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Orai1 and STIM1 have been identified as main components of store-operated Ca2+ entry (SOCE), and their molecular interactions have been extensively studied in heterologous overexpression systems. Here we used molecular and imaging techniques to assess if overexpression of Orai1 and STIM1 may alter some physiological properties of endogenous SOCE pathway, and if there may be a crosstalk between expression levels of different components of SOCE. We found that in contrast to significant inhibition of endogenous SOCE caused by functional or molecular knock down of Ca2+-independent phospholipase A2 (PLA2G6) in naive HEK293 cells, the loss of SOCE was significantly attenuated in cells in which Orai1 or STIM1 were overexpressed. Similarly, overexpression of Orai1 or STIM1 prevented the effects of depletion of plasma membrane cholesterol on SOCE: β-methyl cyclodextrin caused significant inhibition of SOCE in naive, but not Orai1/STIM1 overexpressing cells. Further, we found crosstalk between expression levels of endogenous Orai1 and the plasma membrane variant of PLA2G6, but not STIM1. siRNA-induced knock down of Orai1 caused up to 6 fold increase in expression level of PLA2G6, but caused no change in STIM1 expression in HEK293 cells. Reciprocally, siRNA-induced down-regulation of PLA2G6 caused about 2 fold increase in endogenous Orai1 expression. Knock down of STIM1 had no effect on expression levels of either Orai1 or PLA2G6. Similar results were obtained in human aortic smooth muscle cells. These data suggest that there is an interdependence of expression levels of Orai1 and PLA2G6, and overexpression of either Orai1 or STIM1 may significantly influence the important physiological properties of SOCE. Further studies of endogenous SOCE mechanism are needed to determine the full spectrum of its molecular and functional components that may be required for effective signal transduction in naive cells.

STIM1, Orai1 and iPLA2β have been identified as crucial elements of the store-operated Ca2+ entry (SOCE) pathway, but the mechanism of their functional interaction and molecular requirements for SOCE activation remain controversial. Here we used high resolution imaging, patch clamp, molecular and pharmacological approaches to study functional behavior and mutual relationship between STIM1, Orai1 and iPLA2β in native cells and heterologous systems in which fluorescently tagged STIM1 and/or Orai1 were over-expressed. We found that STIM1 accumulated in puncta equally well in the presence or absence of Orai1, and STIM1 accumulation in puncta is not sufficient for Orai1 accumulation in the same areas. The normal ICRAC could be activated in STIM1-deficient cells. We further found that the effects of C-terminus of STIM1 may be profoundly different in native cells and in cells in which Orai1 and/or full length STIM1 were over-expressed. Also, we found that while inhibition of iPLA2β caused dramatic impairment of endogenous SOCE in native cells, its effects on SOCE in heterologous expressing systems may be less prominent, and may require higher concentrations of inhibitors. Our new data provide first indications of the potential differences in SOCE between native cells and heterologous systems, and challenge the idea of a direct conformational coupling between STIM1 and Orai1 as a mechanism of puncta formation and SOCE activation in native cells.