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

Na+,K+-ATPase is a heterodimer consisting of catalytic alpha 1-alpha 4 and regulatory beta 1-beta 3 subunits. Recently, we reported that transfection with ouabain-resistant alpha 1R-Na+,K+-ATPase rescues renal epithelial C7-MDCK cells exclusively expressing the ouabain-sensitive alpha 1S-isoform from the cytotoxic action of ouabain. To explore the role of alpha 2 subunit in ion transport and cytotoxic action of ouabain, we compared the effect of ouabain on K+(Rb-86) influx and the survival of ouabain-treated C7-MDCK cells stably transfected with alpha 1R- and alpha 2R-Na+,K+-ATPase. alpha 2R mRNA in transfected cells was similar to 8-fold more abundant than alpha 1R mRNA, whereas immunoreactive alpha 2R protein content was 5-fold lower than endogenous alpha 1S protein. A concentration of 10 mu mol/L ouabain led to complete inhibition of Rb-86 influx both in mock- and alpha 2R-transfected cells, whereas maximal inhibition of Rb-86 influx in alpha 1R-transfectd cells was observed at 1000 mu mol/L ouabain. In contrast to the massive death of mock-and alpha 2R-transfected cells exposed to 3 mu mol/L ouabain, alpha 1R-cells survived after 24 h incubation with 1000 mu mol/L ouabain. Thus, our results show that unlike alpha 1R, the presence of alpha 2R-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 alpha 2-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.