Ion Channels and Signaling Mechanisms In T Lymphocytes

描述(申请人提供)：细胞内游离钙浓度升高是控制T淋巴细胞分化和功能的基本信号。这项建议的长期目标是阐明在T细胞中产生和塑造钙信号的分子和细胞机制。钙释放激活的钙通道(CRAC)在这一过程中起着核心作用。内质网(ER)中储存的钙离子耗尽后，这些通道开始开放，但内质网耗竭与通道激活之间的联系机制尚不清楚。此外，通过CRAC通道进入细胞的Ca~(2+)与质膜上的线粒体和Ca~(2+)泵相互作用，产生持久而复杂的钙信号。荧光钙指示剂和荧光显微镜的最新发展使得在亚细胞水平上研究这些机制和可视化CRAC通道活动的微观位置成为可能。我们将使用多种方法的组合来研究CRAC通道与内质网、线粒体和泵之间的功能相互作用，包括膜片钳记录、靶向遗传编码的钙离子指示剂和全内反射荧光显微镜(TIR-FM)。我们将区分CRAC激活的两种模式：与内质网中蛋白质的物理接触，以及通过内质网释放的可扩散信使激活。我们将探讨内质网片段化在钙失活CRAC通道机制中的作用，以及聚集在质膜上的钙泵并使它们接近CRAC通道以进行功能相互作用的机制。这个项目的意义是双重的。首先，这些结果将有助于澄清与各种非兴奋性细胞中的存储操作通道和钙信号有关的基本问题。其次，这些研究的结果可能有助于确定控制免疫反应的新靶点，这些靶点可能有助于治疗自身免疫性疾病或免疫缺陷，并可能有助于解释钙信号机制异常操作导致的免疫功能障碍。

DESCRIPTION (provided by applicant): The elevation of intracellular free Ca2+ concentration is an essential signal controlling the differentiation and functions of T lymphocytes. The long-term goal of this proposal is to elucidate the molecular and cellular mechanisms that generate and shape Ca2+ signals in T cells. The Ca2+ release activated Ca2+ (CRAC) channel plays a central role in this process. These channels open in response to the depletion of the Ca2+ stored in the endoplasmic reticulum (ER), but the mechanism linking store depletion to channel activation is not well understood. In addition, Ca2+ entering the cell through CRAC channels interacts with mitochondria and Ca2+ pumps in the plasma membrane to create prolonged and complex Ca2+ signals. Recent developments in fluorescent Ca2+ indicators and fluorescence microscopy have made it possible to address these mechanisms at a subcellular level and visualize microscopic sites of CRAC channel activity. We will address functional interactions between CRAC channels and the ER, mitochondria, and pumps using a combination of approaches, including patch-clamp recording, targeted genetically-encoded Ca2+ indicators, and total internal reflection fluorescence microscopy (TIR-FM). We will distinguish between two models of CRAC activation: physical contact with proteins in the ER, and activation via a diffusible messenger released from the ER. We will explore the role of ER fragmentation in the mechanism of CRAC channel inactivation by Ca2+, and the mechanisms for clustering Ca2+ pumps in the plasma membrane and bringing them close to CRAC channels for functional interaction. The significance of this project is two-fold. First, the results will help to clarify fundamental issues related to store-operated channels and calcium signaling in a wide variety of non-excitable cells. Second, the results of these studies may help identify novel targets for the control of the immune response that may be beneficial in treating autoimmune disorders or immunodeficiencies, and they may help explain immune dysfunction resulting from aberrant operation of the Ca signaling machinery.