mann, S., and Patrick, J. (1986) Nature 319, 368-374. Boulter, J., O'Shea-Greenfield, A., Duvoisin, R. M., Connolly, J. G.,. Wada, E., Jensen, A., Gardner, P. D., ...
Vol. 266, No. 17, Issue of June 15, pp. 11192-11198.1991 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY (GI 1991 by The American Society for Biochemistry and Molecular Biology, Inc.
Neuronal Nicotinic Acetylcholine Receptors Expressedin Xenopus Oocytes Havea Pentameric Quaternary Structure* (Received for publication, December 24, 1990)
Rene Anand$, William G. Conroys, Ralf Schoepferll, Paul Whiting11 , and Jon LindstromS From The Salk Institute for Biological Studies, San Diego, California 92186-5800
We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer oftwo a4 acetylcholine-binding subunits and three (32 structural subunits. We also show that these expressed receptors bind ~-[~H]nicotine with high affinity, aretransported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (al),Bly6 stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo a1 or human a1 subunits, with Torpedo 81, y, and 6 subunits.
Nicotinic acetylcholine receptors (AChRs)’ are found both in muscle and neurons. Muscle-type AChRs from the electric organ of Torpedo have been well characterized and shown to be pentameric ACh-gated cation channels composed of two ACh-binding subunits termed a1 and one of each of three structural subunits termed(31,y, and 6 (Reynolds and Karlin, 1978; Lindstrom et al., 1979; Raftery et al., 1980). These subunits show extensivesequence homologies and are expected to have similar transmembrane orientations of their polypeptide chains. Based on high-resolution electronmicroscopy, these subunits are thought to be organized like barrel *Research in thelaboratory of J. L. issupportedinpart by the National Institutes of Health (NS11323), the National Science Foundation (BNS8819911), the Muscular Dystrophy Association, the California Chapter of the Myasthenia Gravis Foundation, the Council for Tobacco Research, USA, and the Smokeless Tobacco Research Council. R. S. was supported by a fellowship from the BoehringerIngelheim Fonds.The costs of publication of this articlewere defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” inaccordancewith 18 U.S.C. Section 1734 solely to indicate thisfact. j: Present address: 503 Clinical Research Building, 422 Curie Blvd., University of Pennsylvania, Philadelphia, PA 19104-6140. Tel. 215573-2859; Fax: 215-573-2028. To whom correspondenceshould be addressed. 5 Present address: Dept. of Biology, B-022, University of California, San Diego, La Jolla, CA 92093. B Present address: ZMBH, Molecular Neurobiology, Im Neuenheimer Feld 282, 6900 Heidelberg, FRG. 1) Present address: Merck Sharp & Dohme Laboratories, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, England. ’ The abbreviationsused are: AChR, nicotinic acetylcholine receptor; ACh, acetylcholine; aBgt, abungarotoxin;EGTA,[ethylenebis(oxyethylenenitrile)]tetraacetic acid; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; BAC, bromoacetylcholine; PBS, phosphate-buffered saline.
staves arounda central cation channel, in the order alplalyd (Kubalek et al., 1987) or alyal6(3 (Karlin et al., 1983; Blount and Merlie, 1989; Pedersen et al., 1990). NeuronalnicotinicAChRs,ontheotherhand, have remained poorly characterized until recently. AChRs have been immunoaffinity purifiedfrom brains of chickens (Whiting and Lindstrom, 1986a; Whiting et al., 1987a), rats (Whiting and Lindstrom, 1987a), and cattle (Whiting and Lindstrom, 1988) and shown to be composed of only twotypes of subunits. In the caseof AChRs from rat brains (Whiting et al., 1987b) and chicken brains (Whiting et al., 1991), the ACh-binding subunit of the major subtype was shown by N-terminal amino acid sequencing to beencoded by the a4 gene. Similarly, the structural subunit was shown to be encoded by the 0 2 gene (Schoepfer et al., 1988a). cDNAs for the individual subunits of neuronal AChRs from several different species have been cloned. cDNAs for the ACh-binding subunits a2 (Nef et al., 1988; Wada et al., 1988), a 3 (Boulter et al., 1986; Nef et al., 1988; Fornasari et al., 1990),’ a4 (Goldman et al., 1987; Nef et al., 1988), anda5 (Boulter et al., 1990), aswell as the structural subunits (32 (Deneris et al., 1988; Nef et al., 1988; Schoepfer et al., 1988a; Anand and Lindstrom, 1990), (33 (Deneris et al., 1989), and p4 (Duvoisin et al., 1989; Couturier et al., 1990), have been sequenced. Coexpression of a2, a3, or a4 subunits with 02 or (34subunits inoocytes (Boulter et al.,1987; Ballivet et al., 1988; Deneris et al., 1988, 1989; Wada et al., 1988; Duvoisin et al., 1989; Papke et al., 1989; Bertrand et al., 1988; Couturier et al., 1990) or in transfected fibroblasts3 results in the formation of functional AChRs when assayed electrophysiologically. Based on these studies, neuronal AChRs are currently thought to be composed of only two kinds of subunits: ACh-binding subunits termed a2, a3, etc., and structural subunits termed(32, p3, etc. However, the stoichiometry in which these subunits associate to form functional AChRs is not known. What subunit stoichiometry could be expectedfor neuronal nicotinic AChRs, given that they are composed of only two kinds of subunits? Because neuronal AChR subunits belong to the same gene superfamily as do subunits of muscle AChRs, onemightexpectthatneuronalAChRsexhibitthesame pentameric arrangement of two ACh-binding subunits and threestructuralsubunitsas muscle-typeAChRs. Alternatively, because neuronal AChRs appear to have only one kind of structural subunit permolecule rather than three kinds, as in muscle AChRs, they could assemble in a simple tetrameric arrangement of two ACh-binding andtwo structural subunits around a centralchannel.Otherarrangementsmight also occur. The known constraintsarethatneuronal nicotinic AChRs sediment onsucrose gradients as though theirmolecR. Anand and J. Lindstrom, unpublished. P. Whiting, R. Schoepfer, J. Lindstrom, and T. Priestly, unpublished.
