Activation mechanism for CRAC current and store-operated Ca2+ entry -: Calcium influx factor and Ca2+-independent phospholipase A2β-mediated pathway

Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A(2) (iPLA(2)) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA(2)beta and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA(2)beta when applied to cell homogenates but not intact cells, 4) activation of I-CRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA(2)beta prevents activation of I-CRAC, which could be rescued by cell dialysis with a human recombinant iPLA(2)beta, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA(2)beta activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA(2)beta, could be effectively used for pharmacological inhibition of I-CRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA(2)-dependent mechanism of activation of I-CRAC and store-operated Ca2+ entry.