Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim: To investigate the reverse mode function of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA. Methods: CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca2+ level ([Ca2+](i)) was measured using Ca2+ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na+-free medium. Ca2+ paradox was induced by Ca2+-free EBSS medium, followed by Ca2+-containing solution (1.8 or 3.8 mmol/L CaCl2). Results: The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca2+](i), which was followed by a Ca2+ level plateau at higher external Ca2+ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca2+](i) at higher external Ca2+ concentrations. The PKC activator PMA (0.3-10 mu mol/L) and PKA activator 8-Br-cAMP (10-100 mu mol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 mu mol/L). Conclusion: Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca2+](i) in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.