Molecular cloning of rat brain Na,K-ATPase α-subunit cDNA

Abstract

We have isolated a cDNA clone for the rat brain Na,K-ATPase α subunit. A λgt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, λrb5, containing a 1200-base-pair cDNA insert expressed a β-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the α subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of λrb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney > brain > liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to λrb5 cDNA were amplified ≃40-fold. Analysis of the λrb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase α-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the λrb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase α subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.