Ouabain-resistant OR6 cells express the murine alpha 1-subunit of the Na,K-ATPase with a T797-I797 substitution.
Introduction of the murine "ouabain resistance gene" (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) into African Green monkey CV1 cells reportedly resulted in one cell line, OR6 cells, which was found to be resistant to > 1 mM ouabain (English, L., Epstein, J., Cantley, L., Housman, D., and Levenson, R. (1985) J. Biol. Chem. 260, 1114-1119). The present analysis of the genomic sequence of the alpha 1-subunit of the Na,K-ATPase from OR6 cells reveals copies of the gene for the murine and human alpha 1-subunit of the Na,K-ATPase and not the monkey gene, indicating that the cells have been misidentified. The sequence of the murine alpha 1-subunit from OR6 cells reveals a single point mutation encoding a T797-I797 amino acid substitution at the H6 membrane border. Transfection of CV1 cells with a murine alpha 1 cDNA containing this point mutation resulted in cells with an IC50 for ouabain of approximately 5 mM. These data show that the super ouabain-resistant phenotype of OR6 cells is due to expression of a mutant form of the murine alpha 1-subunit of the sodium pump (rather than the ouabain resistance gene) and further support the recent data suggesting involvement of the H5-H6 domain in ouabain inhibition of the sodium pump.
Cantley
L G
LG
Division of Nephrology, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Cunha
M J
MJ
Zhou
X M
XM
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004351:Drug Resistance / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology*; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase