Structure and Finction of AMPA subtype ionotropic glutamate receptors

项目总结 AMPA(¿-amino-3-hydroxy-5-methyl-4-isoxazolepropionic酸)亚型离子型谷氨酸受体 调节中枢神经系统的大部分兴奋性神经传递。AMPA中的变更 受体功能与包括阿尔茨海默氏症在内的许多破坏性神经疾病有关 疾病、肌萎缩侧索硬化症、癫痫和脑缺血。AMPA受体活性的调节是 因此这是一个重要的临床目标。AMPA受体的功能是打开它们的离子通道，使其 激动剂谷氨酸与受体的结合反应中的阳离子。离子通道的这一过程 开启或激活门控通常伴随着较慢的脱敏过程。脱敏 导致离子通道孔道关闭，表现为AMPA受体减少10-500倍。 在谷氨酸持续存在的情况下介导的电流。脱敏是一种自然的方式 抑制AMPA受体活性，以保护神经元免受兴奋性毒性钙超载的影响。增强或 因此，弱化脱敏作用是调节AMPA受体活性的有效途径。 条件。抑制AMPA受体活性的另一种方法是使用离子 通道阻滞剂，堵塞通道毛孔的分子。在分子水平上，脱敏和离子通道 块预计具有共同的功能，允许他们将离子通道锁定在关闭的 构象。我们计划研究AMPA受体的脱敏和阻断，使用结构和 并揭示这些过程中的异同之处。因此，我们的具体情况 目的是：1)获得脱敏状态的AMPA受体的高分辨结构；2) 建立了离子通道阻滞剂的结构模型。AMPA受体是一个具有挑战性的结构功能靶点 研究是因为它代表了一种多聚体完整膜蛋白，其大小通常较低 表达式级别。为了实现我们的目标，我们将采用结构性和功能性相结合的方法。 包括现代结晶学技术、基于荧光的尺寸排除色谱(FSEC) 和电生理学。我们会使用不同的结晶方法和温度，筛分洗涤剂，油脂 和配体以获得脱敏和阻断剂结合状态下的全长AMPA受体结构。我们 然后将新生的结构信息与电生理记录和动力学建模相结合 找出AMPA受体脱敏和阻断的机制。实现我们的目标将具有重大的意义 对分子神经科学的影响，并将导致一种新的AMPA受体结构/功能模型 可以作为理论预测的动态模板，在硅胶拟合和新的化学合成中 化合物可以在不同的神经疾病模型中进行测试，最终变得安全和 有效的药物。

PROJECT SUMMARY AMPA (¿-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) subtype ionotropic glutamate receptors mediate the majority of excitatory neurotransmission in the central nervous system. Alteration in AMPA receptor function is associated with numerous devastating neurological diseases including Alzheimer's disease, amyotrophic lateral sclerosis, epilepsy and ischemia. Regulation of AMPA receptor activity is therefore an important clinical goal. AMPA receptors function by opening their ion channel for the flow of permeant cations in response to binding of agonist glutamate to the receptor. This process of ion channel opening or activation gating is accompanied by typically slower process of desensitization. Desensitization leads to the closure of the ion channel pore and appears as a 10-500 fold reduction of AMPA receptor- mediated currents in the continuous presence of glutamate. Desensitization represents a natural way of suppressing AMPA receptor activity that protects neurons from the excitotoxic calcium overload. Enhancing or weakening desensitization is therefore an effective way of regulating AMPA receptor activity in pathological conditions. An alternative way to suppress AMPA receptor activity that has therapeutic potential is to use ion channel blockers, molecules that plug the channel pore. At the molecular level, desensitization and ion channel block are expected to have common features that allow them to lock the ion channel in the closed conformation. We plan to study AMPA receptor desensitization and block using a combination of structural and functional approaches and to reveal similarities and differences in these processes. Accordingly, our specific aims are: 1) Obtaining a high-resolution structure of an AMPA receptor in the desensitized state and 2) Building a structural model of ion channel block. AMPA receptor is a challenging target for structure-functional studies because it represents a multimeric integral membrane protein of a large size with typically low expression level. To achieve our goals, we will use a combination of structural and functional approaches including modern crystallographic techniques, Fluorescence-based Size Exclusion Chromatography (FSEC) and electrophysiology. We will use different crystallization methods and temperatures, screen detergents, lipids and ligands to obtain full length AMPA receptor structures in the desensitized and blocker-bound states. We will then combine nascent structural information with electrophysiological recordings and kinetic modeling to figure out mechanisms of AMPA receptor desensitization and block. Achieving our aims will have a significant impact on molecular neuroscience and will result in a new structural/functional model of AMPA receptor that can serve as a dynamic template for theoretical prediction, in silico fitting and chemical synthesis of new compounds that can be tested in different models of neurological diseases and eventually become safe and effective medications.