Two-electrode voltage clamp

Two-electrode voltage clamp (TEVC) recording was performed at room temperature with an OC-725C amplifier (Warner Instruments, Hamden, CT, USA) and pClamp8 software (Molecular Devices, Sunnyvale, CA, USA) 4 to 5 d after cRNA injection as described above. Oocytes were placed in a small-volume oocyte bath (Warner Instruments) and viewed with a dissection microscope. Bath solution was (mM): 96 NaCl, 4 KCl, 1 MgCl₂, 1 CaCl_2, and 10 HEPES (pH 7.6). Preincubations with cytochalasin D or wortmannin (Sigma-Aldrich) were performed in SBB storage solution (Ecocyte). Pipettes were of 1 to 2 MΩ resistance when filled with 3 M KCl. Currents were recorded in response to pulses between −80 and 40 or 60 mV at 10-mV intervals, or a single pulse to +40 mV, from a holding potential of −80 mV, to yield current–voltage relationships and current magnitude, and for quantifying the activation rate. TEVC data analysis was performed with Clampfit (Molecular Devices) and Origin 6.1 (OriginLab, Northampton, MA, USA) software; values are stated as means ± sem. Normalized tail currents were plotted vs. prepulse voltage and fitted with a single Boltzmann function according to the following equation:

where g is the normalized tail conductance, A₁ is the initial value at −∞, A₂ is the final value at +∞, V_½ is the half-maximal voltage of activation, and V_s the slope factor. KCNQ2/3 activation at −40 mV was fit with a single exponential function. Where informative, currents were compared with one another using 1-way ANOVA to assess statistical significance (P < 0.05). If multiple comparisons were performed, a post hoc Tukey’s HSD test was performed after ANOVA.