Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex.
OBJECTIVE
Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes.


METHODS
Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy.


RESULTS
TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1.


CONCLUSIONS
These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.
Eder
P
P
Institute of Pharmaceutical Sciences, Pharmacology and Toxicology, Karl-Franzens-University, Graz, Austria.
Probst
D
D
Rosker
C
C
Poteser
M
M
Wolinski
H
H
Kohlwein
S D
SD
Romanin
C
C
Groschner
K
K
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D006706:Homeostasis; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D018613:Microscopy, Confocal; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*
D019831:Sodium-Calcium Exchanger; D050052:TRPC Cation Channels; D010738:Type C Phospholipases; D002118:Calcium