Structure and Finction of AMPA subtype ionotropic glutamate receptors

描述（由申请人提供）：AMPA（α-氨基-3-羟基-5-甲基-4-异恶唑丙酸）亚型离子型谷氨酸受体介导中枢神经系统中的大多数兴奋性神经传递。AMPA受体功能的改变与许多破坏性神经系统疾病相关，包括阿尔茨海默病、肌萎缩性侧索硬化、癫痫和缺血。因此，AMPA受体活性的调节是重要的临床目标。AMPA受体通过响应激动剂谷氨酸与受体的结合而打开其离子通道以使渗透阳离子流动来发挥作用。这种离子通道开放或激活门控的过程通常伴随着较慢的脱敏过程。脱敏导致离子通道孔的关闭，并且表现为在谷氨酸持续存在下AMPA受体介导的电流减少10-500倍。脱敏是一种抑制AMPA受体活性的自然方式，可保护神经元免受兴奋性毒性钙超载的影响。因此，增强或减弱脱敏是在病理条件下调节AMPA受体活性的有效方法。抑制AMPA受体活性的另一种具有治疗潜力的方法是使用离子通道阻断剂，即堵塞通道孔的分子。在分子水平上，脱敏和离子通道阻滞预期具有共同的特征，使它们能够将离子通道锁定在闭合构象中。我们计划研究AMPA受体脱敏和块使用的结构和功能的方法相结合，并揭示这些过程中的相似性和差异。因此，我们的具体目标是：1）获得AMPA受体在脱敏状态下的高分辨率结构和2）建立离子通道阻断的结构模型。AMPA受体是一个具有挑战性的结构-功能研究的目标，因为它代表了一个大尺寸的多聚体的完整的膜蛋白，通常低表达水平。为了实现我们的目标，我们将使用结构和功能方法的组合，包括现代晶体学技术，基于分子排阻色谱法（FSEC）和电生理学。我们将使用不同的结晶方法和温度，筛选洗涤剂，脂质和配体，以获得在脱敏和阻断剂结合状态下的全长AMPA受体结构。然后，我们将结合联合收割机新生的结构信息与电生理记录和动力学建模，以找出AMPA受体脱敏和阻断的机制。实现我们的目标将对分子神经科学产生重大影响，并将产生AMPA受体的新结构/功能模型，该模型可以作为理论预测，计算机拟合和化学合成新化合物的动态模板，这些新化合物可以在不同的神经系统疾病模型中进行测试，并最终成为安全有效的药物。

DESCRIPTION (provided by applicant): AMPA (α-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.