles (9). Using techniques detailed in this paper it has been possible to purify AChR solubilized in lipid/cholate mixtures and subsequently recon- stitute it into both lipid vesicles (lo), where AChR activity can be assayed by %a+ uptake, and into planar lipid bilayers, where AChR activity can be assayed electrically (11). Unpu- rified AChR-rich membranes have also been reconstituted into planar lipid bilayers (12).

Monomers of Torpedo californica AChR are known to be composed of four kinds of subunits in the mole ratio m2/3y8 (13, 14). AChR from other species contain corresponding subunits (15-17). The a subunits are known to compose part or all of the lf51-aBGT binding site (18). The functions of /3, y , and S are not known, but it seems reasonable to suspect that one or more of these subunits may compose the cation channel, which is an integral component of the AChR monomer (10). The two a subunits are known to be nonequivalent because the affinity alkylating agents MBTA (13) and bro- moacetylcholine (19) bind to a chains, but inhibit only half of aBGT binding.

Here we report five methods by which AChR can be purified while retaining fully functional cation channels. In native membrane vesicles carbamylcholine-induced "Na' flux is lim-

' The abbreviations used are: AChR, acetylcholine receptor; '2ni- aBGT, '2511-labeled a-bungarotoxin; Con A-agarose, Concanavalin A conjugated to Sepharose 4B; toxin-agarose, N. naja siamensis toxin 111 conjugated to Sepharose C14B; MBTA, 4-(N- maleimido)benzyltrimethylarnmonium; SDS, sodium dodecyl sulfate; Trizma, 2-amino-2-hydroxymethyl-1,3-propanediol.

a340

Acetylcholine Receptor Reconstitution 834 1

ited by equilibration of the internal volume of the vesicles with external "Na' due to the high concentration of AChR in the vesicles (20). We show that, by contrast, after reconstitution, the 22Na' flux/AChR is limited by desensitization of the AChR molecule. We use this system in which '?Na' flux is directly proportional to the amount of active AChR to demonstrate that only one of the two toxin binding sites on the AChR monomer, the one labeled by MBTA, controls opening of the cation channel of the AChR.

METHODS

Purification of AChR-rich Vesicles-This procedure followed the general approach of several other laboratories (4, 5, 21) in which AChR-rich membrane vesicles were purified on the basis of their relatively high density due to their high concentration of AChR. Care was taken to use EDTA and iodoacetamide as protease inhibitors. Frozen T. californica electric organ (Pacific Biomarine). typically 400 g, was broken into chunks and mixed with 1.5 volumes of 10 mM Na phosphate pH 7.5, 10 m~ NaN:(, 5 mM EDTA, and agitated occasion- ally over about 15 min while the tissue thawed. All subsequent steps were performed at 4°C. Iodoacetamide (to 5 mM) and phenylmeth- anesulfonylfluoride (to 1 m ~ ) were added and the mixture was homogenized in a I-gallon Waring blendor for 8 X 15 s. The initial homogenate was then centrifuged at low speed, 10 min a t 5,000 rprn in a Beckman JA-21 rotor. The supernatant was strained through wire mesh to remove large fragments. The pellet was rehomogenized 1 X 30 s in an equal volume of the initial buffer, recentrifuged, and the strained supernatant pooled with the first. The low speed pellet was discarded and the supernatant was centrifuged in a Beckman type 19 rotor a t 19,000 rprn for 60 min. This crude membrane pellet was the starting material for solubilized AChR to be further purified on toxin-agarose. In order to prepare membranes more enriched in AChR, the high speed pellet was resuspended in 3 volumes of 10 mM Na phosphate, pH 7.5, 1 mM EDTA, 10 mM NaN.3 in a Virtis homogenizer 4 X 30 s at high speed on ice. Then 4 M NaCl was added to a final concentration of 0.4 M and 60% sucrose to a final concentration of 32% (w/w) and the mixture was homogenized again for 2 X 30 s. This suspension was layered a t 11.5 ml/tube over 14.5 ml of 36% (w/ w) sucrose in 0.4 M NaCI, 10 mM Na phosphate, pH 7.5, 1 mM EDTA, 10 mM NaN:(. After centrifugation for 50 min a t 50,000 rpm in a Beckman Ti-60 rotor, the floating fat pad and pellet were discarded. The band of AChR-rich membranes was collected, diluted to 30% sucrose, and then centrifuged for 45 min a t 50,000 rprn in the Ti-60 rotor. This pellet of membranes partially enriched in AChR was the starting material for solubilizing AChR to be further purified on Con A-agarose or by centrifugation on sucrose gradients. For further purification of AChR in intact membranes, this pellet was resuspended in a minimal volume of flux buffer (145 mM sucrose, 10 mM Na phosphate buffer, pH 7.5, 5 m~ NaNa) using a Polytron homogenizer for 3 X 30 s a t high speed on ice. Then aliquots of approximately 12 ml were layered on 54 ml of linear 31 to 39% sucrose gradients (in 10 mM Na phosphate buffer, pH 7.5, 1 m~ EDTA, 10 m~ NaN:,). These gradients were centrifuged overnight at 25,000 rpm in a Beck- man SW 25 rotor. The upper layer and pellet were discarded and the mid band diluted 1:l with flux buffer and then centrifuged at 50,000 rpm for 50 min in the Ti-60 rotor. The resulting pellet was resuspended in a minimal volume of flux buffer using an 18-gauge needle and syringe, and then homogenized in a motor-driven homogenizer with a Teflon pestle. This was the purified vesicle fraction.

Alkaline Extraction of Vesicles-This method is based on the report that extraction of vesicles with low ionic strength pH 11.0 buffer removes the 43,000-dalton protein (3) and is similar to methods reported by others (6, 8). One to two milligrams of purified vesicle protein were layered on a gradient consisting of 9.5 ml of 15% (w/w) sucrose in water brought to pH 11.0 with NaOH and layered over 1 ml of 33% (w/w) sucrose, 10 mM Na phosphate, pH 7.5, 10 mM NaNs. Gradients were centrifuged for 90 rnin a t 40,000 rpm in a Beckman SW 41 rotor to pass the vesicles through the alkaline layer and pellet them. The supernatant containing the soluble proteins and Some of the lower density membrane proteins was decanted and discarded.

