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Identification of tryptophan 55 as the primary site of [ 3 H]nicotine photoincorporation in the γ‐subunit of the Torpedo nicotinic acetylcholine receptor
Author(s) -
Chiara David C,
Middleton Richard E,
Cohen Jonathan B
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00093-3
Subject(s) - torpedo , photoaffinity labeling , nicotinic acetylcholine receptor , chemistry , agonist , protein subunit , nicotine , nicotinic agonist , acetylcholine receptor , binding site , tryptophan , amino acid , biochemistry , stereochemistry , receptor , biology , neuroscience , gene
[ 3 H]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 [ 3 H]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 [ 3 H]nicotine‐labeled γ‐subunit. Amino‐terminal sequence analysis identified γTrp‐55 as the major site of [ 3 H]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.