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AChRs
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KF, 5 mM EDTA, 5 mM EGTA, 10 mM p-aminobenzamidine, 1 mM ular weights were slightly larger than muscle-type AChRs, and binding of subunit-specific mAbs indicates that there is phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 5 mM N-ethylmaleimide, and 2 mg/ml bovine serum albumin, pH 7.5), incubated more than onecopy of each type of subunit in the assembled a t 4 "C for 30 min, and centrifuged in a microfuge for 30 min to clear neuronal nicotinic AChR (Whiting et al., 1987a; Whiting and the cellular debris. Cleared lysate (250 p l ) from five oocytes was then Lindstrom, 1987a, 1987b, 1988). Determining the stoichiom- incubated with 100 pl of a 1:l slurry of mAb 270 coupled to Acti-Gel etry of neuronal nicotinic AChRs is also important for com- (Sterogene) for immunoaffinity purification, or with 100 p1 of a 1:l parison with other receptors in thegene superfamily, includ- slurry of bromoacetylcholine-Affi-Gel401 resin prepared asdescribed by Ellena et al. (1983), for 2 h a t 4 "C with shaking. The resin was ingthose for glycine, y-aminobutyric acid, and glutamate then washed three times with 1 ml of lysis buffer, and the affinity(reviewed in Barnard et al., 1987; Hollmann et al., 1989). To purified AChR was eluted off the mAb 270-Acti-Gel resin with 70 pl date, only the subunit stoichiometry of the glycine receptor of sample buffer or eluted off the BAC-Affi-Gel401 resin with 40 mM has been investigated. Using protein cross-linking, the glycine carbamylcholine chloride. Triton X-100 lysates of Torpedo and hureceptor hasbeen proposed to have apentameric arrangement man (ul-Torpedo hybrid AChR expressed in oocytes were not purified of three ligand-binding and two structural subunits (Langosch on BAC, but, used directly on sucrose gradients. Sucrose Gradient Sedimentation-AChRs from embryonic day 18 et al., 1988). chicken brains were extracted from crude membranes for 2 h, using In this studywe have determined the subunit stoichiometryan equal volume of 2% Triton X-100 in 50 mM Tris, pH 7.5, 1 mM of the neuronal AChR formed when chicken a4 and (32 sub- EDTA, 1 mM EGTA, 5 mM p-aminobenzamidine, 5 mM iodoacetaunits are coexpressed in Xenopus oocytes, by metabolically mide, and 0.5 mM phenylmethylsulfonyl fluoride. Torpedo AChRs labelingthe expressedAChRwith["S]methionine. These used as internal controls were similarly extracted with Triton X-100 from crude membranesof Torpedo electricorgan. The Torpedo AChRs AChRs were affinity purified and shown to cosediment on (-77 nM) were identified using "'I-labeled nBgt (10 nM) and then sucrose gradients with AChRs solubilized from brain. Quan- diluted into either brain extracts or oocyte lysates to give -20,000titation of the amount of radioactivity in individual subunits 40,000 cpm of Torpedo AChRs marked with '2'II-labeled nBgt per of AChRs, obtained after separation by electrophoresis on gradient. Aliquots (450 p1) of either the brain extracts or the oocyte polyacrylamide gels containing sodiumdodecyl sulfate (SDS- lysates containing chicken neuronal AChRs were layered onto 11-ml PAGE), and correction for the numberof methionines in each sucrose gradients (5-2096 sucrose (w/w), in 10 mM sodium phosphate buffer, pH 7.5, containing 100 mM NaCI, 1 mM NaNn, and 0.5% subunit reveals that these AChRs are composed of two a4 Triton X-100). Similarly, BAC-Affi-Gel-purified [:'"S]methionine-laACh-binding subunits and three p2 structural subunits. Using beled chicken AChRs were layered onto11-ml sucrose gradients, the samemethodology, we also show that muscle-type AChRs except 100 p1 of 77 nM unlabeled Torpedo AChRs was included in the made by coexpression of either Torpedo a l , (31, y, and 6 aliquots. The gradients were centrifuged for 17 h a t 41,000 rpm in a BeckmanSWTi-4lrotor. 26 dropfractions (-300 pl) were subsesubunits or humana1, Torpedo (31, y,and 6 subunitsin quently collected from the bottom of the tubes and used for further oocytesassemble to form the expected pentameric AChRs analysis. Fractions from gradients containingTorpedo AChR tagged composed of two a1 subunits and oneof each of (31, 7,and 6 with '2511-labeled cuBgt were counted in the y counter to determine the subunits. Thus, this method of determining subunit stoichi- sedimentation of the Torpedo AChRs. 