Abstract
The functional role of serine 775, predicted to be located in the fifth transmembrane segment of the alpha subunit of the Na,K-ATPase (YTLTSNIPE), was studied using site-directed mutagenesis, expression, and kinetic analysis. Substitutions S775A, S775C, and S775Y were introduced into an ouabain-resistant alpha 1 sheep isoform and expressed in HeLa cells. cDNAs carrying substitutions S775C and S775A produced ouabain-resistant colonies only when extracellular K+ was increased from 5.4 mM to 10 or 20 mM, respectively. No ouabain-resistant colonies were obtained for substitutions S775Y at any tested K+ concentration. Kinetic characterization of S775C and S775A substituted enzymes showed expression levels higher than control enzyme, reduced Vmax and turnover, and normal phosphorylation and high affinity ATP binding. Dephosphorylation experiments indicated that S775A substituted enzyme is insensitive to ADP but readily dephosphorylated by K+. The K+ K1/2 values for the activation of the Na,K-ATPase were markedly altered, with S775C displaying a 13-fold increase and S775A exhibiting a 31-fold increase. These large changes in the Na,K-ATPase affinity for K+ are consistent with the participation of this amino acid in binding K+ during the translocation of this cation. Substitutions of Ser775 did not change Na+ affinity, indicating that this residue is likely not involved in Na+ binding and occlusion. These data show that the electronegative oxygen and the small side chain of Ser775 are required for efficient enzyme function. Moreover, these results suggest Ser775 plays a distinct role in K+ transport and not in Na+ interactions, revealing a possible mechanism for the enzymatic differentiation of these cations by the Na,K-ATPase.
Highlights
These data show that the electronegative oxygen and the small side chain of Ser775 are required for efficient enzyme function
Epitope localization, selective proteolytic cleavage, and labeling of putative transmembrane segments with hydrophobic probes suggest a topological arrangement of the ␣ subunit involving 10 transmembrane segments (3, 8, see Fig. 3 in Ref. 2)
In the Na,K-ATPase there are probably seven carboxyl residues within the membrane (Glu327, Glu779, Asp804, Asp808, Asp926, Glu953, and Asp954)1 and they have been the target of chemical modification and site-directed mutagenesis [17,18,19,20,21,22,23,24,25,26]
Summary
Mutagenesis and Cloning—The eukaryotic expression vector pKC4 containing the sheep Na,K-ATPase ␣1 subunit cDNA, which was modified by substitutions Q111R and N122D to encode a form of the enzyme with low affinity for ouabain (RD ␣1), was used [30]. A serial dilution of the purified sheep Na,K-ATPase was blotted onto each membrane and served as the standard by which the amount of heterologously expressed protein was determined. Na,K-ATPase Activity Determinations—The standard assay medium for Na,K-ATPase was 0.5 mM EGTA, 130 mM NaCl, 20 mM KCl, 3 mM MgCl2, 3 mM ATP, and 50 mM imidazole, pH (20 °C) 7.2; 0.3 mg/ml BSA, approximately 1 g/ml of membrane protein, and either 10 or 0.01 mM ouabain. Phosphorylation Assays—Naϩ-activated phosphorylation by ATP was carried out in a medium containing 100 mM NaCl, 0.04 mM EGTA, 75 mM Hepes/imidazole, pH (20 °C) 7.2, 1 mM MgCl2, 0.01 mM [32P]ATP, 0.01 mM ouabain, and 0.2 mg/ml membrane protein. The incubations were stopped with acid, pelleted, resuspended, and counted as described above
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