Ca2+ signaling by TRPC3 involves Na+ entry and local coupling to the Na+/Ca2+ exchanger

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca2+ signaling. Here we investigated the role of TRPC3-mediated Na+ entry as a determinant of plasmalemmal Na+/Ca2+ exchange. Ca2+ signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na+, in that carbachol-stimulated Ca2+ entry into TRPC3 expressing cells was significantly suppressed when extracellular Na+ was reduced to 5 mM. Moreover, KB-R9743 ( 5 muM) an inhibitor of the Na+/Ca2+ exchanger (NCX) strongly suppressed TRPC3-mediated Ca2+ entry but not TRPC3-mediated Na+ currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na+ after Na+ loading with monensin resulted in significant rises in intracellular free Ca2+ (Ca-i(2+)) of HEK293 cells. Similar rises in Ca-i(2+) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na+ subsequent to stimulation with carbachol. These increases in Ca-i(2+) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 ( 5 muM), chelation of extracellular Ca2+, or dominant negative suppression of TRPC3 channel function. This suggests that Ca2+ entry into TRPC3-expressing cells involves reversed mode Na+/Ca2+ 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 Ca2+ signaling.