MUTANT GLU781-]ALA OF THE RAT-KIDNEY NA+,K+-ATPASE DISPLAYS LOW CATION AFFINITY AND CATALYZES ATP HYDROLYSIS AT A HIGH-RATE IN THE ABSENCE OF POTASSIUM-IONS

Site-specific mutagenesis was used to replace Glu329, Glu781, Asp806, Thr809, and Asp810 in the transmembrane domain of the ouabain-insensitive alpha(1)-isoform of rat kidney Na+,K+-ATPase. cDNAs encoding any of the mutants Glu329-->Ala, Glu781-->Ala, Asp806-->Asn, Thr809-->Ala, and Asp810-->Asn were transfected into COS cells, and transfectants were grown in the presence of ouabain to inhibit the endogenous COS cell Na+,K+-ATPase. Mutants Glu781-->Ala and Thr809-->Ala were functional as evidenced by their ability to confer ouabain resistance to the cells, whereas mutants Glu329-->Ala, Asp806-->Asn, and Asp810-->Asn were inactive. The apparent Na+ affinities determined by titrations of Na+,K+-ATPase activity, Na+-ATPase activity, and phosphorylation from ATP in mutants Glu781-->Ala and Thr809-->Ala were strongly reduced relative to the affinity of the wild type (6-8-fold increase in K-0.5 for Na+ in the phosphorylation assay for both mutants). The Glu781-->Ala mutant displayed a 3-4-fold reduction in the apparent affinity for K+ and was able to hydrolyze ATP at a high rate in the absence of K+(V-max for Na+-ATPase activity 5-fold higher than that of the wild-type enzyme). The steady-state phosphoenzyme level formed by the Glu781-->Ala mutant was increased 3-fold by addition of oligomycin, whereas only a slight effect of oligomycin was observed for mutant Thr809-->Ala and the wild type. Using the steady-state phosphoenzyme level determined in the presence of oligomycin as a measure of the concentration of active enzyme sites, a maximum molecular turnover number for the Na+,K+-ATPase reaction was calculated to be slightly lower for mutant Glu781-->Ala than the maximum turnover numbers of the wild type and mutant Thr809-->Ala.