Structure and Function of AMPA subtype ionotropic glutamate receptors

AMPA受体介导快速兴奋性神经传递，有助于高级认知过程，如 学习和记忆，并与许多精神和神经退行性疾病有关。在……里面 特别是，ampa受体在癫痫的发生和癫痫的传播中起着关键作用，因此，最近 成为癫痫治疗最有希望的靶点之一。然而，药物靶向的发展 AMPA受体由于缺乏对AMPA受体结构和功能的了解而停滞不前 功能。例如，只有同源四聚体完整的AMPA受体的结构被确定，而 中枢神经系统中绝大多数的AMPA受体是异构体。一批 非竞争性抑制剂和离子通道阻滞剂已被确定为有希望的药物候选药物。 但它们作用于AMPA受体的结构机制在很大程度上仍不清楚。这 信息缺失对于未来基于结构的合理药物设计是绝对关键的。我们计划研究一下 结合生物物理和生物化学方法研究AMPA受体的结构和功能， 包括现代结晶学和低温电子显微镜(Cryo-EM)技术，基于荧光的 方法：电生理学、动力学和分子模拟。我们的具体目标是(1)获得 异构体AMPA受体，(2)建立非竞争性抑制的分子机制，(3)建立 离子通道阻滞剂的结构模型。为了达到我们的目标，我们将优化AMPA受体的结构 结晶和冷冻-EM实验，制定它们的表达和纯化方案，并解决 异四聚体AMPA受体和AMPA受体与非竞争性抑制剂复合体的结构 和离子通道阻滞剂。为了改进我们的结构模型，我们将使用新的结构精化方法 结合分子动力学(MD)模拟。我们还将使用以下组合测试我们的模型 实验和电子诱变、全细胞膜片钳记录和MD模拟。要理解 AMPA受体异构体组装、非竞争性抑制和离子通道的分子机制 BLOCK，我们将在不同的环境中对同型和异型AMPA受体进行广泛的MD模拟 激活状态以及在存在或不存在非竞争性抑制剂和离子通道阻滞剂的情况下。我们会 结合结构、计算、功能和突变实验的结果，提出分子 AMPA受体异构体组装、非竞争性抑制和离子通道阻断模型。触达 我们的研究目标将提供分子水平的必要知识，以极大地促进新的 有可能成为治疗癫痫和其他疾病的安全和更有效的药物的分子 与兴奋性神经传递有关的障碍。

AMPA receptors mediate fast excitatory neurotransmission, contribute to high cognitive processes such as learning and memory and are implicated in numerous psychiatric and neurodegenerative diseases. In particular, AMPA receptors play a key role in epileptogenesis and seizure spread and, thus, have recently emerged as one of the most promising targets for epilepsy therapy. However, development of drugs targeting AMPA receptors has been stalled because of the lack of knowledge about AMPA receptor structure and function. For example, only structures of homotetrameric intact AMPA receptors have been determined, while the overwhelming majority of AMPA receptors in the central nervous system are heterotetramers. A number of noncompetitive inhibitors and ion channel blockers have been identified as promising candidates for drug development but structural mechanisms of their action on AMPA receptors remain largely unexplored. This missing information is absolutely critical for the future structure-based rational drug design. We plan to study structure and function of AMPA receptors using a combination of biophysical and biochemical approaches, including modern crystallographic and cryo-electron microscopy (cryo-EM) techniques, fluorescence-based methods, electrophysiology, kinetic and molecular modeling. Our specific aims are to (1) obtain structures of heteromeric AMPA receptors, (2) establish the molecular mechanism of noncompetitive inhibition, and (3) build a structural model of ion channel block. To reach our goals, we will optimize AMPA receptor constructs for crystallization and cryo-EM experiments, develop protocols of their expression and purification and solve structures of heterotetrameric AMPA receptors and AMPA receptors in complex with noncompetitive inhibitors and ion channel blockers. To improve our structural models, we will use new methods of structural refinement combined with molecular dynamics (MD) simulations. We will also test our models using a combination of experimental and in silico mutagenesis, whole-cell patch-clamp recordings and MD simulations. To understand the molecular mechanisms of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block, we will perform extensive MD simulations of homo- and heteromeric AMPA receptors in different activation states and in the presence or absence of noncompetitive inhibitors and ion channel blockers. We will combine the results of structural, computational, functional and mutagenesis experiments to propose molecular models of AMPA receptor heteromeric assembly, noncompetitive inhibition and ion channel block. Reaching our research goals will provide molecular level knowledge essential to greatly facilitate design of new molecules that will have a potential to become safe and more efficacious drugs to treat epilepsy and other disorders related to excitatory neurotransmission.