The noncompetitive blocker [3H]chlorpromazine labels segment M2 but not segment M 1 of the nicotinic acetylcholine receptor α‐subunit

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 radioactivity incorporated into the AChR subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The α-subunit was purified digested with trypsin and/or CNBr and the resulting fragments fractionated by HPLC. Sequence analysis resulted in the identification of Ser-248 as a major residue labeled by [³H]chlorpromazine in a phencyclidine-sensitive manner. This residue is located in the hydrophobic and putative transmembrane segment M2 of the α-subunit, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the δ-chain [1] and the Ser-254 and Leu-257 in the β-chain [2]. Extended sequence analysis of the hydrophobic segment M 1 further showed that no labeling occurred in this region.