Ouabain-resistant mutants of the rat Na,K-ATPase α2 isoform identified by using an episomal expression vector

Victor Canfield, J. R. Emanuel, N. Spickofsky, Robert Levenson, R. F. Margolskee

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Site-directed mutagenesis was used to identify residues responsible for the > 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase α1 and α2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat α2 subunit with the corresponding residues from the rat α1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat α2 subunit cDNA (Leu111→Arg or Asn-122→Asp) conferred partial resistance (∼10 μM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat al cDNA (∼500 μM ouabain) and the rat α2 cDNA (∼0.2 μM ouabain). A double substitution of the rat α2 cDNA (Leu-111→Arg and Asn-122→Asp) conferred a resistance level equivalent to that obtained with rat α1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the ends of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

Original languageEnglish (US)
Pages (from-to)1367-1372
Number of pages6
JournalMolecular and cellular biology
Issue number4
StatePublished - 1990

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology


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