Structure of GABA-A Receptor Anion-Selective Channel

GABA-A 受体阴离子选择性通道的结构

• 批准号: 7586636
• 负责人: Myles H. Akabas
• 金额: $ 36.31万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-05 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7586636
• 关键词: Accounting; Acetylcholine; Adverse effects; Affinity; Amino Acids; Aminobutyric Acids; Anions; Anxiety Disorders; Binding; Binding Sites; Brain; Caliber; Cations; Charge; Chemical Synapse; Cholinergic Receptors; Clinical Medicine; Communication; Cryoelectron Microscopy; Cysteine; Cytoplasmic Tail; Data; Distant; Disulfides; Drug usage; Electrons; Epilepsy; Equilibrium; Extracellular Domain; Foundations; Free Energy; Future; GABA-A Receptor; Gated Ion Channel; General Anesthesia; Glycine Receptors; Goals; Grant; Homologous Gene; Homology Modeling; Imagery; Inherited; Ions; Kinetics; Knowledge; Lead; Ligand Binding; Location; Maps; Mediating; Medicine; Membrane Lipids; Membrane Potentials; Modeling; Molecular; Motion; Mutation; Nervous System Physiology; Neuraxis; Neurons; Neurosciences; Neurotransmitters; Pattern; Peptides; Pharmaceutical Preparations; Play; Positioning Attribute; Potassium Channel; Property; Proteins; Protons; Reagent; Receptor Gene; Relative (related person); Resolution; Role; Serotonin; Serotonin Receptors 5-HT-3; Side; Signal Transduction; Site; Solutions; Spasm; Structural Models; Structure; Study models; Synaptic Transmission; Testing; Torpedo; Transmembrane Domain; Uncertainty; Vertebral column; base; channel blockers; crosslink; design; drug of abuse; extracellular; gamma-Aminobutyric Acid; insight; member; novel; quantum; receptor; receptor function; receptor structure function; research study; two-dimensional

DESCRIPTION (provided by applicant): The GABAA receptors (GABAAR) are members of the Cys-loop receptor gene superfamily of neurotransmitter-gated ion channels that includes acetylcholine, serotonin 5-HT3 and glycine receptors. The GABAAR mediate fast inhibitory synaptic transmission in the central nervous system. They are targets for drugs used to treat anxiety disorders, insomnia, epilepsy and muscle spasm, as well as for general anesthesia. A long term goal of molecular neuroscience, and of this project, has been to understand the structural bases for the functional properties of these receptors and for their modulation by medicines and drugs of abuse. Cys- loop receptor structure-function studies have benefited from a 4-E resolution structure of the Torpedo acetylcholine receptor (AChR) that was solved from electron diffraction maps obtained by cryo-electron microscopy of two dimensional crystals. This has served as a basis for attempts to model the open state and for construction of homology models of other superfamily members. At 4 E resolution the peptide backbone is well defined but the amino acid side chain positions are uncertain. What is lacking for these modeling studies is experimental data to validate the 4-E resolution AChR model and the GABAAR homology models based on it. This grant will focus on three major aspects of Cys-loop receptor structure. In Aim 1 the hypothesis that the open channel diameter narrows from the extracellular to the cytoplasmic end will be tested by determining the open channel diameter as a function of depth in the channel in GABAA and 5-HT3 receptors. Aims 2 and 3 focus on testing the validity of closed state GABAAR homology models. The most straightforward, testable predictions from a homology model are proximity relationships between neighboring residues that are either distant in the primary sequence or in separate subunits. In Aim 2 we will identify the residues in the M1 and M3 transmembrane segments that form the interface between adjacent subunits. In Aim 3 we will identify the contacts between the M4 segment and the M1 and M3 segments within a subunit. By examining changes in contact patterns in the absence and presence of GABA we will investigate the conformational changes that occur during channel gating. In Aim 4 we will study a newly discovered prokaryotic Cys-loop receptor homologue from Gloeobacter violaceus. This proton-activated, cation-selective channel has many features of eukaryotic Cys-loop receptors but lacks others such as the cysteines responsible for the eponymous Cys-loop and the M3-M4 loop cytoplasmic domain. We will identify residues lining the channel in the closed and open states, the charge selectivity filter location and the channel gate, and the location of channel blocker binding sites. These studies will provide a foundation for understanding the structural basis for function in this novel channel. Successful completion of this project will provide the basis for structural modeling of the GABAAR and the conformational changes they undergo following the binding of GABA and modulatory drugs. PROJECT NARRATIVE The GABAA receptor plays an essential role in communication between nerve cells in the brain and is the target for drugs used to treat anxiety disorders, insomnia, epilepsy and muscle spasm, as well as for general anesthesia. The successful completion of this project will help to elucidate the molecular basis of action of these medicines and possibly provide a basis for the design of more specific drugs with fewer side effects. It will also provide a foundation for a structural understanding of how mutations in GABAA receptor genes result in hereditary epilepsies.

