Molecular Mechanisms of lon Channels in T Lymphocytes

项目摘要 本更新提案的总体目标是更好地了解Orai 1通道在T中的功能 细胞在分子、亚细胞和细胞水平上的变化。奥赖蛋白是一类新的钙通道， 质膜（PM）。它们被ER中的STIM蛋白激活，并与之共定位于内质网的点状区域。 ER腔中Ca 2+耗尽后的ER-PM连接。尽管Orai 1作为一种潜在的 尽管在T细胞中存在治疗靶点，但关键问题仍然存在于单通道、斑点和细胞水平。在很大程度上， 是因为膜片钳技术的单通道记录不能应用于Orai 1或其他奥赖 通道，其具有异常低的电导，并且因为局部信号传导发生在STIM 1：Orai 1 斑点。为了规避这些限制，我们的建议是基于我们最近开发的新型Ca 2 +- 指示剂融合蛋白;报告单通道信号的Orai 1-GCaMP 6 f通道指示剂，和 比率，红/绿色胞质Ca 2+指示剂。在转基因小鼠中实施，这些探针揭示了新的 和非预期的局部Ca 2+信号传导事件，以及第一次有机会监测内源性Orai 1活性， T细胞。结合T细胞特异性Orai 1敲除小鼠，我们使用这些工具来研究：1）Orai 1是如何被 在单分子水平上激活; 2）Orai 1通道活性在斑点和天然T细胞中如何变化;以及3） 局部Ca 2+信号如何调节淋巴结免疫监视过程中T细胞的运动。

Project Summary The overall objective of this renewal proposal is to better understand how Orai1 channels function in T cells at the molecular, subcellular, and cellular levels. Orai proteins are a novel class of Ca2+ channels in the plasma membrane (PM). They are activated by STIM proteins in the ER, with which they colocalize in puncta at ER-PM junctions following depletion of Ca2+ in the ER lumen. Despite the importance of Orai1 as a potential therapeutic target in T cells, crucial questions remain at single channel, puncta and cellular levels. In large part this is because single-channel recording with the patch-clamp technique cannot be applied to Orai1 or other Orai channels, which have exceptionally low conductance, and because local signalling takes place in STIM1:Orai1 puncta. To circumvent these limitations, our proposal is predicated on our recent development of novel Ca2+- indicator fusion proteins; an Orai1-GCaMP6f channel-indicator that reports single-channel signals, and a ratiometric, red/green cytosolic Ca2+ indicator. Implemented in transgenic mice, these probes are revealing new and unanticipated local Ca2+ signalling events and the first chance to monitor endogenous Orai1 activity in native T cells. In conjunction with T cell-specific Orai1 knockout mice, we use these tools to investigate: 1) how Orai1 is activated at the single-molecule level; 2) how Orai1 channel activity varies in puncta and in native T cells; and 3) how local Ca2+ signals modulate T cell motility during immune surveillance in the lymph node.