Structure of the GABA A Receptor Binding Sites

描述(申请人提供)：伽马-氨基丁酸A型受体(GABAARs)介导大脑中的突触抑制和几种临床重要药物的作用，包括苯二氮卓类药物、巴比妥酸盐、乙醇和麻醉药。该受体的几个突变与遗传性癫痫有关。我们研究计划的长期目标是从分子结构的角度了解GABAAR的功能和药理调节。本项目期间的工作极大地促进了我们对GABA和苯二氮卓类(BZD)结合位点的结构和动力学的理解。本文提出的实验建立在这些信息的基础上，以推进我们在结构水平上对GABA结合如何触发通道门控以及BZD结合如何耦合到受体调制的理解。我们建议检验以下假设：1)通过连接刚体蛋白块，亚基内和亚基间盐桥将细胞外区域的GABA结合位点的运动传递到跨膜通道区域的门控运动；2)环2中的残基参与了GABAAR的激活和脱敏；3)非结合位点界面的环9中的残基包含一个对将GABA结合与门控连接至关重要的‘铰链’元件；以及4)BZD结合通过触发细胞外域的运动来调节GABAAR的功能，该运动通过环2、环7、环9、环9、环9、环9、环2、环7、环9、环9的残基转导到跨膜螺旋前M1区和M2-M3区的11个和32个亚基。该方法结合了定点突变、二硫键交联、突变循环分析、取代半胱氨酸可及性方法、膜片钳技术和动力学分析。这些实验将借助最近阐明的原子级结构进行解释，以更深入地了解GABAARs和相关受体功能的分子机制。GABAARs在大脑功能中发挥的核心作用使这一基础研究与人类健康直接相关。本文提出的研究的相关性超出了GABAA受体本身。GABAA受体是受体家族的成员，其功能是配体门控离子通道，它们的活动调节整个大脑的信息流。这些渠道的缺陷导致了各种各样的疾病，它们是大量临床用药的靶点。在分子水平上提高我们对这些通道如何工作的理解，将提高我们预测作用于这些通道的药物的作用、设计更安全有效的药物、开发更好的治疗策略以及了解致病突变的病因学的能力。这里提出的研究将增加我们对一种类型的离子通道--GABAA受体--如何在健康和疾病中发挥作用的理解，并将为阐明其他相关的配体门控离子通道如何发挥作用建立可检验的假说。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid type A receptors (GABAARs) mediate synaptic inhibition in the brain and the actions of several clinically important drugs, including benzodiazepines, barbiturates, ethanol and anesthetics. Several mutations in the receptor are linked to inherited forms of epilepsy. The long-term goal of our research program is to understand the function and pharmacological modulation of the GABAAR in terms of its molecular structure. Work during the current project period significantly advanced our understanding of the structure and dynamics of the GABA and benzodiazepine (BZD) binding sites. Experiments proposed herein build on this information to advance our understanding, on a structural level, of how GABA binding triggers channel gating and how BZD binding is coupled to receptor modulation. We propose to test the following hypotheses: 1) that intra- and inter-subunit salt bridges relay GABA binding site movements in the extracellular domain to gating movements in the transmembrane channel domain by connecting rigid-body protein blocks, 2) that residues in Loop 2 are involved in GABAAR activation and desensitization, 3) that residues in Loop 9 at non-binding site interfaces comprise a `hinge' element important for coupling GABA binding to gating, and 4) that BZD binding modulates GABAAR function by triggering movements in the extracellular domain that are transduced to the transmembrane helices via residues in Loop 2, Loop 7, Loop 9, pre-M1 and M2-M3 regions of the 11 and 32 subunits. The approach combines site-directed mutagenesis, disulfide crosslinking, mutant cycle analysis, substituted cysteine accessibility method, patch-clamping and kinetic analysis. The experiments will be interpreted with the aid of recently elucidated atomic-level structures to gain a deeper understanding of the molecular mechanisms underlying the function of GABAARs and related receptors. The central role played by GABAARs in brain function make this basic research directly relevant to human health. The relevance of the research proposed herein extends beyond the GABAA receptor itself. GABAA receptors are members of a family of receptors that function as ligand-gated ion channels, and their activity regulates information flow throughout the brain. Defects in these channels lead to a wide variety of diseases, and they are the targets of a large number of clinically used drugs. Improvements in our understanding of how these channels work at a molecular level will improve our ability to predict the actions of drugs that act on these channels, to design safer and more effective drugs, to develop better therapeutic strategies, and to understand the etiology of disease-causing mutations. The research proposed here will increase our understanding of how one type of ion channel, the GABAA receptor, functions in health and disease and will establish testable hypotheses for elucidating how other related ligand-gated ion channels function.