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 K0.5 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+ (Vmax 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Vilsen
B
B
Danish Biomembrane Research Centre, Institute of Physiology, University of Aarhus.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002412:Cations / Q000378:metabolism; D002522:Chlorocebus aethiops; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D017354:Point Mutation; D011188:Potassium / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002412:Cations; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium