Structural investigation of a GABA receptor subtype

描述(由申请人提供)：配体门控离子通道(LGICs)是在化学突触中介导快速信号转导的完整膜蛋白。GABAA受体属于LGICs的一个重要家族，称为Cys-loop受体，当神经递质GABA结合时，它允许氯-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而成，从而产生具有不同药理特性的多种受体亚型。具体地说，结合常用处方的苯二氮卓类药物，如安定，增强了观察到的由1/2/3亚基亚基组成的受体的氯离子电导。然而，缺乏高分辨率的结构信息阻碍了对地西潘将GABA结合到正变构调节的分子机制的详细分析。这项建议概述了用X射线衍射法确定11/22/32受体与GABA和安定的复合体中高分辨率晶体结构的方法。高灵敏度的荧光检测尺寸排除层析技术将被用来快速筛选优化的基因结构和条件，以分离出具有功能的GABAA受体。然后将优化后的构建物在Sf9昆虫细胞中表达，获得镁含量的受体，用于结晶实验。与GABA和安定的共结晶实验将被用来确定11/22/32受体的配体结合部位和构象变化。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实验相结合的方式进行评估。这项工作的结果将有助于理解GABAA受体的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.