Abstract
The intramembrane Glu781 residue of the Na,K-ATPase alpha subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native beta 1 subunit in Sf9 insect cells using the baculovirus expression system. All alpha mutants are able to efficiently assemble with the beta 1 subunit and produce catalytically competent Na,K-ATPase molecules with hydrolytic activities comparable to that of the wild-type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild-type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781-->Asp and Glu781-->Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781-->Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781-->Ala mutant displays a Na(+)-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781-->Ala mutant in Na+ medium produces a K(+)-stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase alpha subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781-->Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.
Highlights
Ning, heterodimeric protein that uses the energy from the hydrolysis of ATP to maintain the low intracellular sodium concentration and high intracellular potassium concentration common to most animal cells
The catalytic ␣ subunit contains the binding sites for ATP and the cardiotonic inhibitor, ouabain; it is phosphorylated by ATP, and undergoes the cation-dependent E1 7 E2 conformational transitions associated with cation transport
Previous chemical modification studies using DEAC have implicated a glutamate residue of the Na,K-ATPase ␣ subunit in cation binding (Arguello and Kaplan, 1994)
Summary
Ning, heterodimeric protein that uses the energy from the hydrolysis of ATP to maintain the low intracellular sodium concentration and high intracellular potassium concentration common to most animal cells. The use of hydrophobic, carboxyl-selective reagents to chemically modify and inactivate cation binding of the Na,K-ATPase has proven useful in identifying intramembrane residues in the ␣ subunit that may be involved in cation transport function (Goldshleger et al, 1992; Arguello and Kaplan, 1994). DEAC inhibition appears to be selective for the cation binding site as the modified enzyme is still able to undergo the E1 7 E2 conformational transitions and displays normal levels of ATP/ADP binding. The importance of this residue in Na,K-ATPase function is further evidenced by the findings that (i) substitution of Glu[779] with a leucine or aspartate residue inhibits Na,K-ATPase-dependent. ATPase function, site-directed mutagenesis was used to replace Glu[781] (the equivalent residue in the rat Na,K-ATPase) with an alanine, aspartate, or lysine residue, and the mutant Na,K-
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