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

FEBS Letters

Chiara, David C¹  Cohen, Jonathan B¹  Middleton, Richard E¹ 

[1] Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115, USA

关键词: Nicotinic acetylcholine receptor;    Ligand binding domain;    Photoaffinity labeling;    Nicotine;    Tryptophan;    nAChR;    nicotinic acetylcholine receptor;    dTC;    d-tubocurarine;    ACh;    acetylcholine;    carb;    carbamylcholine;   

DOI  :  10.1016/S0014-5793(98)00093-3

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

[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.

【 授权许可】

Unknown