ION CHANNELS AND SIGNALING MECHANISMS IN T LYMPHOCYTES

描述(改编自调查员的申请)：提升 抗原诱导的细胞内游离钙浓度在血管内皮细胞死亡中起关键作用 决定T淋巴细胞的命运和功能。的长期目标 这项提议是为了阐明产生和调节 钙信号，并了解它如何激活特定的T细胞 激活基因。过去五年的研究表明，钙离子 T细胞中的信号由电容钙离子内流维持，这是一种广泛存在的 细胞内钙离子储存耗竭的信号机制 通过钙释放激活的钙通道(CRAC)激活钙内流。 人们对这些渠道是如何监管的以及如何监管知之甚少 它们的活动决定了T细胞的激活或耐受等反应。 提出了四个具体目标，以阐明其调节和功能 结合显微成像和免疫组织化学技术研究T细胞CRAC通道 膜片钳技术：1)确定CRAC通道的依赖性 对储存内容和细胞内钙离子的激活。2)调查 细胞内钙离子反馈缓慢灭活CRAC的机制 频道。3)确定线粒体在调节钙离子中的作用 发信号并测试它们通过以下方式影响CRAC通道功能假设 干扰慢速灭活。4)研究[Ca~(2+)]i的作用 在提高效率和/或特异性方面的振荡 转录反应，使用两个钙敏感的报告基因驱动 转录因子、NFAT和AP-1。 这些研究的意义在于，它们将首次描述 对控制动态行为的几个细胞过程进行计时 耗竭激活的钙通道。此外，在许多细胞中，特定的 动态钙信号(如振荡)的后果在很大程度上 这些研究将评估它们对可定义的和 生理上重要的终点，基因激活。考虑到要求 对于CRAC通道在T细胞激活中的作用，这些研究可能最终 暴露了许多免疫反应的控制点，导致了新的 具有重要治疗效益的免疫调节策略 预防或治疗疾病，如艾滋病和自身免疫性疾病。

DESCRIPTION (Adapted from the investigator's application): The elevation of intracellular free calcium concentration by antigen plays a pivotal role in determining the fate and functions of T lymphocytes. The long term goals of this proposal are to elucidate the mechanisms that generate and regulate the calcium signal, and to understand how it activates specific T-cell activation genes. Work over the past five years indicates that Ca2+ signaling in T cells is sustained by capacitative Ca2+ entry, a widespread signaling mechanism by which the depletion of intracellular Ca2+ stores activated Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels. Relatively little is known about how these channels are regulated and how their activity determines responses such as T-cell activation or tolerance. Four specifics aims are proposed to elucidate the regulation and functions of CRAC channels in T cells using a combination of microscopic imaging and patch-clamp techniques: 1) Determine the dependence of CRAC channel activation on store content and intracellular Ca2+. 2) Investigate the mechanism by which intracellular Ca2+ feeds back to slowly inactivate CRAC channels. 3) Establish the role of mitochondria in modulating the Ca2+ signal and test the hypothesis that they influence CRAC channel function by interfering with slow inactivation. 4) Examine the role of [Ca2+]i oscillations in increasing the efficiency and/or the specificity of transcriptional responses, using reporter genes driven by two Ca2+-sensitive transcriptions factors, NFAT and AP-1. The significance of these studies is that they will describe for the first time several cellular processes that control the dynamic behavior of depletion-activated Ca2+ channels. Moreover, in many cells the specific consequences of dynamic Ca2+ signals such as oscillations has been largely unexplored, and these studies will assess their impact on a definable and physiologically important end point, gene activation. Given the requirement for CRAC channel function in T-cell activation, these studies may ultimately expose a number of control points for the immune response, leading to new strategies for immunomodulation with important therapeutic benefit in the prevention or treatment of disorders such as AIDS and autoimmune disease.