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

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Abstract
[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 residues, γLeu-109 and δLeu-111. The photolabeling of these amino acids suggests that when the antagonist TDBzcholine occupies the agonist binding sites, the Cys-192-193 disulfide and Pro-194 from the α subunit Segment C are oriented toward the agonist site and are in proximity to γLeu-109/δLeu-111 in Segment E, a conclusion consistent with the structure of the binding site in the molluscan acetylcholine binding protein, a soluble protein that is homologous to the nAChR extracellular domain.