TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) 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 Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) 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(2+) signaling.