Protonation of key acidic residues is critical for the K⁺-selectivity of the Na/K pump

Abstract

The sodium-potassium (Na/K) pump is a P-type ATPase that generates Na(+) and K(+) concentration gradients across the cell membrane. For each hydrolyzed ATP molecule, the pump extrudes three Na(+) and imports two K(+) by alternating between outward- and inward-facing conformations that preferentially bind K(+) or Na(+), respectively. Remarkably, the selective K(+) and Na(+) binding sites share several residues, and how the pump is able to achieve the selectivity required for the functional cycle is unclear. Here, free energy-perturbation molecular dynamics (FEP/MD) simulations based on the crystal structures of the Na/K pump in a K(+)-loaded state (E2·P(i)) reveal that protonation of the high-field acidic side chains involved in the binding sites is crucial to achieving the proper K(+) selectivity. This prediction is tested with electrophysiological experiments showing that the selectivity of the E2P state for K(+) over Na(+) is affected by extracellular pH.

Figure 1

Superposition of instantaneous configurations from MD simulations of the Na/K pump taken at 5ns, 8ns, 11ns, 14ns, 17ns, and 20ns from Simulation B in Table 1 with the protonation states of the binding site residues assigned according to the theoretical prediction (thin lines) superimposed with the 2ZXE crystal structure (thick lines). The average heavy-atom RMSD are 0.5 Å for Glu334, 0.4 Å for Glu786, 0.8 Å for Asp811, and 0.8 Å for Asp815.

Figure 2

a. K⁺-induced mediated outward pump currents from a single Na⁺-loaded oocyte (see METHODS), held at -50 mV in the presence of 125 mM Na⁺, at two different external pH (7.6, top trace and 9.6, bottom trace). Vertical deflection of the current trace represents 50-ms long voltage pulses (in a compressed time scale) used to obtain the half maximally activating [K⁺] (from Hill fits to the [K⁺]-dependence of outward current). Application of 10 mM ouabain inhibits the K⁺-induced outward Na/K pump current. b. Voltage dependence of the K_0.5 for K⁺ activation of outward pump currents at two different pH, in the presence and absence of Na⁺ in the external solution. The measurements were performed at pH 7.6 (circles) and pH 9.6 (triangles) in the presence of 125 mM external Na⁺ (red) or in the presence of 125 mM NMG (black). The voltage range chosen avoids influence of the non-competitive voltage dependent binding of external Na⁺ to the Na-exclusive site-III, on the apparent affinity for external K⁺. Data points are mean standard deviations from 5 oocytes where titration at both pH was evaluated.