Department of Pharmacology, University of Cambridge, Cambridge, CB2 1PD, United Kingdom.
Department of Basic Medical Sciences, College of Medicine, QU Health, Qatar University, Doha, Qatar.
Proc Natl Acad Sci U S A. 2021 Mar 9;118(10). doi: 10.1073/pnas.2010789118.
Increases in cytosolic Ca concentration regulate diverse cellular activities and are usually evoked by opening of Ca channels in intracellular Ca stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP), IP receptors coordinate the contributions of these two Ca sources by mediating Ca release from the endoplasmic reticulum (ER). Loss of Ca from the ER then activates store-operated Ca entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca channel, Orai1, causing its pore to open. The relative concentrations of STIM1 and Orai1 are important, but most analyses of their interactions use overexpressed proteins that perturb the stoichiometry. We tagged endogenous STIM1 with EGFP using CRISPR/Cas9. SOCE evoked by loss of ER Ca was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca stores revealed an average of 14.5 STIM1 molecules within each sub-PM punctum. The fluorescence intensity distributions of immunostained Orai1 puncta were minimally affected by store depletion, and similar for Orai1 colocalized with STIM1 puncta or remote from them. We conclude that each native SOCE complex is likely to include only a few STIM1 dimers associated with a single Orai1 channel. Our results, demonstrating that STIM1 does not assemble clusters of interacting Orai channels, suggest mechanisms for digital regulation of SOCE by local depletion of the ER.
细胞质 Ca 浓度的增加调节着多种细胞活动，通常是通过开放细胞内 Ca 储存库和质膜（PM）中的 Ca 通道引起的。对于许多引发肌醇 1,4,5-三磷酸（IP）形成的信号，IP 受体通过介导内质网（ER）中的 Ca 释放来协调这两个 Ca 源的贡献。然后，ER 中 Ca 的丢失通过导致 STIM1 二聚体聚集并展开与 PM Ca 通道 Orai1 相互作用的细胞质结构域，从而激活储存操作的 Ca 进入（SOCE），导致其孔打开。STIM1 和 Orai1 的相对浓度很重要，但对它们相互作用的大多数分析都使用了扰乱化学计量的过表达蛋白。我们使用 CRISPR/Cas9 将内源性 STIM1 标记为 EGFP。ER Ca 丢失引发的 SOCE 不受标签的影响。对空 Ca 储存库的细胞进行分步光漂白分析显示，每个亚 PM 斑点中平均有 14.5 个 STIM1 分子。免疫染色的 Orai1 斑点的荧光强度分布受储存耗竭的影响很小，并且对于与 STIM1 斑点共定位或远离它们的 Orai1 斑点也是如此。我们得出结论，每个天然 SOCE 复合物可能只包含几个与单个 Orai1 通道相关的 STIM1 二聚体。我们的结果表明，STIM1 不会组装相互作用的 Orai 通道簇，这表明了通过 ER 的局部耗竭对 SOCE 进行数字调节的机制。