Reconstitution of Vesicles-Reconstitution buffer (2% Na cholate, Interchem, Montlucon, France), 25 mg/ml of soybean L-a-phosphatidylcholine (Sigma, commercial grade), 100 mM NaC1, 10 mM phosphate buffer, pH 7.5, 10 m~ NaN:J was prepared by three cycles of stirring the ingredients together at room temperature, freezing, and thawing. The solution was then centrifuged 30 min at 30,000 rpm in

a Beckman No. 30 rotor to remove any unsolubilized lipid. This solution was then aliquoted and stored frozen. Reconstitution buffer was also made by dilution of a 150 mg/ml stock of lipid in water sonicated under argon to clarity in a bath-type sonicator at 4°C. The method by which cholate/lipid mixtures were made had no effect on the activity of AChR in the reconstituted membranes.

Vesicles were resuspended in reconstitution buffer a t concentrations of 0.1 to 6 mg of protein/ml. After blending on a Vortex mixer and stirring to provide a homogeneous suspension, this mixture was dialyzed overnight against at least 100 volumes of 100 mM NaC1, 10 mM Na phosphate, pH 7.5, and 10 mM NaN:t, followed by an additional overnight dialysis against flux buffer.

Purification of Solubilized AChR on Toxin-Agarose-Crude membranes were resuspended with a Virtis homogenizer in 10 mM Na phosphate buffer, pH 7.5, 10 m~ NaN.,, 1 mM EDTA. Sonicated soybean phospholipids (150 mg/ml in water) and 10% sodium cholate were added to the suspension to final concentrations of 5 mg/ml and 2%, respectively. After 30 min shaking at 4°C. the mixture was centrifuged for 30 min a t 30,000 rprn in a Beckman No. 30 rotor. The supernatant was added to toxin-agarose (0.5 mg of N. naja siamensis toxin III/ml of Sepharose C1 4B) a t about 5 nmol of AChR/ml of agarose and shaken gently for 1 h before being poured into a column. The column was washed with 250 to 300 column volumes of cholate/ lipid buffer (2% Na cholate, 5 mg/ml of soybean lipid, 100 mM NaC1, 4 mM Na phosphate, pH 7.5). AChR was eluted by a 4-h incubation at room temperature on a shaker with .K volume of 2 m~ benzoquinonium chloride (gift of Stirling-Winthrop) in cholate/lipid buffer. The toxin-agarose was packed into a column and the eluate adjusted to 25 mg/ml of soybean lipid using a sonicated 150 mg/ml dispersion. Benzoquinonium was removed from the eluate by passage over 1.5 volumes of Bio Rex 70 equilibrated in the same buffer. The eluate was then reconstituted by successive I-day dialyses against 500 volumes of 100 mM NaCl, 10 mM Na phosphate, pH 7.5, and flux buffer, as with AChR purified by alkaline extraction.

Purification of Solubilized AChR on Con A-Agarose-Partially purified vesicles were solubilized in cholate/lipid buffer the same way that the crude membrane fraction was solubilized before use of toxin- agarose. Concanavalin A conjugated to Sepharose 4B (Sigma), equilibrated in cholate/lipid buffer, was then added at about 9 nmol of AChR/ml of agarose. After 1 h on a shaker a t 4'C, the Con A-agarose was poured into a column and rinsed with 250 to 300 volumes of cholate/lipid buffer. Elution was in two steps a t room temperature. First the resin was eluted for 1 h on a shaker with .K volume of 0.2 M a-methyl-D-mannoside, 5 mM NaN:+, 1 mM EDTA in cholate/lipid buffer. This eluate contained about 10% of the AChR and most of the contaminating glycoproteins bound with low affinity. A quick wash with 10 to 20 volumes of the same buffer a t 4°C removed many of the remaining contarninants with low affinity for Con A. A second elution a t room temperature for 4 h with '% volume of 1.0 M a-methyl-D- mannoside, 5 mM NaN3, 1 m~ EDTA in the same buffer yielded 17% of the bound AChR in a more purified form. For reconstitution, the eluates were adjusted to 25 mg/ml of lipid in 2% cholate and dialyzed as above. AChR solubilized in cholate/lipid buffer could also be rapidly frozen and stored before reconstitution by dialysis.

Purification of Solubilized AChR by Sucrose Gradient Centrifu- gation-Purified vesicles were solubilized using 2% Na cholate, 5 mg/ ml of soybean lipid, 100 mM NaCI, 10 mM NaN,(, 10 mM Na phosphate buffer, pH 7.5, and dialyzed overnight against 2% cholate buffer to insure solubilization. The mixture was centrifuged 30 min at 45,000 rprn in a Beckman Ti 50 rotor. The supernatant was trace-labeled with "'11-aBGT and applied in 2.4-ml aliquots to 36 ml of linear 10 to 25% (w/w) sucrose gradients containing 2% Na cholate, 5 mg/ml of soybean lipid, 100 mM NaCI, 10 m~ NaN:t, and 10 mM Na phosphate buffer, pH 7.5. After centrifugation for 5 h a t 50,000 rprn in a Beckman VTi 50 rotor, the gradients were fractionated. '"I-(YBGT in the fractions was determined, and the AChR monomer, dimer, and larger aggregate peaks pooled. After adding sonicated soybean lipid to a final concentration of 25 mg/ml, the sample was reconstituted by dialysis.

