Studies on the structure of gamma-aminobutyric acid type A receptor subtypes

γ-氨基丁酸A型受体亚型的结构研究

• 批准号: 10646275
• 负责人: Stephen J Moss
• 金额: $ 46.28万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-02 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10646275
• 关键词: Ablation; Address; Adult; Affinity Chromatography; Antibodies; Anxiety; Barbiturates; Benzodiazepines; Binding; Brain; Cell membrane; Cell surface; Comprehension; Confocal Microscopy; Coupled; Development; Disease; Drug Targeting; Epilepsy; GABA Receptor; Genes; Image; Individual; Inhibitory Synapse; Integral Membrane Protein; Intravenous Anesthetics; Ion Channel Gating; Knock-in; Knock-out; Ligands; Liquid Chromatography; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Mental Depression; Modification; Molecular Structure; Mus; Mutation; Neurons; Permeability; Phase; Phenotype; Physiological; Play; Polyacrylamide Gel Electrophoresis; Process; Property; Protein Isoforms; Proteins; Reporter; Role; Schizophrenia; Site; Spectrin; Structure; Substance abuse problem; Synapses; System; Testing; Variant; Visualization; autism spectrum disorder; beta Spectrin; efficacious treatment; experimental study; gamma-Aminobutyric Acid; improved; insight; neuronal excitability; neuropsychiatric disorder; neurosteroids; novel; pharmacologic; protein complex; receptor; stoichiometry; synaptic inhibition; tool

Type A γ-aminobutyric receptors (GABAAR) are Cl--preferring ligand gated ion channels that mediate fast phasic synaptic inhibition in the adult brain and are are drug targets for barbiturates, benzodiazepines, intravenous anesthetics, and neurosteroids. Consistent with their essential role in regulating neuronal excitability, modification in GABAAR activity contribute to anxiety, autism, depression, epilepsy, substance abuse and schizophrenia. The receptors are heteropentamers that can be assembled from α(1-6), β(1-3), γ(1-3), δ, ε, θ and π subunits. Surprisingly, to date our comprehension of the molecular structure of native GABAARs subtypes, and how these proteins are targeted to inhibitory synapses is rudimentary. However, these issues are of fundamental importance given the critical role GABAARs play in determining neuronal excitability, in neuropsychiatric diseases and as drug targets. To address these issues, we will determine the structure of the principal GABAAR subtypes assembled from α1 and α2 subunits in the brain. We will then assess which proteins co-purify with these receptor subtypes. The roles that the most abundant “receptor-associated proteins” play in the synaptic targeting of distinct GABAAR subtypes will then be examined. Our studies will be facilitated by the novel mouse lines we have developed in which the N-terminus of the α1 and α2 subunits have been modified with distinct fluorescent reporters; pHlourin/9E10-α2 (pHα2) and mKate/FLAG-α1 (mKα1). These additions are functionally silent but allow sequential affinity purification of individual GABAAR subtypes, and analysis of their structure using blue-native polyacrylamide gel electrophoresis (BN-PAGE) followed by liquid chromatography coupled mass spectroscopy (LC-MS/MS). Preliminary experiments using these new tools have allowed us to formulate a central hypothesis that will be tested here; α2- and α1-subunits are assembled into two distinct GABAAR subtypes; ‘α2/α1’ containing equimolar amounts of both α subunits (α2α1βγ2), and ‘α1/α1’ which does not contain an α2 subunit (α1βγ2). These subtypes are targeted to distinct subsets of synapses by spectrins, which are intimately associated with these GABAAR subtypes. Our experiments will focus on the following specific aims: Aim 1. To test the hypothesis that neurons assemble GABAAR subtypes containing α2α1βγ2 and α1βγ2 subunits. Aim 2. To test the hypothesis that the α2/α1 and α1/α1 GABAAR subtypes are associated with distinct spectrins. Aim 3. To test the hypothesis that spectrins facilitate the synapse-specific targeting of GABAARs. Collectively, our study will provide novel insights into the structure of native GABAARs and the processes that regulate their accumulation at inhibitory synapses, information that may lead to improved understanding of, and treatments for epilepsy and other neuropsychiatric disorders.

A型γ-氨基丁酸受体(GABA)是氯离子偏爱的配基门控离子通道 它们在成人大脑中介导快速时相突触抑制，是巴比妥酸盐的药物靶点， 苯二氮卓类、静脉麻醉药和神经类固醇。与它们在监管中的基本作用一致 神经元的兴奋性、GABAAR活性的改变导致焦虑、自闭症、抑郁、癫痫， 药物滥用和精神分裂症。受体是可由α(1-6)组装而成的杂五聚体， β(1-3)、γ(1-3)、δ，ε，θ和π亚基。令人惊讶的是，到目前为止，我们对天然的分子结构的理解 GABAARs亚型，以及这些蛋白是如何靶向抑制性突触的，目前尚不清楚。然而，这些 鉴于GABA受体在确定神经元兴奋性方面发挥的关键作用，这些问题具有根本性的重要性。 神经精神疾病和药物靶标。 为了解决这些问题，我们将确定组装的主要GABAAR子类型的结构 来自大脑中的α1和α2亚单位。然后，我们将评估哪些蛋白质与这些受体亚型共同纯化。 最丰富的“受体相关蛋白”在不同GABAAR突触靶向中的作用 然后将检查亚型。我们开发的新型小鼠品系将有助于我们的研究 其中α1和α2亚基的N-末端被明显的荧光报告分子修饰； PHlourin/9E10-α2(pHα2)和mKate/FLAG-α1(MKα1)。这些添加在功能上是静默的，但允许 GABAAR亚型的顺序亲和纯化及其结构分析 聚丙烯酰胺凝胶电泳法(BN-PAGE)-液-质联用法 (LC-MS/MS)。使用这些新工具的初步实验使我们能够形成一个中心假设 这将在这里进行测试；α2和α1亚基组装成两个不同的伽巴亚型：α2/α1。 包含α亚基(α2α1βγ2)和不包含α2的‘α1/α1’的等摩尔量 亚基(α1βγ2)。这些亚型通过幽灵蛋白靶向不同的突触亚集，这些亚型是 与这些GABAAR亚型密切相关。我们的实验将集中在以下几个具体方面 目标： 目的1.验证神经元组装包含α-2、α-1、βγ-2和GABAR亚型的假说 α1βγ2亚基。目的2.验证α2/α1和α1/α1 GABAAR亚型相关的假设 带有明显的幽灵。目的3.检验血影蛋白促进突触特异性的假说 目标是GABAAR。 总而言之，我们的研究将为原生GABA的结构和过程提供新的见解 调节它们在抑制性突触的积累，这一信息可能会导致对 以及癫痫和其他神经精神疾病的治疗。