Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: Serine-262 of the δ subunit is labeled by [³H]chlorpromazine

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [³H]chlorpromazine under equilibrium conditions in the presence of agonist. Incorporation of radioactivity into all subunits occurred and was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The δ subunit was purified and digested with trypsin, and the resulting fragments were fractionated by reversed-phase HPLC. The labeled peptide could not be purified to homogeneity because of its marked hydrophobic character, but a combination of differential CNBr subcleavage and consequencing of partially purified fragments enabled us to identify Ser-262 as being labeled by [³H]chlorpromazine. The labeling of this particular residue was prevented by phencyclidine and thus took place at the level of, or in proximity to, the high-affinity site for noncompetitive blockers. Ser-262 is located in a hydrophobic and potentially transmembrane segment termed MII.