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 t...
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Veröffentlicht in: | Biochemistry (Easton) 1999-08, Vol.38 (35), p.11389-11400 |
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Zusammenfassung: | 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 μM 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 μM in the mutant versus 100 μM 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 μM K+ present (increased K+-ATP antagonism), and an 8-fold reduced apparent ouabain affinity (increased K+-ouabain antagonism). |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi990951t |