2410 zyxwvutsrqpon Biochemistry zyxwvu 1987, 26, 2410-2418 Henderson, L. E., Hewetson, J. F., Hopkins, R. F., Sowder, R. C., Neubauer, R. H., zyxwvutsrq & Rabin, H. zyxwvutsr (1 983) J. Immunol. Holley, R. W., Armour, R., & Baldwin, zyxwvut J. H. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 1864-1866. Holley, R. W., Armour, R., & Baldwin, J. H. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5989-5992. Hunkapiller, M. W., & Hood, L. E. (1983) Science (Wash- ington, D.C.) 219, 650-659. Kaighn, M. E., Narayan, K. S., Ohnuki, Y., Lechner, J. F., & Jones, L. W. (1979) Invest. Urol. 17, 16-23. Kaighn, M. E., Lechner, J. F., Babcock, M. S., Marnell, M., Ohnuki, Y., & Narayan, K. S. (1980) in Models for Prostatic Cancer (Murphy, G. P., Ed.) pp 85-109, Alan R. Liss, New York, NY. 131, 810-815. Laemmli, U. K. (1970) Nature (London) 227, 680-685. Marquardt, H., & Todaro, G. J. (1982) J. Biol. Chem. 257, 5220-5225. Articles Merril, C. R., Goldman, D., Sedman, S. A., & Ebert, M. H. (1981) Science (Washington, D.C.) 211, 1437-1438. Seyedin, S. M., Thomas, T. C., Thompson, A. Y., Rosen, D. M., & Piez, K. A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, Seyedin, S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., & Graycar, J. (1987) J. Biol. Chem. 262, Sporn, M. B., & Todaro, G. J. (1980) N. Engl. J. Med. 303, 878-880. Sporn, M. B., & Roberts, A. B. (1985) Nature (London) 313, 745-747. Sporn, M. B., Roberts, A. B., Wakefield, L. M., & Assoian, R. K. (1986) Science (Washington, D.C.) 233, 532-534. Todaro, G. J., De Larco, J. E., Fryling, C., Johnson, P. A., & Sporn, M. B. (1981) J. Supramol. Struct. 15, 287-301. Tucker, R. F., Shipley, G. D., Moses, H. L., & Holley, R. W. (1984) Science (Washington, D.C.) 226, 705-707. 2267-223 1. 1946-1 949. Structure of the High-Affinity Binding Site for Noncompetitive Blockers of the Acetylcholine Receptor: [ 3H]Chlorpromazine Labels Homologous Residues in the and 6 Chains? JCr6me Giraudat,* Michael Dennis,* Thierry Heidmann,t Pierre-Yves Haumont,§ Florence Lederer,§ and Jean-Pierre Changeux*vf Unit2 de Neurobiologie MolPculaire et UnitP AssociZe au Centre National de la Recherche Scientvique UA 041149, Interactions MolPculaires et Cellulaires, DZpartement des Biotechnologies, Institut Pasteur, 75724 Paris CZdex 15, France, and UnitP 25 de l’lnstitut National de la SantP et de la Recherche MPdicale et Laboratoire AssociP au Centre National de la Recherche Scientifique LA 122, H6pital Necker, 75730 Paris CPdex 15, France Received July 22, 1986; Revised Manuscript Received October 10, 1986 ABSTRACT: The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled zy p chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpr~mazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M I1 of the chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the 6 chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83,2719-27231. These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule. x e nicotinic acetylcholine receptor (AcChR)’ from fish electric organ and vertebrate neuromuscular junction is a This work was supported by grants from the Muscular Dystrophy Association of America, the College de France, the Ministere de la Recherche et de I’Enseignement Superieur, the Centre National de la Recherche Scientifique, and the Commissariat zyxwvutsr i I’Energie Atomique. M.D. is the recipient of a fellowship from the Fonds de la Recherche en Sante du Quebec. * Institut Pasteur. Hapita1 Necker. heterologous pentamer (azP$) that both carries the acetyl- choline binding sites at the level of the a chains and contains the agonist-gated ion channel [reviews in Changeux et al. (1984), Anholt et al. (1985) and Stroud and Finer-Moore I Abbreviations: AcChR, acetylcholine receptor; NCB, noncompeti- tive blocker; CPZ, chlorpromazine: NaDodSO,, sodium dodecyl sulfate; PTH-amino acids, phenylthiohydantoin amino acids; HPLC, high-per- formance liquid chromatography; TPCK, N~-tosylphenylalanine chloro- methyl ketone; AUFS, absorbance units full scale. 0006-2960/87/0426-2410$01.50/0 0 1987 American Chemical Society