Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells
Na-K-ATPase is a fundamental component of ion transport. Four α isoforms of the Na-K-ATPase catalytic α subunit are expressed in human cells. The ubiquitous Na-K-ATPase α1 was recently discovered to also mediate signal transduction through Src kinase. In contrast, α2 expression is limited to a few cell types including myocytes, where it is coupled to the Na+/Ca2+ exchanger. To test whether rat Na-K-ATPase α2 is capable of cellular signaling like its α1 counterpart in a recipient mammalian system, we used an α1 knockdown pig renal epithelial cell (PY-17) to create an α2-expressing cell line with no detectable level of α1 expression. These cells exhibited normal ouabain-sensitive ATPase, but failed to effectively regulate Src. In contrast to α1-expressing cells, ouabain did not stimulate Src kinase or downstream effectors such as ERK and Akt in α2 cells, although their signaling apparatus was intact as evidenced by EGF-mediated signal transduction. Additionally, α2 cells were unable to rescue caveolin-1. Unlike the NaKtide sequence derived from Na-K-ATPase α1, which downregulates basal Src activity, the corresponding α2 NaKtide was unable to inhibit Src in vitro. Finally, coimmunoprecipitation of cellular Src was diminished in α2 cells. These findings indicate that Na-K-ATPase α2 does not regulate Src and, therefore, may not serve the same role in signal transduction as α1. This further implies that the signaling mechanism of Na-K-ATPase is isoform specific, thereby supporting a model where α1 and α2 isoforms play distinct roles in mediating contraction and signaling in myocytes.
Xie J, Ye Q, Cui X, Madan N, Yi Q, Pierre SV, Xie Z. Expression of rat Na-K-ATPase α2 enables ion pumping but not ouabain-induced signaling in α1-deficient porcine renal epithelial cells. American Journal of Physiology-Cell Physiology. 2015;309:C373-382.