Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [H-3]DCTA: Identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine

Torpedo marmorata acetylcholine binding sites were photolabeled using 360 n m light, at equilibrium in the desensitized state, with the agonist [H-3]DC TA utilizing the Ce-IV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [H-3]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on th e alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation co nstants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 mu M, respectively. The alpha-chains isolated from receptor-ric h membranes photolabeled in the absence or in the presence of carbamylcholi ne were cleaved with CNBr using an efficient "in gel" procedure. The result ing peptide fragments were purified by HPLC and further submitted to trypsi nolysis. The digest was analyzed by HPLC leading to a single radioactive pe ak which, by microsequencing, revealed two sequences extending from alpha L ys-179 and from alpha His-186, respectively. Radioactive signals could be u nambiguously attributed to positions corresponding to residues alpha Tyr-19 0, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [ H-3]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [H-3]nicotine, belong to lo op C of the ACh binding site. The chemical structure of [H-3]DCTA, together with its well-defined and powerful photochemical reactivity, provides conv incing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.