Modulation of TRPC store‐operated channels by Orai and STIM proteins

Abstract

Store‐operated calcium entry is significant pathway of calcium concentration rise in cytoplasm. The main players in this process are Orai store‐operated channels, which are activated directly by STIM calcium sensors. Data on participation of TRPC channels in store‐operated calcium entry is controversial. Study was focused on interaction between STIM and Orai proteins with endogenous TRPC and its role in pathology. We investigated activity of TRPC channels in a model object — HEK293 cells and in the podocytes of the rat model of diabetic nephropathy, using single channel recording patch camp technique. To eliminate Orai channels activity in HEK293 cells we used several experimental approaches: overexpression of dominant‐negative Orai mutant, Orai genes knockout and knockdown via CRISPR‐Cas 9 system and shRNA. Endogenous TRPC1 channels remain active in cells without functional Orai proteins and become insensitive to store depletion. In experiments with different extracellular calcium concentration we confirmed that calcium entry is needed for TRPC1 activation by store depletion. In experiments with calcium, barium or sodium as a charge carrier we observed similarities in dependence of open channel probability versus membrane potential between TRPC1 and Orai channels. The role of STIM calcium sensors in TRPC1 channels activation was investigated in cells with different expression level of STIM1 and STIM2 proteins. The findings allow us to propose that STIM proteins don’t activate the endogenous TRPC channels directly. TRPC channels are activated downstream to calcium entry through the Orai channels. Support or Funding Information The study was supported by grant of Russian science foundation № 19‐14‐00114 (Glushankova L., Skopin A., Kaznacheyeva E.), and by grant of Russian foundation for basic research № 19‐315‐90065 (Shalygin A., Kolesnikov D.).