Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger.
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.
Rosker
Christian
C
Department of Pharmacology and Toxicology, Karl-Franzens-University Graz, A-8010 Graz, Austria.
Graziani
Annarita
A
Lukas
Michael
M
Eder
Petra
P
Zhu
Michael X
MX
Romanin
Christoph
C
Groschner
Klaus
K
D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006706:Homeostasis; D006801:Humans; D007473:Ion Channels / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050052:TRPC Cation Channels; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; D000889:Anti-Arrhythmia Agents; D007473:Ion Channels; D019831:Sodium-Calcium Exchanger; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; C110374:sodium-calcium exchanger 1; D012964:Sodium; D013890:Thiourea; D002118:Calcium