Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: [3H]chlorpromazine labels homologous residues in the .beta. and .delta. chains

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 beta 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]chlorpromazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M II of the beta chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the delta chain [Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y., & Changeux, J.-P. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2719-2723]. 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.