Regulation of Na,K-ATPase beta 1 subunit gene transcription by low external potassium in cardiac myocytes

Expression of Na,K-ATPase activity is up-regulated in cells incubated for e xtended intervals in the presence of low external K+. Our previous data sho wed that exposure of cardiac myocytes to low K+ increased the steady-state abundance of Na,K-ATPase beta 1 subunit mRNk In the present study we determ ined that incubation of primary cultures of neonatal rat cardiac myocytes w ith low K+ augmented Na,K-ATPase beta 1 gene expression at a transcriptiona l level and that this effect required extracellular Ca2+. The stimulatory e ffect of low K+ on Na,K-ATPase beta 1 gene transcription was not dependent on increased contractile activity of cardiac myocytes, Na,KATPase beta 1 5' -flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides -62 to -42 of the beta 1 p ro meter contained a low K+ response element. Site-directed mutagenesis of a potential GC box core motif GCG in the -58/-56 region of the beta 1 promo ter decreased basal and low K+-mediated transcription. Mutation of the core sequence of a putative GC box element located between nucleotides -101 and -99 further decreased the low K+ effect on beta 1 gene transcription. Elec trophoretic mobility shift assays using oligonucleotides spanning the proxi mal and distal GC box elements of the beta 1 promoter showed enhanced bindi ng of two complexes in response to low K+. The inclusion of a consensus GC box sequence as a competitor in gel shift analysis reduced factor binding t o the low K+ response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding o f nuclear factors was abolished in gel shift studies using GC box mutants. Together these data indicate that enhanced binding. of Sp1 and Sp3 to two G C box elements in the rat Na,K-ATPase beta 1 subunit gene promoter mediates beta 1 gene transcription up-regulation in neonatal rat cardiac myocytes e xposed to low external K+.