Molecular and cellular mechanisms of store-operated calcium channels

钙池操纵的Ca 2+进入（SOCE）产生对许多生理过程至关重要的Ca 2+信号， 从免疫细胞活化和分化到肌肉活动、分泌和运动。钙池操作 通道（SOC）被从ER消耗Ca 2+的受体激活; Ca 2+的损失被STIM 1感知， 然后在ER-质膜（ER-PM）连接处积聚，在那里它结合、捕获和激活 钙选择性奥赖通道在PM中扩散。功能获得性和功能丧失性突变 这两种途径都与严重的人类疾病有关，强调了精确诊断的至关重要性。 调控我们实验室的长期目标是了解SOC特性的分子基础， 调节以及它们的细胞作用。虽然SOCE途径的整体组织现在是已知的， 许多潜在的蛋白质已经被鉴定出来，但在我们对它们如何起作用的理解上仍然存在重大差距 以调节SOCE位置和幅度。在接下来的五年里，我们的目标是研究三个基本的 通过SOC调节钙流入的过程。(1)ER-PM连接的动力学。这些结 ER紧密接近PM的位置是细胞中STIM可以结合和激活奥赖的唯一位点， 因此，它们的大小、丰度和位置决定了Ca 2+进入的幅度和位置。而 已知连接位点有许多束缚蛋白，它们在连接起始与转换中的具体作用是 不清楚通过用荧光标记物监测活细胞中ER-PM连接的出现和去除， 我们将区分不同的贡献已知的拴系蛋白的启动，寿命和营业额 新的连接率，以及他们进行SOCE的能力。(2)STIM 1的激活机制及其在 与Orai 1互动。STIM 1的胞质结构域在ER后经历了巨大的构象变化 Ca 2+耗竭，以暴露和扩展CRAC激活结构域（CAD），从而激活血浆中的奥赖 膜的通过用单分子荧光和交联技术研究STIM 1，我们的目标是鉴定 激活过程中的步骤和可能有助于减轻展开的能量成本的中间状态， STIM 1重折叠。将采用类似的方法来确定STIM-Orai相互作用的基本特征- STIM-Orai复合物的化学计量、CAD在结合状态下的构象以及结合 接口本身-目前还不了解。(3)Ca ~（2+）依赖性失活的分子机制 （土发委会）。尽管在鉴定STIM和奥赖中对CDI至关重要的多个残基和结构域方面取得了进展， 仍然缺乏一个综合机制。我们将使用孔可及性测定来定位 失活门，并探索CDI结构域的功能和物理相互作用，以了解它们如何 合作实现CDI。总的来说，我们的研究结果将揭示基本的细胞和分子 在不同细胞中控制钙库操作的钙信号强度的机制，并可能提示新的 调控它们的策略，以探索细胞功能并开发人类疾病的新疗法。

Store-operated Ca2+ entry (SOCE) generates Ca2+ signals that are critical for many physiological processes, from immune cell activation and differentiation to muscle activity, secretion, and motility. Store-operated Ca2+ channels (SOCs) are activated by receptors that deplete Ca2+ from the ER; the loss of Ca2+ is sensed by STIM1, which then accumulates at ER-plasma membrane (ER-PM) junctions where it binds, traps, and activates calcium-selective Orai channels diffusing in the PM. Gain-of-function and loss-of-function mutations in this pathway have both been connected to serious human diseases, underscoring the critical importance of precise regulation. The long-term goal of our laboratory is to understand the molecular basis of SOC properties and regulation as well as their cellular roles. While the overall organization of the SOCE pathway is now known and many of the underlying proteins have been identified, major gaps still exist in our understanding of how they act to regulate SOCE location and amplitude. Over the next five years we aim to investigate three fundamental processes that regulate calcium influx through SOCs. (1) The dynamics of ER-PM junctions. These junctions where the ER closely approaches the PM are the only sites in the cell where STIM can bind and activate Orai, such that their size, abundance and location determine both the amplitude and location of Ca2+ entry. While a host of tethering proteins at junctional sites is known, their specific roles in junction initiation vs. turnover is unclear. By monitoring the appearance and removal of ER-PM junctions in living cells with fluorescent markers we will distinguish the different contributions of known tethering proteins to the initiation, lifetime and turnover rate of new junctions, as well as their ability to conduct SOCE. (2) The mechanism of STIM1 activation and its interaction with Orai1. The cytosolic domain of STIM1 undergoes a massive conformational change after ER Ca2+ depletion in order to unmask and extend the CRAC activation domain (CAD) to activate Orai in the plasma membrane. By studying STIM1 with single-molecule fluorescence and crosslinking techniques we aim to identify steps in the activation process and intermediate states that may help mitigate the energetic cost of unfolding and refolding STIM1. Similar approaches will be applied to determine basic features of the STIM-Orai interaction - the stoichiometry of the STIM-Orai complex, the conformation of CAD in the bound state, and the binding interface itself – which are currently not understood. (3) A molecular mechanism for Ca2+-dependent inactivation (CDI). Despite progress in identifying multiple residues and domains in STIM and Orai that are critical for CDI, an integrated mechanism is still lacking. We will use a pore accessibility assay to localize the position of the inactivation gate, and explore functional and physical interactions of CDI domains to understand how they cooperate to bring about CDI. Overall, the results of our studies will reveal fundamental cellular and molecular mechanisms that control the strength of store-operated calcium signals in diverse cells, and may suggest new strategies for regulating them to explore cellular functions and develop new treatments for human disease.