50-rl aliquots of fractions from ometryshould have broad applicability to other receptors gradientscontaining[~'5S]methionine-labeled chickenAChRs were added to 2 ml of T2 5% scintillation cocktail, and the radioactivity whose cDNAs have been cloned. was determined by liquid scintillat,ion counting. The brain AChRs and the neuronal AChRsfrom oocytes in the gradient fractionswere quantitated by ~-[:'H]nicotine bindingusing a mAb-immobilization mAbs and cDNAs-mAb 270 to chicken 82 subunits and mAb 289 assay(WhitingandLindstrom, 1986b). Gradientfractions were to chicken a 4 subunits were described by Whiting etai. (1987a). mAb shaken overnight at 4 "C with 25 pl of a 1:l slurry of goat anti-rat Ig210 binds to the main immunogenic region (068-76) of Torpedo n Sepharose and 1 pl of a stock solution of mAb 270. The Sepharose and human a (Das and Lindstrom, 1989; Saedi et ai., 1990). The was washed three times with1 ml of 10 mM sodium phosphate buffer, cDNA forchicken a 4 (Whiting et al.,1991) is equivalent to that pH 7.5, containing 100 mM NaCland 0.5% Triton X-100 (PBSdescribed by Nef et ai. (1988). The cDNAfor chicken 8 2 was described Triton), and incubated with 10 nM ~-[:'H]nicotine for 15 min at room by Schoepfer etal. (1988a). The cDNA for human muscle n l subunits temperature. The Sepharose was then washed three times with 1 ml from the TE671cell line was described by Schoepfer etal. (1988b). of PBS-Triton by repeated centrifugation and resuspension. A soluExpression of Chicken Neuronal AChRs, Torpedo AChRs, and Hu- tion of 2.5% SDS and 5% 2-mercaptoethanol (100 pl) was added to man Muscle al-Torpedo HybridAChRs in Oocytes-The cDNAs the Sepharose pellets, the entire contentswere then added to 5 ml of corresponding to chicken a4 (Nef et al., 1988; Whiting et ai., 1991) T2 5% scintillationcocktail,andthe radioactivity determined by and 82 (Schoepfer et al., 1988b) and human a1 (Schoepfer et al., liquid scintillationcounting.Controltubescontaining onlybuffer 1988a) were cloned into amodified SP64T(Meltonet ai.,1984) solution were included in each assay to determine nonspecific L-[.'H] expression vector using standard DNA cloning techniques. Torpedo nicotine binding. The extent of contamination of each tube by residc u l , 81, y, and 6 cDNA cloned into SP64T were kindly provided by ual '"'I-labeled tvBgt was determined by y counting the tubes before Dr. Toni Claudio (Yale University). RNA was synthesized in vitro the ~-[,"H]nicotine binding assaywas performed. In no case did the according to Melton et ai. (1984) using the SP6 RNA polymerase. gradient fractions contain >lo0 cpm of '"I, thus indicating minimal Oocytes were prepared for microinjection as describedbyColman contamination by '"I when counting "H. The sedimentation of Tor(1984) and injected with -1.6 ng of cRNA of each of the chicken pedo AChRs in gradients containing [:"S]methionine-labeled AChRs neuronal a4 and 82 or Torpedo a l , 81, y,6 and human a1 subunits. was determined by radioimmunoassays. 10-pl aliquots of each fraction Oocytes were incubated for 4-5 days afterinjection in Barth's solution were incubated at 4 "C overnight with 5 p1 of normal rat serum, 5-pl (Colman, 1984) a t 19 "C. For metabolic labeling of expressed neuronal stocksolution of mAb 210, and 2 nM '""I-labeled nB@in afinal AChR, oocytes were incubatedinBarth'ssolutioncontaining 0.5 volume of 100 p1 PBS-Triton. Following a 2-h incubation at 4 "C with mCi/ml of [:"S]methionine (-1,000 Ci/mmol, Amersham) immedi100 pl of goat anti-rat IgG, the samples were diluted with 1 ml of ately after injection, for 4-5 days, and 2 days for muscle AChRs. PBS-Triton, centrifuged, thepellet washed with 1 ml of PBS-Triton, Surface expression of neuronal AChRs in oocytes was determined by and the radioactivity in thepellet measured by y counting. For SDSincubating oocytes in 200 p1 of Barth's solution containing 10 p1 of PAGE, [:'"S]methionine-labeled AChR was affinity-purified from normal rat serum and 10 nm of '"I-mAb 270 for 2 h at,19 "C. Surface pooled fractions containing assembled AChR by the addition of 100 expression of muscle AChR was determined by incubating oocytes p1 of a 1:l slurry of either BAC-Affi-Gel 401, mAb 270-Acti-Gel, or with 2 nM '"'I-labeled n-bungarotoxin (aBgt) in 200 p1 of Barth's mAb 210-Acti-Gel. After a 2-hincubation a t 4"C, the resin was solution containing normal fetalcalf serum for 2 h at 19 "C. Nonspe- washedtwo timeswith 1 ml of PBS-Triton and the immobilized cific binding was determined by incubating noninjectedoocytes under AChR was eluted from the resin with 70 p1 of sample buffer. The similar conditions. samples were electrophoresed on 10% polyacrylamide gels in SDS at Purification of AChR from Oocytes-Oocytes were homogenized in 30 mA for 2-3 h, and the gels were fixed, treated for fluorography lysis buffer (1.5% TritonX-100, 50 mM Tris, 100 mM NaC1, 100 mM with Enlighten (Du Pont-New Englaad Nuclear), dried, and exposed MATERIALS AND METHODS
