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: 8847340
• 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/8847340
• 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(申请人提供)：本研究的总体目标是获得完整的异源多聚体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，介导哺乳动物脑内大部分兴奋性突触传递。功能失调的NMDAR与各种神经疾病和疾病有关，包括精神分裂症、抑郁症、阿尔茨海默病和帕金森氏病。NMDAR的一个独特之处在于，它们是由GluN1和GluN2(A-D)或GluN3(A-B)亚基组成的必需的异四聚体或更高的寡聚体。NMDAR离子通道的开放需要甘氨酸与GluN1和GluN3结合，谷氨酸与GluN2结合。到目前为止，对NMDARs的结构研究仅限于GluN1和GluN2胞外区的异二聚体结构。因此，亚基和结构域是如何排列形成异质多聚体离子通道的，以及跨膜离子通道孔是如何形成的，从而赋予NMDAR离子通道的特殊性质，包括高钙电导和电压依赖的镁阻塞，目前还没有明确的知识。尽管有各种技术突破，但由于样品的异质性和不稳定性导致表达、纯化和结晶的困难，真核膜蛋白的结晶学研究的成功受到了限制。重要的是，到目前为止还没有重组生产的真核异源多聚体膜蛋白的晶体结构。许多与神经系统疾病有关的离子通道、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源多聚体的形式存在，这表明对异源多聚体膜蛋白的结构研究是非常必要的。为了获得异质多聚体离子通道的第一个晶体结构，了解NMDAR的结构与功能的关系，我们将进行以下两个目标的研究：目的1是利用我们的新方法制备完整的异质多聚体NMDAR蛋白，并对纯化的蛋白质进行生化表征；目标2是应用膜蛋白质结晶学的前沿技术完成完整的NMDAR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说。拟议研究的成功完成有望产生第一个异质多聚体离子通道的晶体结构，并提供对NMDAR的机制理解，NMDAR在大脑生理学和发育中至关重要。重要的是，这里获得的结构信息也将为开发对神经疾病和疾病具有治疗效果的化合物提供策略。此外，这些关于NMDARs的研究将为异质多聚膜蛋白的结晶学建立基本的指导方针。