Purification of Solubilized AChR using Toxin-Agarose and Con A-Agarose-AChRs were solubilized as described for toxin-agarose purification. Extracts were mixed with toxin-agarose in the ratio 4 nmol of "'1-aBGT binding sites/ml of toxin-agarose and gently shaken for 1 h a t 4°C. The toxin-agarose was then poured into a chromatography column and washed as described for toxin-agarose purification. Then a small column containing one-tenth the toxin- agarose volume of Con A-agarose was attached below the toxin- agarose column. AChR was eluted from the toxin agarose by IO-.' M

8342 Acetylcholine Receptor Reconstitution

benzoquinonium in 2% cholate, 5 m g / d lipid buffer recirculated through the two columns overnight by means of a peristaltic pump. AS the AChR was eluted from toxin-agarose by benzoquinonium, it was bound to the Con A-agarose. The Con A-agarose was then washed free of benzoquinonium, and AChR was eluted a t room temperature by four (45-min) elutions with 1 column volume of 1 M a-methyl-D- mannoside, 5 mM NaN:I, 1 mM EDTA in cholate/lipid buffer.

Assay of Agonist-dependent "Nu+ Influx-An ion exchange resin was used to bind external "Na+ essentially as described by Epstein and Racker (7). Dowex 50W-X8-100 (Sigma) was first washed with distilled water by decantation until the supernatant was clear. Trizma base (Sigma) was then added to equal volumes of resin and water a t the ratio of 440 g/kg of resin. After stirring for an hour, additional Trizma was added if necessary to bring the pH to 9.8. Then the Tris was decanted and the beads washed with many changes of water until the pH approached that of the water. Disposable columns (-0.5 X 8 cm) were poured in short Pasteur pipettes plugged with glass wool. A small plastic funnel was attached to the top of each column with Tygon tubing. The capacity of these columns was sufficiently large to allow repeated use for a t least five assays, but were usually used once. Immediately before the fvst use, each column was washed with 3 ml of 170 mM sucrose, 3.3 m g / d of bovine serum albumin.

The z2Na+ influx assay was conducted a t room temperature behind suitable lead shielding. In microfuge tubes, 'lNa+ (5 p1 of 0.2 mCi/ml) and water (5 pl) or carbamylcholine (5 pl of e.g. 1 X lo-'' M) were mixed. The assay was initiated by pipetting in 40 p1 of vesicles in flux buffer with a Pipetman pipetter. After mixing by five up-and-down strokes of the pipetter, the mixture was transferred to a column in a total elapsed time of about 20 s. Sucrose solution (3 ml of 175 mM) was carefully added to elute the vesicles. The tube containing the eluate of each column was then placed in the y counter and counted a t 38% efficiency. For each sample, triplicate carbamylcholine responses and triplicate water blanks were measured. The agonist- induced y2Na+ influx was defined as the difference between the counts per min in the water samples and those containing carbamylcholine.

The apparent internal volume of the vesicles was measured in triplicate by mixing 40 p1 of vesicles, 5 p1 of "Na+, and 5 p1 of Hz0 as above, but incubating at 4°C for 24 h before application to the columns.

Measurement of AChR Concentration-AChR concentration was measured by a radioimmunoassay which was more rapid than that previously used (22). Aliquots (1 ml) of vesicles diluted to about 1 X IO-" M in aBGT binding sites in 0.5% Triton X-100, 100 m~ NaCI, 10 mM Na phosphate, pH 7.5, and 10 mM NaN:J were incubated with 3 X M I2'II-aBGT for 10 min. Then 5 pl of a 3-fold concentration of a 40% ammonium sulfate cut of anti-AChR serum was added for 30 min. Thereafter, anti-IgC was added for 30 min. The immune precip- itate was pelleted by 2 min centrifugation in an Eppendorf microfuge. The washed pellets were counted in a y counter. Counts of in petlets from reaction mixtures without AChR were subtracted from all samples.

Measurement ofsidedness-The fraction of AChR exposed on the external surface of native or reconstituted vesicles was measured by comparing the total AChR concentration observed after solubilization in Triton X-100 (as described above) with the AChR concentration observed when the vesicles were kept intact so that "'11-aBGT had access to only the external surface by conducting the assay described above in flux buffer.

Measurement of Protein-The method of Lowry et al. (23) was used with bovine serum albumin as standard.

Electrophoresis-Acrylamide slab gels in SDS using the discontin- uous buffer system of Laemmli (24) were run as previously described (15, 25).

Freeze-Fracture Electron Microscopy-Suspensions of crude, purified, or reconstituted vesicles were mixed with glycerol to a final concentration of 20% (v/v). Two-microliter aliquots of the mixture were deposited on Balzer's specimen carriers and flash-frozen in Freon-22 chilled by liquid nitrogen. The specimens were fractured by microtomy in a Balzer's 300 instrument at 116'c and at 2 X 10" millibar. The fracture face was shadowed immediately with a 2-nm- thick deposit of platinum-carbon and backed with a 20-nm-thick deposit of carbon emitted from electron beam guns. Thickness of the deposited films was monitored with a quartz crystal device. The replicas were retrieved and cleansed by conventional methods, mounted on 300-mesh uncoated copper grids, and examined in a Phillips 300 electron microscope.

Inhibition of "Nu+ Flux by Toxin-Purified native AChR-rich vesicles (3.5 x M in "'1-aBGT binding sites, 89% external) were

incubated overnight in 190-pl aliquots plus 10 p1 of either flux buffer or a dilution of N. naja siamensis toxin I11 calculated to block 10 to 100% of the AChR present. Reconstituted vesicles of AChR purified by alkaline extraction and diluted 3-fold (6.2 X 10.' M "'I-aBGT binding sites, 67% external) were similarly incubated with toxin. The

M carbamylcholine-induced ."Na+ flux was then determined on quadruplicate 40- 1 aliquots.

Inhibition of Na' Flux by MBTA-Purified native AChR-rich vesicles (3.5 X M "'I-aBGT binding sites) and a 3-fold dilution of reconstituted vesicles prepared from AChR purified by alkaline extraction (6.2 X IO-' M) were used in 500-pl aliquots, two for each preparation. MBTA was synthesized as described by Karlin et al. (26) and the fundamental methods of Karlin (18) were used for reduction and subsequent affinity alkylation. Dithiothreitol (IO pl of 1 X 10" M) was added to all samples. After 20 min at room temperature, 20 pJ of acetonitrile added to control aliquots and 20 yl of 1 X 10.' M MBTA in acetonitrile were added to the aliquots to be alkylated. The control and MBTA-treated samples were then dialyzed separately overnight a t 4°C against two changes of 10' volumes of flux buffer. Specific carbamylcholine-induced 22Na+ flux was then determined on quadruplicate samples. Both external and total "'I-aBGT toxin binding sites were determined in triplicate for each sample.

