Structure and function of hetero-multimeric ligand-gated ion channels

异多聚配体门控离子通道的结构和功能

• 批准号: 10593042
• 负责人: Hiroyasu Furukawa
• 金额: $ 55.76万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-15 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10593042
• 关键词: Address; Agonist; Alternative Splicing; Alzheimer' s Disease; Architecture; Binding; Binding Sites; Biochemistry; Brain; Cells; Complex; Cryoelectron Microscopy; Data; Development; Electrodes; Electrophysiology (science); Enhancers; Exons; Family; Glean; Glutamate Receptor; Glutamates; Glycine; Goals; Heat shock proteins; Insecta; Ion Channel; Ion Channel Gating; Knowledge; Laboratories; Length; Ligand Binding Domain; Ligands; Mediating; Membrane; Mental Depression; Methods; Molecular; Mutagenesis; N-Methyl-D-Aspartate Receptors; NMDA receptor A1; Neurologic Process; Neurotransmitters; Parkinson Disease; Pattern; Play; Polyamines; Protein Conformation; Protein Subunits; Public Health; RNA Splicing; Rattus; Research; Resolution; Role; Schizophrenia; Seizures; Serine; Speed; Spermine; Stroke; Structure; Synaptic Transmission; Testing; Time; Transmembrane Domain; Untranslated Regions; Variant; X-Ray Crystallography; antagonist; conformational alteration; desensitization; experimental study; mutant; nervous system disorder; particle; patch clamp; promoter; protein structure; receptor; receptor function; stoichiometry; structural biology; therapeutic development; voltage/patch clamp

PROJECT SUMMARY The goal of this project is to gain an in-depth mechanistic understanding about multi-heteromeric ion channels, N-methyl-D-aspartate receptors (NMDARs), regulated by different compounds, splice variants, and subtypes. These receptors belong to the family of ionotropic glutamate receptors (iGluRs) that mediate the majority of excitatory synaptic transmission and play significant roles in basic brain functions, development, and neurological disorders such as seizures, strokes, depression, and schizophrenia, as well as Parkinson’s and Alzheimer’s diseases. NMDARs are hetero-multimeric ligand-gated ion channels composed of GluN1 and GluN2 and/or GluN3 subunits. The GluN1 and GluN3 subunits bind co-agonists including glycine and D-serine, whereas the GluN2 subunits bind the neurotransmitter, glutamate. Each subunit protein is composed of an amino terminal domain (ATD), a ligand-binding domain (LBD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD). The GluN1-GluN2 NMDARs open their transmembrane ion channels upon binding of glycine and glutamate, whereas the GluN1-GluN3 NMDARs activate by glycine alone. We recently solved the first structure of the intact hetero-tetrameric rat GluN1-GluN2B NMDARs, initially by x-ray crystallography and later by cryo- electron microscopy. Despite recent advances, there are many fundamental questions remaining regarding the mechanism of activation, desensitization, inhibition by competitive antagonists, and functional diversity elicited by alternative splicing in GluN1 and different subunit combinations (GluN1-GluN2A-D and GluN3A-B). The structure of the GluN3 NMDARs is limited to that of the GluN3A LBD, thus, restricting our understanding about how this under-studied subtype form ion channels and mediate functions. Thus, the goal of the proposed project is to investigate patterns of compound bindings, protein conformations and subunit arrangements in various functional states and subtypes of NMDARs. To achieve these goals, we will conduct research aimed at: Aim 1 determining the mechanisms of activation, desensitization, and inhibition by agonists and antagonists; Aim 2 defining the underlying mechanism for altered pH-sensitivity and deactivation speeds by polyamines in a splice- variant-specific manner; and Aim 3 revealing the architecture of the GluN1-GluN3 NMDAR for the first time. These three aims will be achieved by obtaining the structural information of the GluN1-GluN2B NMDAR in the presence of agonists and antagonists, splice variants with and without spermine, and the GluN1-GluN3A NMDAR by a combination of x-ray crystallography and cryo-EM. The structure-based functional hypotheses will be tested by experiments involving electrophysiology. Successful completion of the proposed studies will provide in-depth information into the sophisticated function of NMDAR subtypes and splice variants mediated by compound bindings followed by rearrangement of multiple subunits and domains. These findings will support the development of therapeutic strategies used to treat the devastating neurological disorders and diseases mentioned above.

项目概要 该项目的目标是深入了解多异质离子通道的机制， N-甲基-D-天冬氨酸受体 (NMDAR)，受不同化合物、剪接变体和亚型调节。 这些受体属于离子型谷氨酸受体 (iGluR) 家族，介导大多数 兴奋性突触传递，在基本大脑功能、发育和神经系统中发挥重要作用 癫痫、中风、抑郁症和精神分裂症等疾病，以及帕金森病和阿尔茨海默病 疾病。 NMDAR 是由 GluN1 和 GluN2 组成的异多聚配体门控离子通道和/或 GluN3 亚基。 GluN1 和 GluN3 亚基结合共激动剂，包括甘氨酸和 D-丝氨酸，而 GluN2 亚基与神经递质谷氨酸结合。每个亚基蛋白质由氨基末端组成 结构域 (ATD)、配体结合结构域 (LBD)、跨膜结构域 (TMD) 和羧基末端结构域 （CTD）。 GluN1-GluN2 NMDAR 在与甘氨酸结合后打开其跨膜离子通道 谷氨酸，而 GluN1-GluN3 NMDAR 仅由甘氨酸激活。我们最近解决了第一个结构 完整的异源四聚体大鼠 GluN1-GluN2B NMDAR，最初通过 X 射线晶体学，后来通过冷冻- 电子显微镜。尽管最近取得了进展，但仍然存在许多关于 激活、脱敏、竞争性拮抗剂抑制的机制以及引发的功能多样性 通过 GluN1 和不同亚基组合（GluN1-GluN2A-D 和 GluN3A-B）中的选择性剪接。这 GluN3 NMDAR 的结构仅限于 GluN3A LBD 的结构，因此限制了我们对 这种尚待研究的亚型如何形成离子通道并介导功能。因此，拟议项目的目标 是研究各种化合物结合、蛋白质构象和亚基排列的模式 NMDAR 的功能状态和亚型。为了实现这些目标，我们将开展旨在实现以下目标的研究： 目标 1 确定激动剂和拮抗剂的激活、脱敏和抑制机制；目标2 定义了剪接中多胺改变 pH 敏感性和失活速度的基本机制 特定于变体的方式；目标 3 首次揭示了 GluN1-GluN3 NMDAR 的架构。 这三个目标将通过获得 GluN1-GluN2B NMDAR 的结构信息来实现 激动剂和拮抗剂的存在、含或不含精胺的剪接变体以及 GluN1-GluN3A NMDAR 结合 X 射线晶体学和冷冻电镜。基于结构的功能假设将 通过涉及电生理学的实验进行测试。成功完成拟议的研究将提供 深入了解 NMDAR 亚型和剪接变异体的复杂功能 复合结合，然后是多个亚基和结构域的重排。这些发现将支持 开发用于治疗破坏性神经系统疾病的治疗策略 上面提到过。