Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the delta 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 delta 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 cosequencing 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.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002746:Chlorpromazine / Q000378:metabolism*; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000378:metabolism*; D012694:Serine; D014101:Torpedo
D000345:Affinity Labels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D012694:Serine; D002746:Chlorpromazine