Regulation of Na,K-ATPase alpha1 subunit gene transcription in response to low K(+): role of CRE/ATF- and GC box-binding proteins.

Na,K-ATPase expression is upregulated in mammalian cells as a consequence of persistent inhibition of Na,K-ATPase enzymatic activity by low external K(+). We previously demonstrated that exposure of neonatal rat cardiac myocytes to low K(+) increased Na,K-ATPase alpha1 subunit mRNA content and promoter activity. In this work, we utilized transient transfection studies with rat Na,K-ATPase alpha1 subunit 5'-flanking region deletion plasmids to identify DNA sequences required for low K(+)-mediated stimulation of alpha1 subunit promoter expression in cardiac myocytes. Maximal low K(+)-responsiveness of the alpha1 promoter was found to be dependent on nucleotides from -102 to -62 and a downstream region from +53 to +261. Further analysis of the upstream low K(+)-responsive region using mutant constructs revealed that a CRE/ATF site at -70 to -63 and a GC box motif at -57 to -48 were both required for the effect of low K(+) on alpha1 subunit gene transcription. Electrophoretic mobility shift assays revealed that low K(+) increased binding of transcription factors to the GC box and, to a lesser extent, to the CRE/ATF site. Western blot analysis demonstrated that exposure of cardiac myocytes to low K(+) resulted in increased nuclear content of Sp1, Sp3 and CREB-1. Finally, a selective increase in phosphorylation of Sp1 was found in nuclear extracts from low K(+)-treated cells. We conclude that low K(+)-mediated upregulation of Na,K-ATPase alpha1 subunit gene expression in neonatal rat cardiac myocytes is dependent, in part, on CRE/ATF- and GC box-binding transcription factors.