Gay Elaine A, Giniatullin Rashid, Skorinkin Andrei, Yakel Jerrel L
Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services PO Box 12233, Research Triangle Park, NC 27709, USA.
J Physiol. 2008 Feb 15;586(4):1105-15. doi: 10.1113/jphysiol.2007.149492. Epub 2007 Dec 20.
The rat alpha7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat alpha7 nAChR subunit, which lies within the beta2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the alpha7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype alpha7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (alpha7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for alpha7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat alpha7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions.
大鼠α7烟碱型乙酰胆碱受体（nAChR）可迅速发生脱敏；然而，脱敏机制在很大程度上尚不清楚。位于β2链内的大鼠α7 nAChR亚基第55位的色氨酸（W）残基的作用，通过将其突变为其他疏水和/或芳香族氨基酸进行了研究，随后在非洲爪蟾卵母细胞中进行了电压钳实验。当突变为丙氨酸时，α7-W55A nAChR的脱敏速度比野生型α7 nAChR慢，且从脱敏状态恢复得更快。通过动力学建模验证了脱敏的作用。将W55突变为其他芳香族残基（苯丙氨酸或酪氨酸）对脱敏动力学没有显著影响，而突变为各种疏水残基（丙氨酸、半胱氨酸或缬氨酸）则显著降低了起始速率并增加了从脱敏状态恢复的速率。为了深入了解脱敏过程中可能的结构重排，我们通过将W55突变为半胱氨酸（α7-W55C）并测试各种巯基试剂与该半胱氨酸反应的能力，来探究W55的可及性。几种带正电荷的巯基试剂阻断了α7-W55C nAChR的ACh诱导反应，而一种中性巯基试剂增强了反应；在脱敏状态下，残基C55不可被修饰。这些数据表明，W55在大鼠α7 nAChR的脱敏起始和恢复过程中均起重要作用，且第55位的芳香族残基对于维持快速脱敏至关重要。此外，这些数据表明W55可能是通过疏水相互作用起作用的调节剂的潜在靶点。
