RANDOM MUTAGENESIS OF THE SHEEP NA,K-ATPASE-ALPHA-1 SUBUNIT GENERATESA NOVEL T797N MUTATION THAT RESULTS IN A OUABAIN-RESISTANT ENZYME

Site-directed mutagenesis of the Na,K-ATPase has provided a rational a pproach to identify several amino acids that appear to be involved in ouabain sensitivity. In order to identify additional amino acids that play a role in ouabain binding, we used formic acid to randomly mutage nize a cDNA cassette encoding amino acids 691-946 of the sheep Na,K-AT Pase alpha subunit. This was then used to replace the wild type cDNA c assette in the full-length cDNA, and pools of mutants were electropora ted into HeLa cells. Ouabain-resistant cells were selected in 0.5 muM ouabain, and the polymerase chain reaction was used to amplify the reg ion corresponding to the mutagenized cassette from the genomic DNA of the resistant cells. Sequence analysis of the polymerase chain reactio n product revealed a single amino acid substitution, T797N. Site-direc ted mutagenesis was used to reproduce this change in the wild type she ep alpha subunit, and this construct was able to confer resistance to HeLa cells, verifying the role of the mutation in ouabain resistance. The mutant sheep enzyme was found to be as resistant to ouabain as is the rat Na,K-ATPase. These data suggest that T797N, predicted to be in the fifth putative transmembrane domain, is involved in the interacti on between ouabain and the Na,K-ATPase.