Ca 2 Signaling by TRPC 3 Involves Na Entry and Local

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca signaling. Here we investigated the role of TRPC3-mediated Na entry as a determinant of plasmalemmal Na /Ca exchange. Ca signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na , in that carbachol-stimulated Ca entry into TRPC3 expressing cells was significantly suppressed when extracellular Na was reduced to 5 mM. Moreover, KB-R9743 (5 M) an inhibitor of the Na /Ca exchanger (NCX) strongly suppressed TRPC3-mediated Ca entry but not TRPC3-mediated Na currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3overexpressing HEK293 cells, and reduction of extracellular Na after Na loading with monensin resulted in significant rises in intracellular free Ca (Ca i) of HEK293 cells. Similar rises in Ca i were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na subsequent to stimulation with carbachol. These increases in Ca i were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 M), chelation of extracellular Ca , or dominant negative suppression of TRPC3 channel function. This suggests that Ca entry into TRPC3expressing cells involves reversed mode Na /Ca exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca signaling.