Abstract
Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca(2+) entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca(2+)-independent phospholipase A(2) β (iPLA(2)β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA(2)β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA(2)β but not STIM1. These data confirm the role of iPLA(2)β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.
Highlights
Store-operated Ca2ϩ entry (SOCE) is essential for cell function
Functional Inhibition of iPLA2 Blocks SOCE in Naïve Cells, but Not in Cells Overexpressing Orai1 or STIM1—To compare the role of iPLA2 in endogenous SOCE in naïve HEK293 cells with its role in SOCE mediated by overexpressed Orai1 or STIM1, we started with testing the effects of inhibition of catalytic activity of iPLA2 on SOCE evoked by either buffering free Ca2ϩ in ER with TPEN or by depleting ER stores as a result of SERCA inhibition with thapsigargin (TG, Fig. 2)
These results indicate that the overexpression of either Orai1 or STIM1 significantly changes the properties of endogenous SOCE and can make it
Summary
Store-operated Ca2ϩ entry (SOCE) is essential for cell function. Results: We discovered cross-talk between expression of molecules that determine SOCE and demonstrated that the role of endogenous mediators may be altered in overexpressed system. 2H3 cells [29, 30], neuroblastoma/glioma cells [22], as well as in astrocytes [23], keratinocytes [24], skeletal muscle cells [25], fibroblasts [26], prostate cancer cells [27], and endothelial cells [28] In all of these studies, inhibition of the catalytic activity or molecular knockdown of iPLA2 caused dramatic impairment of endogenous SOCE, leaving little doubt about its important role in the SOCE process (for review, see Ref. 20). We tested a novel idea that there may be a cross-talk between expression levels of the major molecular determinants of endogenous SOCE and found that expression of endogenous Orai determines and depends upon expression levels of a specific plasma membrane variant of iPLA2 but not STIM1 These findings highlight the complexity of the SOCE mechanism and the role of its endogenous mediators, which may be lost in overexpression systems
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