P

RESULTS

Purification of Functional AChR-Torpedo AChR solubilized in 1% cholate and purified by affinity chromatography on toxin-agarose using 0.2% cholate buffer contains endogenous lipid (and traces of high molecular weight proteins) (14). After mixing with 2% cholate, 25 mg/ml of lipid, and dialyzing to form vesicles, immunoprecipitation experiments using anti- AChR antibody, '"I-aBGT-labeled AChR, and ['4C]phosphatidylcholine trace-labeled lipid showed that AChR was associated with lipid (data not shown). However, carbamylcholine- induced Z2Na+ flux was never observed. These results suggested that cholate-solubilized AChRs could reincorporate into vesicle membranes, but were inactive. Treatment of AChRs while bound to toxin-agarose with a single column volume of 0.5% Triton X-100 removes both the lipid and high molecular weight contaminants. Attempted reconstitution of this material was also unsuccessful. The resulting vesicles had a low apparent internal volume (5.9 pl/ml), similar to that of vesicles formed in the absence of any protein. Freeze-fracture electron microscopy of these vesicles revealed that they were small and contained no intramembranous particles, and therefore resembled lipid vesicles formed in the absence of any protein (Fig. 1, A and B). This result suggested that Triton X- 100 either caused a conformation change in the AChR or remained associated with the AChR in an annulus that pre- vented reincorporation into vesicle membranes.

We c o n f i e d t h e results of Wu and Raftery (6) showing that alkaline extraction of highly purified AChR-rich vesicles gave a preparation consisting primarily of the a, ,8, y, and 6 bands characteristic of purified AChR (Fig. 2, Gel I). However, we could never obtain preparations entirely free of high molecular weight material. Immunochemical evidence suggests that a prominent component of torpedo membrane preparations with an apparent molecular weight of 1 x lo5 may be a component of the Na+/K'-dependent ATPase (15). The alkaline-extracted vesicles could be reconstituted into soybean lipid vesicles which showed carbamylcholine-sensitive "Na+ flux (Table I). Freeze-fracture electron microscopy of these vesicles revealed AChR as particles incorporated in the reconstituted membranes (Fig. 1C). Electron microscopy of sec- tioned samples revealed that most vesicles were unilamellar (data not shown). The AChRs in the vesicles shown in Fig. 1c are unusually closely packed, because reconstitution in this case was at 6.4 x M AChR and 25 mg/ml of lipid. For the rest of the experiments reported here, we reconstituted at 5 1 x M AChR in the same amount of lipid, thereby giving far fewer AChR per vesicle. The concentration of AChR in

Acetylcholine Receptor Reconstitution . . -, " .

8343

FIG. 1. Electron microscopy of freeze-fracture replicas of vesicles formed by dialysis of cholate/lipid mixtures in the absence of AChR or in the presence of AChR purified by conventional aftinity chromatography. The bars indicate 1 pm. A, control reconstitution mixture dialyzed in the absence of AChH. B. reconstituton mixture dialyzed in the presence of 5.1 X IO"' M torpedo AChH purified by conventional affinity chromatography methods which exposed it to Triton X-100. No intramembranous particles (Le. AChH) are seen in either A or B. C, reconstituted alkaline-extracted AChH-rich vesicles showing fractured AChH as intramembranous particles.

native vesicle membranes (Fig. 3A) was much greater than in tamination and free of unbound lipids or other unrecognized reconstituted vesicles (Fig. 3B) . components of the native membranes, we wanted to solubilize

These results had shown that the reconstitution technique AChR and then purify it. Fig. 4 shows that our previous of Epstein and Racker (7) could be applied to AChR purified experiments with cholate-solubilized purified AChR had failed in membranes and then solubilized in cholate/lipid mixtures. because the cation channel of AChR was denatured irrevers- In order to obtain AChR free of high molecular weight con- ibly by cholate. Only 10% of the initial cation channel activity


6

Y P a!

I 2 3 4 5

FIG. 2. Acrylamide gel electrophoresis in SDS of purified functionally active AChH preparations. ( I ) AChH purified by alkaline extraction of highly purified native AChH-rich vesicles and then reconstituted. (2 ) AChH purified by solubilization of crude nmemhrane fractions in cholate/lipid mixtures and affinity chromatography on toxin-agarose. l'he sample was concentrated before electrophoresis by dialysis against water followed by lyophilization. ( 3 ) AChH purified by soluhilization of partially purified AChH-rich mem- hranv fractions in cholate/lipid mixtures, adsorption t o Con A-agarose. and elution with tr-methylmannoside and then reconstituted. ( 4 ) AChH dimers purified hv solubilization of highly purified AChH-rich vesicles in cholate/lipid mixtures. sucrose gradient centrifugation. and then reconstituted. 'l'he sample was concentrated before electrophoresis by dialysis against water followed hy lyophilization and extrac- t i o n with chlorofomm/melhanol ( 2 1 ) t o remove excess lipid. (.5) AChH purified hy soluhilization of crude membrane fractions in cholate/ lipid mixtures and cnmhined affinity chromatography on toxin-agarose and Con A-agarose. From a different gel than Lanes I to 4.

survived for 16 h in 2% cholate in extracts of crude vesicles a t 1.67 mg of protein/ml. This residual activity was probably protected by endogenous lipids in the crude extract. However, the presence of relatively low amounts of soybean lipid in 2%. cholate solutions effectively preserved channel activity (Fig. 4). T h e binding of "'I-aRGT was not affected by the presence of supplementary lipid (data not shown).