Pentameric Neuronal Nicotinic AChRs
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to x-ray films a t -70 "C for 1-16 h. The gels were then aligned with the x-ray film and gel slices containingthe individual subunits excised, incubated with 5 mlof T 2 5% liquid scintillation cocktail overnight, andtheamount of radioactivity determined by liquid scintillation counting. Western Blots with mAb 270 and 289"AChRs affinity purified from oocytes on mAb 270-Acti-Gel resin were resolved into subunits by SDS-PAGE, blotted as previously described(Whitinget a/., 1987a), and probed overnight a t 4 "C with either "'I-labeled mAb 270 (-2 X 101'cpm/mol, 2 nM) or with mAb 289 (2 nM). Blots probed with mAb 289 were washed with PBS-Triton andprobed with '"JI-labeled mouse anti-rat IgG (-2 X 10" cpm/mol, 1 nM) for 2 ha t 4 "C. After washing, hinding of mAb 270 or 289 was visualized by autoradiography a t -70 "C. Treatment of ["SIMethionine-labeled AChR Subunits with Endo~lycosidases-Aliquots of affinity-purified[:%]methionine-labeled AChRs from oocytes were digested a t 37 "C overnight with either 10 milliunits of endoglycosidase H, 1 unit of endoglycosidase F, or 4 units of glycopeptidase F, as described by the manufacturer (Boehringer Mannheim Biochemicals), after denaturation of the AChRs in 1% SDS at90 "C for 2 min and dilution of the samples such that the final concentration of SDS was 0.1% in the presence of 0.5% Triton x-100.
methionine-labeled AChR also revealed two sets of doublet bands corresponding to the approximate molecular weightsof the a4 and p2 subunits obtained by Western blotting. Both endoglycosidase H and endoglycosidase F failed to completely deglycosylate these individual subunits, andwe obtained multiple bands corresponding to partially deglycosylated subunits (results not shown). These results probably reflect the inability of both of these enzymes to completely digest the complex carbohydrate moiety of the glycosylated subunits in oocytes. However, glycopeptidase F deglycosylated both of the subunits, resulting in the closely spaced doublets for each of the subunits coalescing into single bands for each of the subunits (Fig. 2). To quantitate theexpression of AChRs on theoocyte outer membrane, we determined the surfacebinding of ""I-labeled mAb 270. Oocytes injected with a4 and p2 cRNAs showed -15,000 cpm of ""I-labeled mAb 270 specifically bound per oocyte (after subtraction of -500 cpm of nonspecific binding obtained from noninjectedoocytes).Based on the specific cpm/mol),and activity of the '"I-labeled mAb 270 (2 X assuming three binding sites for mAb 270 per AChR molecule, RESULTS we obtained -2-3 fmol of neuronal AChR expressed on the Biochemical Characterization of Chicken a4p2 AChRs Ex- surface of each oocyte. ~-[:'H]nicotine binding assays perpressed in Xenopus Oocytes-Proper expression of chicken formed with neuronal AChR affinity purified on mAb 270AChRalso neuronal AChRs in oocytes injected with RNAs for a4 and Acti-Gel andassuming two bindingsitesper p2 subunitswas verified by Western blotting. Chicken AChRs indicate that a total of-2-3 fmol of AChR is expressed in expressed in oocytes were solubilized and then immunoaffin- each oocyte. Thus AChRs expressed in oocytes are efficiently assembled and transported to the oocyte outer membrane ity purified on mAb 270-Acti-Gel resin, eluted with sample surface and attain high-affinity binding of mAb 270 and Lbuffer, and fractionated by SDS-PAGE. Following electrophoretic transferof the proteins ontoImmobilon membranes, ["Hlnicotine. Additionally, the binding of mAb 270 confirms the blots were incubated with either ""I-labeled mAb 270 our previous observation that the epitope for this mAb is (specific for the p2 subunit) or with mAb 289 (specific for the located on the extracellular surface of AChRs expressed in a4 subunit), followed by '2'II-labeled mouse anti-rat IgG, and PC12 cells (Whiting et al., 1987~).We did not attempt a visualized by autoradiography (Fig. 1).Using mAb 270, we precise quantitative comparison of the number of binding obtained a doublet band of -50 kDa, a molecular mass which sites for mAb 270 and nicotine on oocyte membranes because of measurement of the corresponded approximately to the expected size of the glyco- it seemed likely thattheerrors amounts of binding and the specific activities of the ligands sylated p2 subunit deduced from its cDNA sequence. Similarly, mAb289 identified a closely spaced doublet band of would exceed those necessary for meaningful estimation of by '2sII-labeledmAb -75 kDa, a molecular mass also corresponding closely to the the stoichiometryof p2 subunits detected a4 subunits detected by ~-[:'H]nicotine. 270 and expected size deduced from the a4 cDNA sequence. In both Having shown expression of substantial amounts of neucases we suspected that the doublets for each subunit were ronal AChRswith the expected subunit composition and the result of heterogeneous glycosylation. T o verify this, we nicotine binding properties in oocytes injected with chicken metabolically labeled the expressed AChR with ["S]methionine and attempted deglycosylate to the expressed AChR with three different endoglycosidases. Affinity purification of ["'SS] Glycopeptidase F -
