Rescue of Na+ affinity in aspartate 928 mutants of Na+,K+-ATPase by secondary mutation of glutamate 314

Abstract

The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.

Keywords: Alternating Hemiplegia of Childhood; Membrane Transport; Na+,K+ Pump; Na+/K+-ATPase; Neurological Disease; P-type ATPase; Rapid-onset Dystonia Parkinsonism; Second-site Revertant; Site-directed Mutagenesis; Sodium Transport.

SCHEME 1.

Post-Albers model of the Na⁺,K⁺-ATPase reaction cycle. E₁ and E₂ represent the main conformational states of the enzyme. Occluded Na⁺ and K⁺ ions are shown in brackets, and free ions are labeled c and e for cytoplasmic and extracellular, respectively. P indicates phosphorylation. Boxed ATP indicates ATP bound in a non-phosphorylating mode, enhancing the rate of K⁺ deocclusion, and accompanying E₂-E₁ conformational change.

FIGURE 1.

Disposition of Na⁺ binding residues in protomers of Na⁺,K⁺-ATPase E₁(Na₃)·AlF₄⁻·ADP crystal structures. Shown is the view from the extracellular side. Relevant transmembrane helices are shown as cylinders with blue letters and numbers (M4C and M4E, cytoplasmic and extracellular parts of M4), and Na⁺ ions as spheres numbered I, II, and III. Broken lines with numbers indicate distances in Å. Atomic models (Protein Data Bank codes 3WGV (with oligomycin) and 3WGU (without oligomycin) (3)) were aligned with the M7–M10 transmembrane helices. Upper and middle panels show protomers A and B, respectively, of the 3WGV structure. Selected residues are shown as lines colored according to the elements (carbon, green or gray; oxygen, red; nitrogen, blue) and numbered according to the rat α1-isoform. The lower panel shows all four different protomers superposed, with the side chain of Asp-928 in stick representation. Color codes: green, 3WGV protomer A; gray, 3WGV protomer B; blue, 3WGU protomer A; pink, 3WGU protomer B.

FIGURE 2.

Na⁺ and K⁺ dependences of M8 aspartate mutants. A, phosphorylation was carried out for 10 s at 0 °C in 20 mm Tris (pH 7.5), 3 mm MgCl₂, 1 mm EGTA, 2 μm [γ-³²P]ATP, 10 μm ouabain, 20 μg oligomycin/ml, and the indicated concentration of Na⁺, added as NaCl with various concentrations of N-methyl-d-glucamine to maintain the ionic strength. Each line represents the best fit of a Hill function (see Equation 1 of “Experimental Procedures”) with extracted K_0.5 values and Hill coefficients being listed in Table 1. B, ATPase activity was measured at 37 °C in 40 mm NaCl, 3 mm ATP, 3 mm MgCl₂, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and K⁺ concentrations as indicated, added as KCl. K_0.5 values for the rising parts of the curves are listed in Table 1. For direct comparison, the dotted line in the left panel reproduces the data for α1 wild type from the corresponding right panel. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 3.

ATP and vanadate dependences of Na⁺,K⁺-ATPase activity of M8 aspartate mutants. ATPase activity was measured at 37 °C in 130 mm NaCl, 20 mm KCl, 3 mm MgCl₂, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and the indicated ATP concentrations (upper panels) or 3 mm ATP and the indicated vanadate concentrations (lower panels). Each line represents the best fit of a Hill function (see Equations 1 and 2 of “Experimental Procedures”) with Hill coefficient ranging between 0.9 and 1.1. Extracted K_0.5 values are listed in Table 1. The dotted line in the left panel reproduces the data corresponding to wild type from the corresponding right panel for direct comparison in each panel. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 4.

Rescue of Na⁺ affinity, but not of Na⁺ selectivity, in the double mutants E314D/D928A and E314D/D928N. A, Na⁺ dependence of phosphorylation was studied as described for Fig. 2A. The dotted and broken lines reproduce wild type and mutant data from Fig. 2A. B, rapid kinetic measurements of phosphorylation time course at 25 °C in the presence of 150 mm NaCl, 40 mm Tris (pH 7.5), 3 mm MgCl₂, 2 μm [γ-³²P]ATP, 10 μm ouabain, and 20 μg of oligomycin/ml. Each line represents the best fit of a mono-exponential rise to max function (see Equation 3 of “Experimental Procedures”). The dotted and broken lines in the right panel reproduce wild type and mutant data from the left panel. C, K⁺ dependence of Na⁺,K⁺-ATPase activity was studied as described for Fig. 2B. The dotted and broken lines reproduce wild type and mutant data from Fig. 2B. For all panels, the extracted parameters are listed in Table 1. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 5.

