Understanding Spatial Interactions Between STIM and Orai

了解 STIM 和 Orai 之间的空间相互作用

• 批准号: 9538071
• 负责人: Robert M Nwokonko
• 金额: $ 3.1万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9538071
• 关键词: A kinase anchoring protein; Address; Adenylate Cyclase; Affect; Amino Acids; Binding; Binding Proteins; Biological Assay; C-terminal; CREB1 gene; Calcium; Cell Differentiation process; Cell membrane; Cell physiology; Cells; Characteristics; Complex; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diffusion; Endoplasmic Reticulum; Eukaryotic Cell; Event; FOS gene; Fluorescence Recovery After Photobleaching; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Genetic Transcription; Goals; Immune; Immunoprecipitation; Ion Channel; Label; Ligands; Link; Mammals; Measures; Mediating; Membrane; Membrane Lipids; Metabolism; Microscopy; Modeling; Molecular Conformation; Mutation; Nature; Neurons; Pancreas; Pathway interactions; Positioning Attribute; Protein Isoforms; Proteins; RNA Splicing; Regulation; Reporter; Reporting; Research; Resolution; Rest; Role; STIM1 gene; Sarcoplasmic Reticulum; Serine; Signal Pathway; Signal Transduction; Signaling Protein; Structural Models; Testing; Three-Dimensional Image; Transcript; Variant; base; cell type; crosslink; dimer; functional mimics; image reconstruction; novel; prevent; public health relevance; scaffold; sensor

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.

项目摘要/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机制 对于介导长期 Ca2+ 信号和恢复内质/肌浆网 Ca2+ 至关重要 SOCE 中的关键操纵子是 Ca2+ 选择性 PM 六聚体 Orai 通道。 （主要是 Orai1）和 ER/SR 驻留二聚体跨膜钙传感器蛋白 STIM1 和 当 ER/SR 管腔 Ca2+ 耗尽时，STIM2 被激活，诱导其展开并与 Orai1 结合。 PM 中的活跃 Orai1 通道在 ER-PM 连接内产生高 Ca2+ 的离散微域。 新的 EM 研究表明，Ca2+ 浓度比静息细胞质水平高出大约 100 倍。 揭示 ER-PM 连接内的 Orai1 通道间隔很近（9-13 nm），大致相当于 尽管 Orai 通道的聚集对于二聚体 STIM1 的 STIM-Orai 激活区域 (SOAR) 至关重要。 产生这种 Ca2+ 饱和微区，聚类发生的实际机制并不是 我的研究解决了 Orai1 通道的聚集如何局部控制 SOCE 介导的 Ca2+。 通过交联 Orai1 通道“集中”或空间限制 Ca2+ 进入，从而在连接处发出信号 由 STIM1 中的二聚体 SOAR 结构域介导 该研究使用含有 SOAR 的可溶性二聚体。 阻止 SOAR 与 Orai1 通道结合的关键 F394H 突变，我将研究新的方法。 发现并广泛表达的 STIM2 同种型 (STIM2.1) 剪接变体，包含 8 个氨基酸 插入到 SOAR 中关键的 F394 STIM-Orai1 结合域附近，功能上模仿 SOAR1(F394H) 突变的目标是解决我们对 Orai1 聚类方式理解上的一个关键差距。 我的总体假设是 STIM2.1 在空间上控制 Ca2+ 信号及其下游效应器。 作为 STIM1 的重要负调节因子，可防止 Orai1 通道聚集，并控制空间 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创建 YFP-SOAR2.1 串联体相互作用的 3D 图像重建 使用 Orai1-CFP 和光漂白后荧光恢复 (FRAP) 来评估膜扩散速率 和与 Orai1 结合的 YFP-OAR2.1 连接体的大小；(2) 使用基因编码的 Ca2+ 指示剂 测量簇中的局部 Ca2+ 信号，以及基于 FRET 的循环 AMP 传感器 Epac2，以进行功能报告 下游 Ca2+ 依赖性腺苷酸环化酶 (AC8) 与 Orai1 通道密切相关。 研究将确定 SOAR2.1 如何通过阻止来介导重要的控制 Orai1 通道聚类 Orai1 交联，并将评估 STIM2.1 如何调节 Orai1 间距和局部 Ca2+ 微域 这些研究汇集了对 AC8 下游活性的新认识。 局部 Ca2+ 和环 AMP 信号的相互依赖性在胰腺、神经和神经系统中具有重要意义 免疫细胞功能。