Low efficiency of functional translation of ouabain-resistant α2-Na+,K+-ATPase mRNA in C7-MDCK epithelial cells

Abstract

Na+,K+-ATPase is a heterodimer consisting of catalytic α1–α4 and regulatory β1–β3 subunits. Recently, we reported that transfection with ouabain-resistant α1R-Na+,K+-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive α1S-isoform from the cytotoxic action of ouabain. To explore the role of α2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K+(86Rb) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with α1R- and α2R-Na+,K+-ATPase. α2R mRNA in transfected cells was ∼8-fold more abundant than α1R mRNA, whereas immunoreactive α2R protein content was 5-fold lower than endogenous α1S protein. A concentration of 10 µmol/L ouabain led to complete inhibition of86Rb influx both in mock- and α2R-transfected cells, whereas maximal inhibition of86Rb influx in α1R-transfectd cells was observed at 1000 µmol/L ouabain. In contrast to the massive death of mock- and α2R-transfected cells exposed to 3 µmol/L ouabain , α1R-cells survived after 24 h incubation with 1000 µmol/L ouabain. Thus, our results show that unlike α1R, the presence of α2R-Na+,K+-ATPase subunit mRNA and immunoreactive protein does not contribute to Na+/K+pump activity, and does not rescue C7-MDCK cells from the cytotoxic action of ouabain. Our results also suggest that the lack of impact of transfected α2-Na+,K+-ATPase on Na+/K+pump activity and cell survival can be attributed to the low efficiency of its translation and (or) delivery to the plasma membrane of renal epithelial cells.