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: 9905566
• 负责人: Hiroyasu Furukawa
• 金额: $ 58.29万
• 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/9905566
• 关键词: Address; Agonist; Alpha Particles; Alternative Splicing; Alzheimer' s Disease; Architecture; Binding; Binding Proteins; 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; Mental Depression; Methods; Molecular; Molecular Structure; 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; base; conformational alteration; desensitization; experimental study; mutant; nervous system disorder; patch clamp; promoter; protein structure; receptor; receptor function; stoichiometry; structural biology; therapeutic development; voltage 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.

项目摘要 本项目的目标是深入了解多异聚体离子通道的机制， 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，其介导大多数的谷氨酸受体。 兴奋性突触传递，并在基本的脑功能，发育和神经系统中发挥重要作用 癫痫、中风、抑郁症、精神分裂症、帕金森氏症和阿尔茨海默氏症等疾病 疾病NMDAR是由GluN 1和GluN 2组成的异源多聚体配体门控离子通道，和/或 GluN 3亚基。GluN 1和GluN 3亚基结合包括甘氨酸和D-丝氨酸的共激动剂，而GluN 3亚基结合包括甘氨酸和D-丝氨酸的共激动剂。 GluN 2亚基结合神经递质谷氨酸。每个亚基蛋白由一个氨基末端 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）。GluN 1-GluN 2 NMDAR在结合甘氨酸后打开其跨膜离子通道， 谷氨酸，而GluN 1-GluN 3 NMDAR仅由甘氨酸激活。我们最近解决了第一个 的完整的异源四聚体大鼠GluN 1-GluN 2B NMDAR，最初通过X射线晶体学，后来通过冷冻， 电镜尽管最近取得了一些进展，但在这方面仍然存在许多基本问题。 激活、脱敏、竞争性拮抗剂抑制和功能多样性的机制 通过GluN 1和不同亚基组合（GluN 1-GluN 2A-D和GluN 3A-B）的选择性剪接。的 GluN 3 NMDAR的结构限于GluN 3A LBD的结构，因此，限制了我们对GluN 3 NMDAR的理解。 这一研究不足的亚型如何形成离子通道和介导功能。因此，拟议项目的目标 是为了研究在不同的细胞中化合物结合，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： 确定激动剂和拮抗剂的激活、脱敏和抑制机制;目的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， Aim 3首次揭示了GluN 1-GluN 3 NMDAR的结构。 这三个目标将通过获得GluN 1-GluN 2B NMDAR在细胞中的结构信息来实现。 存在激动剂和拮抗剂，有和没有精胺的剪接变体，以及GluN 1-GluN 3A 通过X射线晶体学和cryo-EM的组合进行NMDAR。基于结构的功能假说将 可以通过电生理学实验进行测试。成功完成拟议的研究将提供 深入了解NMDAR亚型和剪接变体介导的复杂功能， 复合结合，随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障碍和疾病的治疗策略 上面提到的。