Molecular engineering of the TRPC3 pore structure identifies Ca²⁺ permeation through TRPC3 channels as a key determinant of cardiac calcineurin/NFAT signaling

Background

TRPC channels have been identified as key players in cardiac remodeling and as crucial upstream components of NFAT signaling. The linkage between non-selective TRPC conductances and calcineurin/NFAT signaling may involve either direct TRC-mediated Ca²⁺ entry or indirect mechanisms involving crosstalk with other cardiac Ca²⁺ transport systems.

Methods

The pore structure of TRPC3 was analyzed by site-directed mutagenesis guided by a molecular modeling approach combined with patch-clamp measurements in the HEK293 expression system. TRPC3-mediated Ca²⁺ entry as well as NFAT translocation was investigated by fluorescence microscopy using Fura-2 and expression of a GFP-NFAT fusion protein in HEK293 as well as in HL1 cells.

Results

Elimination of Ca²⁺ permeation through TRPC3 abrogated its ability to trigger NFAT translocation in both HEK293 cells and in HL-1 atrial myocytes. Wild-type TRPC3 was found capable of initiating NFAT translocation in atrial myocytes by a small, homogenous elevation of cytoplasmic Ca²⁺ that was independent of voltage-gated Ca_V1.2 channels. By contrast, a Ca²⁺ impermeant TRPC3 mutant strongly promoted endothelin-induced Ca²⁺ signals in HL1 cells via enhanced activity of Ca_V1.2 channels without concomitant NFAT translocation.

Conclusions

Our results demonstrate two strictly separated Ca²⁺ signaling functions of cardiac TRPC3 channels as well as a tight and efficient link between TRPC3-mediated Ca²⁺ permeation and calcineurin/NFAT signaling.