Structural investigation of a GABA receptor subtype

描述（由申请人提供）：配体门控离子通道（LGIC）是介导化学突触快速信号转导的整合膜蛋白。GABAA受体属于LGIC的一个主要家族，称为Cys环受体，其允许Cl-在结合神经递质GABA时通过中心孔快速扩散。传导通道由相同或同源亚基的五聚体组装形成，产生具有多种药理学性质的多种受体亚型。具体而言，结合常用处方的苯二氮卓类药物，如地西泮，增强了观察到的由1/2/3亚基亚型组成的受体的Cl-电导。然而，缺乏高分辨率的结构信息，防止详细分析的分子机制，耦合GABA结合积极变构调制地西泮。该提案概述了使用经过验证的策略通过X射线衍射方法确定11/22/32受体与GABA和地西泮复合物的高分辨率晶体结构。将采用高度灵敏的荧光检测尺寸排阻色谱技术来快速筛选优化的基因构建体和条件，以分离功能性GABAA受体。然后将优化的构建体在Sf 9昆虫细胞中表达以获得mg量的受体用于结晶试验。GABA和地西泮的共结晶实验将用于鉴定11/22/32受体结构中的配体结合位点和构象变化。这些构象变化与受体功能特性的关系将通过诱变、配体结合试验和电生理学实验的组合来评估。本工作的结果将大大有助于理解GABAA受体结构和配体结合对通道行为的变构效应。

DESCRIPTION (provided by applicant): Ligand-gated ion channels (LGICs) are integral membrane proteins that mediate fast signal transduction at chemical synapses. GABAA receptors belong to a major family of LGICs, called Cys-loop receptors, which allow rapid diffusion of Cl- through a central pore upon binding of the neurotransmitter GABA. The conducting channel is formed from a pentameric assembly of identical or homologous subunits giving rise to multiple receptor subtypes with a diverse array of pharmacological properties. Specifically, binding of commonly prescribed benzodiazepines, such as diazepam, enhances the observed Cl- conductance of receptors composed of 1/2/3 subunit isoforms. However, the lack of high resolution structural information prevents a detailed analysis of the molecular mechanism that couples GABA binding to positive allosteric modulation by diazepam. This proposal outlines the use of proven strategies to determine a high resolution crystal structure of the 11/22/32 receptor in complex with GABA and diazepam by x-ray diffraction methods. The highly sensitive technique of fluorescence detection size exclusion chromatography will be employed to rapidly screen for optimized gene constructs and conditions to isolate a functional GABAA receptor. Then optimized constructs will be expressed in Sf9 insect cells to obtain mg quantities of receptor for crystallization trials. Co-crystallization experiments with GABA and diazepam will be used to identify the ligand binding sites and conformational changes in the structure of the 11/22/32 receptor. The relationship of these conformational changes to the functional properties of the receptor will be assessed by a combination of mutagenesis, ligand binding assays and electrophysiological experiments. The results of this work will substantially contribute to the understanding of GABAA receptor structure and the allosteric effects of ligand binding on channel behavior.