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 该项目的目的是获得有关多型离子离子通道的深入机理理解， N-甲基-D-天冬氨酸受体（NMDAR），由不同化合物，剪接变体和亚型调节。 这些受体属于介导大多数的离子型谷氨酸受体（iglurs）家族 兴奋性合成传播并在基本的大脑功能，发育和神经系统中起重要作用 癫痫发作，中风，抑郁和精神分裂症等疾病，以及帕金森氏症和阿尔茨海默氏症 疾病。 NMDAR是由Glun1和Glun2和/或 Glun3亚基。 Glun1和Glun3亚基结合了包括甘氨酸和D-丝氨酸在内的联合使，而 Glun2亚基结合神经递质，谷氨酸。每个亚基蛋白由氨基末端组成 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在结合甘氨酸和 谷氨酸，而glun1-glun3 NMDAR仅通过甘氨酸激活。我们最近解决了第一个结构 完整的杂型四聚大鼠glun1-glun2b nmdar，最初是X射线晶体学，后来由冷冻 电子显微镜。尽管最近进步，但仍有许多基本问题 激活机制，脱敏，竞争性拮抗剂的抑制以及引起的功能多样性 通过glun1和不同亚基组合（Glun1-Glun2a-d和glun3a-b）中的替代剪接。这 Glun3 NMDAR的结构仅限于Glun3a LBD的结构，因此限制了我们对 这种研究不足的亚型如何形成离子通道并介导功能。那是拟议项目的目标 是为了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，我们将进行针对的研究：目标1 确定激动剂和拮抗剂的激活，脱敏和抑制的机制；目标2 定义了多胺在剪接中改变pH值和失活速度的基本机制 特定于变体的方式； AIM 3首次揭示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，带有和不带有精子的剪接变体以及Glun1-Glun3a NMDAR结合了X射线晶体学和冷冻EM。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。拟议研究的成功完成将提供 深入了解NMDAR亚型的复杂函数和由由剪接变体介导的 化合物结合物，然后重新排列多个亚基和域。这些发现将支持 用于治疗毁灭性神经系统疾病和疾病的理论策略的发展 上面提到的。