Structure and Function of NMDA Receptors

描述（由申请人提供）：该项目的总体目标是揭示n -甲基- d -天冬氨酸（NMDA）受体亚型特异性和变构功能调节的分子决定因素。NMDA受体属于嗜离子性谷氨酸受体（iGluRs）家族，参与哺乳动物大脑中大多数兴奋性突触传递。NMDA受体是由NR1和NR2亚基组成的多聚体配体门控离子通道，分别在细胞外结构域（ATD/S1S2）与甘氨酸和l -谷氨酸结合。跨膜离子通道的门控或打开是由甘氨酸和l -谷氨酸结合到配体结合域（S1S2）介导的，并通过包括苯乙醇胺、多胺、质子和Zn2+在内的调节剂化合物结合到氨基末端结构域（ATD）进行变构调节。NMDA受体的功能特性取决于它们的亚型，这些亚型由四个不同的NR2亚基（A到D）定义。尽管先前已经从NR1和NR2A中确定了S1S2的几种结构，但由于缺乏其他NR2亚基的结构信息，亚型特异性配体结合和功能的分子基础尚不清楚，包括含有NMDA受体的NR2D异常缓慢的失活动力学。此外，NR2亚基的激活和抑制的一般机制仍然是一个悬而未决的问题，因为NR2 S1S2与任何部分激动剂或拮抗剂的复合物的结构尚未阐明。最后，由于完全缺乏ATD结构，调节化合物与ATD结合介导的变构调节的分子机制仍然难以捉摸。因此，我们的目标是获得NMDA受体ATD和S1S2的原子视图，以揭示ATD中通过调节剂结合介导的亚型特异性和变构调节的分子机制。实验计划结合了x射线晶体学、电生理学和生化技术。NMDA受体胞外结构域配体结合核（S1S2）和氨基末端结构域（ATD）的结构信息将通过诱变结合电生理和生化实验来补充，以建立NMDA受体的结构-功能关系。这项建议有两个具体目的。目的1是了解通过NR2 S1S2介导的亚型特异性配体结合、激活、抑制和失活的结构机制。目的2是破解由变构调节剂（包括Zn2+、伊芬普罗地尔、质子和多胺）与NR1和NR2B ATDs结合介导的NMDA受体活性变构调节的分子机制。NMDA受体一直是药理学研究的主要目标，因为它们在脑功能和发育中起着关键作用。NMDA受体的功能障碍与神经和精神健康相关的疾病和损伤有关，包括癫痫、精神分裂症、阿尔茨海默病和帕金森病。本研究的结果有望帮助设计出具有高特异性和效力的靶向ATD和S1S2的新型化合物，具有重要的治疗价值。

DESCRIPTION (provided by applicant): The overall goal of this project is to uncover molecular determinants for subtype specificity and allosteric functional modulations of N-methyl-D-aspartate (NMDA) receptors. NMDA receptors belong to a family of ionotropic glutamate receptors (iGluRs) involved in the majority of excitatory synaptic transmission in the mammalian brain. NMDA receptors are multimeric ligand-gated ion channels composed of NR1 and NR2 subunits that bind to glycine and L-glutamate at the extracellular domain (ATD/S1S2), respectively. Gating or opening of transmembrane ion channels is mediated by binding of both glycine and L-glutamate to the ligand- binding domain (S1S2) and are allosterically modulated by binding of modulator compounds including phenylethanolamines, polyamines, protons, and Zn2+ to the amino terminal domain (ATD). The functional properties of NMDA receptors differ significantly depending on their subtypes that are defined by four distinct NR2 subunits (A though D). Although several structures have been previously determined for the S1S2 from NR1 and NR2A, the molecular basis for subtype specific ligand-bindings and functions including exceptionally slow deactivation kinetics of the NR2D containing NMDA receptors is unknown due to a lack of structural information on the other NR2 subunits. Furthermore, the general mechanism of activation and inhibition in the NR2 subunits remains an open question because the structure of the NR2 S1S2 in complex with any partial agonist or antagonist has yet to be elucidated. Finally, the molecular mechanism for allosteric modulation mediated by the binding of modulator compounds to the ATD remains elusive due to a complete lack of the ATD structures. Thus, our goal is to obtain the atomic view of the ATD and S1S2 of the NMDA receptors to reveal the molecular mechanism for subtype specificity and allosteric modulation mediated through modulator binding in the ATD. The experimental plan combines x-ray crystallography, electrophysiology, and biochemical techniques. The structural information of the extracellular domain of the NMDA receptors, ligand-binding core (S1S2), and amino terminal domain (ATD), will be complemented by mutagenesis coupled with electrophysiology and biochemical experiments to establish the structure-function relationships of the NMDA receptors. There are two specific aims in this proposal. Aim 1 is to understand the structural mechanism for the subtype specific ligand-bindings, activation, inhibition, and deactivation mediated through NR2 S1S2. Aim 2 is to decipher the molecular mechanism underlying allosteric modulation of the NMDA receptor activity mediated by the binding of allosteric modulators, including Zn2+, ifenprodil, proton, and polyamines, to the NR1 and NR2B ATDs. The NMDA receptors have been a major target for pharmacological studies because they play pivotal roles in brain function and development. Dysfunction of the NMDA receptors is implicated in neurological and mental health related diseases and injuries, including seizure, schizophrenia, Alzheimer's disease, and Parkinson's disease. The results of this research are expected to help design novel compounds that target the ATD and S1S2 with high specificity and potency and with significant therapeutic values. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to uncover the molecular basis for the function of NMDA receptors, which are crucial in normal brain function and development. The studies are relevant to public health because NMDA receptors are implicated in various neurological and mental health related diseases and disorders including seizure, stroke, schizophrenia, as well as Parkinson's and Alzheimer's diseases. Defining the molecular structure of the extracellular region of NMDA receptors is expected to help in the design of new therapeutics.