Xie Y, Cohen J B
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2001 Jan 26;276(4):2417-26. doi: 10.1074/jbc.M009085200. Epub 2000 Oct 30.
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 alpha-gamma and alpha-delta subunit interfaces. For Torpedo nAChR, photoaffinity-labeling studies with the competitive antagonist d-[(3)H]tubocurarine (dTC) identified two tryptophans, gammaTrp-55 and deltaTrp-57, as the primary sites of photolabeling in the non-alpha subunits. To characterize the importance of gammaTrp-55 and deltaTrp-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 deltaW57L and gammaW55L mutations decreased acetylcholine (ACh) binding affinity by 20- and 7,000-fold respectively. For the wild-type, gammaW55L, and deltaW57L nAChRs, the concentration dependence of channel activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the gammaW55L mutant, dTC binding at the alpha-gamma site acts not as a competitive antagonist but as a coactivator or partial agonist. These results establish that interactions with gamma 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.
亲和标记研究和突变分析的结果提供了证据，表明烟碱型乙酰胆碱受体（nAChR）的激动剂结合位点位于α-γ和α-δ亚基界面。对于电鳐nAChR，用竞争性拮抗剂d-[(3)H]筒箭毒碱（dTC）进行的光亲和标记研究确定了两个色氨酸，γTrp-55和δTrp-57，是非α亚基中光标记的主要位点。为了表征γTrp-55和δTrp-57对激动剂和拮抗剂相互作用的重要性，在非洲爪蟾卵母细胞中表达电鳐nAChR，并使用平衡结合测定和电生理记录来检查当一个或两个色氨酸突变为亮氨酸时的功能后果。两种取代都没有改变dTC的平衡结合。然而，δW57L和γW55L突变分别使乙酰胆碱（ACh）结合亲和力降低了20倍和7000倍。对于野生型、γW55L和δW57L nAChR，通道激活的浓度依赖性由希尔系数1.8、1.1和1.7表征。对于γW55L突变体，α-γ位点处的dTC结合不是作为竞争性拮抗剂，而是作为共激活剂或部分激动剂。这些结果表明，与电鳐nAChR的γTrp-55相互作用在激动剂结合以及导致通道开放的激动剂诱导的构象变化中起关键作用。
