The store-operated Ca²⁺ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

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.