Interaction with extracellular ions of WT and Y780F. (A) Representative currents at −50 mV from oocytes expressing WT and Y780F mutant. Application of K⁺ in NMDG⁺ external solution at the millimolar concentrations indicated over the grey lines activates Na⁺/K⁺ pump current. At −50 mV all current induced by ≤10 mM K⁺ is Na⁺/K⁺ pump current (Meyer et al., 2020; Stanley et al., 2015). (B) Pump current amplitude induced by 10 mM K⁺ in NMDG⁺, number of oocytes indicated in parentheses. (C) K_0.5.K from Hill plots of dose-response data in NMDG⁺ and in 125 mM Na⁺. Individual data points are shown as grey spheres. (D and E) Representative ouabain-sensitive currents from two oocytes expressing WT (D) or Y780F (E). The currents were elicited by the pulse protocol illustrated in the box in two conditions: 125 mM Na⁺ (top) and 62.5 mM Na⁺ (bottom). The pulse protocol was repeated in the absence and then in the presence of ouabain, to obtain the subtracted ouabain-sensitive current displayed. (F and G) Q–V curves for the integral of the currents during the pulse (Q_ON), at 125 mM Na⁺, and upon return to −50 mV (Q_OFF) at 125 mM Na⁺ (solid) and 62.5 mM Na⁺ (open) for WT (F) and Y780F (G) expressing oocytes displayed in D and E. Line plots are fits by Boltzmann distribution (Eq. 4) with parameters V_1/2 = −49.1 mV, kT/z_qe = 43.6 mV, Q_tot = 39.7 nC (Q_ON at 125 mM Na⁺), V_1/2 = −38.2 mV, kT/z_qe = 39.1 mV, Q_tot = 35.7 nC (Q_OFF at 125 mM Na⁺) and V_1/2 = −60.5 mV, kT/z_qe = 39.1 mV, Q_tot = 34 0.1 nC (Q_OFF at 62.5 mM Na⁺) for WT, and V_1/2 = −119.6 mV, kT/z_qe = 56.8 mV, Qt_ot = 16.8 nC (ON at 125 mM Na⁺, fit to −160 mV only), V_1/2 = −121 mV kT/z_qe = 48 mV Q_tot = 16.3 nC (OFF at 125 mM Na⁺) and V_1/2 = −131 mV kT/z_qe = 48 mV Q_tot = 14.5 nC (OFF at 62.5 mM Na⁺), for Y780F. (H) Mean Q_OFF−V curves (normalized to Q_tot from Boltzmann equations) from oocytes expressing WT (black, n = 5) or Y780F (red, n = 8) where transient charge movement was evaluated in both 125 and 62.5 mM Na⁺. Line plots are Boltzmann curves with parameters: V_1/2 = −42.0 mV, kT/z_qe = 35 mV at 125 mM Na⁺ and V_1/2 = −67.6 mV, kT/z_qe = 35 mV for WT and V_1/2 = −113.5 mV, kT/z_qe = 51.8 mV at 125 mM Na⁺ and V_1/2 = −144.8 mV, kT/z_qe = 51.8 mV at 62.5 mM Na⁺, for Y780F. A shared slope factor (kT/z_qe) was used for the two concentrations in each mutant.