描述（由申请人提供）：GABAA受体（GABAAR）是神经递质门控离子通道的Cys环受体基因超家族的成员，其包括乙酰胆碱、血清素5-HT3和甘氨酸受体。 GABAAR 介导中枢神经系统中的快速抑制性突触传递。它们是用于治疗焦虑症、失眠、癫痫和肌肉痉挛以及全身麻醉的药物的目标。分子神经科学和该项目的长期目标是了解这些受体的功能特性及其通过药物和滥用药物的调节的结构基础。 Cys环受体结构功能研究受益于鱼雷乙酰胆碱受体（AChR）的4-E分辨率结构，该结构是通过二维晶体的冷冻电子显微镜获得的电子衍射图解算出来的。这已成为尝试模拟开放状态和构建其他超家族成员的同源模型的基础。在 4 E 分辨率下，肽主链明确，但氨基酸侧链位置不确定。这些建模研究缺乏的是验证4-E分辨率AChR模型和基于它的GABAAR同源模型的实验数据。这笔资助将重点关注半胱环受体结构的三个主要方面。在目标 1 中，开放通道直径从细胞外到细胞质末端变窄的假设将通过确定开放通道直径作为 GABAA 和 5-HT3 受体通道深度的函数来测试。目标 2 和 3 重点测试封闭状态 GABAAR 同源模型的有效性。来自同源模型的最直接、可测试的预测是在一级序列中较远或在单独的亚基中的相邻残基之间的邻近关系。在目标 2 中，我们将鉴定 M1 和 M3 跨膜片段中形成相邻亚基之间界面的残基。在目标 3 中，我们将识别亚基内 M4 片段与 M1 和 M3 片段之间的接触。通过检查 GABA 不存在和存在时接触模式的变化，我们将研究通道门控过程中发生的构象变化。在目标 4 中，我们将研究一种新发现的来自紫色胶杆菌的原核半胱环受体同源物。这种质子激活的阳离子选择性通道具有真核 Cys 环受体的许多特征，但缺乏其他特征，例如负责同名 Cys 环和 M3-M4 环胞质结构域的半胱氨酸。我们将识别关闭和打开状态下通道内衬的残基、电荷选择性过滤器位置和通道门以及通道阻断剂结合位点的位置。这些研究将为理解这一新通道功能的结构基础奠定基础。该项目的成功完成将为 GABAAR 的结构建模及其在 GABA 与调节药物结合后发生的构象变化提供基础。 项目叙述 GABAA 受体在大脑神经细胞之间的通讯中发挥着重要作用，是用于治疗焦虑症、失眠、癫痫和肌肉痉挛以及全身麻醉的药物的靶标。该项目的成功完成将有助于阐明这些药物作用的分子基础，并可能为设计更特异性、副作用更少的药物提供基础。它还将为从结构上理解 GABAA 受体基因突变如何导致遗传性癫痫奠定基础。