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 (Leu-111→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 α1 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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology