Structure, Activation, and Modulation of AMPA/Glutamate Receptors

描述（由申请人提供）：过度刺激嗜离子性谷氨酸受体（iGluRs），包括AMPA受体，可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，它们并不是简单的目标，因为iglur在大脑发育和正常的神经元过程（包括学习和记忆）中也起着重要作用。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是相似亚基的四聚体，每个亚基由一组模块组成，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为研究全息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作为可溶性蛋白质产生，并且它以与完整受体大致相同的亲和力结合激动剂和拮抗剂。我们之前已经确定了AMPA受体LBD与激动剂、拮抗剂和变构调节剂结合的GluA2和GluA3亚型的晶体结构，并使用NMR波谱检查了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描述了野生型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。我们拟结合x射线晶体学、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、多通道贴片快速药物应用等方法，从两个方面研究GluA2和GluA3的门控。第一个目标涉及信号从结合位点到通道域的传输。我们的核磁共振研究表明，LBD的叶2具有相当大的动力学和静电相互作用需要保持刚性结构。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，功能后果将通过单通道和多通道补丁记录进行分析。假设是，几个离散的相互作用是必要的有效激活通道和独特的动力学AMPA受体相对于其他谷氨酸受体亚型。第二个目标建立在我们对AMPA受体的变构调节剂的研究基础上，并将研究这些调节剂的结合机制和反应机制中的步骤。虽然同质受体可能局限于少数神经元，但GluA2/GluA3异质受体通道可能代表了新皮层中相当数量的突触后AMPA受体。这些实验的结果将揭示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重要信息。

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.