Structure, Activation, and Modulation of AMPA/Glutamate Receptors

AMPA/谷氨酸受体的结构、激活和调节

• 批准号: 9093854
• 负责人: ROBERT E OSWALD
• 金额: $ 33.91万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9093854
• 关键词: AMPA Receptors; Affect; Affinity; Agonist; Alanine; Alzheimer' s Disease; Aspartate; Attention Deficit Disorder; Back; Bacteria; Behavior; Binding; Binding Sites; Biological; Biological Models; Brain; Calorimetry; Clinical Trials; Cognition; Complex; Coupling; Crystallography; Data; Degenerative Disorder; Development; Disease; Diuretics; Drug Design; Drug Targeting; Electrophysiology (science); Electrostatics; Epilepsy; Event; Glutamate Receptor; Glutamates; Goals; Health; Hydrogen Bonding; Ion Channel; Kainic Acid Receptors; Kinetics; Knowledge; Learning; Ligand Binding; Ligand Binding Domain; Light; Link; Lobe; Mediating; Memory; Modeling; Molecular Structure; Mutagenesis; Mutation; N-Methyl-D-Aspartate Receptors; N-Methylaspartate; NMR Spectroscopy; Neocortex; Neuraxis; Neurodegenerative Disorders; Neurons; Peptides; Pharmaceutical Preparations; Process; Property; Proteins; Reaction; Recovery; Roentgen Rays; Role; Side; Signal Transduction; Sodium Chloride; Stroke; Structure; Synapses; Synaptic Transmission; Synaptic plasticity; Therapeutic; Titrations; Work; X-Ray Crystallography; base; cyclothiazide; desensitization; dimer; drug development; flexibility; kainate; multidisciplinary; mutant; postsynaptic; receptor; receptor function; release of sequestered calcium ion into cytoplasm; research study; response; therapeutic target; transmission process

DESCRIPTION (provided by applicant): Overstimulation of ionotropic glutamate receptors (iGluRs), including AMPA receptors contributes to a number of neurodegenerative diseases, notably stroke and epilepsy. Thus, these receptors are drug targets of considerable therapeutic value. However, they are not simple targets since iGluRs also have essential roles in brain development and normal neuronal processes including learning and memory. Our goal is to investigate the structural basis of AMPA receptor function to develop a clearer understanding of how AMPA receptor-channels activate and desensitize. iGluRs are tetramers of similar subunits and each subunit is made up of a set of modules including the ligand binding domain (LBD) that is the focus of our structure-function studies. A full crystal structure of an AMPA receptor provides the framework for studying molecular interactions in the holoreceptor, but the receptor LBD is a particularly useful model system as it can be produced in bacteria as a soluble protein, and it binds agonists and antagonists with approximately the same affinity as the intact receptor. We have previously determined crystal structures of the GluA2 and GluA3 subtypes of AMPA receptor LBD bound to agonists, antagonists, and allosteric modulators, and, using NMR spectroscopy, examined the dynamic behavior of the GluA2 LBD in the presence of full and partial agonists. We have characterized the complex kinetic behavior of wildtype GluA3 AMPA receptors in single channel studies and generated mutations in the LBD of GluA3 receptor-channels that affect channel activation and gating properties. We propose to combine X-ray crystallography, NMR spectroscopy, small angle X-ray scattering, single channel recording, isothermal titration calorimetry, and rapid drug application to multi-channel patches to investigate two aspects of GluA2 and GluA3 gating. The first aim involves the transmission of the signal from the binding site to the channel domain. Our NMR studies suggest that Lobe 2 of LBD has considerable dynamics and electrostatic interactions are required to maintain a rigid structure. Mutations at residues affecting these interactions will be analyzed by NMR (sidechain dyanmics and H/D exchange) and the functional consequences will be analyzed by single channel and multi-channel patch recording. The hypothesis is that several discrete interactions are necessary for efficient activation of the channel and for the unique kinetics of AMPA receptors relative to other glutamate receptor subtypes. The second aim builds on our work on allosteric modulators of AMPA receptors and will investigate the binding mechanism and the steps in the reaction mechanism that are modified by these agents. Although homomeric receptors are likely to be confined to a small number of neurons, GluA2/GluA3 heteromeric receptor-channels may represent a significant number of postsynaptic AMPA receptors in the neocortex. The results from these experiments will shed light on the structure and function of an important glutamate receptor and provide essential information for further drug development.

描述（由申请人提供）：过度刺激离子型谷氨酸受体（IGLURS），包括AMPA受体会导致许多神经退行性疾病，尤其是中风和癫痫病。因此，这些受体是具有相当可观治疗价值的药物靶标。但是，它们不是简单的目标，因为iGlurs在大脑发育和正常神经元过程中也具有重要作用，包括学习和记忆。我们的目标是研究AMPA受体功能的结构基础，以更清楚地了解AMPA受体通道如何激活和脱敏。 iGlurs是类似亚基的四聚体，每个亚基都由一组模块组成，包括配体结合域（LBD），这是我们结构功能研究的重点。 AMPA受体的完整晶体结构提供了研究整体受体中分子相互作用的框架，但是受体LBD是一个特别有用的模型系统，因为它可以在细菌中作为可溶性蛋白质产生，并且它可以结合具有与完整受体相同的亲和力的激动剂和拮抗剂。我们先前已经确定了与激动剂，拮抗剂和变构调节剂结合的AMPA受体LBD的GLUA2和GLUA3亚型的晶体结构，并使用NMR光谱检查了GLUA2 LBD在充分和部分偏向的激动剂的情况下的动态行为。我们已经表征了野生型GLUA3 AMPA受体在单个通道研究中的复杂动力学行为，并在GLUA3受体通道的LBD中产生了突变，从而影响通道激活和门控性能。我们建议将X射线晶体学，NMR光谱，小角度X射线散射，单个通道记录，等温滴定量热法和快速药物应用于多通道斑块，以研究Glua2和Glua3 Gating的两个方面。第一个目的涉及信号从结合位点传输到通道域。我们的NMR研究表明，LBD的Lobe 2具有相当大的动力学，需要静电相互作用才能维持刚性结构。 NMR（Sidechain Dyanmics和H/D交换）将分析影响这些相互作用的残基的突变，功能后果将通过单个通道和多通道贴片记录来分析。假设是，相对于其他谷氨酸受体亚型的通道有效激活和AMPA受体的独特动力学，几种离散相互作用是必需的。第二个目的是基于我们在AMPA受体的变构调节剂上的工作，并将研究这些药物修饰的反应机制的结合机制和步骤。尽管同源受体可能仅限于少数神经元，但GLUA2/GLUA3异源受体通道可能代表新皮层中大量突触后AMPA受体。这些实验的结果将阐明重要的谷氨酸受体的结构和功能，并为进一步的药物开发提供必不可少的信息。