文章基本信息

标题：A conserved salt bridge critical for GABAA receptor function and loop C dynamics
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作者：Srinivasan P. Venkatachalan ; Cynthia Czajkowski
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：2008
卷号：105
期号：36
页码：13604-13609
DOI：10.1073/pnas.0801854105
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Chemical signaling in the brain involves rapid opening and closing of ligand gated ion channels (LGICs). LGICs are allosteric membrane proteins that transition between multiple conformational states (closed, open, and desensitized) in response to ligand binding. While structural models of cys-loop LGICs have been recently developed, our understanding of the protein movements underlying these conformational transitions is limited. Neurotransmitter binding is believed to initiate an inward capping movement of the loop C region of the ligand-binding site, which ultimately triggers channel gating. Here, we identify a critical intrasubunit salt bridge between conserved charged residues ({beta}E153, {beta}K196) in the GABAA receptor (GABAAR) that is involved in regulating loop C position. Charge reversals (E153K, K196E) increased the EC50 for GABA and for the allosteric activators pentobarbital (PB) and propofol indicating that these residues are critical for channel activation, and charge swap (E153K-K196E) significantly rescued receptor function suggesting a functional electrostatic interaction. Mutant cycle analysis of alanine substitutions indicated that E153 and K196 are energetically coupled. By monitoring disulfide bond formation between cysteines substituted at these positions (E153C-K196C), we probed the mobility of loop C in resting and ligand-bound states. Disulfide bond formation was significantly reduced in the presence of GABA or PB suggesting that agonist activation of the GABAAR proceeds via restricting loop C mobility.
关键词：disulfide trapping ; electrostatic ; ligand-gated ion channel ; mutant cycle