Store operated calcium influx in cells of the immune system

描述(由申请人提供)：我们和其他人对CRAC通道的鉴定和克隆(CRACM1/Orai1)已经确定了商店操作的钙内流所需的基本分子成分。突变分析表明，CRACM1/Orai1均聚合，构成了CRAC通道的钙选择孔。重要的是，使用基因捕获技术产生了CRACM1-/-小鼠，我们已经证明CRACM1在体内肥大细胞效应功能和过敏反应中是必不可少的。令人惊讶的是，我们发现这些小鼠的T细胞发育和增殖相对没有受到影响。为了全面了解T细胞中的SOCE，我们现在正在研究CRACM2和CRACM3。我们的初步数据表明，这些同源物可能在T细胞中具有不同的功能。我们将通过产生和表征每个基因的基因敲除小鼠来剖析它们各自的作用(目标1)。电压操纵型钙通道(VOCC)在T细胞中表达，影响NFAT转位，调节细胞因子的产生。但他们的作用机制仍不清楚。我们已经建立了一个可诱导的CaV1.2 T细胞基因敲除小鼠，并观察到CaV1.2缺失显著抑制T细胞中细胞因子的产生。有趣的是，我们的最新数据显示，CaV1.2和CRACM2的C末端结构域在静止的T细胞中相关。这些新数据揭示了至少一个VOCC亚基与CRAC之间出人意料的直接联系，并为研究T细胞中所有VOCC功能提供了一个新的框架。我们将充分描述这种相互作用，剖析CaV1.2功能的机制，并进一步确定其在体内T细胞中的作用(目标2)。最后，我们发现相关的VOCC通道CaV1.3定位于内质网，并与STIM1形成稳定的蛋白质复合体。这一新的观察结果表明，VOCC通道和CRAC之间存在着另一个基本的交集。我们假设CaV1.3在内质网中作为STIM1的钙传感器。我们提出了实验来测试这一想法，并全面剖析了CaV1.3在T细胞中的功能(目标3)。与公共卫生相关：CRAC通道的鉴定和克隆已经确定了储存操作的钙离子进入(SOCE)所需的基本分子成分。我们最近证实，CRACM1缺乏会导致CRACM1缺陷小鼠出现轻微的T细胞异常。我们现在建议使用缺失小鼠模型来研究CRACM2和CRACM3的功能，并确定它们在T细胞中的活体作用(目标1)。此外，我们还将研究VOCC通道CaV1.2(Aim 2)和CaV1.3(Aim 3)如何相互作用和调制CRAC通道。这些研究将对T细胞内SOCE的分子调控产生新的见解，使开发治疗自身免疫性疾病的新方法成为可能。

DESCRIPTION (provided by applicant): The identification and cloning of the CRAC channel by us and others (CRACM1/Orai1) has defined the basic molecular components required for store-operated calcium influx. Mutational analysis demonstrated that CRACM1/Orai1 homopolymerizes and that it constitutes the calcium selective pore of the CRAC channel. Importantly, using gene-trap technology to generate the Cracm1-/- mouse, we have demonstrated that CRACM1 is essential for mast cell effector functions and allergic responses in vivo. Surprisingly, we found that T cell development and proliferation were relatively unaffected in these mice. In order to gain a comprehensive view of SOCE in T cells, we are now studying CRACM2 and CRACM3. Our preliminary data suggest that these homologs may have distinct functions in T cells. We will dissect their respective roles by generating and characterizing knockout mice for each of these genes (Aim 1). Voltage operated calcium channels (VOCCs) are expressed in T cells, impact NFAT translocation and regulate cytokine production. But their mechanism of action is still unclear. We have generated an inducible CaV1.2 T cell knockout mouse and have observed that CaV1.2 loss significantly inhibits cytokine production in T cells. Intriguingly, our most recent data show that C-terminal domain of CaV1.2 and CRACM2 associate in resting T cells. These new data reveal an unexpectedly direct connection between at least one VOCC subunit and CRAC and provide a new framework in which to study all VOCC functions in T cells. We will fully characterize this interaction, dissect the mechanism of CaV1.2 function, and further define its role in T cells in vivo (Aim 2). Finally, we have discovered that the related VOCC channel CaV1.3 is localized to the ER and forms a stable protein complex with STIM1. This novel observation suggests another fundamental intersection between VOCC channels and CRAC. We hypothesize that CaV1.3 acts as a calcium sensor for STIM1 in the ER. We propose experiments to test this idea and fully dissect CaV1.3 function in T cells (Aim 3). PUBLIC HEALTH RELEVANCE: The identification and cloning of the CRAC channel has defined the basic molecular components required for store-operated calcium entry (SOCE). We have recently demonstrated that CRACM1 deficiency results in minor T cell abnormalities in CRACM1-deficient mice. We now propose to study CRACM2 and CRACM3 function and determine their in vivo roles in T cells using deletion mouse models (Aim 1). In addition we will study how VOCC channel CaV1.2 (Aim 2) and CaV1.3 (Aim 3) interact and modulate CRAC channels. These studies will yield new insights into the molecular regulation of SOCE within T cells, enabling the development new approaches to treat autoimmune diseases.