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，并涉及许多精神和神经退行性疾病。在 特别地，AMPA受体在癫痫发生和癫痫扩散中起关键作用，因此，最近， 成为癫痫治疗最有前途的靶点之一。然而，药物靶向的发展 由于缺乏对AMPA受体结构的了解， 功能例如，仅确定了同源四聚体完整AMPA受体的结构，而 中枢神经系统中绝大多数AMPA受体是异源四聚体。一些 非竞争性抑制剂和离子通道阻断剂已被确定为药物治疗的有希望的候选物。 但是它们对AMPA受体的作用的结构机制仍然在很大程度上未被探索。这 缺失信息对于未来基于结构的合理药物设计是绝对关键的。我们计划学习 AMPA受体的结构和功能，使用生物物理和生物化学方法的组合， 包括现代晶体学和低温电子显微镜（cryo-EM）技术、基于荧光的 方法，电生理学，动力学和分子建模。我们的具体目标是（1）获得 异聚AMPA受体，（2）建立非竞争性抑制的分子机制，和（3）建立 离子通道阻滞的结构模型。为了达到我们的目标，我们将优化AMPA受体结构， 结晶和冷冻EM实验，开发其表达和纯化的方案，并解决 异源四聚体AMPA受体和与非竞争性抑制剂复合的AMPA受体的结构 和离子通道阻断剂。为了改进我们的结构模型，我们将使用新的结构细化方法 结合分子动力学（MD）模拟。我们还将使用以下组合来测试我们的模型 实验和计算机诱变，全细胞膜片钳记录和MD模拟。了解 AMPA受体异聚体组装、非竞争性抑制和离子通道的分子机制 块，我们将进行广泛的分子动力学模拟同源和异源AMPA受体在不同的 激活状态和存在或不存在非竞争性抑制剂和离子通道阻断剂。我们将 联合收割机的结构，计算，功能和诱变实验的结果，提出分子 AMPA受体异聚体组装、非竞争性抑制和离子通道阻断的模型。达到 我们的研究目标将提供必要的分子水平的知识，大大促进新的 这些分子将有可能成为治疗癫痫和其他疾病的安全和更有效的药物。 与兴奋性神经传递有关的疾病。

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.