Using 5 mg/ml of lipid in 2% cholate throughout the solubilization and purification of AChR, we succeeded in starting from a crude membrane fraction, using affinity chromatography on toxin-agarose, and obtaining pure AChH consisting only of a, y, and 8 chains (Fig. 2. Gel 2) which retained full cation channel activity (Table I). However, the presence of lipid interfered with the purification. When purifying AChR solubilized in cholate alone, we use a small DEAE-column connected to a large toxin-agarose column and elute AChR by recycling many column volumes of a benzoquinonium solution through both columns overnight. As the AChR is slowly competitively eluted from the toxin agarose, it accu- mulates on the DEAE. The DEAE is then easily washed free of the positively charged benzoquinonium, and the bound negatively charged AChR is eluted as a concentrated solution with high salt at a total yield of 40 to 80%. However, lipid bound to the DEAE, depriving us of a way of accumulating and concentrating AChIi eluted from the toxin-agarose and

I'urifird vesicles I<econstitutcd pu-

rified vesicles 1kconstitute.d al-

kaline-extracted vesicles

toxin-agarose- purified AChH

Con A-agarose- purified AChR

Heconstituted sucrose gradient- purified AChH

toxin-agarose/ Con A-agarose- purified AChH

Reconstituted

Reconstituted

Reconstituted

~ ~~~

Yield"

I5 15

I O

:<

5

5

20

0.685 5 0.14 2.75 ? 0 2 2

4.24 5 0.45

4.57 5 0.32

4.56 f 0.84

4.06 f 1.2

4.27 5 0.51

- ~~

" Per cent electric organ AChH recovered. I' Not determined.

eliminating the benzoquinonium. We therefore used a batch procedure for benzoquinonium elution, which was much less efficient than recycling the eluant and binding AChH as it was eluted. Then we used Hio Rex 7 0 to bind the benzoquinonium. leaving the eluted AChR in a dilute solution that defied accurate protein determinations.

After partial purification of membrane vesicles, AChR could be solubilized in cholate/lipid mixtures and further purified by adsorption to Con A-agarose and elution with a-methvl- D-mannoside. This procedure gave better yields and a more concentrated product than affinity chromatography on toxin- agarose, and was fully active on reconstitution (Table I), but although the AChR was completely freed of the 43,000 molecular weight component found in membranes (which does not periodic acid-Schiff stain for carbohydrate, data not shown), substantial contamination of high molecular weight material remained (Fig. 2, Gel 3). The high molecular weight material was glycoprotein, but bound somewhat less avidly to Con A- agarose than did AChR.

When torpedo electric organ membranes are prepared in the presence of iodoacetamide and EDTA, the disulfide bond between 6 chains is preserved and most of the AChR are present as dimers ( 2 7 ) . The large size of these dimers (13 S) permits them to be well resolved from the bulk of the protein by centrifugation on gradients containing 2% cholate. Starting from purified vesicles, pure AChR can be obtained in this way (data not shown). However, in gradients containing lipid as well as cholate, AChR is well resolved from the 43,000 molecular weight protein (which remains near the top of the gradient) but only partially resolved from high molecular weight material (which sediments less rapidly than AChR) (Figs. 2, Gel 4 and 5). It is worth noting that in the presence of cholate/ lipid mixtures, but not cholate alone, a substantial fraction of the AChR sediments as aggregates larger than dimers. The monomers ( lo) , dimers (Table I), and aggregates (data not shown) are all equally active after reconstitution.

Although AChR purified by all of the four methods described thus far was equally active (see below) after reconstitution, none of these four methods was fully satisfactory. Alkaline extraction of AChR-rich vesicles required the labo-

Acetylcholine Receptor Reconstitution a345

FIG. 3 . Electron microscopy of freeze-fracture replicas of native and reconstituted vesicles. The hnrs indicate 1 prn. A, purified AChH-rich vesicles. Note the dense packing of intramernbra- nous particles (ie. AChH). This sample had a protein concentration of 9.2 mg/ml and an AChH concentration of 3.5 X 10 .' M . Its carharnylcholine (10 '' M)-induced "Na' flux was 0.87 X IO1,' cpm/rnol of AChH and its apparent internal volume was 1.41 X 10'" cpm/mol

2 40001

14 16 IE 20 rng/rnl llpld In 2% CHOLATE

FIG. 4. Protection of ion channel function during solubilization of AChK by supplementary soybean lipids. I'elleted AChH-rich vesicles (1.67 mg of protein) were solubilized in 0.5 ml of 2 7 cholate. 1 0 0 r n M NaCI. 4 mM Na phosphate, pH 7.5, in the presence of the indicated amounts of soybean lipid added as a dispersion sonicated to clarity. After gentle agitation for 16 h at 4°C. the lipid in all samples was adjusted to 25 mg in a final volume of 1 ml. After 10 min agitation, samples were centrifuged for 30 min at 10" X g and then reconstituted by dialysis. :"Na' uptake in response to 1 0 ~ ' M carbamvlcholine was assayed.

rious purification of vesicles, and the final product contained endogenous lipids and some high molecular weight protein contamination. Toxin-agarose eluted batchwise easily gave a pure product, but at low vield. Con A-agarose was rather non- specific, requiring partially purified vesicles, and even then producing a product contaminated with high molecular weight glycoprotein. Sucrose gradient centrifugation gave pure dimers, but these did not differ functionally from mon-

of AChH. R. reconstituted alkaline-extracted AChH-rich vesicles. Note the less dense packing of AChH in this 25 rng/rnl lipid mixture. This sample had a protein concentration of 0.94 mg/rnl and an AChH concentration of 4.78 X 10 I' M . I t s 10 ' M carbarnvlcholine-induced flux was 3.43 X IO'.' cpm/mol of AChH and i t s apparent internal volume 12.3 X IO1, ' cpm/mol of AChH.

