期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：27
页码：10195-10200
DOI：10.1073/pnas.0301885101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the {beta}19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by ({Delta}F/F) {approx}10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for {Delta}F agreed well with those for epibatidine-induced currents, but were shifted {approx}100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the {alpha}{gamma}-binding site than to the {alpha}{delta} site and (ii) ACh binds with reverse-site selectivity, these data suggest that {Delta}F monitors an event linked to binding specifically at the {alpha}{delta}-subunit interface. In experiments with flash-applied agonists, the earliest detectable {Delta}F occurs within milliseconds, i.e., during activation. At low [ACh] ([≤] 10 {micro}M), a phase of {Delta}F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from {Delta}F is complete before the slowest phase of recovery from desensitization (time constant {approx}250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the {alpha}{delta}-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes.
