Jung Sangwook, Harris R Adron
Cell and Molecular Biology Program and Waggoner Center for Alcohol and Addiction Research, University of Texas at Austin, TX, USA.
J Neurochem. 2006 Feb;96(3):885-92. doi: 10.1111/j.1471-4159.2005.03617.x. Epub 2006 Jan 9.
Abstract gamma-Aminobutyric acid type A (GABA(A)) receptors are molecular targets for alcohols. Previous work suggests that S270 and A291 residues in the transmembrane (TM) 2 and 3 domains of the GABA(A) receptor alpha subunit are components of an alcohol-binding pocket, and S270I and A291W mutants abolished ethanol potentiation. Our results showed that A295C and F296C residues in the TM3 of the GABA(A) receptor alpha1 subunit are accessible to hexylmethanethiosulfonate (HMTS) in the alcohol-bound state, but not in the resting state. Thus, the A295C and F296C sites become water-accessible as a result of alcohol-induced conformational changes. If S270 or A291 residues are sites of alcohol binding, then S270I or A291W mutations should prevent alcohol-induced conformational movements within the TM3 domain. To investigate this question, the accessibility of HMTS reagent to double mutants (A291W/A295C, A291W/F296C, S270I/A295C or S270I/F296C) in the presence of ethanol or hexanol was tested. The A291W or S270I mutations markedly reduced the accessibility of HMTS to all the double mutants in the ethanol-bound state, and to S270I/F296C, A291W/A295C and A291W/F296C double mutants in the hexanol-bound state, suggesting that the A291 or S270 residues are critical sites for alcohol binding and alcohol-induced conformational changes.
摘要 γ-氨基丁酸A型（GABA(A)）受体是酒精的分子靶点。先前的研究表明，GABA(A)受体α亚基跨膜（TM）2和3结构域中的S270和A291残基是酒精结合口袋的组成部分，而S270I和A291W突变体消除了乙醇增强作用。我们的结果表明，GABA(A)受体α1亚基TM3中的A295C和F296C残基在酒精结合状态下可被己基甲硫代磺酸盐（HMTS）接近，但在静息状态下则不能。因此，由于酒精诱导的构象变化，A295C和F296C位点变得可被水接近。如果S270或A291残基是酒精结合位点，那么S270I或A291W突变应该会阻止酒精诱导的TM3结构域内的构象运动。为了研究这个问题，测试了在乙醇或己醇存在下HMTS试剂对双突变体（A291W/A295C、A291W/F296C、S270I/A295C或S270I/F296C）的可及性。A291W或S270I突变显著降低了HMTS在乙醇结合状态下对所有双突变体以及在己醇结合状态下对S270I/F296C、A291W/A295C和A291W/F296C双突变体的可及性，这表明A291或S270残基是酒精结合和酒精诱导的构象变化的关键位点。
