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

基本信息

批准号：
9812999
负责人：
Stephen J Moss
金额：
$ 48.72万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-02 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9812999
关键词：
Address Adult Affinity Chromatography Antibodies Anxiety Barbiturates Benzodiazepines Binding Brain Cell membrane Cell surface Comprehension Confocal Microscopy Coupled Development Disease Drug Targeting Epilepsy GTP-Binding Protein alpha Subunits, Gs Gated Ion Channel Genes Image Imagery Individual Inhibitory Synapse Integral Membrane Protein Intravenous Anesthetics Ion Channel Gating Knock-in Knock-out Lead Ligands Liquid Chromatography LoxP-flanked allele Mass Spectrum Analysis Mediating Mental Depression Modification Molecular Structure Mus Mutation Neurons Permeability Pharmacology 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 autism spectrum disorder beta Spectrin experimental study gamma-Aminobutyric Acid improved insight neuronal excitability neuropsychiatric disorder neurosteroids novel protein complex receptor stoichiometry synaptic inhibition tool

项目摘要

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 (GABAAR) 是 Cl 偏好的配体门控离子通道介导成人大脑中的快速阶段性突触抑制，是巴比妥类药物的靶标，苯二氮卓类药物、静脉麻醉药和神经类固醇与它们在调节中的重要作用一致。神经元兴奋性、GABAAR活性的改变会导致焦虑、自闭症、抑郁症、癫痫症、药物滥用和精神分裂症受体是可以由 α(1-6) 组装而成的异五聚体，令人惊讶的是，迄今为止我们对天然分子结构的理解。 GABAAR 亚型以及这些蛋白质如何靶向抑制性突触尚不成熟。鉴于 GABAAR 在决定神经兴奋性方面发挥的关键作用，问题至关重要，神经精神疾病和药物靶点。为了解决这些问题，我们将确定组装的主要 GABAAR 亚型的结构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 的突触靶向中发挥的作用然后我们将通过我们开发的新型小鼠品系来检查亚型。其中α1和α2亚基的N末端已被不同的荧光产生者修饰； pHlourin/9E10-α2 (pHα2) 和 mKate/FLAG-α1 (mKα1) 这些添加在功能上是沉默的，但允许。单个 GABAAR 亚型的连续亲和纯化，并使用 blue-native 分析其结构聚丙烯酰胺凝胶电泳 (BN-PAGE)，随后进行液相色谱联用质谱分析 (LC-MS/MS) 使用这些新工具的初步实验使我们能够提出一个中心假设。将在这里进行测试；α2-和α1-亚基被组装成两种不同的“α2/α1”；含有等摩尔量的两个 α 亚基 (α2α1βγ2)，以及不含 α2 的“α1/α1” 亚基（α1βγ2）。这些亚型通过血影蛋白针对不同的突触子集。与这些 GABAAR 亚型密切相关，我们的实验将集中于以下特定的方面。目标：目的 1. 检验神经元组装含有 α2α1βγ2 和 α2α1βγ2 的 GABAAR 亚型的假设 α1βγ2 亚基。目标 2. 检验 α2/α1 和 α1/α1 GABAAR 亚型相关的假设。目标 3. 检验血影蛋白促进突触特异性的假设。 GABAAR 的靶向。总的来说，我们的研究将为本地 GABAAR 的结构和过程提供新的见解调节它们在抑制性突触处的积累，这些信息可能会导致更好的理解，以及癫痫和其他神经精神疾病的治疗。