Molecular and cellular mechanisms of store-operated calcium channels

存储操作的钙进入(SOCE)产生对许多生理过程至关重要的钙信号， 从免疫细胞的激活和分化到肌肉的活动、分泌和运动。门店运营的钙 通道(SOC)由耗尽内质网钙离子的受体激活；钙离子的丢失由STIM1感知， 然后聚集在内质网-质膜(ER-PM)连接处，在那里它结合、捕获和激活 钙离子选择性Orai通道在PM中的扩散在这种情况下，功能获得和功能丧失突变 这两种途径都与严重的人类疾病有关，强调了精确的 监管。我们实验室的长期目标是了解SOC属性和 监管以及它们的细胞作用。虽然SOCE途径的总体组织现在是已知的， 许多潜在的蛋白质已经被识别出来，但我们对它们如何发挥作用的理解仍然存在重大差距 调节SOCE的位置和幅度。在接下来的五年里，我们的目标是调查三个基本问题 通过SOC调节钙离子内流的过程。(1)ER-PM结的动力学。这些交汇点 在ER接近PM的情况下是细胞中STIM可以结合和激活ORAI的唯一位置， 使得它们的大小、丰度和位置决定了钙离子进入的幅度和位置。当一个 连接位点上的大量系留蛋白是已知的，它们在连接起始和转换中的特定作用是 不清楚。通过用荧光标记物监测活细胞中ER-PM连接的出现和移除 我们将区分已知的拴系蛋白在启动、寿命和周转中的不同作用。 新结点的比率，以及它们进行SOCE的能力。(2)STIM1的激活机制及其作用机制 与Orai1的互动。内质网后STIM1的胞液结构域发生了巨大的构象变化 去掩蔽和扩展CRAC激活结构域(CAD)以激活血浆中的ORAI 薄膜。通过用单分子荧光和交联技术研究STIM1，我们的目标是鉴定 激活过程中的步骤和中间状态，可能有助于降低展开和 重新折叠STIM1。将采用类似的方法来确定STIM-ORAI互动的基本特征-- STIM-Orai络合物的化学计量、结合态CAD的构象和结合 接口本身--目前还不能理解。(3)钙依赖失活的分子机制 (CDI)。尽管在鉴定STIM和ORAI中对CDI至关重要的多个残基和结构域方面取得了进展， 目前仍缺乏一套完整的机制。我们将使用毛孔可及性分析来定位 失活门，并探索CDI结构域的功能和物理相互作用，以了解它们是如何 合作实现CDI。总体而言，我们的研究结果将揭示基本的细胞和分子 控制不同细胞中储存操作的钙信号强度的机制，并可能提出新的 调节它们的策略，以探索细胞功能和开发治疗人类疾病的新方法。

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.