- R

h 4 0

I

n 5 30 4

o w 20

m U ..I

z IO 0

H

5 0

FIG. 5. Centrifugation of solubilized vesicles on a sucrose gradient containing 2 8 cholate and 5 mg/ml of lipid. Purified AChH-rich vesicles were solubilized and then centrifuged on a 5 to 2% linear sucrose gradient in cholate/lipid buffer as described under "Methods." Fractions numbered from the bottom of the gradient to the top were assayed for ""I-aBCT added to trace label AChR (-1 and for protein (M). The indicated fractions (25-pI aliquots) were selected for electrophoresis, as shown below.


omers (lo), highly purified vesicles were required, the yield was not high, and some high molecular weight protein contamination remained.

A satisfactory method for purifying solubilized AChR was developed by combining the toxin-agarose and Con A-agarose techniques. The toxin-agarose was highly specific, and Con A- agarose could be used (like DEAE was used in the absence of lipid) to bind AChR eluted from toxin-agarose during elution with benzoquinonium recycled overnight first through the toxin-agarose column and then through the Con A-agarose column. The Con-A agarose was washed free of benzoquinonium and then eluted with a-methyl-D-mannoside. The resulting product was pure AChR (Fig. 2, Gel 5) , fully functional, and obtained with ease in a reasonable yield (Table I).

Specific activities of purified and reconstituted AChR for binding '"'I-aBGT were measured and values ranging from 2000 to 8000 nmol/g of protein were obtained, but despite the use of appropriate cholate/lipid blanks in the Lowry protein assay, the measurements of protein concentration were often spuriously high. We therefore considered the appearance of samples after electrophoresis a more reliable index of purity.

Properties of Reconstituted AChR-Several lines of evidence suggest that there is a precise nucleation event as AChR and lipid begin to interact when the cholate is removed by dialysis. The apparent internal volume of vesicles formed by dialysis of a cholate/lipid mixture in the presence of increasing amounts of AChR increases, indicating active participation of the AChR in the vesicle-forming process (Fig. 7). Reconsti- tuted vesicles containing AChR are larger than most vesicles formed from soybean lipid alone, as shown by electron microscopy and equilibrium volume (2.2 pl/ml for vesicles made with only lipid versus 11 pl/ml for vesicles made in parallel with 4 X M AChR). Most AChRs in reconstituted vesicles, like those in native vesicles, have their toxin binding sites oriented toward the external surface (Table I). This indicates preferential orientation as well as active participation of the AChR in the vesicle-forming process.

Purified AChR functioned equally well after reconstitution in carbamylcholine-induced "Na' flux, independent of the method by which it was purified, or the amount of contaminating high molecular weight material present (Table I). The amount of "Na' flux observed (-4 x 1013 cpm/mol of "'I- aBGT binding sites) corresponds to a net inward flux of 64 mol of Na'/mol of AChR monomer. In frog muscle (28), 50,000 ions flow/channel opening; but under our conditions, Na' concentration is low (23 mM), and there is no membrane potential to drive Na' into the vesicles (29). AChR monomers functioned as well as dimers (10) or aggregates (data not shown). All the purified preparations shared the four subunits a, p, y, 6 recognized in conventionally purified AChR (Fig. 2). These results indicate that the functional unit is the AChR monomer composed of two a subunits and one each of /3, y, and 6 (13, 14).

Dose-response curves for reconstituted AChR purified by all methods were biphasic, like those for AChR in intact membranes (Fig. 6). The decrease in 'jNa' flux at high concentrations of carbamylcholine is due to desensitization. De- sensitization of AChR is demonstrated by the loss of response to carbamylcholine which occurs after a 10-s pre-exposure to carbamylcholine before the addition of "Na' (Fig. 6). AChR is thought to exist in a resting state which can be rapidly activated by agonist binding and then relaxes at a rate which increases with agonist concentration into a desensitized state with increased affinity for agonists (30, 31). The average concentration of carbamylcholine giving a half-maximal response was 2.8 f 0.6 X M. Since we have measured "2Na' amounts rather than flux rates, and since the response in

native vesicles is limited by both desensitization and equilibration (as will be discussed below), this may not accurately represent the K,, of carbamylcholine for the active form of T. californica AChR.

l A

I B

I C loot

I E

I F

0

Log concentration of CARBAMYLCHOLINE (M) -8 -7 -6 -5 -4 -3 -2

influx of "Na+. Carbamylcholine-induced influx of '>Na+ was meas- FIG. 6. Dose-response curves for carbamylcholine-induced

ured as described under "Methods." W, response to carbamylcholine; -, response after a 10-s preincubation with the same concentration of carbamylcholine before addition of 22Na+, demon- strating blockage of the response due to desensitization of the AChR. A, native AChR-rich vesicles; B, reconstituted native AChR-rich vesicles; C, reconstituted alkaline-extracted vesicles; D, reconstituted vesicles containing AChR purified on toxin-agarose; E , reconstituted vesicles containing AChR purified on Con-A agarose; F, reconstituted vesicles containing AChR purified by sucrose gradient centrifugation.

Acetylcholine Receptor Reconstitution 8347

Reconstitution of carbamylcholine-induced "lNa' flux was very efficient. In fact, the influx of "Na+ per AChR was much greater after reconstitution than before (Table I). Also, the flux/AChR was essentially a constant in reconstituted AChR, independent of the method of purification, and always at least 6-fold greater than the flux/AChR in native vesicles (Table I). A hypothesis which would explain these observations is that in native AChR-rich vesicles the concentration of AChR is so great that the "Na+ influx is not proportional to the amount of AChR, but instead the influx of "Na+ is limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles, where the concentration of AChR in the membrane is much lower, the influx of z2Na+ is proportional to the amount of AChR and influx is limited by desensitization of the AChR molecule. There is much evidence to support this hypothesis. We know that the lipid/protein ratio is much higher in reconstituted (>25) than native vesicles (-0.4) (32). And by direct observation we know that AChR are more densely packed in native membranes (Fig. 3). Moore et al. (20) have shown that 2"Na' flux in native vesicles is not proportional to the amount of AChR, but that excess AChR are present, and we have. confirmed this observation (Fig. 8). The amount of ""a+ I

influx in reconstituted vesicles, however, is directly proportional to the amount of AChR present. This can be seen either by reconstituting vesicles with increasing amounts of AChR present (Fig. 7), or by inhibition of AChR reconstituted in vesicles with toxin (Fig. 8). It is consistent with this hypothesis that the equilibrium volume of the reconstituted vesicles is much larger (3- to 4-fold) than the carbamylcholine-induced influx. However, the excess in equilibrium volume over carbamylcholine-induced flux in reconstituted vesicles is to a substantial extent due to the presence of lipid vesicles which contain no AChR. These may correspond to the very small vesicles lacking intramembranous particles in Figs. 1C and

