Mechanisms of Ion Channels in Calcium Signaling

项目总结 这项研究计划的目的是了解离子渗透的分子机制。 在真核细胞离子通道中产生或响应细胞内钙信号的选择性和门控。这个 正在研究的离子通道包括钙释放激活钙通道(CRAC)ORAI，钙离子通道ORAI。 激活的氯通道Bestrophin(BEST)和线粒体钙单转运体(MCU)。航道 在细胞生理学中起着至关重要的作用，并受到严格的调控。CRAC通道是激活所必需的 T细胞中免疫反应基因的突变；突变会导致严重的联合免疫缺陷样疾病。 最佳通道形成受细胞内钙离子变化调节的阴离子选择性孔道 浓度，通过磷酸化，通过细胞体积的变化。最佳频道中的突变导致 遗传性视网膜退行性疾病(肥厚性视网膜病变)，包括幼年型视网膜病变 变性(最佳卵黄样黄斑营养不良)。MCU是钙离子进入人体的主要手段 线粒体。MCU的线粒体钙摄取调节ATP的产生，形成胞浆钙 信号，并控制导致细胞死亡的线粒体通透性转变。我们结合了各种方法 确定三维结构(X射线结晶学和低温电子显微镜)的功能 分析(电生理学和基于荧光的方法)来剖析其分子机制 这些离子通道。我们已完成的对这些通道的结构和功能的研究表明，每个通道都有 与其他离子通道相比具有独特的体系结构，并调节离子渗透、离子选择性和 以独特的方式选门。对于CRAC频道Orai，当前的目标是识别频道如何 在关闭和打开状态之间的转换，通道的激活器STIM的绑定如何驱动这一转换 过程，该通道如何巧妙地区分钙与其他离子，以及该通道如何催化离子 渗透，而不会使细胞充满钙。对于最佳渠道，我们的目标是捕获 代表不同的通道门控状态，并辨别钙的功能和分子基础- 依赖活化、失活和阴离子选择性。更进一步，我们试图辨别分子和 哺乳动物BEST1-4亚型之间的功能差异。关于MCU，我们的目标是研究三个方面- 维度结构，研究钙和蛋白质-蛋白质相互作用是如何调节通道的， 并探讨了离子选择性的分子基础。拟议的研究将揭示CRAC、BEST、 和MCU通道的功能，从而对我们理解离子通道和 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.