+
Probed with:
anti-P2 anti-a4 mAb 270
mAb289
MW (kD)
13075 -
50 -
I
a
m
P
m
39 FIG. 1. Western blots of chicken AChRs purified from oocytes. Chicken AChR from oocytes were purified on mAb 270-Actiof the Gel andfractionated by SDS-PAGE.TheWesternblots fractionated AChR were probed with either ","I-labeled mAb 270 or mAb 289 followed by "'I-labeled anti-rat IgG. The binding of the mAbs was visualized by autoradiography.
FIG. 2. Fluorographs of glycopeptidase F digestion of ["SI methionine-labeled chicken AChRs expressed in oocytes. ["SI Methionine-labeledchickenAChRsaffinity purified on mAb 270Acti-Gel were digested with glycopeptidase F, fractionated by SDSPAGE, and the subunitsvisualized by fluorography.
Pentameric Neuronal Nicotinic AChRs
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4500 a4 and p2 cRNAs,we compared the sedimentation behavior A of Triton X-100-solubilized AChRsisolatedfromchicken brain and fromoocytes expressing neuronal AChRs on 5-20% sucrose gradients, using Torpedo AChR as an internal standard. As can be seen in Fig. 3, AChRs isolated from chicken brain cosediment with neuronal AChRsexpressed in oocytes, both of which sediment faster than Torpedo AChR monomers. This suggests that the size andsubunitstoichiometry of AChRs expressed in oocytes is the same as those expressed in chicken brain. Subunit Stoichiometry of Expressed ChickenNeuronal a4P2 AChRs-We determinedthesubunitstoichiometry of the chicken neuronal AChR expressed in oocytes by quantitating the relative amounts of radioactivity in subunits metabolically labeled with [%]methionine. Oocytes injected with chicken for 4a 4 and p2 cRNAs were incubated with [35S]methionine 5 days, following which Triton X-100-solubilized AChR was affinity purified on bromoacetylcholine (BAC)-Affi-Gel 401 resin and eluted off using 40 mM carbamylcholine chloride. T h e affinity purification step was introduced to purify the properly assembled AChR from the unassembled subunits. T h e BAC-Affi-Gel-purified AChR was then sedimented on a 5-20% sucrose gradient to further separate the assembled ..? 1000 AChR from any assembly intermediates which might have ii .affinity for BAC. The sedimentation of the assembled ["SI methionine-labeled AChR was determined by liquid scintil2 500 lation counting of aliquots from each of the sucrose gradient fractions, the resultsof which are shown inFig. 3C. The peak fractions containing the [35S]methionine-labeled assembled 4 0 AChR correspondsexactly to thepeak fractionsof the L - [ ~ H ] 14000 nicotine binding activity from both chicken brain and AChRs expressed in oocytes. The two peak fractions (fractions 17 .$ 12000 and 18, Fig. 3C) were collected andthe[3sS]methioninec e labeled AChRs were either affinity purified using mAb 270p. 10000 Acti-Gel or BAC-Affi-Gel resin. This step served two pur3 8000 poses: l) to concentrate the AChR from the sucrose fractions, allowing for quantitative loading of the samples for SDSwoo PAGE; and2) t o provide two independent meansof purifying .-g c the sedimented AChRs to determine if there were any bias in 4000 the stoichiometry in favor of one of the subunits. We expected the assembled AChRs that bound BAC to attain that capacity 5 2000 through the a4 subunit, and the assembled AChRs that bound 0 , mAb 270 to be due to the /32 subunit's affinity for mAb 270. 0 5 10 15 20 25 30 35 40 Thus, we expected to obtain an altered ratio of a4 to /32 Fraction Number subunits using the two purification methods if we failed t o purify the assembled AChRs from assembly intermediates or FIG. 3. Sucrose gradient sedimentation analysis of chicken oligomerized subunits that may have cosedimented with the neuronal AChRs. ChickenbrainAChRsandchickenneuronal oocytes were sedimentedon 5-20% sucrose assembled AChRs. Fig. 4 shows the fluorographsof the [3sS] AChRsexpressedin methionine-labeled AChR subunitsafterfractionation by gradients asdescribed under "Materials andMethods." Fractions are SDS-PAGE. We obtained very similar ratios for both BAC- numbered from the bottom of the gradients. Torpedo AChR tagged with 12sII-labeledaBgt was included in the