Structural dynamics underlying rho1 GABAC receptor activation and antagonism

描述（申请人提供）：3-氨基丁酸（GABA）门控氯通道（GABAA和GABAC受体）在哺乳动物大脑的快速突触抑制中起重要作用。它们也是许多临床有用的神经活性化合物的主要靶点。这些gaba门控离子通道的功能障碍可导致癫痫和其他神经系统疾病。GABA与其受体的结合被认为会引发从结合位点开始的构象波，并传播到门控机制以打开孔。因此，深入了解这些构象重排的动态结构基础是理解GABA受体激活和拮抗机制的关键一步。然而，尽管过去进行了大量的结构-功能关系研究，但GABA受体激活的结构动力学仍未完全了解。本项目旨在通过采用在本实验室成功适应的技术，为GABAC受体功能的结构基础提供新的见解。第一个目标是使用位点特异性荧光结合替代半胱氨酸可及性分析和电生理记录来定义激动剂诱导的亚基界面中通道激活的结构重排。第二个目的是利用荧光共振能量转移（FRET）来检测不位于亚基界面的构象变化，并推断其在受体激活过程中的移动方向。第三个目的是使用位点特异性荧光和FRET来定义非竞争性拮抗剂可拮抗的激动剂诱导的运动。第四个目标是通过单通道分析验证之前目标识别的移动残基的功能意义，并通过突变周期分析探索耦合机制。总的来说，这项工作将使我们能够检测GABA受体激活和拮抗的亚基界面内外的构象变化。这些研究将验证该项目的中心假设，即GABA受体激活涉及激动剂诱导的n端结构域旋转，然后传播到门控机制以打开孔。从这项工作中获得的见解将阐明受体激活和拮抗的机制，这些机制可能更普遍地适用于整个配体门控离子通道。这些发现也将为开发针对GABA受体的新疗法或研究工具提供结构基础。

DESCRIPTION (provided by applicant): 3-aminobutyric acid (GABA)-gated chloride channels (GABAA and GABAC receptors) play important roles in fast synaptic inhibition in the mammalian brain. They are also the major targets of many clinically useful neuroactive compounds. Dysfunction of these GABA-gated ion channels can result in epilepsy and other neural disorders. Binding of GABA to its receptor is thought to initiate a conformational wave starting from the binding site(s) and propagating to the gating machinery to open the pore. Gaining insights into the dynamic structural basis of these conformational rearrangements therefore is the key step to understanding the mechanism of GABA receptor activation and antagonism. However, despite intensive structure-function relationship studies in the past, structural dynamics underlying GABA receptor activation is still not fully understood. This project seeks to provide new insights into structural bases for GABAC receptor function by employing techniques successfully adapted in this laboratory. The first aim is to use site-specific fluorescence combined with substituted cysteine accessibility analysis and electrophysiological recording to define agonist-induced structural rearrangements in the subunit interface underlying channel activation. The second aim is to use fluorescence resonance energy transfer (FRET) to detect conformational changes not located in subunit interface and to deduce their moving direction during receptor activation. The third aim is to use site-specific fluorescence and FRET to define agonist-induced movements antagonizable by noncompetitive antagonists. The fourth aim is to validate the functional significance of the moving residues identified by previous aims by single channel analysis and explore coupling mechanism by mutant cycle analysis. Collectively, this work will allow us to detect conformational changes in and out of subunit interface underlying GABA receptor activation and antagonism. These studies will test the project's central hypothesis that GABA receptor activation involves an agonist-induced rotation of the N-terminal domain, which then propagates to the gating machinery to open the pore. Insights gained from this work will illuminate mechanisms of receptor activation and antagonism that have potential applicability more generally to the entire class of ligand-gated ion channels. The findings also will provide a structural basis for development of new therapeutics or research tools targeting GABA receptors.