Functional consequences of mutation Asn 326→Leu in the 4th transmembrane segment of the a-subunit of the rat kidney Na +,K +-ATPase

Abstract

Site-specific mutagenesis was used to replace Asn 326 in transmembrane segment M4 of the ouabain-insensitive α 1-isoform of rat kidney Na +,K +-ATPase. Mutant Asn 326 → 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 Asn 326 → 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 Asn 326 → Len 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.