Structure-function relationships in the sodium-potassium ATPase .alpha. subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme

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Abstract
Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the .alpha.1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat .alpha.1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase .alpha. subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep .alpha.1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep .alpha.1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Futhermore, similar results were observed when HeLA cells were transfected with a sheep .alpha.1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111 .fwdarw. Arg and Asn-122 .fwdarw. Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat .alpha.1 cDNA, the rat/sheep chimera, or the mutant sheep .alpha.1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity. These results demonstrate that the presence of arginine and aspartic acid on the amino end and carboxyl end, respectively, of the H1-H2 extracellular domain of the Na,K-ATPase .alpha. subunit together is responsible for the ouabain-resistant character of the rat enzyme and the corresponding residues in the sheep .alpha.1 subunit (glutamine and asparagine) and somehow involved in ouabain binding.