Store operated calcium influx in cells of the immune system

储存操作的钙流入免疫系统细胞

• 批准号: 7583065
• 负责人: JEAN-PIERRE M KINET
• 金额: $ 42.5万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7583065
• 关键词: Allergic; Autoimmune Diseases; C-terminal; Calcium; Calcium Channel; Cell Nucleus; Cells; Cloning; Confocal Microscopy; Data; Defect; Development; Dominant-Negative Mutation; Event; Genes; Genetic Transcription; Homologous Gene; Human; Immune; Immune system; Knockout Mice; Length; Mediating; Minor; Molecular; Mus; Mutation; Nature; Neurons; Nuclear; Pathway interactions; Production; Proteolysis; Regulation; Rest; Role; STIM1 gene; Signal Transduction; Synapses; System; T-Cell Activation; T-Cell Development; T-Cell Proliferation; T-Lymphocyte; Tamoxifen; Technology; Testing; Transgenic Mice; cytokine; extracellular; immune function; in vivo; insight; mast cell; mouse model; mutant; novel; novel strategies; protein complex; public health relevance; research study; response; sensor; voltage

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.

描述（由申请人提供）：我们和其他人对 CRAC 通道（CRACM1/Orai1）的鉴定和克隆已经定义了钙池操作的钙流入所需的基本分子成分。突变分析表明 CRACM1/Orai1 均聚并构成 CRAC 通道的钙选择性孔。重要的是，使用基因捕获技术生成 Cracm1-/- 小鼠，我们证明了 CRACM1 对于体内肥大细胞效应器功能和过敏反应至关重要。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不受影响。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。我们将通过生成和表征每个基因的敲除小鼠来剖析它们各自的作用（目标 1）。电压控制钙通道 (VOCC) 在 T 细胞中表达，影响 NFAT 易位并调节细胞因子的产生。但它们的作用机制仍不清楚。我们已经培育出可诱导的 CaV1.2 T 细胞敲除小鼠，并观察到 ​​CaV1.2 缺失显着抑制 T 细胞中细胞因子的产生。有趣的是，我们最新的数据显示 CaV1.2 和 CRACM2 的 C 末端结构域在静息 T 细胞中相关联。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，并提供了一个新的框架来研究 T 细胞中所有 VOCC 功能。我们将充分描述这种相互作用，剖析 CaV1.2 功能的机制，并进一步明确其在体内 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ER并与STIM1形成稳定的蛋白质复合物。这一新颖的观察表明 VOCC 通道和 CRAC 之间存在另一个基本交叉点。我们假设 CaV1.3 充当 ER 中 STIM1 的钙传感器。我们提出实验来测试这个想法并全面剖析 T 细胞中的 CaV1.3 功能（目标 3）。公共健康相关性：CRAC 通道的识别和克隆定义了钙库操作钙进入 (SOCE) 所需的基本分子成分。我们最近证明，CRACM1 缺陷会导致 CRACM1 缺陷小鼠的轻微 T 细胞异常。我们现在建议使用缺失小鼠模型研究 CRACM2 和 CRACM3 功能并确定它们在 T 细胞中的体内作用（目标 1）。此外，我们将研究 VOCC 通道 CaV1.2（目标 2）和 CaV1.3（目标 3）如何相互作用和调节 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，从而有助于开发治疗自身免疫性疾病的新方法。