Structural investigation of a GABA receptor subtype

GABA 受体亚型的结构研究

基本信息

批准号：
8254694
负责人：
Derek P Claxton
金额：
$ 4.92万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-02-01 至 2014-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8254694
关键词：
Adverse effects Affinity Agonist Amnesia Anxiety Behavior Benzodiazepines Binding Binding Sites Biological Assay Cells Chemical Synapse Chimeric Proteins Complex Couples Cryoelectron Microscopy Crystallization Crystallography Detection Detergents Development Diazepam Diffusion Drug Binding Site Drug Design Electrodes Electrophysiology (science)Environment Epilepsy Fab Immunoglobulins Family Fluorescence Functional disorder GABA Receptor Gated Ion Channel Genes Goals Homologous Gene Individual Insecta Integral Membrane Protein Investigation Ion Channel Knowledge Laboratories Length Ligand Binding Ligands Link Measurement Mediating Membrane Methods Micelles Modeling Molecular Analysis Molecular Conformation Molecular Sieve Chromatography Monitor Mutagenesis Neuraxis Neurotransmitters Nicotinic Receptors Oocytes Pattern Pharmaceutical Preparations Phase Process Property Protein Isoforms Proteins Radiolabeled Receptor Gene Resolution Role Screening procedure Shapes Signal Transduction Site-Directed Mutagenesis Structural Models Structure Techniques Therapeutic Therapeutic Agents Torpedo Work Xenopus oocyte base comparative gamma-Aminobutyric Acid milligram nervous system disorder neurotransmission postsynaptic prevent radiotracer receptor receptor function research study stoichiometry three dimensional structure voltage clamp

项目摘要

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. PUBLIC HEALTH RELEVANCE: The dysfunction of GABAA receptor activity has been linked to a variety of neurological disorders, such as epilepsy, which can be treated with potent receptor modulators like diazepam. However, the broad interaction spectrum of diazepam (and other benzodiazepines) with multiple GABAA receptor subtypes leads to undesired side effects including amnesia. The results of this application will significantly increase the knowledge of the therapeutic value of diazepam-GABAA receptor interactions and will inform rational drug design strategies for the development of receptor subtype-specific drugs with reduced side effects.

描述（由申请人提供）：配体门控离子通道（LGIC）是介导化学突触中快速信号转导的整体膜蛋白。 GABAA受体属于一个称为Cys-Loop受体的主要LGIC家族，在神经递质GABA结合后，CL-通过中心孔快速扩散。导电通道由相同或同源亚基的五聚体组装形成，产生具有多种药理特性的多种受体亚型。具体而言，常见规定的苯二氮卓类药物（例如地西epa）的结合增强了观察到的受体的CL-由1/2/3亚基同工型组成的受体。然而，缺乏高分辨率结构信息阻止了对GABA结合与地西epa的阳性变构调节的分子机制的详细分析。该提案概述了使用验证的策略来确定与GABA复合物中11/22/32受体的高分辨率晶体结构，并通过X射线衍射方法来确定与GABA和地西epa的复合物。荧光检测大小排除色谱的高度敏感技术将用于快速筛选优化的基因构建体和条件，以隔离功能性GABAA受体。然后，在SF9昆虫细胞中将表达优化的构建体，以获得用于结晶试验的Mg量的受体。与GABA和地西epa的共结晶实验将用于识别11/22/32受体结构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，配体结合测定和电生理实验的结合来评估。这项工作的结果将大大有助于理解GABAA受体结构以及配体结合对通道行为的变构影响。公共卫生相关性：GABAA受体活性的功能障碍与多种神经系统疾病（例如癫痫病）有关，可以用有效的受体调节剂（例如地西epa）治疗。然而，与多个GABAA受体亚型的地西ep（和其他苯二氮卓类）的广泛相互作用光谱导致包括健忘症在内的不良副作用。该应用的结果将大大提高地西epa受体相互作用的治疗价值的知识，并将为有理药物设计策略提供副作用降低的受体亚型特异性药物的开发。