Structure and Function of NMDA Receptors

描述(由申请人提供)：本项目的目标是确定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，介导哺乳动物大脑中大部分的快速兴奋性突触传递。NMDARs的异常活动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。这些受体是主要由GluN1和GluN2亚单位组成的多配体门控离子通道，分别与甘氨酸和L-谷氨酸在胞外区域结合。跨膜离子通道的门控是由甘氨酸和L-谷氨酸同时结合到配体结合域(LBD)介导的，并受包括苯乙醇胺和锌离子在内的调制化合物与氨基末端域(ATD)结合的变构调节。重要的是，NMDARs亚型的功能特性由四个不同的GluN2亚基(A到D)定义，表现出截然不同的功能特性。不同的NMDAR亚型在特定发育阶段的大脑不同区域表达，也与不同的神经疾病和障碍有关。因此，为了开发治疗上述神经系统疾病的特异性试剂，了解亚型特异性的分子基础是必要的。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，它针对GluN1/GluN2B NMDAR，但在用于治疗时与非靶点效应有关。针对其他亚型，如GluN1/GluN2A的试剂的开发受到阻碍，因为关于NMDARs亚型的结构信息有限，这将使全面的结构比较成为可能。为了从机制上了解NMDARs的亚型特异性，促进针对GluN1/GluN2A型和GluN1/GluN2B型NMDARs亚型特异性试剂的开发，我们将进行以下方面的研究：目的1深入了解GluN1/GluN2B ATD和GluN1/GluN2ALBD中的配体结合部位；目的2确定GluN1/GluN2AATD中亚型特异性变构抑制的分子机制；以及目的3确定我们最近开发的抑制性抗体对GluN1/GluN2B亚型特异性变构抑制的结合和抑制机制。这三个目标将通过获取ATD和LBD的结构信息并通过电生理学测试基于结构的假说来实现。这项拟议研究的成功完成将提供对ATD、LBD的配体结合位点以及亚型特异性的分子元件的前所未有的见解，并展示一种使用抑制性抗体以亚型特异性方式抑制NMDAR的新方法。这些发现将促进亚型特异性试剂的开发，以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.