Ion Channels and Signaling Mechanisms In T Lymphocytes

描述（申请人提供）：细胞内游离Ca2+浓度升高是控制T淋巴细胞分化和功能的重要信号。该建议的长期目标是阐明在T细胞中产生和形成Ca2+信号的分子和细胞机制。Ca2+释放激活Ca2+ (CRAC)通道在这一过程中起核心作用。这些通道打开响应Ca2+储存在内质网（ER）的损耗，但机制连接存储损耗通道激活尚不清楚。此外，通过CRAC通道进入细胞的Ca2+与质膜中的线粒体和Ca2+泵相互作用，产生长时间和复杂的Ca2+信号。荧光Ca2+指示剂和荧光显微镜的最新发展使得在亚细胞水平上解决这些机制和可视化CRAC通道活性的微观位点成为可能。我们将解决CRAC通道和内质网，线粒体和泵之间的功能相互作用，使用组合的方法，包括膜片钳记录，靶向基因编码的Ca2+指标，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：与内质网中的蛋白质物理接触，以及通过内质网释放的扩散信使激活。我们将探讨内质网碎裂在Ca2+作用下CRAC通道失活机制中的作用，以及Ca2+泵在质膜上聚集并使其靠近CRAC通道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不可兴奋细胞中的储存操作通道和钙信号传导相关的基本问题。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，这可能有助于治疗自身免疫性疾病或免疫缺陷，并且它们可能有助于解释由Ca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.