Mutant Phe788 --> Leu of the Na+,K+-ATPase is inhibited by micromolar concentrations of potassium and exhibits high Na+-ATPase activity at low sodium concentrations.
Mutant Phe788 --> Leu of the rat kidney Na+,K(+)-ATPase was expressed in COS cells to active-site concentrations between 40 and 60 pmol/mg of membrane protein. Analysis of the functional properties showed that the discrimination between Na+ and K+ on the two sides of the system is severely impaired in the mutant. Micromolar concentrations of K+ inhibited ATP hydrolysis (K(0.5) for inhibition 107 microM for the mutant versus 76 mM for the wild-type at 20 mM Na+), and at 20 mM K+, the molecular turnover number for Na+,K(+)-ATPase activity was reduced to 11% that of the wild-type. This inhibition was counteracted by Na+ in high concentrations, and in the total absence of K+, the mutant catalyzed Na(+)-activated ATP hydrolysis ("Na(+)-ATPase activity") at an extraordinary high rate corresponding to 86% of the maximal Na+,K(+)-ATPase activity. The high Na(+)-ATPase activity was accounted for by an increased rate of K(+)-independent dephosphorylation. Already at 2 mM Na+, the dephosphorylation rate of the mutant was 8-fold higher than that of the wild-type, and the maximal rate of Na(+)-induced dephosphorylation amounted to 61% of the rate of K(+)-induced dephosphorylation. The cause of the inhibitory effect of K+ on ATP hydrolysis in the mutant was an unusual stability of the K(+)-occluded E2(K2) form. Hence, when E2(K2) was formed by K+ binding to unphosphorylated enzyme, the K(0.5) for K+ occlusion was close to 1 microM in the mutant versus 100 microM in the wild-type. In the presence of 100 mM Na+ to compete with K+ binding, the K(0.5) for K+ occlusion was still 100-fold lower in the mutant than in the wild-type. Moreover, relative to the wild-type, the mutant exhibited a 6-7-fold reduced rate of release of occluded K+, a 3-4-fold increased apparent K+ affinity in activation of the pNPPase reaction, a 10-11-fold lower apparent ATP affinity in the Na+,K(+)-ATPase assay with 250 microM K+ present (increased K(+)-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K(+)-ouabain antagonism).
Vilsen
B
B
Department of Physiology, University of Aarhus, Denmark. bv@fi.au.dk
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000235:genetics; D007668:Kidney / Q000201:enzymology; D007930:Leucine / Q000037:antagonists & inhibitors / Q000235:genetics*; D010649:Phenylalanine / Q000037:antagonists & inhibitors / Q000235:genetics*; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology* / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D012964:Sodium / Q000502:physiology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D011994:Recombinant Proteins; D010649:Phenylalanine; D000255:Adenosine Triphosphate; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D007930:Leucine; D011188:Potassium