Mechanisms of Ion Channels in Calcium Signaling

钙信号传导中离子通道的机制

• 批准号: 9898393
• 负责人: Stephen Barstow Long
• 金额: $ 94.12万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9898393
• 关键词: Adolescent; Anions; Architecture; Asthma; Binding; Calcium; Calcium Channel; Calcium Signaling; Cell Death; Cell Volumes; Cell physiology; Cells; Chloride Channels; Cryoelectron Microscopy; Cystic Fibrosis; Disease; Electrophysiology (science); Fertilization; Fluids and Secretions; Fluorescence; Foundations; Hypersensitivity; Immune; Immune Response Genes; Immune response; Inherited; Ion Channel; Ions; Macular degeneration; Malignant Neoplasms; Mitochondria; Molecular; Molecular Probes; Molecular Structure; Muscle Contraction; Mutation; Permeability; Phosphorylation; Physiological Processes; Play; Process; Production; Protein Isoforms; Research; Retinal Degeneration; Rheumatoid Arthritis; Role; Severe Combined Immunodeficiency; Shapes; Structure; T-Lymphocyte; Vitelliform macular dystrophy; X-Ray Crystallography; acute pancreatitis; base; calcium uniporter; cell growth; cell motility; disease-causing mutation; immune activation; inhibitor/antagonist; programs; protein protein interaction; public health relevance; three dimensional structure; uptake

PROJECT SUMMARY The objectives of this research program are to understand the molecular mechanisms of ion permeation, ion selectivity, and gating in eukaryotic ion channels that generate or respond to intracellular calcium signals. The ion channels under study include the calcium release-activated calcium (CRAC) channel Orai, the calcium- activated chloride channel bestrophin (BEST), and the mitochondrial calcium uniporter (MCU). The channels play vital roles in cellular physiology and are tightly regulated. CRAC channels are necessary for the activation of immune response genes in T cells; mutations cause severe combined immunodeficiency-like disorders. BEST channels form anion-selective pores that are regulated by changes in the intracellular calcium concentration, by phosphorylation, and by changes in cell volume. Mutations in BEST channels cause inherited retinal degenerative diseases (bestrophinopathies) including a juvenile-onset form of retinal degeneration (Best vitelliform macular dystrophy). MCU is the primary means for calcium entry into mitochondria. Mitochondrial calcium uptake by MCU regulates ATP production, shapes cytosolic calcium signals, and controls a mitochondrial permeability transition that leads to cell death. We combine approaches to determine three-dimensional structures (X-ray crystallography and cryo-electron microscopy) with functional analyses (electrophysiology and fluorescence-based approaches) to dissect the molecular mechanisms of these ion channels. Our accomplished structural and functional studies of these channels reveal that each has a distinct architecture in comparison to other ion channels and regulates ion permeation, ion selectivity and gating in unique ways. For the CRAC channel Orai, the current aims are to discern how the channel transitions between closed and open states, how the binding of STIM, the channel's activator, drives this process, how the channel exquisitely discriminates calcium from other ions, and how the channel catalyzes ion permeation without overwhelming the cell with calcium. For BEST channels, we aim to capture structures that represent different gating states of the channel and discern the functional and molecular bases for calcium- dependent activation, inactivation, and anion selectivity. Further, we seek to discern the molecular and functional differences among mammalian BEST1-4 isoforms. Regarding MCU, we aim to study three- dimensional structure, investigate how the channel is regulated by calcium and protein-protein interactions, and probe the molecular basis of ion selectivity. The proposed studies will reveal principles of CRAC, BEST, and MCU channel function, thereby making significant contributions to our understandings of ion channels and the physiological processes they control.

项目摘要 该研究计划的目标是了解离子渗透的分子机制，离子 选择性和对细胞内钙信号产生或反应的真核离子通道中的门控。这 所研究的离子通道包括钙释放激活的钙（CRAC）通道ORAI，钙 - 活化的氯化物通道BESTRophin（最佳）和线粒体钙Uniporter（MCU）。频道 在细胞生理学中发挥至关重要的作用，并受到严格调节。 CRAC通道是激活所必需的 T细胞中的免疫反应基因；突变会导致严重的联合免疫缺陷样疾病。 最佳通道形成阴离子选择性孔，受细胞内钙的变化调节 浓度，通过磷酸化以及细胞体积的变化。最佳渠道中的突变原因 遗传性视网膜退行性疾病（最佳嗜性疾病），包括视网膜的少年发作形式 变性（最佳卵形黄斑营养不良）。 MCU是进入钙进入的主要手段 线粒体。 MCU调节ATP产生的线粒体钙摄取，形成胞质钙 信号，并控制导致细胞死亡的线粒体通透性过渡。我们结合了方法 确定具有功能的三维结构（X射线晶体学和冷冻电子显微镜） 分析（电生理学和基于荧光的方法），以剖析分子机制 这些离子通道。我们对这些渠道的完成的结构和功能研究表明，每个渠道都有 与其他离子通道相比，一种独特的结构，并调节离子渗透，离子选择性和 以独特的方式进行门控。对于CRAC频道Orai，目前的目的是辨别频道 封闭状态和开放状态之间的过渡，刺激器的结合如何驱动这一点 过程，通道如何精确区分钙与其他离子以及通道如何催化离子 渗透而不会用钙压倒细胞。对于最佳渠道，我们旨在捕获 代表通道的不同门控状态，并辨别钙的功能和分子碱基 依赖激活，灭活和阴离子的选择性。此外，我们试图辨别分子和 哺乳动物最佳1-4同工型之间的功能差异。关于MCU，我们的目标是研究三个 尺寸结构，研究通道如何受钙和蛋白质 - 蛋白质相互作用的调节， 并探测离子选择性的分子基础。拟议的研究将揭示CRAC的原则，最好， 和MCU渠道功能，从而为我们对离子渠道的理解做出了重大贡献 他们控制的生理过程。