gradients shown in A ( 0 ) Affi-Gel- and mAb 270-Acti-Gel-purified AChR. By quanti- and E , and unlabeled Torpedo AChR was included in the gradient tation of the amount of radioactivity by liquid scintillation shown in C. Neuronal AChRs were quantitated by ~-[:'H]nicotine counting in each of the doublet bands corresponding to the binding in an immunoimmobilization assay using mAb 270 coupled to agarose to bind the AChRs through their structural subunits(m). individual subunits and after normalizing each subunit for the number of methionine residues per subunit (chicken a4 The sedimentation of the unlabeled Torpedo AChR was determined radioimmunoassays asdescribed under "Materials and Methods." has 17 methionine residues and chicken ,82 has 13 methionine by The sedimentation of the ["'SS]methionine-labeled AChR was deterresidues) we obtained a ,8/a ratio of 1.46 & 0.13 (Table I). mined by counting 50-pl aliquots of the gradient fractionsin a liquid Since this ratio corresponds closely to the theoretical value of scintillation counter (A).A , Triton X-100 extracts of chicken brain and Torpedo AChRs; E , Triton X-100 extracts of chicken neuronal 1.5 for an AChR composed of three structural subunits and two ACh-binding subunits, we conclude that coexpression of AChRs expressed in oocytes; C, Triton X-100 extracts of [%]methichickenneuronalAChRsexpressedin oocytes and chicken neuronal a4 and p2 subunits in oocytes results in the onine-laheled affinity purified on BAC-Affi-Gel. formation of pentameric neuronal AChRs. Subunit Stoichiometry of Expressed Muscle-type AChRsTo test the validity of this method, we coexpressed combina- AChR with ["sS]methionine for 2 days. We obtained -2-3 tions of either Torpedo a1 or humana1 subunits withTorpedo fmol surface expression of muscle-type AChR, as determined Dl, y , and 6 subunits inoocytes and metabolically labeled the from binding of lZ5I-labeledaBgt. Triton X-100-solubilized
rt
h
-6 3 s
UI
11196
Pentameric Neuronal NicotinicAChRs
FIG.4. Fluorographs of [""Slmethionine-labeled chicken neuronal AChRs fractionated by SDS-PAGE. ["'SIMethioninelaheled chicken neuronal AChRs were affinity purified from pooled fractions 17 and 18 (Fig. 3C) using either RAC-Affi-Gelor mAb 270Acti-Gel andfractionated by SDS-PAGE, and the subunits were visualized by fluorography.
AChRs were then sedimented on 5-20% sucrose gradients without any furtherpurification. This was necessary because we found that both Torpedo and human al-Torpedo hybrid AChRs had low affinityfor BAC-Affi-Gel 401. Since both Torpedo and human al-Torpedo AChRs synthesized in oocytescosedimentwith native Torpedo AChR monomers (Saedi et al., 1990; Conroy et QL, 1990), we collected fractions corresponding to the native Torpedo AChR monomers included in these gradients as internal controls (Fractions 18, 19, and 20, Fig. 1).AChRs were immunoaffinity purified from these fractions using mAb 210-Acti-Gel (which binds to the main immunogenic region a68-76 on both Torpedo a1 and human a1 subunits; Das and Lindstrom, 1989). Fig. 5 shows the fluorographs of [""Slmethionine-labeled AChR subunits after fractionation by SDS-PAGE. After quantitation of the amount of radioactivity in each band corresponding to the individual subunits and normalizationsof these values forthe number of methionine residues per subunit (Torpedo a l , Dl, y, and 6 subunits have 15, 9, 8, and 11 methionines, respectively; human a1 subunit has 14 methionine residues), we obtained a al:/3l:y:fi ratio of 2.101.13:1.14:1.00 for the Torpedo AChR and 1.94:1.09:1.19:1.00 for the human a1-Torpedo hybrid AChR. In both cases the values obtained are close to the theoretical ratio of 2:l:l:l expectedformuscle-type AChRs, confirming the validity of this method. DISCUSSION
We have used a novel approach to determine the subunit stoichiometry of chicken neuronal AChRs composed of a 4
and 62 subunits expressedinXenopus oocytes. First,the expressed AChRswere shown to correspond to nativeAChRs. Then, by metabolically labeling the expressed AChRs with [%]methionine and quantitating therelative amount of label in each of the subunits, we could use the known number of methionines in each subunit sequence to calculate the ratio of /32/a4 subunits in an AChR monomer. From the molecular weights of the subunits and the apparent molecular weights of the AChRmonomers, thesubunit composition of the monomers was calculated. By a similar process, the subunit stoichiometry of other members of the ligand-gated ion channel gene superfamily of known subunit composition could be determined. Here we also used the method successfully on muscle-type AChRs. Coexpression of a4 and p2 subunitswas shown to produce