OUABAIN-RESISTANT OR6 CELLS EXPRESS THE MURINE ALPHA(1)-SUBUNIT OF THE NA,K-ATPASE WITH A T-797-I-797 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 rep ortedly resulted in one cell line, OR6 cells, which was found to be re sistant to >1 mM ouabain (English, L., Epstein, J., Cantley, L., Housm an, D., and Levenson, R. (1985) J. Biol. Chem. 260, 1114-1119). The pr esent 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 a nd 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 mutatio n encoding a T-797-I-797 amino acid substitution at the H-6 membrane b order. Transfection of CV1 cells with a murine alpha(1) cDNA containin g this point mutation resulted in cells with an IC50 for ouabain of ap proximately 5 mM. These data show that the super ouabain-resistant phe notype of OR6 cells is due to expression of a mutant form of the murin e alpha(1)-subunit of the sodium pump (rather than the ouabain resista nce gene) and further support the recent data suggesting involvement o f the H-5-H-6 domain in ouabain inhibition of the sodium pump.