Molecular Mechanisms of lon Channels in T Lymphocytes

项目摘要 此更新建议的总体目标是更好地了解Orai1通道如何在T 分子、亚细胞和细胞水平的细胞。Orai蛋白是细胞内一类新的钙通道。 质膜(PM)。它们被内质网中的STIM蛋白激活，与之共存于 内质网腔内钙离子耗竭后的内质网-PM连接。尽管Orai1作为一种潜在的 针对T细胞的治疗靶点，关键问题仍然存在于单通道、点状和细胞水平。这在很大程度上是 是因为膜片钳技术的单通道记录不能应用于Orai1或其他Orai 具有极低电导的通道，并且因为本地信令发生在STIM1：ORAI1中 标点符号。为了绕过这些限制，我们的建议是基于我们最近开发的新型钙离子- 指示器融合蛋白；Orai1-GCaMP6f通道指示器，报告单通道信号；以及 比率测量法，红色/绿色胞浆钙指示剂。在转基因小鼠身上实施的这些探针揭示了新的 和意想不到的局部钙信号事件以及首次有机会监测天然的内源性Orai1活性 T细胞。结合T细胞特异性Orai1基因敲除小鼠，我们使用这些工具来研究：1)Orai1是如何 在单分子水平上激活；2)Orai1通道活性在斑点和天然T细胞中的变化；以及3) 局部Ca~(2+)信号在淋巴结免疫监测中如何调节T细胞运动

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.