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，在与神经递质 GABA 结合后，Cl- 可以通过中央孔快速扩散。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特性的多种受体亚型。具体来说，常用的苯二氮卓类药物（例如地西泮）的结合增强了由 1/2/3 亚基异构体组成的受体的观察到的 Cl-电导。然而，由于缺乏高分辨率结构信息，无法详细分析 GABA 结合与地西泮正向变构调节之间的分子机制。该提案概述了使用经过验证的策略，通过 X 射线衍射方法确定与 GABA 和地西泮复合的 11/22/32 受体的高分辨率晶体结构。高度灵敏的荧光检测尺寸排阻色谱技术将用于快速筛选优化的基因构建体和分离功能性 GABAA 受体的条件。然后优化的构建体将在Sf9昆虫细胞中表达以获得毫克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、配体结合测定和电生理学实验的组合来评估。这项工作的结果将极大地有助于理解 GABAA 受体结构和配体结合对通道行为的变构效应。公共健康相关性：GABAA 受体活性功能障碍与多种神经系统疾病有关，例如癫痫，可以用地西泮等强效受体调节剂进行治疗。然而，地西泮（和其他苯二氮卓类药物）与多种 GABAA 受体亚型的广泛相互作用谱会导致不良副作用，包括健忘症。该应用的结果将显着增加对地西泮-GABAA受体相互作用的治疗价值的认识，并将为开发具有减少副作用的受体亚型特异性药物的合理药物设计策略提供信息。