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

Abstract

[3H]nicotine has been used as a photoaffinity agonist to identify amino acids within the Torpedo nicotinic acetylcholine receptor (nAChR) γ-subunit that contributes to the structure of the agonist binding site. UV irradiation (254 nm) of nAChR-rich membranes equilibrated with [3H]nicotine results in covalent incorporation into α- and γ-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 [3H]nicotine-labeled γ-subunit. Amino-terminal sequence analysis identified γTrp-55 as the major site of [3H]nicotine photoincorporation in γ-subunit. Thus γTrp-55 is the first amino acid within a non-α-subunit to be identified by affinity labeling in direct contact with a bound agonist