native AChRs. Previously, by N-terminal aminoacid sequencing of thecomponentsubunits, we showed that the two subunits that compose the most abundant subtype of AChR with high affinity for nicotine in the brains of chickens or rats correspond to the cDNAs a4 and /32 (Whiting et al., 1987b; Schoepfer et QL, 1988a; Whitinget al., 1991). By affinity labeling, we showed that the a4 subunits formed part or all of the ACh binding sites of these AChRs (Whiting and Lindstrom, 198713). Coexpression of a4 and /32 cDNAsin Xenopus oocytes or mouse fibroblasts produced AChRs with ACh-gated cationchannel activity and cholinergicligandbinding properties expected of native AChRs (Papke et al., 1989; Ballivet et al., 1988).:'Here we showed that coexpression of a4 and p2cDNAs in Xenopus oocytes efficiently produced AChRs of the same subunit composition which we had observed in AChRs purified from brain and which could bind nicotine and were of the same apparent molecular size as AChRs extracted from brain. Metabolic labeling permitted determination of the ratio of a4 to /32 subunits in an AChR monomer. The expressed AChRs were labeled with [""Slmethionine over a periodof 45 daysto provideuniformlabeling of the 17 methionine 13 methionine residues in /32. The large residues in a4 and the number of methionines provides a large signal and buffers against peculiarities of any single residue. Our preliminary attempts to determine subunit stoichiometry used '"I labeling of tyrosines in purified AChRs (Whiting et al., 1989). This method gave the expected results for Torpedo AChRs if both SDS and ureawere used to denature theAChRs and provide access to buried tyrosines; but with purifiedbrain AChRs this method suggested that there were equal numbers of a and p subunits, presumably because with brain AChRs, some tyrosine residues remained inaccessible under these conditions. We had previously observed an excess of an unassembled a1 subunit with immatureligand-binding properties in a muscle cell line (Conroy et dl., 1990) and had observed that some partially assembled subunit combinations could be detected when Torpedo AChRs were expressedinXenopus oocytes
TABLE I Computation of subunit ratios for [""Slmethionine-labeled ~ 4 0 AChRs 2 purified by two methods Affinity column RAC-Aff-Gel
270-Acti-Gel
mAb
1
2
3
4
6
7
02 (cpm)
1,.140
3,UbY
2,834
14,~4n
13.1 I I
8,b IY
tu4 (cpm)
1,720
z,n33 2,482
2,891
2,644
14,731
12,730
6,720
1.39 1.32
1.50
1.41
1.33
1.56
1.68
Experiment
Normalized ratio (32 (cpm) x '7 tu4 (cpm) 13 ratio Mean f S.E. ( n = 7)
5
1.46 f 0.13
Pentameric Neuronal NicotinicAChRs
AChRs are thought tobe oriented like barrel staves arounda central cation channel, and the two a1 subunits are thought to be separated by structural subunits (Karlin et ~ l . ,1983; Kubalek et ~ l . 1987; , Blount andMerlie, 1989; Pedersen et al., 1990). Neuronal AChRsprobably exhibit a similar apaPP arrangement. It may be that a similar pentameric arrangement of subunits will be exhibited by all of the neuronal AChR subtypes andby other members of the ligand-gated ion channel gene superfamily. Protein cross-linking methods usingpurified glycine receptors have been used to suggest that this receptor is also a pentamer. However, an arrangement of three ligand-binding subunits and two structural subunits was proposed for these receptors (Langosch et d . , 1988). Using the method of metabolic labeling of expressed subunit cDNAs described here, including careful comparison with native receptors and purification of fully assembled receptors, it should be possible to determine the subunit stoichiometry of other receptorsin the ligand-gated ion channel gene superfamily.
6-
&I a1
11197
-
FIG. 5. Fluorographs of[36S]methionine-labeled muscletype AChRs fractionatedby SDS-PAGE. ['"SjMethionine-laheled Torpedo AChR or human al-Torpedo AChR expressed in oocytes were affinity purified on mAb 210-Acti-Gel from pooled fractions corresponding to where Torpedo AChR monomers sedimenton a 5-20?; sucrose gradient (fractions 28, 19, and 20, as in Fig. 1) and fractionated by SDS-PAGE, and the subunits were visualized by fluorography.
Acknowledgments-We thank Kathy Havilan, Edwin Ozawa, and Sheri Laughon for excellent technical assistance and Lisa ChurchillRoth for her unending patience during the preparation of this manuscript. Note Added in Proof-After submission of this paper,Ellis Cooper, et al. also concluded that chicken neuronal AChRs have the subunit stoichiometry (n4)2(P2)3using an indirect method dependingon the different conductancesof wild type and in vitro-mutagenized AChRs expressed in oocytes(Cooper, E., Couturier, S., and Ballivet, M. (1991) Nature 350, 235-238).
