Structures and reagents of NMDA receptors

NMDA受体的结构和试剂

• 批准号: 10467766
• 负责人: Hiroyasu Furukawa
• 金额: $ 71.98万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10467766
• 关键词: Achievement; Active Sites; Affect; Agonist; Allosteric Regulation; Alzheimer' s Disease; Antibodies; Anxiety Disorders; Autoantibodies; Autoimmune; Binding; Binding Sites; Brain; Cells; Cerebellum; Chemosensitization; Complex; Coupled; Cryoelectron Microscopy; Cycloserine; Development; Disease; Electrophysiology (science); Elements; Encephalitis; Engineering; Epilepsy; Excitatory Amino Acid Antagonists; Exhibits; Family; Glutamate Receptor; Glutamates; Glycine; Goals; Grant; IgG Receptors; Insecta; Ion Channel; Ion Channel Gating; Kainic Acid Receptors; Ketamine; Kinetics; Lead; Libraries; Ligand Binding Domain; Ligands; Measures; Mediating; Memantine; Membrane Proteins; Mental Depression; Methods; Molecular; Molecular Conformation; Movement; Mutagenesis; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMDA receptor A1; Neurotransmitters; Patients; Pattern; Pharmacology; Phencyclidine; Play; Property; Psychoses; Public Health; RNA Splicing; Reagent; Recombinants; Regulation; Research; Resolution; Roentgen Rays; Role; Schizophrenia; Series; Serine; Site; Site-Directed Mutagenesis; Specificity; Stroke; Structure; Surface Plasmon Resonance; Synaptic Transmission; Technology; Therapeutic; Time; Transmembrane Domain; Variant; X-Ray Crystallography; Yeasts; Zinc; antagonist; antibody engineering; base; biophysical tools; channel blockers; clinical research site; clinically relevant; desensitization; excitatory neuron; experimental study; improved; insight; mental health related disorder; nanobodies; nervous system disorder; novel; particle; patch clamp; protein complex; receptor function; screening; spatiotemporal; stoichiometry; therapeutic development

Project Summary The goal of this project is to unravel molecular details of pharmacologically active sites in N-methyl-D-aspartate receptors (NMDARs) with a scope to develop subtype-specific reagents against mental health-related disorders and diseases including depression, psychosis, schizophrenia, epilepsy, Alzheimer’s disease, and stroke where dysfunctional NMDARs are implicated. NMDARs belong to the ionotropic glutamate receptor (iGluR) family which mediates the majority of excitatory synaptic transmission in mammalian brains. They are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind glutamate and NMDA. Each NMDAR subunit contains an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their TMD ion channels upon binding of glycine and glutamate. NMDARs subtypes, defined by four distinct GluN2 subunits (A through D) or two distinct GluN3 (A and B) in combination with GluN1, exhibit different functional properties and spatio-temporal expression patterns. In the previous grant cycles, we unraveled the fragment structures of GluN1-2A and GluN1-2B ATDs, GluN1-2A and GluN1-2D LBDs, and intact structures of GluN1-2B by utilizing x-ray crystallography and single-particle electron cryo-microscopy (cryo-EM). Our findings provided molecular insights into channel assembly and basic patterns of conformational movements leading to activation, inhibition, and allosteric regulation. Despite these advances, there are still many fundamental questions remaining unanswered, including the completely unexplored structures of the GluN2C-containing NMDARs, unknown/uncharacterized allosteric and channel blockade sites with clinical relevance, and novel regulation by antibodies. We will now conduct research aimed at fulfilling these shortfalls. Aim 1 will determine the first structure of GluN2C-containing NMDARs; Aim 2 will identify and characterize pharmacological sites of clinically relevant compounds including ketamine, phencyclidine, memantine, and Glyx13. Aim 3 will unravel the mechanism of binding and functional regulation by novel engineered antibodies, and the auto-immune antibody for the first time. These three aims will be achieved by obtaining the structural information of intact NMDAR and domain fragments in the presence and absence of reagents by utilizing x-ray crystallography and single-particle cryo-EM. Structure-based mechanistic hypotheses will be examined mainly by electrophysiology. Successful completion of the proposed research aims will provide a detailed understanding about NMDAR subtypes- specificity, the regulatory mechanism by different reagents, and novel means to control NMDAR subtypes, which will help develop therapeutic strategies for neurological disorders and diseases.

项目概要 该项目的目标是揭示 N-甲基-D-天冬氨酸药理活性位点的分子细节 受体（NMDAR），可开发针对精神健康相关疾病的亚型特异性试剂 以及抑郁症、精神病、精神分裂症、癫痫症、阿尔茨海默病和中风等疾病 功能失调的 NMDAR 属于离子型谷氨酸受体 (iGluR) 家族。 介导哺乳动物大脑中的大部分兴奋性突触传递。它们是异源多聚体。 由 GluN1 和 GluN2 和/或 GluN3 亚基组成的配体门控离子通道。 GluN2 亚基结合甘氨酸和 D-丝氨酸等共激动剂，而 GluN2 亚基结合谷氨酸和 NMDA。 每个 NMDAR 亚基包含一个氨基末端结构域 (ATD)、一个配体结合结构域 (LBD)、一个 GluN1-GluN2 NMDAR 打开其跨膜结构域 (TMD) 和羧基末端结构域 (CTD)。 甘氨酸和谷氨酸结合后的 TMD 离子通道，由四种不同的 GluN2 定义。 亚基（A 至 D）或两个不同的 GluN3（A 和 B）与 GluN1 组合，表现出不同的功能 在之前的资助周期中，我们解开了该片段。 GluN1-2A 和 GluN1-2B ATD 的结构、GluN1-2A 和 GluN1-2D LBD 以及 GluN1-2B 的完整结构 通过利用 X 射线晶体学和单粒子电子冷冻显微镜 (cryo-EM)，我们提供了研究结果。 对通道组装和导致激活的构象运动基本模式的分子洞察， 尽管取得了这些进展，但仍然存在许多基本问题。 仍然没有答案，包括完全未探索的含有 GluN2C 的 NMDAR 结构， 具有临床相关性的未知/未表征的变构和通道阻断位点，以及新的调节 我们现在将进行旨在弥补这些不足的研究，目标1将确定第一个。 含 GluN2C 的 NMDAR 的结构；目标 2 将识别和表征临床药理学位点 包括氯胺酮、苯环己哌啶、美金刚和 Glyx13 在内的相关化合物将揭开谜团。 新型工程抗体和自身免疫抗体的结合和功能调节机制 这三个目标将首次通过获得完整的 NMDAR 的结构信息来实现。 利用 X 射线晶体学和单颗粒分析在存在和不存在试剂的情况下的结构域片段 基于结构的机制假设将主要通过电生理学进行检验。 完成拟议的研究目标将提供对 NMDAR 亚型的详细了解 - 特异性、不同试剂的调节机制以及控制 NMDAR 亚型的新方法，这些 将有助于制定神经障碍和疾病的治疗策略。