RANDOM MUTAGENESIS OF THE SHEEP NA,K-ATPASE ALPHA-1 SUBUNIT GENERATING THE OUABAIN-RESISTANT MUTANT L793P

The polymerase chain reaction was used to randomly mutagenize a cDNA c assette encoding amino acids 691-946 of the sheep Na,K-ATPase alpha su bunit, The mutagenized cassettes were used to replace the wild type re gion in the full-length cDNA, and pools of mutants were transfected in to HeLa cells, After the generation of resistant cells via selection i n 0.5 mu M ouabain, polymerase chain reaction was used to amplify the mutagenized cassette from the genomic DNA of the stable transfectants, Sequence analysis of the polymerase chain reaction product revealed t hree amino acid substitutions: I729V, L793P, and K836R, Subsequent sit e-directed mutagenesis experiments showed that only L793P was importan t for resistance, To elucidate the role of L793 in ouabain inhibition, additional mutations at this position were prepared. L793A and L793I mutants were constructed and expressed in HeLa cells, Only L793A survi ved selection using ouabain, which suggested that resistance is not du e to the specific substitution of leucine with proline. To explore the mechanism of resistance, apparent affinities of the L793P mutant for sodium and potassium were compared to the wild-type HeLa pump, Althoug h the apparent affinities were comparable for sodium, the mutant had a a-fold higher apparent affinity for potassium, This suggests that the mechanism of ouabain insensitivity of L793P is due to a perturbation in the region of the enzyme that may include the K+ binding site.