Mutation of Gly-94 in transmembrane segment M1 of Na+,K+-ATPase interferes with Na+ and K+ binding in E2P conformation

Abstract

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na+,K+-ATPase for interaction with Na+ and K+ was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca2+-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K+-induced dephosphorylation allowed determination of the affinity of the E2P phosphoenzyme intermediate for K+. In Gly-94-Ala, the K+ affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E1P-E2P equilibrium to Na+, with accumulation of E2P even at 600 mM Na+, indicating that interaction of E2P with extracellular Na+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K+ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na+ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca2+-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na+,K+-ATPase mutant Gly-94-Ala on the basis of the Ca2+-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.

Fig. 1.

Sequence alignment (5) of the M1 region of Ca²⁺-ATPase (Upper) and Na⁺,K⁺-ATPase (Lower). The residues studied here (Gly-93 and Gly-94) and the corresponding residues in the Ca²⁺-ATPase are highlighted in yellow, and conserved residues are shown in red.

Fig. 2.

Na⁺ dependence (A) and time course (B) of phosphorylation from [γ-³²P]ATP. (A) Phosphorylation at 0°C for 15 s in 20 mM Tris (pH 7.5)/3 mM MgCl₂/2 μM [γ-³²P]ATP/10 μM ouabain/20 μg/ml oligomycin, and the indicated concentrations of NaCl with N-methyl-d-glucamine added to maintain the ionic strength. Each line shows the best fit of the Hill equation, and the extracted K_0.5 and Hill number (n_H) values are listed in Table 1: filled squares, wild type; open triangles pointing downward, Gly-93-Ala; open squares, Gly-94-Ala. (B) Phosphorylation of E₁Na₃ at 25°C in the presence of 100 mM NaCl/40 mM Tris (pH 7.5)/3 mM MgCl₂/1 mM EGTA/10 μM ouabain/20 μg/ml oligomycin/2 μM[γ-³²P]ATP. Each line shows the best fit of a monoexponential function, and the extracted rate constants are listed in Table 1. Symbols as for A.

Fig. 3.

Distribution of phosphoenzyme between E₁P and E₂P intermediates at 20 mM Na⁺ (A) and 600 mM Na⁺ (B) and dephosphorylation at 600 mM Na⁺ (C). Symbols as for Fig. 2. (A) Phosphorylation was performed at 0°C for 15 s in 20 mM NaCl/130 mM choline chloride/20 mM Tris (pH 7.5)/3 mM MgCl₂/1mM EGTA/10 μM ouabain/2 μM[γ-³²P]ATP. Dephosphorylation was studied upon chase with 1 mM ATP and 2.5 mM ADP. Each line shows the best fit of a biexponential decay function. (B) Twenty millimolar NaCl plus 130 mM choline chloride was replaced by 600 mM NaCl. The initial amounts of E₁P and E₂P, corresponding to the amplitudes of the rapid and slow phases, respectively, are listed in Table 1. (C) Phosphoenzyme was formed at 0°C for 15 s in 600 mM NaCl/20 mM Tris (pH 7.5)/3 mM MgCl₂/1 mM EGTA/10 μM ouabain/2 μM [γ-³²P]ATP. Dephosphorylation was studied upon addition of 1 mM ATP and 20 mM KCl. The phosphoenzyme half-lives are: wild-type, 3.85 s; Gly-93-Ala, 6.52 s; and Gly-94-Ala, 0.83 s.

Fig. 4.

K⁺ dependence of Na⁺,K⁺-ATPase activity (A) and Na⁺ and K⁺ dependencies of E₂P dephosphorylation (B and C). (A) Na⁺,K⁺-ATPase activity measured at 37°C in 40 mM NaCl/3 mM ATP/3 mM MgCl₂/30 mM histidine buffer (pH 7.4)/1 mM EGTA/10 μM ouabain, and the indicated concentrations of KCl. Symbols as for Fig. 2. Each line shows the best fit of the Hill equation, and the extracted K_0.5 and n_H values are listed in Table 1. (B) Phosphorylation was performed at 25°C for 5 s in 20 mM NaCl/130 mM choline chloride/20 mM Tris (pH 7.5)/3 mM MgCl₂/1 mM EGTA/10 μM ouabain (1 μM ouabain was used with Gly-94-Ala, because of the increased ouabain affinity of this mutant)/2 μM [γ-³²P]ATP. Dephosphorylation was studied upon addition of 1 mM unlabeled ATP and 200 mM NaCl (dashed lines with circles), or 1 mM unlabeled ATP and 1 mM KCl (solid lines with squares and triangles). Each line shows the best fit of a monoexponential decay function, and the extracted rate constants are: filled circles, wild-type, 3.4 s^–1; open circles, Gly-94-Ala, 1.1 s^–1; filled squares, wild-type, 62 s^–1; open triangles pointing downward, Gly-93-Ala, 64 s^–1; open squares, Gly-94-Ala, 15 s^–1; open triangles pointing upward, Glu-329-Gln, 8 s^–1.(C) The experiments were performed as in B (solid lines), except that various concentrations of KCl with choline chloride (to maintain ionic strength) were added, and the rate constants are shown as a function of the K⁺ concentration. Each line shows the best fit of the Hill equation, and the extracted K_0.5 and n_H values are listed in Table 1. Symbols as for B. The maximal rate of dephosphorylation corresponding to infinite K⁺ concentration is: wild-type, 210 s^–1; Gly-93-Ala, 192 s^–1; Gly-94-Ala, 258 s^–1; and Glu-329-Gln, 186 s^–1.

Fig. 5.

View from the cytoplasmic side of transmembrane segments M1–M4 of the crystal structure of the Ca²⁺-ATPase E₂ form with bound MgF₄^2– [mimicking phosphate (18)], with alanine replacement of Asp-59 and Leu-60 (M1) and isoleucine replacement of Gly-257 (M3) to model the Na⁺,K⁺-ATPase mutants Gly-93-Ala and Gly-94-Ala. The residue numbering shown corresponds to Na⁺,K⁺-ATPase. The side chain of the alanine replacing Gly-94 in the mutant comes within3Åof Ile-287, leading to steric problems.