Structure, Activation, and Modulation of AMPA/Glutamate Receptors

AMPA/谷氨酸受体的结构、激活和调节

基本信息

批准号：
8894107
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
项目类别：
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/8894107
关键词：
AMPA Receptors Affect Affinity Agonist Alanine Alzheimer&apos 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 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 Relative (related person)Roentgen Rays Role Side Signal Transduction Sodium Chloride Stroke Structure Synapses Synaptic Transmission Synaptic plasticity Therapeutic Titrations Trichlormethiazide 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

项目摘要

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）：离子型谷氨酸受体（iGluR）（包括 AMPA 受体）的过度刺激会导致许多神经退行性疾病，特别是中风和癫痫。因此，这些受体是具有相当大治疗价值的药物靶点。然而，它们并不是简单的目标，因为 iGluR 在大脑发育和正常神经元过程（包括学习和记忆）中也发挥着重要作用。我们的目标是研究 AMPA 受体功能的结构基础，以便更清楚地了解 AMPA 受体通道如何激活和脱敏。 iGluR 是相似亚基的四聚体，每个亚基由一组模块组成，其中包括配体结合域 (LBD)，这是我们结构功能研究的重点。 AMPA 受体的完整晶体结构为研究全感受器中的分子相互作用提供了框架，但受体 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，并且它以大约与完整受体具有相同的亲和力。我们之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 在完全和部分激动剂存在下的动态行为。我们在单通道研究中表征了野生型 GluA3 AMPA 受体的复杂动力学行为，并在 GluA3 受体通道的 LBD 中产生了影响通道激活和门控特性的突变。我们建议将 X 射线晶体学、核磁共振波谱、小角 X 射线散射、单通道记录、等温滴定量热法和快速药物应用于多通道贴片相结合，以研究 GluA2 和 GluA3 门控的两个方面。第一个目标涉及将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，并且需要静电相互作用来维持刚性结构。影响这些相互作用的残基突变将通过 NMR（侧链动力学和 H/D 交换）进行分析，并通过单通道和多通道补丁记录来分析功能后果。该假设认为，为了通道的有效激活以及 AMPA 受体相对于其他谷氨酸受体亚型的独特动力学，需要几种离散的相互作用。第二个目标建立在我们对 AMPA 受体变构调节剂的研究基础上，并将研究这些药物修饰的结合机制和反应机制中的步骤。尽管同聚受体可能仅限于少数神经元，但 GluA2/GluA3 异聚受体通道可能代表新皮质中大量的突触后 AMPA 受体。这些实验的结果将揭示重要谷氨酸受体的结构和功能，并为进一步的药物开发提供重要信息。