Two-electrode voltage clamp

Two-electrode voltage clamp is routinely used to characterize functional properties of Na⁺/K⁺-ATPase pumps (Horisberger and Kharoubi-Hess, 2002; Li et al., 2006; Poulsen et al., 2010; Vedovato and Gadsby, 2014; Hilbers et al., 2016). Data were collected with a GeneClamp 500B amplifier, Digidata 1200 digitizer and pCLAMP^™ software (Molecular Devices) at room temperature. Currents were measured 2–4 days after injection in oocytes preincubated (>30 min) with a Na⁺-loading buffer (Poulsen et al., 2010), unless otherwise mentioned. Electrode resistance was 0.2–0.7 MΩ when filled with NaCl 3 M to allow fast voltage clamp. Currents were elicited with 200-ms test steps from −160 to +60 mV, at 20-mV increments, from a holding potential of −30 mV. Recordings were sampled at 5 kHz and filtered at 1 kHz. Ouabain 1 µM was included in all recording solutions to block endogenous pumps.

Transient sodium-dependent currents were recorded in 0 [K⁺]_o (in mM: NaOH 115, sulphamic acid 110, MgCl₂ 1, CaCl₂ 0.5, BaCl₂ 5, HEPES 10, pH 7.4) with and without 10 mM ouabain, and isolated offline by subtraction. Scale was maximized to minimize signal clipping, and 10 traces were averaged to improve signal-to-noise ratio. The integral of transient ouabain-sensitive currents at −30 mV following the test voltage steps was plotted against test voltage and fitted to a Boltzmann function:

yielding the mid-point potential (V_1/2), slope factor (dV) and top (A1) and bottom (A2) asymptotes. Total charge transfer, Q_tot, represents the span of the Boltzmann fit (A1 − A2). Rate constants of Na⁺ binding/unbinding reactions derived from single (wild-type) or double (p.S779N) exponential fits to transient currents at the onset of the pulse. For p.S779N, the fast time constant, describing >90% of charge transfer, was analysed. Boltzmann fits to mean relaxation-voltage curves yielded the maximal forward and backward rate constants.

Leak currents were measured as the mean ouabain-sensitive currents in the last 50 ms of the test pulse_. In some experiments, pH was altered or Na⁺ was replaced by NMDG⁺ as monovalent cation in a 3:1 ratio. In experiments with 145 mM Na⁺, 30 mM NaOH was added to 0 [K⁺]_o solution. Reversal potential and slope conductance were obtained from linear fits to the current-voltage data.

Forward pumping activity was measured in various [K⁺]_o concentrations before and after addition of 10 mM ouabain. Records were subtracted offline. In these experiments, extracellular NaOH was replaced with equimolar KOH. Concentration-response curves were fit with a Hill equation:

yielding [K⁺] EC₅₀ values, Hill coefficient (h) and top (A1) and bottom (A2) asymptotes.