Structure, Activation, and Modulation of AMPA/Glutamate Receptors

描述(申请人提供)：过度刺激离子谷氨酸受体(IGluRs)，包括AMPA受体，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治疗价值的药物靶点。然而，它们并不是简单的靶点，因为iGluRs在大脑发育和正常的神经过程中也扮演着重要的角色，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，以便更清楚地了解AMPA受体通道是如何激活和脱敏的。IGluRs是由相似亚基组成的四聚体，每个亚基由一组模块组成，其中包括配体结合结构域(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受体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以可溶性蛋白质的形式产生，它以与完整受体大致相同的亲和力结合激动剂和拮抗剂。我们以前已经确定了AMPA受体LBD的GluA2和GluA3亚型与激动剂、拮抗剂和变构调节剂结合的晶体结构，并使用核磁共振光谱研究了GluA2 LBD在全部和部分激动剂存在时的动态行为。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中产生了突变，这些突变影响通道激活和门控特性。我们建议结合X射线结晶学、核磁共振光谱、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来研究GluA2和GluA3门控的两个方面。第一个目的涉及将信号从结合部位传输到通道结构域。我们的核磁共振研究表明，LBD的叶2具有相当大的动力学，需要静电相互作用才能保持刚性结构。影响这些相互作用的残基突变将通过核磁共振(Sideechain Dyanmics和H/D交换)进行分析，功能后果将通过单通道和多通道斑片记录进行分析。这个假设是，几个离散的相互作用是有效激活通道和AMPA受体相对于其他谷氨酸受体亚型的独特动力学所必需的。第二个目标建立在我们对AMPA受体变构调节剂的工作基础上，并将研究这些试剂修饰的结合机制和反应机制中的步骤。虽然同构体受体可能仅局限于少数神经元，但GluA2/GluA3异构体受体通道可能代表新皮质中大量突触后AMPA受体。这些实验的结果将阐明一种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要的信息。

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.