ION CHANNELS AND SIGNALING MECHANISMS IN T LYMPHOCYTES

描述（改编自申请人的描述）： 细胞内游离 Ca2 浓度是控制细胞内钙离子浓度的重要信号 T淋巴细胞的分化和功能。本次活动的长远目标 建议是阐明负责产生的分子机制 T 细胞中的 Ca2 信号塑造。 T 细胞中的 Ca2 信号产生至 很大程度上是由 Ca2 释放激活 Ca2 (CRAC) 通道的活性决定的。 这些通道随着细胞内 Ca2 储备的耗尽而打开， 但门店耗尽与渠道开放之间的联系机制并不完善 明白了。我们将测试 CRAC 通道激活的两种可能机制 结合使用电生理学和荧光成像 方法：通过调节开放通道插入质膜 囊泡融合，以及通过物理耦合来控制 CRAC 通道门控 储存膜蛋白。线粒体在维持生命活动中发挥着重要作用 通过 CRAC 通道的 Ca2 流入率高。为了进一步了解这是如何 函数执行后，我们将检查之间的函数交互 线粒体与 CRAC 通道和 Ca2 激活的 K 通道，并相关 这可以控制膜电位和 Ca2 流入。最后，钙 质膜 (PMCA) 中的 ATP 酶主要负责 T 细胞中 Ca2 的清除，其活性受到以下因素的缓慢调节 [Ca2 ] 的变化，使它们有助于 Ca2 的复杂性 信号动态。我们将应用一种新型的胞质钙钳技术 表征 PMCA 调制及其分子的 Ca2 和时间依赖性 机制。 这些研究的意义有两方面。一、CRAC通道、KCa通道、 线粒体和泵在非兴奋性细胞中以各种形式广泛表达， 以便更好地了解它们在 T 细胞中的运作和相互作用将有助于揭示 许多细胞类型中的 Ca2 信号传导机制。二、Ca2信号的复杂性 T 细胞中的 T 细胞已知是下游选择性调节的重要决定因素。 免疫反应期间的反应，例如基因表达和细胞激活。因此， 这项研究的结果可能有助于确定控制免疫反应的新靶点。 因此，这项研究的结果可能有助于确定免疫控制的新靶点。 可能有益于治疗免疫缺陷的自身免疫性疾病的反应， 它们可能有助于解释因异常运作而导致的免疫功能障碍 通道、泵和线粒体。

DESCRIPTION (Adapted from applicant's Description): 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 mechanisms responsible for generating the shaping Ca2+ signals in T Cells. Ca2+ signals in T cells are generated to a great extent by the activity of Ca2+ release-activated Ca2+ (CRAC) channels. These channels open in response to the depletion of intracellular Ca2+ stores, but the mechanism linking store depletion to channel opening is not well understood. We will test two possible mechanisms of CRAC channel activation using a combination of electrophysiological and fluorescence imaging approaches: regulated insertion of open channels into the plasma membrane by vesicle fusion, and control of CRAC channel gating by physical coupling to store membrane proteins. Mitochondria play an essential role in maintaining a high rate of Ca2+ influx through CRAC channels. To further understand how this function is carried out, we will examine the functional interactions between mitochondria and both CRAC channels and Ca2+-activated K+ channels, and relate this to the control of membrane potential and Ca2+ influx. Finally, Ca2+ ATPases in the plasma membrane (PMCA) are primarily responsible for the clearance of Ca2+ from T cells, and their activity is modulated slowly by changes in [Ca2+], allowing them to contribute to the complexity of Ca2+ signaling dynamics. We will apply a novel cytosolic calcium clamp technique to characterize the Ca2+- and time-dependence of PMCA modulation and its molecular mechanism. The significance of these studies is two-fold. First, CRAC channels, KCa channels, mitochondria, and pumps are widely expressed in various forms among non-excitable cells, so that a better understanding of their operation and interactions in T cells will shed light on Ca2+ signaling mechanisms in many cell types. Second, the complex nature of Ca2+ signals in T cells is known to be an important determinant for the selective regulation of downstream responses such as gene expression and cell activation during the immune response. Thus, the results of this study may help identify novel targets for the control of the immune response. Thus, the results of this study may help identify novel targets for the control of the immune response that may be beneficial in treating autoimmune disorders of immunodeficiencies, and they may help to explain immune dysfunctions resulting from the abberrant operation of channels, pumps and mitochondria.