(Saedi et d., 1991). Therefore, subunits of expressed AChRs were carefully purifiedto select for functional AChRs capable REFERENCES of binding toa cholinergic ligandaffinity column and selected for cosedimentation with brain AChRs to ensure proper sub- Anand, R., and Lindstrom, J. (1990) Nucleic Acids Res. 18, 4272 unit assembly before immunoprecipitating with subunit-spe- Ballivet, M., Nef, P., Couturier, S., Rungger, D., Bader, C., Bertrand, D., and Cooper, E. (1988) Neuron 1,847-852 cific mAbs to avoid any precipitationof unassembled subunits that could affect the apparent subunit ratio. The ratio of [""SI Barnard, E., Darlison, M., and Seeburg, P. (1987) Trends Neurosci. 10,502-509 methionine in the subunits was measured to avoid difficult Bertrand, D., Cooper, E., Bader, C., Rungger, D., Nef, P., Couturier, quantitative comparisonswith the amount of ligand binding S., and Ballivet, M. (1988) in Nicotinic Acetylcholine Receptors in sites involving precise specific activitymeasurements, etc. the Nervous System, NATO-ASI Series H2.5 (Clementi, F., Gotti, After normalizing for the numberof methionines per subunit, C., and Sher, E., eds) Springer-Verlag, Berlin/Heidelberg this method gave the expected a&6 stoichiometric ratio for Blount, P., and Merlie, J. (1989) Neuron 2,349-357 Torpedo AChR of a:P:y:6,2.1:1.1:1.1:1.0. For neuronalAChRs, Boulter, J., Connolly, J., Deneris, E., Goldman, D., Heinemann, S., and Patrick, J. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 7763the subunit ratiofor a4$2 was 1:1.46 0.13. 7767 Determination of the apparent molecular weights of neu- Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heineronal AChR monomers permitted calculation of the number mann, S., and Patrick, J . (1986) Nature 319, 368-374 of a4 andP2 subunits in an AChR monomer. We found that Boulter, J., O'Shea-Greenfield, A., Duvoisin, R. M., Connolly, J. G., Wada, E., Jensen, A., Gardner, P. D., Ballivet, M., Deneris, E. S., AChRs from chicken brains or oocytes expressing a4 and p2 McKinnon, D., Heinemann, S., and Patrick,J. (1990) J. Riol. Chem. subunits sedimented on sucrose gradients slightly more rap265,4472-4482 idly than monomersof Torpedo AChR. The proteinmolecular Colman, A. (1984) inTranscriptionand Translation, A Practical weights of Torpedo AChRmonomers calculated from the Approach. (Hames, B. D., and Higgins, S. J., eds) pp. 271-302, IRL subunit composition ( c ~ l ) ~ P l y 6 a n d t h calculated e subunit Press, Oxford molecular weights a1 = 50,116; D l = 53,681; y = 56,279; and Conroy, W. G., Saedi, M. S., and Lindstrom, J. (1990) J . Riol. Chem. 265,21642-21651 6 = 57,565 is 267,757 (Noda et al., 1983). Addition of two '"IS., Erkman, L., Valera, S., Rungger, D., Bertrand, S., labeled aBgt molecules results inamolecularweight of Couturier, Boulter, J., Ballivet, M., and Bertrand, D. (1990) J. Riol. Chem. 283,757. Glycosylation of individual subunits adds an addi265, 17560-17567 , for atotal of 303,366. The Das, M., and Lindstrom, J. (1989) Riochem. Riophys. Res. Commun. tional 19,609 (Nomoto et ~ l . 1986), 165,865-871 molecular weight of a chicken AChR, composed of two a 4 subunits of 68,400 deduced protein molecular weightand three Deneris, E., Boulter, J., Swanson, L., Patrick, J., and Heinemann, S. (1989) J . Riol. Chem. 264,6268-6272 p2 subunitsof 54,000 deduced molecular weight (Schoepferet Deneris, E., Connolly, J., Boulter, J., Wada, E., Wada, K., Swanson, ~ l . 1988) , and a similar amount of glycosylation, would be L., Patrick, J., and Heinemann,S. (1988) Neuron 1 , 4 5 4 4 318,409. This corresponds very well with the observed sedi- Duvoisin, R., Deneris,E., Patrick, J., andHeinemann, S. (1989) Ncuron 3, 487 40G montltinn proportioo. Thus, wo concludo that tho oubunit Ellena, J. F., Blazing, M. A., and McNamee, M. G. (1983) Biochemstoichiometry of these neuronal AChRs is ( ( ~ 4 ) ~ ( @ 2 ) : ~ . istry 22,5523-5535 Neuronal AChRs with an ( ~ ~ 4 ) ~ (subunit 8 2 ) ~ stoichiometry Fornasari, D., Chini, B., Taroconi, P., and Clementi, F. (1990) Neuthus seem to share the same pattern of two ACh-binding and rosci. Lett. 111, 351-356 three structural subunits observed in the (al)r/31y6 subunit Goldman, D., Deneris, E., Luyten, W., Kochhar, A., Patrick, J., and stoichiometry of muscle-type AChRs. The subunitsof muscle Heinemann, S. (1987) Cell 48, 965-973
*
11198
Pentameric Neuronal Nicotinic AChRs
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