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.

项目摘要 该项目的目的是揭示N-甲基-D-天冬氨酸中药物活性位点的分子细节 受体（NMDAR）具有针对心理健康相关疾病的亚型特异性试剂的范围 以及包括抑郁症，精神病，精神分裂症，癫痫，阿尔茨海默氏病和中风在内的疾病 暗示功能失调的NMDAR。 NMDAR属于离子型谷氨酸受体（Iglur）家族，该家族 介导哺乳动物大脑中的大多数兴奋性突触传播。他们是异性杀手 配体门控离子通道由Glun1和Glun2和/或Glun3亚基组成。 glun1和glun3 亚基结合了包括甘氨酸和D-丝氨酸在内的共振，而Glun2亚基结合了谷氨酸和NMDA。 每个NMDAR亚基都包含一个氨基终端结构域（ATD），一个配体结合域（LBD），A 跨膜结构域（TMD）和羧基末端结构域（CTD）。 glun1-glun2 nmdars打开 甘氨酸和谷氨酸结合后TMD离子通道。 NMDARS亚型，由四个不同的Glun2定义 亚基（A至D）或两个不同的Glun3（A和B）与Glun1结合使用，暴露了不同的功能 性质和时空表达模式。在上一个赠款周期中，我们揭开了片段 Glun1-2a和Glun1-2b ATD，Glun1-2a和Glun1-2d LBD的结构以及Glun1-2b的完整结构 通过使用X射线晶体学和单粒子电子冷冻微镜（Cryo-EM）。我们提供的发现 对通道组件的分子见解和构象运动的基本模式，导致激活， 抑制和变构调节。尽管有这些进步，但仍然有许多基本问题 仍未解决，包括含Glun2C的NMDAR的完全出乎意料的结构 未知/未表征的变构和通道阻滞位点具有临床相关性，并通过 抗体。现在，我们将进行旨在履行这些缺口的研究。 AIM 1将确定第一个 含Glun2C的NMDAR的结构； AIM 2将识别和表征临床上的药物位点 相关化合物，包括氯胺酮，苯基二肽，美灵和Glyx13。 AIM 3将揭开 新型工程抗体和自身免疫抗体的结合和功能调节机制 首次。这三个目标将通过获得完整的NMDAR的结构信息来实现 在存在和不存在试剂的情况下，使用X射线晶体和单粒子的域碎片 冷冻em。基于结构的机械假设将主要通过电生理学研究。成功的 拟议的研究目的的完成将为NMDAR子类型提供详细的了解 - 特异性，不同试剂的调节机制以及控制NMDAR亚型的新型手段，这些方法 将有助于制定神经系统疾病和疾病的理论策略。