Abstract
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.
Highlights
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)
In-gel fluorescence, Western blotting with an anti-GFP antibody, and knockdown of stromal interaction molecule 1 (STIM1) expression using short hairpin RNA confirmed that STIM1-EGFP was the only EGFP-tagged protein expressed in the STIM1
We used cells with one copy of endogenous STIM1 tagged with EGFP (Fig. 1) to define the number of STIM1 molecules within the puncta that regulate Orai1
Summary
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). Loss of Ca2+ from the ER activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai, causing its pore to open. In generating the cytosolic Ca2+ signals that regulate cellular activities, cells call upon two sources of Ca2+: the extracellular space, accessed through Ca2+ channels in the plasma membrane (PM), and Ca2+ sequestered within intracellular stores, primarily within the endoplasmic reticulum (ER). The reduction in ER free-Ca2+ concentration causes Ca2+ to dissociate from the luminal Ca2+-binding sites of stromal interaction molecule 1 (STIM1), a dimeric protein embedded in ER membranes This loss of Ca2+ causes STIM1 to unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai, causing its pore to open and Ca2+ to flow into the cell through the SOCE pathway (Fig. 1A) [4, 5]. We suggest that SOCE may be digitally regulated by local ER depletion, and that local SOCEevoked Ca2+ fluxes are small enough to allow substantial intracellular redistribution of Ca2+ through ER tunnels
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