Abstract
Mutations comprising either deletion of 32 amino acids from the NH2 terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase result in a shift in the steady-state E1 left arrow over right arrow E2 conformational equilibrium toward E1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant alpha1M32E233K double mutant, growth was markedly reduced unless the K+ concentration in the culture medium was increased to at least 10 mM. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (approximately 10-fold) K+ activation of the Na-ATPase activity measured at micromolar ATP under which condition the E2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in Vmax and a compensatory approximately 3-fold increase in expressed alpha1M32E233K protein. In contrast to the behavior of either alpha1M32 or E233K, alpha1M32E233K also showed alterations in apparent cation affinities. K'Na was decreased approximately 2-fold and K'K was increased approximately 2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233 substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.
Highlights
The Na,K-ATPase couples the hydrolysis of one ATP molecule to the translocation of 3 Naϩ and 2 Kϩ ions against their electrochemical gradients, maintaining the normally high Kϩ and low Naϩ concentrations inside animal cells
We showed that the consequence of deletion of residues 1–32, as well as the nonconservative mutation Glu233 3 Lys (E233K) in the M2-M3 cytoplasmic loop, is the displacement of the E1-E2 conformational equilibrium in favor of E1
Catalytic Turnover of Single and Double Mutations: Effect of Charge of Residue 233—We showed previously that the ␣1M32 and E233K mutants as well as the ␣2 isoform have reduced catalytic turnovers, which probably reflects the poise in the steady-state E1-E2 equilibrium in favor of E1
Summary
Mutagenesis, Transfection, and Cell Culture—HeLa cell lines expressing pRc/CMV (Invitrogen) constructs of rat ␣1, ␣1M32, and E233K are the same as those described previously [11, 12]. Membrane Preparation—NaI-treated microsomal membranes were prepared from the mutant cells as earlier described [14, 16], and the protein concentration was determined with a detergent-modified Lowry assay [17]. Enzyme Assays—Assays of Na,K-ATPase activity were carried out as described earlier by measuring the release of 32Pi from [␥-32P]ATP [18]. A final concentration of 10 mM ouabain was used to determine the baseline hydrolysis activity. The Na-ATPase activities were measured with 1 mM MgSO4, 20 mM histidine (pH 7.4), 5 mM EGTA (pH 7.4), 20 mM NaCl, 1 M ATP, 10 M ouabain and KCl concentrations as indicated. For base-line activities, Naϩ was omitted and 20 mM KCl included, with choline chloride added to maintain a constant (40 mM) chloride concentration. Experiments were carried out with two different clones, except for ␣1M32E233D, in which case the results are from at least two different membrane preparations of the same clone
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