Ion Channels and Signaling Mechanisms in T Lymphocytes

描述(申请人提供)：储存操作的钙通道(SOC)产生对许多生理过程至关重要的钙信号，从免疫细胞的激活和分化到肌肉的活动、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是如何激活的方面取得了显着进展。内质网钙离子的耗竭导致内质网钙离子感受器STIM1寡聚，导致其在内质网-质膜(PM)连接处聚集。在这些位点，STIM1与钙释放激活的钙通道(CRAC)的造孔亚单位Orai1结合，从而捕获它并激活跨PM的局部钙内流然而，在STIM-Orai复合体形成后，控制CRAC通道信号强度的几个过程相对较少；这些过程包括钙依赖失活(CDI)的反馈抑制，STIM-Orai结合的化学计量比对通道开放概率的限制，以及STIM1和Orai1在ER-PM连接处的保留动力学。这一建议应用电生理学、STIM1和四聚体Orai1级联通道的突变以及超分辨单粒子跟踪技术来了解这三个过程是如何通过CRAC通道调节钙离子进入的。最近的发现揭示了STIM1、钙调蛋白(CaM)和Orai1细胞内II-III环在CDI机制中所需的作用。在目标1中，我们将构建CaM和STIM1结合位点减少的串联Orai1通道，以探讨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，即使当内质网钙离子储备完全耗尽时，内质网-PM连接处只有一小部分CRAC通道是活跃的，而药物2-氨基乙基二苯硼酸酯(2-APB)可以动员这大量休眠通道。在目标2中，我们将使用具有可变数量的STIM1结合位点的2-APB和CRAC通道来确定通道如何变得休眠以及2-APB如何将它们招募到激活状态。最后，光活化研究表明STIM1和Orai1在ER-PM连接处的停留时间相当短，这限制了STIM1和Orai1可以积累形成活性CRAC通道复合体的数量。在目标3中，我们将应用单分子跟踪技术以纳米精度表征STIM1和Orai1在不同条件下的扩散和限制，并确定控制STIM1和Orai1在ER-PM连接处迁移和保留的关键蛋白质-蛋白质和蛋白质-脂质相互作用。总体而言，这些研究的结果将增加我们对生理条件下存储操作信号强度如何控制的理解，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.