TRPC3 Expression Modulates Store-Operated Currents in RBL-2H3 Cells

Abstract

TRPC3 was repeatedly discussed as a potential component of store-operated signaling pathways and is expressed in mast cells, which display the classical Orai1-mediated CRAC conductance. To elucidate if TRPC3 interferes with this store-operated conductance, we overexpressed TRPC3 in RBL-2H3 mast cells and characterized currents activated by passive store depletion (20mM EGTA). TRPC3 overexpression diminshed the inwardly rectifying CRAC current component significantly when free intracellular Mg2+ was kept at a level of 1.6 mM, and this inhibitory effect was more pronounced when free intracellular Mg2+ was elevated to 8 mM. Moreover, store depletion-induced currents were completely abolished with expression of a pore-dead mutant of TRPC3 (E630K). By contrast, the potential STIM-binding deficient mutant, E697K/E698K lacked effects on CRAC current amplitude. Importantly, at low (1.6 mM) intracellular Mg2+, we observed a reduced Ca2+ selectivity of the store depletion-activated conductance with appearance of a clearly outward rectifying I-V relation. This nonselective conductance was absent at high (8 mM) Mg2+, eliminated by co-expression of the permeation deficient Orai1 E106Q mutation and displayed sensitivity to the TRPC3 blocker Pyr3 (3 μM) as well as to the TRPM7 inhibitor NDGA (10 μM). Biophysical and pharmacological features of the nonselective conductance, which was activated by store depletion at low (physiologic) intracellular Mg2+ levels, favor the concept of generation of a unique membrane conductance by TRPC3 overexpression in RBL cells. Our findings suggest a complex interaction of TRPC3 with Orai1 and probably also TRPM7 in plasma membrane microdomains of mast cells.Supported by FWF, DK-MCD