Mechanisms of Ion Channels in Calcium Signaling

项目摘要 本研究计划的目标是了解离子渗透的分子机制，离子 选择性和真核细胞离子通道中产生或响应细胞内钙信号的门控。的 正在研究的离子通道包括钙释放激活的钙（CRAC）通道奥赖、钙- 激活的氯离子通道雌激素（BEST）和线粒体钙单向转运体（MCU）。通道 在细胞生理学中发挥重要作用，并受到严格调控。CRAC通道是激活所必需的 T细胞中免疫反应基因的突变;突变导致严重的联合免疫缺陷样疾病。 BEST通道形成阴离子选择性孔，其由细胞内钙离子的变化调节 浓度、磷酸化和细胞体积的变化。BEST通道中的突变导致 遗传性视网膜变性疾病（bestrophinopathies），包括幼年型视网膜变性， 最佳卵黄状黄斑营养不良（Best vitelliform macular dystrophy）。MCU是钙进入的主要途径， 线粒体MCU对线粒体钙的摄取调节ATP的产生，塑造胞浆钙 信号，并控制线粒体通透性转换，导致细胞死亡。我们将联合收割机的方法 确定三维结构（X射线晶体学和低温电子显微镜）， 分析（电生理学和基于荧光的方法），以剖析 这些离子通道我们完成的这些通道的结构和功能研究表明，每一个都有 与其他离子通道相比，它具有独特的结构，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通道奥赖，当前的目标是辨别通道如何 关闭和打开状态之间的转换，如何结合STIM，通道的激活剂，驱动这一点 过程，通道如何巧妙地将钙与其他离子区分开来，以及通道如何催化离子 渗透，而不压倒细胞与钙。对于BEST通道，我们的目标是捕获 代表通道的不同门控状态，并辨别钙的功能和分子基础， 依赖性活化、失活和阴离子选择性。此外，我们试图辨别分子和 哺乳动物BEST 1 -4同种型之间的功能差异。关于MCU，我们的目标是研究三个- 三维结构，研究通道是如何调节钙和蛋白质-蛋白质相互作用， 探索离子选择性的分子基础。拟议的研究将揭示CRAC，BEST， 和MCU通道功能，从而为我们对离子通道的理解做出了重大贡献， 它们控制的生理过程。

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.