The cooperativity is not rescued by E314D. Data and fitted lines from Figs. 2A and 4A are shown on an expanded abscissa scale to demonstrate the difference in cooperativity but similar apparent Na⁺ affinities of the wild type and E314D/D928A. As in the other figures, the error bars indicating S.E. values are seen only when larger than the size of symbols.

FIGURE 6.

ATP and vanadate dependences of Na⁺,K⁺-ATPase activity of E314D/D928A and E314D/D928N. Data were obtained and analyzed as for Fig. 3. Extracted K_0.5 values are listed in Table 1. The dotted and broken lines reproduce wild type and mutant data from Fig. 3 for direct comparison. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 7.

Na⁺ dependence of ATPase activity in absence of K⁺. ATPase activity was measured at 37 °C in 3 mm ATP, 3 mm MgCl₂, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and the indicated concentrations of Na⁺ added as NaCl. The catalytic turnover rate shown on the ordinate was calculated as the ratio between the specific ATPase activity and the active site concentration. For direct comparison, the dotted line in the left panel reproduces the data corresponding to wild type in the right panel. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 8.

K⁺ uptake in intact cells under physiological conditions. Uptake of the K⁺ congener ⁸⁶Rb⁺ at 37 °C in intact COS-1 cells stably expressing either wild type or mutants was determined following incubation for the indicated time intervals. K⁺ uptake was calculated by multiplying the relative ⁸⁶Rb⁺ uptake per mg of protein by the K⁺ concentration in the uptake medium (see “Experimental Procedures”). The uptake mediated by expressed mutant or wild type was calculated by subtracting data obtained at 10 mm ouabain from those obtained at 5 μm ouabain. The ordinate shows the data normalized to represent identical expression levels of wild type and mutants. The indicated background corresponds to non-transfected COS-1 cells. Error bars indicate mean ± S.E. (seen only when larger than the size of the symbols).

FIGURE 9.

Interaction network around Asp-928 and residues replacing Asp-928 in mutants studied. Shown is the view from the cytoplasmic side. This figure illustrates likely positions based on the high-resolution crystal structure of the wild type (Protein Data Bank code 3WGV) with Asp-928 being replaced. Selected residues (rat α1 numbering) are shown as sticks and colored according to the elements (carbon, gray; oxygen, red; nitrogen, blue), except that in mutants, the substituent is highlighted by the yellow color of the carbon atoms. The Na⁺ ions bound at sites I and III are shown as blue spheres. Broken lines with numbers indicate distances in Å. A, wild type. B, D928A. C, D928L. D, D928N. E, D928E. F, D928H. For each mutant, the most probable rotamer of the introduced substituted side chain is shown based on energy minimization of the side-chain orientation carried out using the PyMOL software.

FIGURE 10.

Structural relations of Glu-314 and Asn-124 to M4 and the Na⁺ and K⁺ binding region. Shown is the side view of the transmembrane domain, cytoplasmic side upwards (left panels) and view from the extracellular side toward the membrane surface. Transmembrane helices are shown as cylinders with blue letters and numbers, with selected residues indicated (rat α1 numbering; M4C and M4E refer to cytoplasmic and extracellular parts of M4, respectively). Broken lines with numbers indicate distances between Glu-314 and Asn-124 in Å. Putative bonds between selected residues and Na⁺ or K⁺ ions are also shown as broken lines in the right panels, but without numbers. A, Na⁺-bound form (E₁(Na₃)·AlF₄⁻·ADP, Protein Data Bank code 3WGV (3)). Na⁺ ions are shown as blue spheres numbered I, II, and III. B, K⁺-bound form (E₂(K₂)·MgF₄²⁻, Protein Data Bank code 2ZXE (5)). K⁺ ions are shown as purple spheres numbered I and II.