Understanding Spatial Interactions Between STIM and Orai

项目概要/摘要： 描述：钙库操纵的钙内流（SOCE）是真核细胞中普遍存在的信号传导机制 对于介导长期Ca 2+信号和恢复内质网/肌浆网Ca 2+至关重要， 配体诱导的耗竭。SOCE中的关键操纵子是Ca 2+选择性PM六聚体奥赖通道 （主要是Orai 1）和ER/SR驻留的二聚跨膜钙传感器蛋白STIM 1和 STIM 2.当ER/SR腔Ca 2+耗尽时，STIM 1被激活，诱导其展开并结合Orai 1 PM的频道。活性Orai 1通道在ER-PM连接中产生高Ca 2+的离散微区 其含有比静息细胞质水平高大约100倍的Ca 2+浓度。新EM研究 揭示ER-PM结内的Orai 1通道间隔很近（9-13 nm），大致为 二聚体STIM 1的STIM-Orai激活区（SOAR）。尽管奥赖通道的聚类对于 产生这种Ca 2+饱和的微区，聚集发生的实际机制不是 明白我的研究解决了Orai 1通道的聚集如何局部控制SOCE介导的Ca 2 + 通过交联Orai 1通道“集中”或空间限制Ca 2+进入连接中的信号 由STIM 1中的二聚体SOAR结构域介导。这些研究使用含有可溶性SOAR二聚体的 关键的F394 H突变，阻止SOAR与Orai 1通道结合。我将调查一个新的 发现并广泛表达的STIM 2同种型的剪接变体（STIM2.1），含有8个氨基酸 插入邻近SOAR中的关键F394 STIM-Orai 1结合结构域，功能上模拟 SOAR 1（F394 H）突变。我的目标是解决我们对Orai集群如何理解的一个关键差距1 在空间上控制Ca 2+信号及其下游效应物。我的总体假设是STIM2.1的功能 作为STIM 1的重要负调节因子，防止Orai 1通道的聚集，并控制空间 Ca 2+进入信号的特征。利用新的SOAR2.1串联二聚体作为探针，我们的目标是：（1） 使用超分辨率显微镜创建YFP-SOAR 2.1串联体相互作用的3D图像重建 使用Orai 1-CFP和光漂白后荧光恢复（FRAP）来评估膜扩散速率 和与Orai 1结合的YFP-OAR 2.1多联体的大小;（2）使用遗传编码的Ca 2+指示剂， 测量簇中的局部Ca 2+信号，以及基于FRET的环AMP传感器Epac 2，以功能性地报告 下游Ca 2+依赖性腺苷酸环化酶（AC 8）与Orai 1通道密切相关。总体而言，这些 研究将确定SOAR2.1如何通过阻止重要的控制Orai 1通道聚集来介导 Orai 1交联，并将评估STIM2.1如何调节Orai 1间距和局部Ca 2+微区 通过下游AC 8活性介导。这样的研究汇集了对 局部Ca 2+和cAMP信号的相互依赖性在胰腺、神经元和 免疫细胞功能

Project Summary/Abstract: DESCRIPTION: Store-operated calcium entry (SOCE) is a ubiquitous signaling mechanism in eukaryotic cells crucial for mediating longer–term Ca2+ signals and restoring endoplasmic/sarcoplasmic reticulum Ca2+ after ligand induced depletion. The key operators in SOCE are the Ca2+ selective PM hexameric Orai channels (predominantly Orai1) and the ER/SR resident, dimeric transmembrane calcium sensor proteins, STIM1 and STIM2. STIM1 is activated when ER/SR luminal Ca2+ is depleted, inducing it to unfold and bind to Orai1 channels in the PM. Active Orai1 channels create discrete microdomains of high Ca2+ within ER-PM junctions that contain roughly 100-fold greater Ca2+ concentrations than resting cytoplasmic levels. New EM studies reveal Orai1 channels within ER-PM junctions are closely spaced (9-13 nm), roughly the distance spanned by the STIM-Orai Activating Regions (SOAR) of dimeric STIM1. Although clustering of Orai channels is critical for generating such Ca2+-saturated microdomains, the actual mechanism by which clustering occurs is not understood. My studies address how clustering of Orai1 channels locally controls SOCE-mediated Ca2+ signals in junctions by “concentrating” or spatially confining Ca2+ entry through cross-linking Orai1 channels mediated by the dimeric SOAR domains in STIM1. The studies use soluble SOAR-dimers containing the critical F394H mutation that prevents SOAR binding to the Orai1 channels. I will investigate how a newly discovered and widely expressed splice variant of the STIM2 isoform (STIM2.1), containing an 8 amino acid insert adjacent to the critical F394 STIM-Orai1 binding domain in SOAR, functionally mimics the SOAR1(F394H) mutation. My aims address a critical gap in our understanding of how clustering of Orai1 spatially controls Ca2+ signals and their downstream effectors. My overall hypothesis is that STIM2.1 functions as an important negative regulator of STIM1 that prevents clustering of Orai1 channels, and controls the spatial characteristics of Ca2+ entry signals. Using new SOAR2.1 concatemeric dimers as probes, my aims are: (1) to use super-resolution microscopy to create 3D-image reconstructions of YFP-SOAR2.1 concatemer interactions with Orai1-CFP, and fluorescence recovery after photobleaching (FRAP) to assess membrane diffusion rates and size of YFP-OAR2.1 concatemers bound to Orai1; (2) to use genetically encoded Ca2+ indicators to measure localized Ca2+ signals in clusters, and a FRET-based cyclic AMP sensor, Epac2, to functionally report downstream Ca2+-dependent adenylyl cyclase (AC8) closely associated with Orai1 channels. Overall, these studies will determine how SOAR2.1 may mediate important control Orai1 channel clustering by preventing Orai1 cross-linking, and will assess how STIM2.1 may regulate Orai1 spacing and local Ca2+ microdomains mediated through downstream AC8 activity. Such studies bring together new understanding of the interdependence of local Ca2+ and cyclic AMP signals of major importance in pancreatic, neuronal, and immune cell function.