- E, u I

+ Z O

N N

7000r /' 6000 -

'ooo-/o 0

40001 LC 0

concentration AChR ( x M 1 FIG. 7. Effect of AChR concentration on the uptake of "Na+

in reconstituted lipid vesicles. AChR was diluted in 2% cholate, 25 mg/ml soybean lipid buffer to the indicated concentrations of I2'I- aBGT binding sites. Then the samples were reconstituted by dialysis and 4O-pl aliquots assayed for M carbamylcholine-induced "Na+ influx (M) and apparent internal volume (M).

3B. Evidence for the presence of reconstituted vesicles which do not contain AChR is of two types. Immune precipitation of all AChR in a reconstituted vesicle sample by anti-AChR serum followed by anti-antibody left 63% of the equilibrium volume in solution (while 92% of the equilibrium volume remained in solution in a normal serum control sample in which no AChR was precipitated). Also, immune precipitation of AChR reconstituted in the presence of trace amounts of ['4C]phosphatidylcholine as well as soybean lipids precipitated only 26% of the labeled lipid, The equilibrium volume of native purified vesicles is also somewhat larger (2-fold) than the carbamylcholine-induced influx (Table I). This may be explained by the observation that purified vesicles contain substantial amounts of high molecular weight proteins (part of which may be Na'/K+-dependent ATPase) (15). These may be located in membrane fragments from parts of the cell which do not contain AChR and form vesicles which slowly take up "Na+ but do not respond to carbamylcholine.

The observation that the moles of unlabeled toxin required to block all the carbamylcholine-induced "Na' influx in reconstituted vesicles is essentially the same as the moles of Iz5I- aBGT binding sites present on the exterior surface of the vesicles is consistent with but does not prove that all of the reconstituted AChR have active cation channels (Fig. 8).

The simple linear nature of the blockage of flux produced by adding toxin is especially interesting (Fig. 8). I t is known that there are two kinetically equivalent Iz5I-aBGT binding sites on the AChR monomer located on its two a subunits (13). It is known that affinity alkylation of the AChR with MBTA blocks only half of the "'I-cIBGT binding sites (13). Table I1 shows that blockage of half of the L'51-~BGT binding sites in reconstituted AChR with MBTA blocks all of the carbamylcholine-induced 22Na+ flux. This is consistent with the observation that MBTA can block all of the carbamylcholine response in intact Electrophorus electric organ cells (33).

In order to account for these data, we considered four

T T

00 - 70 -

60 - 50 -

40 - 30 - 20.

I\ \$

0 IO 20 30 40 50 60 70 00 90 100 PERCENT EXTERNAL TOXIN BINDING SITES BLOCKED

FIG. 8. Inhibition of carbamylcholine-induced influx of "Na+ in native and reconstituted vesicles by addition of toxin. As described under "Methods," aliquots of native AChR-rich vesicles (M) or reconstituted vesicles (U) were incubated with N . naja siarnensis toxin to block agonist binding and then assayed for

M carbamylcholine-induced "Na' influx. The per cent external toxin binding sites blocked was calculated from the ratio of moles of toxin added to the moles of AChR facing the external surface of these vesicles.


TABLE I1 Effect of affinity alkylation with MBTA on toxin binding and "Nu' flux in native and reconstituted vesicles

"Na' Influx Total toxin binding External toxin bind- t?: ::- Of ex- induced bv slte concentratlon ing site concentration ward exter- terns' 'Oxin lo ' car-

"Na* Influx/mol binding bamylcho- AChR

line

M M < cpm cpm/moI Native vesicles 4.40 x 4.08 X IO"' 93 Native vesicles + MBTA 2.41 x

E O 698 4.28 x 10''

Reconstituted vesicles 6.28 x lo-' 4.85 X IOd7 77 50 2322 Reconstituted vesicles + MBTA

120 x 1Ol2

2.05 x 49.8 409 4.99 x 10"

4.02 X 10-~ 2.39 X 50.6 0 0

Monomer S t a t e s : RO R 1 R 2

MBTA site." e O t h e r t o x i n s i t e

y = degree of tox in occupancy of a l l sites = "1 + "2 2n3 2

where n o . . . 3 = f r a c t i o n i n e a c h state

I = f r a c t i o n of i n a c t i v e AChR monomers

Model RO R1 R2 R3

C o n s i s t e n t C o n s i s t e n t With Toxin w i t h I n h i b i t i o n MBTA I n h i b i t i o n

A a c t i v e i n a c t i v e active i n a c t i v e I = y t t

B a c t i v e 1 / 2 a c t i v e 1/2 active i n a c t i v e I = y + C a c t i v e i n a c t i v e i n a c t i v e i n a c t i v e I = 2y-y

D a c t i v e a c t i v e a c t i v e a c t i v e I = y

2 - + 2 -

FIG. 9. Predictions of different models for the effect of toxin binding on AChR function. Model A, each monomer has one channel and two toxin binding sites, but only the site at which MBTA reacts controls the channel. Model E , each monomer has two channels, each of which is controlled by a toxin binding site. Model C,

reasonable models for the structure and function of an AChR monomer with two toxin binding sites: Model A, each monomer has one channel and only the site at which MBTA reacts controls the channel; Model B, each monomer has two channels, each of which is controlled by a site; Model C, each monomer has one channel and toxin bound to either site blocks the channel; Model D, each monomer has one channel and both sites must be blocked to block the channel. Fig. 9 shows that the shape of the curve for inhibition of carbamylcholine-stimulated "Na+ flux by toxin predicted by each model can be calculated by a probabilistic approach similar to that used by Damle and Karlin (13) to analyze competition between toxin and MBTA binding. Fig. 10 shows the predicted curves for each model. It is clear that only Model A is consistent with both the toxin and MBTA data. Thus, we conclude that in the a2/3y6 subunit structure of the AChR monomer, there are two toxin binding sites and one cation channel, and that occupancy by toxin of only the site on the (Y subunit labeled by MBTA can prevent agonist-induced opening of the channel.

