Albers-Post kinetic scheme of the Na/K pump shown superimposed with a cartoon representation of transitions involving binding and release of ions. Na⁺ ions (black), K⁺ ions (gray). The box encloses states involved in Na⁺-dependent charge movement.

Steady-state Na/K pump-related currents. (A) Continuous TEVC trace from a Na⁺-loaded oocyte (V_h = −50 mV, 3 days postinjection) expressing RDΔKESYY. Increasing [K_o⁺] induced an outward current, both in the presence and absence of 125 mM Na_o⁺ (replaced with 125 mM NMG_o⁺). Vertical deflections correspond to 50-ms square voltage pulses, where the average current during the last 5 ms was measured. K_o⁺-induced currents (i.e., difference between K_o⁺ and no K_o⁺) at each voltage were plotted as a function of [K_o⁺] and fitted with a Hill equation to obtain K_0.5app values. (B) K_0.5 vs. voltage curves from RD injected oocytes in NMG_o⁺ (filled circles) and Na_o⁺ (filled squares), and from RDΔKESYY injected ooctyes in NMG_o⁺ (open circles) and Na_o⁺ (open squares). The Hill coefficients for the fits were: RD n_H = 1.2 ± 0.2 in NMG_o⁺, n_H = 1.4 ± 0.1 in Na_o⁺; RDΔKESYY, n_H = 1.2 ± 0.04 in NMG_o⁺, n_H = 1.5 ± 0.1 in Na_o⁺. (C) Current-voltage relationships measured in 125 mM Na_o⁺. Normalized maximum K_o⁺-induced current from Hill fits (I_max, squares), together with ouabain-sensitive currents at 10 mM K_o⁺ (I_ouab,10K, circles) and 0 mM K_o⁺ (I_ouab,0K, triangles) are shown from oocytes injected with RD (filled symbols) and RDΔKESYY pumps (open symbols). In all cases endogenous pumps were inhibited by application of 10 μM ouabain in the Na⁺-loading solution (see Methods).

Voltage-dependent inhibition of Na/K pump current by guanidinium_o⁺ in RD pumps. (A) Continuous TEVC trace from a Na⁺-loaded oocyte at V_h = −50 mV. K_o⁺-induced outward currents and ouabain inhibited Na/K pump current. To prevent Na_i⁺ depletion because of diffusion, the long recovery following pump inhibition was made in Na_o⁺ solution. (B) Steady-state I–V relationships obtained in 10 mM K_o⁺, in the absence (open symbols) or presence (open crossed symbols) of 10 mM ouabain with either 120 mM Na_o⁺ solution (squares), 120 mM gua_o⁺ solution (circles), or 125 mM NMG_o⁺ (triangles). The ouabain-sensitive currents are presented as filled symbols. (C) I_P (ouabain-sensitive currents in 10 mM K_o⁺) vs. voltage plot, normalized to the current observed at 0 mV with five different external ionic conditions: 120 mM Na_o⁺ (black squares), 120 mM gua_o⁺ (gray circles), 60 mM Na_o⁺ (black stars), 60 mM gua_o⁺ (gray stars), and 125 mM NMG (light gray triangles).

Transient charge movement in the presence of Na_o⁺ and absence of K_o⁺. (A and B) Ouabain-sensitive (10 mM) currents were measured in 125 mM Na⁺ solution via TEVC from Na⁺-loaded oocytes injected with RD-α1 cRNA (A) and RDΔKESYY-α1 cRNA (B) two days postinjection. Currents elicited by 100-ms pulses from V_h = −50 mV to voltages ranging from −160 mV to +40 mV in 40-mV increments. Endogenous pumps were inhibited by preincubation with 10 μM ouabain (see Methods). The speed of the clamp was slower (τ = 340 μs) for the experiment in A than for that in B (τ = 150 μs). (C and D) Na_i⁺-induced currents were measured in giant inside-out patches. All measurements were performed in presence of 4 mM MgATP at 4–6 days postinjection with either RD-α1 cRNA (C) or RDΔKESYY-α1 cRNA (D). The 125 mM Na_o⁺ (intrapipette) solution contained 1 μM ouabain to inhibit endogenous pumps. Traces are the difference between currents observed in the absence and presence of the indicated [Na_i⁺]. (E) Voltage dependence of normalized Q_OFF from ouabain-sensitive currents in TEVC or 40 mM Na_i⁺-induced currents in patches. Data from whole oocytes injected with RD (n = 6) (black open squares) or RDΔKESYY (n = 8) (red open circles) were fitted to a Boltzmann distribution (dotted lines). RD parameters were: V_1/2 = −39 mV, k_B = 33 mV, with a Q_tot = 8.6 ± 1.7 nC and I_3K,NMG = 135 ± 24 nA (i.e., current activated by 3 mM K in NMG). RDΔKESYY parameters were: V_1/2 = −130 mV, k_B = 33 mV, with a Q_tot = 14.8 ± 2.6 nC and I_3K,NMG = 195 ± 30 nA. Data from inside-out patches from oocytes expressing RD (black filled squares; n = 8) or RDΔKESYY (red filled circles; n = 7) pumps were also fitted to Boltzmann distribution (solid lines). RD parameters were: V_1/2 = −41 mV, k_B = 32 mV, with a Q_tot = 491 ± 136 fC. RDΔKESYY parameters were: V_1/2 = −135 mV and k_B = 33 ± 2 mV, with a Q_tot = 442 ± 70 fC. (F) Q_tot was calculated from the Boltzmann fit to the Q–V curves at different Na_i⁺, normalized to the value at 5 mM Na_i⁺ (RD), or 40 mM (RDΔKESYY), and plotted as a function of [Na_i⁺]. Data were fitted to the Hill equation with parameters in Results.

Ouabain-sensitive guanidinium⁺-dependent currents. (A) Ouabain-sensitive currents elicited by indicated voltage pulses, from V_h = −50 mV. TEVC traces are from an oocyte injected with RDΔKESYY in 120 mM gua_o⁺ (Upper) or 120 mM Na_o⁺ (Lower). (B) Voltage dependence of the relative amplitude (filled circles) and τ (open circles) of the slow component from biexponential fits to the traces in 120 mM gua_o⁺. (C) Same as in A but from an RD-injected oocyte. (D) Steady-state current (normalized to the 3 mM K_o⁺-induced current at −50 mV in NMG_o⁺) measured from RD (filled black squares) and RDΔKESYY (open red circles, estimated from biexponential fits) expressing oocytes in 120 mM gua_o⁺.