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. 1990 Apr;10(4):1367–1372. doi: 10.1128/mcb.10.4.1367

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

V Canfield 1, J R Emanuel 1, N Spickofsky 1, R Levenson 1, R F Margolskee 1
PMCID: PMC362238  PMID: 2157137

Abstract

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 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 alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end 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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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