The store-operated Ca(2+) entry complex comprises a small cluster of STIM1 associated with one Orai1 channel

Increases in cytosolic Ca(2+) concentration regulate diverse cellular activities and are usually evoked by opening of Ca(2+) channels in intracellular Ca(2+) stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP(3)), IP(3) receptors coordinate the contributions of these two Ca(2+) sources by mediating Ca(2+) release from the endoplasmic reticulum (ER). Loss of Ca(2+) from the ER then activates store-operated Ca(2+) entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca(2+) 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(2+) was unaffected by the tag. Step-photobleaching analysis of cells with empty Ca(2+) 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.