Properties of transient currents and hyperpolarization-induced inward currents of the ΔYY construct. (A and B) Ouabain-sensitive presteady-state currents ^NaI_(ouab) at [Na⁺]_ext = 100 mM and [K⁺]_ext = 0, pH_ext 7.4, upon voltage steps from −30 mV to potentials between +60 and −140 mV (in 20-mV decrements) for WT (A) and the ΔYY construct (B). (C and D) Voltage dependence of reciprocal time constants from ON transient currents for ATP1A2 WT (C) and ΔYY (D). Data for WT and ΔYY are means ± SE from 14 cells out of three batches. Results from a fit of a polynomial function to the WT or ΔYY data are superimposed in C and D as a dashed or dotted line, respectively. (E) Q-V curves obtained from Q_OFF charges of transient currents from 14 cells expressing ATP1A2 WT. The corresponding Q_ON charges were equivalent (not depicted). The fit of a Boltzmann function to Q-V distribution is superimposed as a dashed line (fit parameters: Q_min = −6.65 ± 0.12 nC; Q_max = 2.34 ± 0.04 nC; V_0.5 = −0.5 ± 1.0 mV; z_q = 0.76 ± 0.02). (F) Q-V curves obtained from Q_ON and Q_OFF charges of 14 cells expressing the ΔYY construct. The fit of a Boltzmann function to the Q_OFF values is superimposed as a dashed line (fit parameters: Q_min = −1.14 ± 0.17 nC; Q_max = 4.72 ± 0.63 nC; V_0.5 = −83.4 ± 7.9 mV; z_q = 0.81 ± 0.06). The short dashed line represents the inverted Q_OFF fit curve for comparison with Q_ON values. Q_ON and Q_OFF values of the ΔYY construct were significantly different at hyperpolarizing potentials (*, P > 0.05; **, P > 0.005; Student’s t test). The WT Q-V curve from E is included as a dotted line in F after appropriate scaling. (G) Transient currents in response to ON voltage steps to +60 and −140 mV from WT data in A and ΔYY data in B on an expanded time scale. The voltage step occurred at 58 ms (dashed line). Fits of the sum of two exponential functions to the data (from t₀ = 59 ms) yielded time constants for the fast rise, τ₁, and the slower decay, τ₂. The peak of the transient current of ΔYY at −140 mV appeared with a delay of ∼2 ms (gray bars). (H) [Na⁺]_ext dependence of normalized ouabain-sensitive inward currents of the ΔYY construct at [K⁺]_ext = 0. The shallow dashed line shows the I-V curve of ^NaI_(ouab) currents of ATP1A2 WT at [Na⁺]_ext = 100 mM and [K⁺]_ext = 0. For each construct, data are means ± SE from 14 cells of four batches. (I) [Na⁺]_int dependence of ouabain-sensitive ^NaI_(ouab) currents (at [Na⁺]_ext = 100 mM and [K⁺]_ext= 0), measured on an oocyte coexpressing the ΔYY construct and the amiloride-sensitive Na⁺ channel ENaC. Na⁺ loading was achieved by exposing oocytes to [Na⁺]_ext-containing solutions in the absence of amiloride, and [Na⁺]_int was determined after each loading step from the amiloride-sensitive reversal potential shift (see Materials and methods). Data from one out of three experiments with a similar [Na⁺]_int range are shown. (J) Temperature dependence (Arrhenius plots) of Na⁺/K⁺ pump currents (^NaI_10K) at 0 mV (filled squares) and hyperpolarization-induced inward currents (^NaI_(ouab)) at −140 mV (open squares) of the ΔYY construct. Data are from one out of four experiments across a similar temperature range (18–30°C). For both data sets, the amplitudes were normalized to the respective current at 21°C. Activation energies were derived from linear fits to the data.