This very interesting insight into AChR function required the use of reconstituted vesicles in which the carbamylcholine- induced flux was directly proportional to the amount of active AChR, and could not have been obtained using native AChR- rich vesicles. The excess of AChR in native vesicles limits the carbamylcholine-induced flux/AChR in this experiment (Ta- ble 11) to 4.3 x 10l2 cpm/mol of AChR. In these reconstituted vesicles, carbamylcholine-induced flux was 120 X 10" cpm/ mol of AChR. (This value is higher than those reported

each monomer has one channel and two toxin binding sites, and toxin bound to either site blocks the channel. Model D, each monomer has one channel and two toxin binding sites and both sites must be blocked to block the channel.

% TOXIN BINDING SITES OCCUPIED

FIG. 10. Predicted shapes of curves for toxin blockage of carbamylcholine-stimulated *'Na+ flux, according to the models in Fig. 9.

elsewhere in this paper primarily due to the high efficiency of the y counter used only for this experiment.) This probably indicates the maximum flux which could occur per AChR before desensitization. At this rate, only 1.5% of the AChR in the native vesicles would have to remain active in order to

Acetylcholine Receptor Reconstitution a349

produce the 41% initial flux response observed after MBTA blockage. This explains the observations of Delegeane and McNamee (34) who observed that carbamylcholine-induced "Na flux remained after MBTA treatment of native vesicles and, therefore, concluded that both toxin binding sites control the opening of the channel.

DISCUSSION

By contrast with the situation several years ago (1, 2), AChR reconstitution is now a reproducible, easy, and efficient process. As long as the cation channel was protected by cholate/lipid mixtures, any of the purification methods described produced equally active AChR. Thus exposure to the antagonists N . naja siamensis toxin and benzoquinonium during purification had no lasting effect on AChR function. Purification of solubilized AChR shows that components of the native membrane which do not remain tightly associated with the AChR monomer during purification are not required for its function. The mechanism of the protective effect of soybean lipids in cholate is not clear. I t may provide a critical lipid cofactor or function in a less specific way to provide a pseudomembrane environment around the AChR. It is evi- dent, and not surprising, that the transmembrane cation channel of the AChR is lipid-dependent, whereas the ligand binding sites on the external surface of the AChR molecule are not.

Although some of the purification methods which we inves- tigated yielded preparations somewhat contaminated with high molecular weight material, all preparations contained the four subunit bands characteristic of affinity-purified AChR. This agrees with the results of Wu and Raftery (6) who observed four bands in their preparations, but contrasts with Huganir et al. (9) who observed no y and an a doublet, and Changeux et al. (8) who observed only a. The conflicting reports on subunit composition are resolved by the observation that proteolysis can nick /3, y, and S into small fragments so that only a is apparent on electrophoresis, but that the nicked fragments of /3, y, and 6 remain both associated with AChR and functional on reconstitution (40, 41). The observations that reconstituted AChR monomers exhibit as much carbamylcholine-induced "Na' flux as the naturally occurring covalently linked dimer, and that the reconstituted monomers are not noncovalently associated, have shown that the cation channel regulated by agonist binding is a part of the a&S subunit structure of the AChR monomer rather than a result of interacting monomers (10).

Because reconstituted AChR have much more carbamylcholine-induced flux/AChR than native vesicles and because the flux response to 10" M carbamylcholine is proportional to the amount of active AChR, reconstituted AChR provide a sensitive system for biochemical studies of AChR function. It has recently become possible to use these reconstituted vesicles to form planar bilayers (11). Although the planar bilayers still face technical problems which make them more difficult to study than vesicles, they have the potential of the great sensitivity and time resolution necessary to measure single AChR channels.

We have observed that monomers rather than dimers are the AChR functional unit (10). Here we provide evidence that there is a single cation channel per monomer and that blockage of only one of the two toxin binding sites, the one labeled by MBTA, can prevent agonist-induced opening of the channel. This means that when the other toxin binding site is occupied by toxin, binding of agonist to only the MBTA site can cause the channel to open. This is consistent with the conclusion that a single molecule of covalent agonist bound a t the MBTA site can cause opening of the channel in intact

Electrophorus electric organ cells (35). The S-shape of agonist dose-response curves (36) and the potentiating effect of car- bamylchofne on subsequent acetylcholine responses (37) had previously led electrophysiologists to believe that although liganding of a single AChR binding site can cause channel opening, liganding of two sites is much more effective. Our results suggest two possible models. One, only the MBTA site exerts primary control over opening or closing of the channel, and the other site has a weak allosteric interaction with it associated with cooperative activation and/or desensitization and perhaps the effects of local anesthetics. Two, either site can open the channel and they can cooperatively interact, but toxin binding or alkylation with MBTA of only the MBTA site prevents agonist binding at the other site from opening the channel.

Although they may differ in function, there is currently no evidence that the two a subunits per monomer differ in structure. At least the NH2-terminal 25 amino acid residues appear to be the same in all a chains (38, 39). It may be that the two a chains differ elsewhere in their amino acid sequence, or in carbohydrate or other covalent modification. Alterna- tively, the a chains may differ only in conformation, due to the asymmetric subunit structure of the monomer.

Acknowledgments-We thank Beth Bussett and especially Vernita Hudson for technical assistance, and Jan Littrell for typing the manuscript.

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Documents

Purification of Acetylcholine Receptors, Reconstitution ... · affinity chromatography using either Naja naja sia- mensis toxin I11 or concanavalin A coupled to agarose. Sucrose gradient