3D Structure and Function of Ligand-Gated Ion Channels

配体门控离子通道的 3D 结构和功能

• 批准号: 10584469
• 负责人: James E Gouaux
• 金额: $ 33.69万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-19 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10584469
• 关键词: AMPA Receptors; Agonist; Alzheimer' s Disease; Amino Acid Sequence; Antibodies; Applications Grants; Autoimmune Diseases; Binding; Biochemical; Brain; Breathing; Cations; Cell Communication; Cells; Cerebellum; Chemical Synapse; Chemicals; Complex; Cryoelectron Microscopy; Cysteine; Detergents; Development; Electrophysiology (science); Encephalitis; Epilepsy; Extracellular Structure; Fab Immunoglobulins; Family; Foundations; Glutamate Receptor; Glutamates; Glycine; Hand; Hippocampus; Human; Immunoglobulin Fragments; Individual; Investigation; Ion Channel; Ion Channel Gating; Kainic Acid Receptors; Knowledge; Lead; Learning; Ligands; Maps; Mediating; Membrane; Memory; Mental Depression; Methods; Molecular; Molecular Structure; Mood Disorders; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Nervous System; Nervous System Immune Disorders; Neurons; Neurotransmitters; Pathway interactions; Pharmacology; Physiological; Physiology; Play; Positioning Attribute; Predisposition; Property; Protein Subunits; Proteins; Receptor Activation; Recombinants; Research; Research Support; Rodent; Role; Schizophrenia; Signal Pathway; Signal Transduction; Site; Structure; Styrenes; Synapses; Testing; Therapeutic Agents; Time; Transmembrane Domain; Zinc; brain tissue; copolymer; crosslink; design; experimental study; glutamatergic signaling; heart function; ifenprodil; ion channel blocker; maleic acid; millisecond; mutant; nervous system development; nervous system disorder; neurotransmission; neurotransmitter release; novel therapeutics; particle; receptor; receptor function; small molecule; stoichiometry; three dimensional structure; trafficking

Ionotropic glutamate receptors (iGluRs) are glutamate-activated, cation-selective ion channels that mediate the majority of millisecond-time scale signaling in the nervous system. The iGluR family of receptors is composed of three major subtypes, the AMPA, NMDA and kainate receptors, each of which plays a crucial role in nervous system development and function. The disruption of normal glutamatergic signaling underpins multiple neurological disorders, including Alzheimer’s disease, depression, schizophrenia and epilepsy. Accordingly, iGluRs are the targets for numerous therapeutic agents to treat seizure and mood disorders, as well as schizophrenia. Several immunological disorders of the nervous system also involve iGluRs, with the most prevalent being NMDA receptor encephalitis, a severe autoimmune disease for which there are few promising treatments. Despite the widespread distribution of iGluRs throughout the human nervous system, and even though they are involved in many debilitating neurological diseases and disorders, the molecular mechanisms of iGluR function are not yet fully elucidated. Moreover, iGluRs are not isolated at their sites of function, typically the synapses, but rather are found in complexes with multiple auxiliary proteins. Indeed, the organization and molecular function of these auxiliary proteins also remains to be fully elucidated. Thus, the major focus of the research proposed in this grant application is to elucidate the molecular structure and mechanisms of function for AMPA and NMDA receptors, alone and in complexes with key auxiliary proteins. Here we will develop methods to isolate native AMPA receptors from whole brain and from the hippocampus and cerebellum, as complexes with their auxiliary proteins. We will then elucidate the structures of these receptor complexes by single particle cryo-EM, using subunit specific antibody fragments to tag subunits. With the structures in hand, we will design mutants to test important aspects of structure and mechanisms, carrying out biochemical and electrophysiological experiments to probe receptor function. In parallel, we will also study the NMDA receptors, here focusing on a structure-based mechanism for receptor gating, investigating the structure of the GluN1/GluN2A receptor in complexes with the agonists glycine and glutamate, as well as with allosteric modulators and ion channel blockers. Through these studies we aim to answer the question of how neurotransmitters activate the receptor and how allosteric modulators increase or decrease receptor activity. In parallel with AMPA receptor studies, we will also isolate native GluN1-containing NMDA receptors from rodent brain tissue, with the aim being to elucidate the first structure of native NMDA receptors, mapping the positions of the GluN1 and GluN2 subunits in the receptor complex. Taken together, our studies will define structure-based, biochemical mechanisms for iGluR function and they will lay the foundation for development of new therapeutic agents.

离子型谷氨酸受体（iGlurs）是谷氨酸激活的阳离子选择性离子通道，可介导 神经系统中的大多数毫秒尺度信号传导。伊格尔接收者家族组成 在三种主要亚型的AMPA，NMDA和海藻酸盐接收器中，它们在神经中都起着至关重要的作用 系统开发和功能。正常谷氨酸能信号传导的破坏基于多个 神经系统疾病，包括阿尔茨海默氏病，抑郁症，精神分裂症和癫痫。 iGlurs是许多治疗癫痫和情绪障碍的众多治疗剂的目标，也是 精神分裂症。神经系统的几种免疫障碍也涉及iGlurs，最多 普遍是NMDA受体脑炎，这是一种严重的自身免疫性疾病，几乎没有承诺 治疗。尽管在整个人类神经系统中，甚至 尽管它们参与了许多使神经系统疾病和疾病的衰弱，但分子机制 iglur功能尚未完全阐明。此外，iGlurs在其功能部位不隔离， 通常是突触，而是在具有多种辅助蛋白的复合物中发现。确实， 这些辅助蛋白的组织和分子功能也尚待充分阐明。那， 本赠款应用中提出的研究的主要重点是阐明分子结构和 单独和具有关键辅助蛋白的复合物中AMPA和NMDA受体的功能机理。 在这里，我们将开发方法，将本机AMPA接收器与全脑和海马隔离 和小脑，作为辅助蛋白的复合物。然后，我们将阐明这些结构 通过亚基特异性抗体片段来标记亚基的接收器复合物通过单个粒子冷冻em。和 我们手头的结构将设计突变体以测试结构和机制的重要方面 探测探测受体功能的生化和电生理实验。同时，我们还将学习 NMDA受体，此处着重于一种基于结构的受体门控机制，研究了 glun1/glun2a受体在与激动剂甘氨酸和谷氨酸的复合物中的结构，以及 变构调节器和离子通道阻滞剂。通过这些研究，我们旨在回答如何 神经递质激活受体以及变构调节剂如何增加或降低受体活性。 与AMPA受体研究并行，我们还将与含天然Glun1的NMDA受体分离 啮齿动物脑组织，目的是阐明天然NMDA受体的第一个结构，绘制 Glun1和Glun2亚基在接收器复合物中的位置。综上所述，我们的研究将定义 基于结构的iGlur功能的生化机制，它们将为发展奠定基础 新的治疗剂。