Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex

Abstract

Objective: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca 2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na + /Ca 2+ 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. Coimmunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GSTpulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca 2+ entry, which was dependent on TRPC3-mediated Na + loading of myocytes. This NCX-mediated Ca 2+ 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. Conclusion: 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 Ca 2+ homeostasis.

Vascular Bioactivation of Nitroglycerin Is Catalyzed by Cytosolic Aldehyde Dehydrogenase-2

“…[17][18][19] Cells were infected with 2ϫ10 7 PFU AdV for 24 hours (AdV-cALDH2) or 30 hours (AdV-mALDH2), fixed, and permeabilized. 20,21 Anti-ALDH2 antibody and the probe MitoTrackerRed (Invitrogen GmbH, Lofer, Austria) were used to localize ALDH2 and mitochondria, respectively. Microscopy was performed using a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany).…”

Diacylglycerol Kinase ζ Inhibits Ventricular Tachyarrhythmias in a Mouse Model of Heart Failure

“…10, 30 In addition, activation of phospholipase C in GPCR signaling pathway stimulates recruitment of a TRPC3-sodium-calcium exchanger 1 (NCX1) complex into the plasma membrane in cardiac myocytes. 31 Moreover, Rosker et al suggested a functional and physical interaction of TRPC channels with NCX proteins. 32 NCX mRNA and protein expression and function are increased in HF.…”

SR Ca2+refilling upon depletion and SR Ca2+uptake rates during development in rabbit ventricular myocytes

“…Our previous results indicated that SOCE load SR was intimately modulated by NCX (13), which we suggested resulted from the SOCE influx pathway and NCX being in the same microdomain. More recent evidence from two different laboratories has strongly supported the concept that TRP-C3 channels and NCX colocalize in heart tissue (7,8). Although not the focus of this study, it is tempting to speculate that TRP-C3 channels may contribute to the observed SOCE.…”