Understanding Spatial Interactions Between STIM and Orai

项目摘要/摘要： 描述：钙离子通道是真核细胞中普遍存在的一种信号转导机制 对介导更长时间的钙信号和恢复内质网/肌浆网至关重要 配基引起的耗竭。SOCE中的关键操纵子是钙离子选择性PM六聚体Orai通道 (主要是Orai1)和ER/SR驻留的二聚体跨膜钙感受器蛋白STIM1和 STIM2。当ER/SR腔内钙离子耗尽时，STIM1被激活，诱导其解离并与Orai1结合 PM中的频道激活的Orai1通道在内质网-质膜连接中产生高钙的离散微区 这些细胞含有的钙离子浓度大约是静止的细胞质水平的100倍。新的新兴市场研究 揭示ER-PM连接中的Orai1通道间隔很近(9-13 nm)，大致相当于 二聚体STIM1的STIM-Orai激活区(SOAR)。尽管ORAI频道的集群对于 产生这样的钙饱和微域，集群发生的实际机制并不是 明白了。我的研究涉及Orai1通道的聚集是如何局部控制SOCE介导的钙离子的 通过交联型Orai1通道“集中”或空间限制Ca~(2+)进入连接中的信号 由STIM1中的二聚体Soar结构域介导。这项研究使用了含有可溶性SOAR-二聚体的 关键的F394H突变，阻止SOAR与Orai1通道结合。我将调查一个新的 发现并广泛表达STIM2亚型的剪接变异体(STIM2.1)，含有8个氨基酸 在SOAR中关键的F394 STIM-Orai1结合结构域附近插入，在功能上模拟 SOAR1(F394H)突变。我的目标是解决我们对Orai1集群如何理解的一个关键差距 在空间上控制钙信号及其下游效应器。我的总体假设是STIM2.1起作用 作为STIM1的重要负性调节因子，阻止Orai1通道的聚集，并控制空间 钙离子进入信号的特征。使用新的SOAR2.1串联二聚体作为探针，我的目标是：(1) 使用超分辨率显微镜重建YFP-SOAR2.1级联体相互作用的3D图像 用Orai1-CFP和光漂白后荧光恢复(FRAP)来评估膜扩散速度 和与Orai1结合的YFP-OAR2.1串联体的大小；(2)使用遗传编码的钙指示剂 测量集群中的局部钙信号，并使用基于FRET的循环AMP传感器Epac2进行功能报告 下游钙依赖的腺苷酸环化酶(AC8)与Orai1通道密切相关。总的来说，这些 研究将确定SOAR2.1如何通过阻止Orai1通道聚集来调节重要的控制 ORAI1交联，并将评估STIM2.1如何调节Orai1间距和局部钙微域 通过下游的AC8活性进行调节。这样的研究汇集了对 胰腺、神经元和胰腺局部钙离子和环磷酸腺苷信号的相互依赖 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.