Structure and Function of NMDA Receptors

描述（由申请方提供）：本项目的目的是确定N-甲基-D-天冬氨酸受体（NMDAR）亚型特异性的分子机制，以促进控制NMDAR活性的亚型特异性试剂的开发。NMDAR属于离子型谷氨酸受体家族，其介导哺乳动物脑中的大部分快速兴奋性突触传递。NMDAR的异常活性涉及各种神经障碍和疾病，包括精神分裂症、抑郁症、阿尔茨海默病和帕金森病。这些受体是多聚体配体门控离子通道，主要由GluN 1和GluN 2亚基组成，分别在细胞外结构域与甘氨酸和L-谷氨酸结合。跨膜离子通道的门控通过甘氨酸和L-谷氨酸与配体结合结构域（LBD）的同时结合来介导，并且通过包括苯乙醇胺和Zn 2+的调节剂化合物与氨基末端结构域（ATD）的结合来变构调节。重要的是，由四个不同的GluN 2亚基（A至D）定义的NMDAR亚型的功能特性表现出显着不同的功能特性。不同的NMDAR亚型在给定的发育阶段在大脑的离散区域中表达，并且还与不同的神经系统疾病和病症相关。因此，了解亚型特异性的分子基础将是必要的，以便开发用于治疗上述神经系统疾病的特异性试剂。尽管有很多热情，但该领域仅限于一种有用的亚型特异性化合物，苯乙醇胺，其靶向GluN 1/GluN 2B NMDAR，但在治疗上使用时与脱靶效应相关。靶向其他亚型（如GluN 1/GluN 2A）的试剂的开发受到阻碍，因为NMDAR亚型的结构信息有限，这将允许进行全面的结构比较。为了获得NMDARs亚型特异性机制的理解，并促进GluN 1/GluN 2A和GluN 1/GluN 2B NMDARs亚型特异性试剂的开发，我们将进行以下研究：目的1获得对GluN 1/GluN 2B ATD和GluN 1/GluN 2A LBD中配体结合位点的深入理解;目的2确定GluN 1/GluN 2A ATD中亚型特异性变构抑制的分子机制;目的3确定GluN 1/GluN 2A ATD中GluN 1/GluN 2A LBD中GluN 1/GluN 2A LBD的配体结合位点。目的3利用我们新近研制的抑制性抗体，确定GluN 1/GluN 2B NMDAR的结合及抑制机制。这三个目标将通过获得ATD和LBD的结构信息并通过电生理学测试基于结构的假设来实现。成功完成所提出的研究将为ATD、LBD中的配体结合位点和亚型特异性的分子元件提供前所未有的见解，并证明使用抑制性抗体以亚型特异性方式抑制NMDAR的新方法。这些发现将有助于开发亚型特异性试剂来研究和治疗上述精神健康相关疾病。

DESCRIPTION (provided by applicant): The goal of this project is to define the molecular mechanism underlying subtype-specificity in N-methyl-D- aspartate receptors (NMDARs) to facilitate development of subtype-specific reagents for controlling NMDAR activities. NMDARs belong to the family of ionotropic glutamate receptors, which mediate the majority of fast excitatory synaptic transmission in mammalian brains. Abnormal activity of NMDARs is implicated in various neurological disorders and diseases including schizophrenia, depression, Alzheimer's disease, and Parkinson's disease. Those receptors are multimeric ligand-gated ion channels composed mainly of GluN1 and GluN2 subunits that bind to glycine and L-glutamate at the extracellular domain, respectively. Gating of transmembrane ion channels is mediated by concurrent binding of glycine and L-glutamate to the ligand- binding domain (LBD) and is allosterically regulated by binding of modulator compounds including phenylethanolamines and Zn2+ to the amino terminal domain (ATD). Importantly, functional properties of NMDARs subtypes, which are defined by four distinct GluN2 subunits (A though D), exhibit dramatically different functional properties. Different NMDAR subtypes are expressed in discrete regions of the brain at given developmental stages and are also associated with distinct neurological diseases and disorders. Thus, understanding the molecular basis for subtype-specificity will be necessary in order to develop specific reagents for treatment of the above neurological diseases. Despite much enthusiasm, the field is limited to one useful subtype-specific compound, phenylethanolamine, which targets GluN1/GluN2B NMDARs but is associated with off-target effects when used therapeutically. Development of reagents targeting other subtypes such as GluN1/GluN2A has been hampered due to limited amount of structural information on subtypes of NMDARs, which would allow comprehensive structural comparison. To obtain a mechanistic understanding of subtype-specificity in NMDARs and to facilitate development of subtype-specific reagents for GluN1/GluN2A and GluN1/GluN2B NMDARs, we will conduct research aimed at: Aim 1 obtaining an in-depth understanding of the ligand-binding site in GluN1/GluN2B ATD and GluN1/GluN2A LBD; Aim 2 defining the molecular mechanism of subtype-specific allosteric inhibition in GluN1/GluN2A ATD; and Aim 3 determining the binding and inhibition mechanism of GluN1/GluN2B NMDAR by inhibitory antibody that we recently developed. These three goals will be achieved by obtaining the structural information of ATD and LBD and testing structure- based hypotheses by electrophysiology. Successful completion of the proposed studies will provide unprecedented insights into ligand-binding sites in ATD, LBD, and molecular elements underlying subtype- specificity, and to demonstrate a novel approach to inhibit NMDAR in a subtype-specific manner using inhibitory antibodies. These findings will facilitate development of subtype-specific reagents to study and treat the mental health related disorders above.