Functional consequences of mutation Asn326-->Leu in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+, K(+)-ATPase.

Site-specific mutagenesis was used to replace Asn326 in transmembrane segment M4 of the ouabain-insensitive alpha 1-isoform of rat kidney Na+, K(+)-ATPase. Mutant Asn326-->Leu was functional as demonstrated by the ability of COS cells expressing the mutant enzyme to grow in the presence of ouabain. In three independent assays encompassing Na+ titrations of Na+,K(+)-ATPase activity, Na(+)-ATPase activity, and phosphorylation from ATP, the Asn326-->Leu mutant displayed a reduced apparent affinity for Na+. By contrast, this mutant exhibited a slightly increased apparent affinity for K+ relative to the wild-type enzyme. In the presence of Na+ without K+, the Asn326-->Leu mutant hydrolyzed ATP at a high rate corresponding to 32% of the maximal Na+,K(+)-ATPase activity, and the rate of dephosphorylation of the phosphoenzyme intermediate was enhanced in the mutant relative to that of the wild-type enzyme. Oligomycin, known to stabilize the Na(+)-occluded phosphoenzyme intermediate, reduced the dephosphorylation rate of the mutant and increased the steady-state phosphoenzyme level formed by the mutant at least 3-fold, whereas an increase in the steady-state phosphoenzyme level of only 10-15% was determined for the wild-type enzyme. The molecular turnover number for the Na+,K(+)-ATPase reaction, calculated when the steady-state phosphoenzyme level obtained in the presence of oligomycin was taken as a measure of the concentration of active sites, was slightly reduced relative to that of the wild-type enzyme. The data are discussed in terms of a role for Asn326 in binding of cytoplasmic Na+ and in mediation of inhibition of dephosphorylation.