Structure and Function of Hetero-multimeric Glutamate Receptors

异多聚谷氨酸受体的结构和功能

• 批准号: 9034604
• 负责人: Hiroyasu Furukawa
• 金额: $ 36.48万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-08 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9034604
• 关键词: AMPA Receptors; Agonist; Allosteric Regulation; Alzheimer' s Disease; Architecture; Baculoviruses; Binding; Biochemical; Brain; Calcium; Complex; Crystallization; Crystallography; Cytosol; Detection; Development; Disease; Early Promoters; Electrophysiology (science); Elements; Extracellular Domain; Family; G-Protein-Coupled Receptors; Gated Ion Channel; Glutamate Receptor; Glutamates; Glycine; Goals; Guidelines; Health; Heterogeneity; Ion Channel; Knowledge; Ligand Binding Domain; Ligands; MK801; Magnesium; Mammals; Mediating; Memantine; Membrane; Membrane Proteins; Mental Depression; Methodology; Methods; Molecular; Molecular Conformation; Molecular Structure; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurotransmitters; Parkinson Disease; Pattern; Peptide Hydrolases; Permeability; Phosphotransferases; Physiology; Play; Production; Property; Protein Subunits; Proteins; Public Health; RNA Splicing; Recombinants; Regulation; Research; Resolution; Role; Sampling; Schizophrenia; Seizures; Shapes; Stroke; Structure; Structure-Activity Relationship; Synaptic Transmission; Synaptic plasticity; System; Techniques; Therapeutic; Transmembrane Domain; Treatment Efficacy; Variant; base; conformational alteration; design; dimer; inhibitor/antagonist; insight; nervous system disorder; novel; postsynaptic; presynaptic; protein complex; receptor; research study; screening; stem; structural biology; success; voltage

DESCRIPTION (provided by applicant): The overall goal of the research studies proposed here is to obtain high-resolution structures of intact hetero- multimeric N-methyl-D-aspartate receptors (NMDARs). NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of excitatory synaptic transmission in mammalian brains. Dysfunctional NMDARs are implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. A unique aspect of NMDARs is that they are obligatory hetero- tetramers or higher oligomers composed of GluN1 and GluN2 (A-D) or GluN3 (A-B) subunits. Opening of NMDAR ion channels requires binding of glycine to GluN1 and GluN3 and glutamate to GluN2. To date, structural studies of NMDARs have been limited to the hetero-dimeric structures of the GluN1 and GluN2 extracellular domains. Thus, there is no clear knowledge on how subunits and domains are arranged to form hetero-multimeric ion channels and how transmembrane ion channel pores are shaped to confer specific properties of NMDAR ion channels including high calcium conductance and voltage-dependent magnesium block. Despite various technological breakthroughs, success in crystallographic studies on eukaryotic membrane proteins has been limited due to difficulties in expression, purification, and crystallization stemming from sample heterogeneity and instability. Importantly, there has been no crystal structure of eukaryotic hetero- multimeric membrane proteins that are recombinantly produced to date. The fact that numerous ion channels, G protein-coupled receptors, receptor kinases, and intramembrane proteases implicated in neurological diseases exist as hetero-multimers in native states points to the great need for structural studies on hetero- multimeric membrane proteins. To obtain the first crystal structure of hetero-multimeric ion channels and to understand the structure-function relationship of NMDARs, we will conduct research with the following two aims: Aim 1 is to produce intact hetero-multimeric NMDAR proteins using our novel methodology and to biochemical characterize the homogeneously purified proteins; and Aim 2 is to complete structural analysis of intact NMDARs in complex with various ligands reflecting different functional states by applying cutting-edge techniques in membrane protein crystallography and validate structure-based functional hypotheses by biochemical and electrophysiological experiments. Successful completion of the proposed studies is expected to result in the first crystal structure of a hetero-multimeric ion channel and to provide a mechanistic understanding of NMDARs that are critical in brain physiology and development. Importantly, the structural information obtained here will also provide strategies to develop compounds with therapeutic efficacy in neurological disorders and diseases. Furthermore, these studies on NMDARs will establish fundamental guidelines for crystallography on hetero-multimeric membrane proteins.

描述（由申请人提供）：此处提出的研究的总体目标是获得完整的异多聚体 N-甲基-D-天冬氨酸受体（NMDAR）的高分辨率结构。 NMDAR 属于离子型谷氨酸受体家族，介导哺乳动物大脑中的大部分兴奋性突触传递。功能失调的 NMDAR 与多种神经系统疾病和疾病有关，包括精神分裂症、抑郁症、阿尔茨海默病和帕金森病。 NMDAR 的一个独特之处在于它们是由 GluN1 和 GluN2 (A-D) 或 GluN3 (A-B) 亚基组成的必需异四聚体或高级寡聚体。 NMDAR 离子通道的打开需要甘氨酸与 GluN1 和 GluN3 结合以及谷氨酸与 GluN2 结合。迄今为止，NMDAR 的结构研究仅限于 GluN1 和 GluN2 胞外结构域的异二聚体结构。因此，对于亚基和结构域如何排列形成异源多聚体离子通道以及跨膜离子通道孔如何形成以赋予 NMDAR 离子通道的特定性质（包括高钙电导和电压依赖性镁块）尚无明确的了解。尽管取得了各种技术突破，但由于样品异质性和不稳定性导致表达、纯化和结晶困难，真核膜蛋白晶体学研究的成功受到限制。重要的是，迄今为止还没有重组产生的真核异多聚体膜蛋白的晶体结构。与神经系统疾病有关的众多离子通道、G蛋白偶联受体、受体激酶和膜内蛋白酶在天然状态下以异多聚体形式存在，这一事实表明非常需要对异多聚体膜蛋白进行结构研究。为了获得异源多聚体离子通道的第一个晶体结构并了解 NMDAR 的结构与功能关系，我们将进行以下两个目标的研究：目标 1 是使用我们的新方法生产完整的异源多聚体 NMDAR 蛋白，并对均质纯化的蛋白质进行生化表征；目标2是应用膜蛋白晶体学的前沿技术，完成完整NMDAR与反映不同功能状态的各种配体复合物的结构分析，并通过生化和电生理实验验证基于结构的功能假设。成功完成拟议的研究预计将产生异多聚离子通道的第一个晶体结构，并提供对脑生理学和发育至关重要的 NMDAR 的机制理解。重要的是，这里获得的结构信息还将提供开发对神经系统疾病和疾病具有治疗功效的化合物的策略。此外，这些关于 NMDAR 的研究将为异源多聚体膜蛋白的晶体学建立基本指南。