Increases in cytosolic Ca²⁺ concentration regulate diverse cellular activities and are usually evoked by opening of Ca²⁺ channels in intracellular Ca²⁺ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP₃), IP₃ receptors coordinate the contributions of these two Ca²⁺ sources by mediating Ca²⁺ release from the endoplasmic reticulum (ER). Loss of Ca²⁺ from the ER then activates store-operated Ca²⁺ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca²⁺ 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 Ca²⁺ was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca²⁺ 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.

细胞溶质 Ca ²⁺浓度的增加调节多种细胞活动，通常由细胞内 Ca ²⁺储存和质膜 (PM)中 Ca ²⁺通道的打开引起。对于引起肌醇 1,4,5-三磷酸 (IP ₃ ) 形成的许多信号，IP ₃受体通过介导内质网 (ER) 释放Ca ²⁺来协调这两种 Ca ²⁺源的贡献。ER中 Ca ²⁺的损失然后通过导致 STIM1 的二聚体聚集和展开与 PM Ca ^{2+相互作用的胞质结构域来激活存储操作的 Ca 2+进入 (SOCE)}通道，Orai1，导致其毛孔打开。STIM1 和 Orai1 的相对浓度很重要，但大多数对它们相互作用的分析都使用了过度表达的蛋白质，这些蛋白质会扰乱化学计量。我们使用 CRISPR/Cas9 用 EGFP 标记内源性 STIM1。^{由 ER Ca 2+}损失引起的 SOCE不受标签的影响。^{空 Ca 2+}细胞的分步光漂白分析商店显示每个亚 PM 泪点内平均有 14.5 个 STIM1 分子。免疫染色的 Orai1 puncta 的荧光强度分布受储存耗尽的影响最小，与与 STIM1 puncta 共定位或远离它们的 Orai1 相似。我们得出结论，每个原生 SOCE 复合体可能仅包含与单个 Orai1 通道相关的少数 STIM1 二聚体。我们的结果，证明 STIM1 不组装相互作用的 Orai 通道簇，表明通过局部消耗 ER 对 SOCE 进行数字调节的机制。