Orai and TRPC channel characterization in FcRI-mediated calcium signaling and mediator secretion in human mast cells.

Inappropriate activation of mast cells via the FcRI receptor leads to the release of inflammatory mediators and symptoms of allergic disease. Calcium influx is a critical regulator of mast cell signaling and is required for exocytosis of preformed mediators and for synthesis of eicosanoids, cytokines and chemokines. Studies in rodent and human mast cells have identified Orai calcium channels as key contributors to FcRI-initiated mediator release. However, until now the role of TRPC calcium channels in FcRI-mediated human mast cell signaling has not been published. Here, we show evidence for the expression of Orai 1,2, and 3 and TRPC1 and 6 in primary human lung mast cells and the LAD2 human mast cell line but, we only find evidence of functional contribution of Orai and not TRPC channels to FcRI-mediated calcium entry. Calcium imaging experiments, utilizing an Orai selective antagonist (Synta66) showed the contribution of Orai to FcRI-mediated signaling in human mast cells. Although, the use of a TRPC3/6 selective antagonist and agonist (GSK-3503A and GSK-2934A, respectively) did not reveal evidence for TRPC6 contribution to FcRI-mediated calcium signaling in human mast cells. Similarly, inactivation of STIM1-regulated TRPC1 in human mast cells (as tested by transfecting cells with STIM1-KKEE - TRPC1 gating mutant) failed to alter FcRI-mediated calcium signaling in LAD2 human mast cells. Mediator release assays confirm that FcRI-mediated calcium influx through Orai is necessary for histamine and TNF release but is differentially involved in the generation of cytokines and eicosanoids.