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J. Biol. Chem., Vol. 275, Issue 31, 24173-24184, August 4, 2000

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Regulation of Na,K-ATPase 1 Subunit Gene Transcription by Low External Potassium in Cardiac Myocytes
ROLE OF Sp1 AND Sp3^*

Yong Zhuang, Christine Wendt^§, and Gregory Gick^¶

From the  Department of Biochemistry and Center for Cardiovascular and Muscle Research, State University of New York Health Science Center at Brooklyn, Brooklyn, New York 11203 and the ^§ University of Minnesota Medical School, Minneapolis, Minnesota 55455

Expression of Na,K-ATPase activity is up-regulated in cells incubated for extended intervals in the presence of low external K⁺. Our previous data showed that exposure of cardiac myocytes to low K⁺ increased the steady-state abundance of Na,K-ATPase 1 subunit mRNA. In the present study we determined that incubation of primary cultures of neonatal rat cardiac myocytes with low K⁺ augmented Na,K-ATPase 1 gene expression at a transcriptional level and that this effect required extracellular Ca²⁺. The stimulatory effect of low K⁺ on Na,K-ATPase 1 gene transcription was not dependent on increased contractile activity of cardiac myocytes. Na,K-ATPase 1 5'-flanking region deletion plasmids used in transient transfection analysis demonstrated that the region between nucleotides 62 to 42 of the 1 promoter contained a low K⁺ response element. Site-directed mutagenesis of a potential GC box core motif GCG in the 58/56 region of the 1 promoter 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 1 gene transcription. Electrophoretic mobility shift assays using oligonucleotides spanning the proximal and distal GC box elements of the 1 promoter showed enhanced binding 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 to the low K⁺ response elements. Antibodies to transcription factors Sp1 and Sp3 interacted with components of both DNA-binding complexes and binding of 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 GC box elements in the rat Na,K-ATPase 1 subunit gene promoter mediates 1 gene transcription up-regulation in neonatal rat cardiac myocytes exposed to low external K⁺.

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