Abstract
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.
Highlights
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
Naϩ entry and membrane depolarization as a typical result of TRPC3 activation is expected to exert substantial impact on Na؉/Ca2؉ exchanger (NCX), leading to either reduced Ca2ϩ extrusion or to Ca2ϩ entry upon shift of the exchanger to reversed mode. Both TRPC3 and NCX proteins appear to be tightly linked to Ca2ϩ homeostasis of the endoplasmic reticulum and mitochondria and may both be targeted to a domain of the plasma membrane that communicates with these intracellular Ca2ϩ stores [5, 11, 12]
TRPC3-mediated Ca2ϩ Entry into HEK293 Cells Is Naϩ-dependent and Inhibited by KB-R7943—Transient or stable overexpression of TRPC3 in HEK cells has repeatedly been shown to result in enhanced phospholipase-dependent Ca2ϩ entry, which is clearly discernable in classical Ca2ϩ readdition protocols
Summary
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. Similar rises in Ca2؉i were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na؉ subsequent to stimulation with carbachol These increases in Ca2؉i were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 M), chelation of extracellular Ca2؉, or dominant negative suppression of TRPC3 channel function. Naϩ entry and membrane depolarization as a typical result of TRPC3 activation is expected to exert substantial impact on NCX, leading to either reduced Ca2ϩ extrusion or to Ca2ϩ entry upon shift of the exchanger to reversed mode Both TRPC3 and NCX proteins appear to be tightly linked to Ca2ϩ homeostasis of the endoplasmic reticulum and mitochondria and may both be targeted to a domain of the plasma membrane that communicates with these intracellular Ca2ϩ stores [5, 11, 12].
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