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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Reduced ouabain inhibition of Na,K-activated adenosine triphosphatase in cultured cell recipients of the ouabain-resistance gene.

Using sequential DNA-mediated gene transfer, a mouse DNA sequence (ouaR) that confers resistance to the cytotoxic effects of ouabain in cultured cells was recently isolated. To determine the basis of this resistance, we examined the ouabain-resistant phenotype of cells from each stage of the transfers that led to isolation of the ouaR gene, as well as the recipient of the isolated gene. Membranes prepared from the original DNA donor, the two intermediate stages of the sequential gene transfer, and the final isolated gene recipient contained at least two functionally distinct forms of the Na,K-ATPase. In addition to a form indistinguishable in ouabain affinity from that in the parental lines, these ouabain-resistant cells contained a form characterized by a low affinity for the glycoside. That the low-affinity form contributed to ouabain resistance was suggested by the correlation between its relative abundance and the ability to limit the increase in intracellular Na+ content when exposed to ouabain. Maintenance of the final recipient of the isolated ouaR gene for 24 h in 10 microM ouabain had no effect on the specific activity of the resistant form of the Na,K-ATPase, implying that the low-affinity form was not uniquely inducible by ouabain. These results suggest that the ouabain-resistant phenotype conferred by ouaR is attributable to expression of a Na,K-ATPase with a low affinity for the glycoside.[1]


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