Identification of tryptophan 55 as the primary site of [3H]nicotine photoincorporation in the γ-subunit of the Torpedo nicotinic acetylcholine receptor

[H-3]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) gamma-subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR-rich membranes equilibrated with [H-3]nicotine results in covalent incorporation into alpha- and gamma-subunits that is inhibitable by agonists and competitive antagonists, but not by non-competitive antagonists (Middleton, R.E. and Cohen, J.B. (1991) Biochemistry 30, 6887-6897). To identify sites of specific incorporation, SDS-PAGE and reversed-phase HPLC were used to isolate proteolytic fragments of [H-3]nicotine-labeled gamma-subunit. Aminoterminal sequence analysis identified gamma Trp-55 as the major site of [H-3]nicotine photoincorporation in gamma-subunit. Thus yTrp-55 is the first amino acid within a non-alpha-subunit to be identified by affinity labeling in direct contact with a bound agonist. (C) 1998 Federation of European Biochemical Societies.