Ion Channels and Signaling Mechanisms in T Lymphocytes

描述（由申请人提供）：储存操作的Ca2+通道（SOC）产生Ca2+信号，这些信号对许多生理过程至关重要，从免疫细胞激活和分化到肌肉活动、分泌和运动，人类SOC功能的丧失会导致毁灭性的严重联合免疫缺陷，并伴有额外的肌病和外胚层发育不良。最近在描述扩散陷阱机制以解释这些通道如何被激活方面取得了显著进展。内质网（ER） Ca2+的消耗导致内质网Ca2+传感器STIM1寡聚，导致其在内质膜（PM）连接处积累。在这些位点上，STIM1与Ca2+释放激活Ca2+ (CRAC)通道的成孔亚基Orai1结合，以捕获它并激活局部Ca2+进入PM。然而，在STIM-Orai复合物形成后，对通过CRAC通道控制信号强度的几个过程知之甚少；其中包括通过Ca2+依赖性失活（CDI）的反馈抑制，STIM-Orai结合的化学计量对通道开放概率的限制，以及STIM1和Orai1在ER-PM连接处保留的动力学。本研究应用电生理学、STIM1和四聚体Orai1串联通道的诱变以及超分辨率单粒子跟踪技术来了解这三个过程如何通过CRAC通道调节Ca2+的进入。最近的研究结果揭示了STIM1、calmodulin （CaM）和Orai1的细胞内II-III环在CDI机制中的作用。在Aim 1中，我们将构建具有减少CaM和STIM1结合位点数量的连接Orai1通道，以探索CaM结合、STIM1结合以及与II-III环的相互作用如何影响CDI。一些证据表明，即使在ER Ca2+储存完全耗尽时，ER- pm连接处只有一小部分CRAC通道是活跃的，并且这种休眠通道的大库可以被2-氨基乙基二苯硼酸盐（2- apb）药物调动。在Aim 2中，我们将使用具有可变数量STIM1结合位点的2- apb和CRAC通道来确定通道如何进入休眠状态以及2- apb如何将它们招募到活性状态。最后，光活化研究表明，STIM1和Orai1在ER-PM交界处停留的时间相当短，这限制了STIM1和Orai1积累形成活性CRAC通道复合物的数量。在Aim 3中，我们将应用单分子跟踪技术以纳米精度表征STIM1和Orai1在各种条件下的扩散和限制，并确定控制STIM1和Orai1在ER-PM连接处的迁移和保留的关键蛋白质-蛋白质和蛋白质-脂质相互作用。总的来说，这些研究的结果将增加我们对生理条件下如何控制储存操作信号强度的理解，并提出上调或下调这些信号的新策略，为自身免疫和免疫缺陷综合征提供新的治疗方法。

DESCRIPTION (provided by applicant): Store-operated Ca2+ channels (SOCs) generate Ca2+ signals that are critical for many physiological processes ranging from immune cell activation and differentiation to muscle activity, secretion, and motility, and loss of SOC functio in humans leads to a devastating severe combined immunodeficiency with additional myopathy and ectodermal dysplasia. Remarkable progress has been made recently in delineating a diffusion-trap mechanism to explain how these channels are activated. Depletion of Ca2+ from the endoplasmic reticulum (ER) causes STIM1, an ER Ca2+ sensor, to oligomerize, leading to its accumulation at ER-plasma membrane (PM) junctions. At these sites STIM1 binds to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, to trap it and activate local Ca2+ entry across the PM. However, comparatively little is known about the several processes that control the strength of signaling through the CRAC channel after the STIM-Orai complex has formed; these include feedback inhibition via Ca2+-dependent inactivation (CDI), limits on channel open probability imposed by the stoichiometry of STIM-Orai binding, and the dynamics of STIM1 and Orai1 retention at ER-PM junctions. This proposal applies electrophysiology, mutagenesis of STIM1 and tetrameric Orai1 concatemer channels, and superresolution single-particle tracking techniques to understand how these three processes regulate Ca2+ entry through CRAC channels. Recent findings have revealed required roles for STIM1, calmodulin (CaM) and the intracellular II-III loop of Orai1 in the CDI mechanism. In Aim 1, we will construct concatemeric Orai1 channels with reduced numbers of CaM and STIM1 binding sites to explore how CaM binding, STIM1 binding, and interactions with the II-III loop impact CDI. Several lines of evidence suggest that only a small fraction of CRAC channels at ER-PM junctions are active, even when ER Ca2+ stores are fully depleted, and this large reservoir of dormant channels can be mobilized by the drug 2- aminoethyldiphenyl borate (2-APB). In Aim 2 we will use 2-APB and CRAC channels with variable numbers of STIM1 binding sites to determine how channels become dormant and how 2-APB recruits them to the active state. Finally, photoactivation studies show that the residence time of STIM1 and Orai1 at the ER-PM junction is rather short, placing limits on the amounts of STIM1 and Orai1 that can accumulate to form active CRAC channel complexes. In Aim 3 we will apply single-molecule tracking techniques to characterize the diffusion and confinement of STIM1 and Orai1 under various conditions with nanometer precision, and identify the critical protein-protein and protein-lipid interactions that control the mobility and retention of STIM1 and Orai1 at ER-PM junctions. Overall, the results of these studies will increase our understanding of how the strength of store-operated signals is controlled under physiological conditions, and suggest new strategies for up- or down-regulating these signals to provide new treatments for autoimmune and immunodeficiency syndromes.