Structure and mechanism of extrasynaptic GABAA receptors

项目摘要： A型γ-氨基丁酸受体（GABAA）是一种五聚体配体门控离子通道， 大量存在于中枢神经系统中GABA，主要的脑抑制性神经递质， GABAA受体占大脑中快速突触信号的约30%。这些受体很重要 用于感觉和运动处理、中枢自主控制和认知。虽然大部分的研究在 GABAA受体的结构和功能集中在突触上发现的亚基组装上，这是一个巨大的挑战。 在突触外发现了多种重要的GABAA受体亚型。的目标 这项建议是确定和验证突触外GABAA受体的高分辨率结构， 与神经系统疾病、麻醉、成瘾和药物滥用有关的不同药物类别。这些 受体被轻度致醉浓度的乙醇增强，对神经类固醇具有高亲和力 并且是麻醉剂、镇痛剂和抗惊厥药物的靶点。在两个目标中，我建议优化 确定的异聚GABAA受体组装体的表达和纯化条件，精制样品 冷冻电子显微镜的准备，收集和处理单粒子冷冻EM数据集，并建立和 完善受体-药物复合物的原子模型。高分辨率的结构信息， 重要但研究不足的亚基组装，与变构和正构位点配体复合，将广泛地 告知配体识别和亚基组装的原理。 !

Project Summary: Type A γ-aminobutyric acid receptors (GABAA) are pentameric ligand-gated ion channels found abundantly in the central nervous system. The binding of GABA, the major brain inhibitory neurotransmitter, to GABAA receptors, accounts for ~30% of the fast synaptic signaling in the brain. These receptors are important for sensory and motor processing, central autonomic control, and cognition. While much of the research in GABAA receptor structure and function has focused on the subunit assemblies found at synapses, a tremendous diversity of pharmacologically important GABAA receptor subtypes are found outside of synapses. The goal of this proposal is to determine and validate high-resolution structures of extrasynaptic GABAA receptors bound to distinct drug classes of relevance to neurological disorders, anesthesia, addiction and drug abuse. These receptors are potentiated by mildly intoxicating concentrations of ethanol, have high affinity for neurosteroids and are targets of anesthetics, analgesics, and anticonvulsant drugs. In two Aims, I propose to optimize conditions for expression and purification of defined heteromeric GABAA receptor assemblies, refine sample preparation for cryo-electron microscopy, collect and process single particle cryo-EM datasets, and build and refine atomic models of receptor-drug complexes. High-resolution structural information for physiologically important but understudied subunit assemblies, in complex with allosteric and orthosteric-site ligands will broadly inform on principles of ligand recognition and subunit assembly. !