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Contributions of Torpedo Nicotinic Acetylcholine Receptor γTrp-55 and δTrp-57 to Agonist and Competitive Antagonist Function

2001; Elsevier BV; Volume: 276; Issue: 4 Linguagem: Inglês

10.1074/jbc.m009085200

1083-351X

Yu Xie, Jonathan B. Cohen,

Insect and Pesticide Research

Results of affinity-labeling studies and mutational analyses provide evidence that the agonist binding sites of the nicotinic acetylcholine receptor (nAChR) are located at the α-γ and α-δ subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d -[ 3 H]tubocurarine (dTC) identified two tryptophans, γTrp-55 and δTrp-57, as the primary sites of photolabeling in the non-α subunits. To characterize the importance of γTrp-55 and δTrp-57 to the interactions of agonists and antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and electrophysiological recordings were used to examine the functional consequences when either or both tryptophans were mutated to leucine. Neither substitution altered the equilibrium binding of dTC. However, the δW57L and γW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, γW55L, and δW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the γW55L mutant, dTC binding at the α-γ site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with γ Trp-55 of the Torpedo nAChR play a crucial role in agonist binding and in the agonist-induced conformational changes that lead to channel opening.