3D Structure and Function of Ligand-Gated Ion Channels

离子型谷氨酸受体(IGluRs)是谷氨酸激活的、阳离子选择性的离子通道，介导 神经系统中大多数毫秒级的信号。IGluR受体家族由 在三种主要的亚型中，AMPA，NMDA和海人藻酸受体，每一种都在神经 系统的开发和功能。正常谷氨酸能信号的中断支持多发性 神经疾病，包括阿尔茨海默病、抑郁症、精神分裂症和癫痫。因此， IGluRs是许多治疗药物治疗癫痫和情绪障碍的靶点，以及 精神分裂症。神经系统的几种免疫紊乱也与iGluRs有关，其中最多的 流行的是NMDA受体脑炎，这是一种严重的自身免疫性疾病，对此几乎没有什么希望 治疗。尽管iGluR广泛分布在人类神经系统中，甚至 尽管它们与许多使人衰弱的神经系统疾病和障碍有关，但其分子机制 目前还没有完全阐明iGluR的功能。此外，iGluR并不是在其功能位点上分离出来的， 通常是突触，但在含有多种辅助蛋白的复合体中发现。事实上， 这些辅助蛋白的组织结构和分子功能也有待于充分阐明。因此， 这项拨款申请的主要研究重点是阐明分子结构和 AMPA和NMDA受体单独和与关键辅助蛋白的复合体的作用机制。 在这里，我们将开发从整个大脑和海马体中分离天然AMPA受体的方法 和小脑，作为它们的辅助蛋白的复合体。然后我们将阐明这些分子的结构 受体复合体通过单颗粒冷冻-EM，使用亚单位特异性抗体片段来标记亚基。使用 在手头的结构中，我们将设计突变体来测试结构和机制的重要方面，携带 进行生化和电生理实验，探讨受体功能。同时，我们还将研究 NMDA受体，这里侧重于受体门控的基于结构的机制，研究 GluN1/GluN2A受体在与激动剂甘氨酸和谷氨酸以及与甘氨酸和谷氨酸的复合体中的结构 变构调节剂和离子通道阻滞剂。通过这些研究，我们旨在回答这样一个问题： 神经递质激活受体，以及变构调节剂如何增加或降低受体活性。 在AMPA受体研究的同时，我们还将从 啮齿动物脑组织，目的是阐明天然NMDA受体的第一结构，绘制 GluN1和GluN2亚基在受体复合体中的位置。总而言之，我们的研究将界定 IGluR功能的基于结构的生化机制，它们将为开发奠定基础 新的治疗药物。

Ionotropic glutamate receptors (iGluRs) are glutamate-activated, cation-selective ion channels that mediate the majority of millisecond-time scale signaling in the nervous system. The iGluR family of receptors is composed of three major subtypes, the AMPA, NMDA and kainate receptors, each of which plays a crucial role in nervous system development and function. The disruption of normal glutamatergic signaling underpins multiple neurological disorders, including Alzheimer’s disease, depression, schizophrenia and epilepsy. Accordingly, iGluRs are the targets for numerous therapeutic agents to treat seizure and mood disorders, as well as schizophrenia. Several immunological disorders of the nervous system also involve iGluRs, with the most prevalent being NMDA receptor encephalitis, a severe autoimmune disease for which there are few promising treatments. Despite the widespread distribution of iGluRs throughout the human nervous system, and even though they are involved in many debilitating neurological diseases and disorders, the molecular mechanisms of iGluR function are not yet fully elucidated. Moreover, iGluRs are not isolated at their sites of function, typically the synapses, but rather are found in complexes with multiple auxiliary proteins. Indeed, the organization and molecular function of these auxiliary proteins also remains to be fully elucidated. Thus, the major focus of the research proposed in this grant application is to elucidate the molecular structure and mechanisms of function for AMPA and NMDA receptors, alone and in complexes with key auxiliary proteins. Here we will develop methods to isolate native AMPA receptors from whole brain and from the hippocampus and cerebellum, as complexes with their auxiliary proteins. We will then elucidate the structures of these receptor complexes by single particle cryo-EM, using subunit specific antibody fragments to tag subunits. With the structures in hand, we will design mutants to test important aspects of structure and mechanisms, carrying out biochemical and electrophysiological experiments to probe receptor function. In parallel, we will also study the NMDA receptors, here focusing on a structure-based mechanism for receptor gating, investigating the structure of the GluN1/GluN2A receptor in complexes with the agonists glycine and glutamate, as well as with allosteric modulators and ion channel blockers. Through these studies we aim to answer the question of how neurotransmitters activate the receptor and how allosteric modulators increase or decrease receptor activity. In parallel with AMPA receptor studies, we will also isolate native GluN1-containing NMDA receptors from rodent brain tissue, with the aim being to elucidate the first structure of native NMDA receptors, mapping the positions of the GluN1 and GluN2 subunits in the receptor complex. Taken together, our studies will define structure-based, biochemical mechanisms for iGluR function and they will lay the foundation for development of new therapeutic agents.