3D Structure and Function of Ligand-Gated Ion Channels

离子型谷氨酸受体（iGluR）是谷氨酸激活的阳离子选择性离子通道，其介导谷氨酸的代谢。 神经系统中毫秒级的信号传递。iGluR受体家族由以下组成 三种主要亚型，AMPA，NMDA和红藻氨酸受体，其中每一种都在神经系统中起着至关重要的作用。 系统开发和功能。正常的多巴胺能信号传导的中断 神经系统疾病，包括阿尔茨海默病、抑郁症、精神分裂症和癫痫。因此，委员会认为， iGluR是许多治疗剂的靶标，用于治疗癫痫发作和情绪障碍，以及 精神分裂症神经系统的几种免疫学病症也涉及iGluRs，其中最常见的是iGluRs。 流行的是NMDA受体脑炎，一种严重的自身免疫性疾病， 治疗。尽管iGluRs广泛分布在整个人类神经系统中，甚至 尽管它们与许多使人衰弱的神经系统疾病和紊乱有关， iGluR的功能尚未完全阐明。此外，iGluR在其功能位点不是分离的， 通常是突触，而是在与多种辅助蛋白的复合物中发现的。持续增 这些辅助蛋白的组织和分子功能也有待充分阐明。因此 在这项资助申请中提出的研究的主要重点是阐明分子结构， AMPA和NMDA受体的功能机制，单独和与关键辅助蛋白的复合物。 在这里，我们将开发从整个大脑和海马体分离天然AMPA受体的方法。 和小脑，作为与它们的辅助蛋白的复合物。然后，我们将阐明这些结构 受体复合物的单颗粒冷冻EM，使用亚基特异性抗体片段标记亚基。与 我们将设计突变体来测试结构和机制的重要方面， 进行生物化学和电生理实验来探测受体功能。与此同时，我们还将研究 NMDA受体，这里集中在基于结构的受体门控机制，研究 GluN 1/GluN 2A受体与激动剂甘氨酸和谷氨酸以及与 变构调节剂和离子通道阻断剂。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减少受体活性。 在AMPA受体研究的同时，我们还将从小鼠中分离天然的含GluN 1的NMDA受体。 啮齿类动物脑组织，目的是阐明天然NMDA受体的第一结构，绘制 GluN 1和GluN 2亚基在受体复合物中的位置。综合起来，我们的研究将定义 iGluR功能的基于结构的生化机制，它们将为开发奠定基础 新的治疗剂。

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.