Identification of amino acids in the nicotinic acetylcholine receptor agonist binding site and ion channel photolabeled by 4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine, a novel photoaffinity antagonist.

[3H]4-[(3-trifluoromethyl)-3H-diazirin-3-yl]benzoylcholine (TDBzcholine) was synthesized and used as a photoaffinity probe to map the orientation of an aromatic choline ester within the agonist binding sites of the Torpedo nicotinic acetylcholine receptor (nAChR). TDBzcholine acts as a nAChR competitive antagonist that binds at equilibrium with equal affinity to both agonist sites (KD ∼ 10 μM). Upon UV irradiation (350 nm), nAChR-rich membranes equilibrated with [3H]TDBzcholine incorporate 3H into the α, γ, and δ subunits in an agonist-inhibitable manner. The specific residues labeled by [3H]TDBzcholine were determined by N-terminal sequence analysis of subunit fragments produced by enzymatic cleavage and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high-performance liquid chromatography. For the α subunit, [3H]TDBzcholine photoincorporated into αCys-192, αCys-193, and αPro-194. For the γ and δ subunits, [3H]TDBzcholine incorporated into homologous leucine re...