Whole Cell Electrophysiology

Transfected CHO cells were recorded at room temperature (20–22°C), 1–3 days post-transfection, using an Axopatch 200B (Molecular Devices) amplifier, pCLAMP v.9, a cFlow perfusion controller and mPre8 manifold (Cell MicroControls), and glass micropipettes (VWR International) with 1–4 MΩ resistance. The extracellular solution consisted of (in mM): 138 NaCl, 5.4 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, 10 HEPES, pH 7.4 with NaOH (Miceli et al., 2013). Pipette solution contained (in mM): 140 KCl, 2 MgCl₂, 10 EGTA, 10 HEPES, and 5 Mg-ATP, pH 7.4 with KOH. Series resistance was compensated by 70% after compensation using Axopatch 200B fast and slow capacitance controls. Currents were digitally sampled at 5–10 kHz, depending on the protocol, and filtered at 5 kHz using a low-pass Bessel filter. For voltage-activation experiments, cells were held at −80 mV and depolarized in 10 mV incremental steps from −80 to +40 mV for 1 s, then stepped to 0 mV for 60 ms. Tail currents at 0 mV were fitted using the Boltzmann function: GGmax=11+eV12−Vmk to obtain the half-maximum activation voltage (V_1/2) and the slope factor (k). Activation and deactivation rates were measured in SigmaPlot or Clampfit using a single exponential function: I = Ae^−t/τ + C. TEA sensitivity experiments were performed as previously described (Soldovieri et al., 2016). Currents were elicited by 3 s ramp depolarizations from −80 to 40 mV. To monitor inhibition by Oxo-M, cells were pulsed every 2 s for 200 ms to +40 mV from a holding potential of −80 mV before, during and after perfusion with drug solution. In experiments using ciVSP to study recovery, cells were held at −80 mV, then pre-pulsed for 2 s at −20 mV before stepping to +100 mV for 10 s, and then held at −20 mV for 30 s to monitor recovery. Kinetic parameters for current depletion and recovery were determined by non-linear regression models in SigmaPlot. Rates of current decline after ciVSP activation were estimated by fitting to a sigmoidal equation: y=11+e−t−t0/b. For WT Kv7.2 + Kv7.3 heteromers, current recovery kinetics after the end of a ciVSP-activating step were fit using the squared exponential function: y2=1–2e−tτ+e−2t/τ as described previously (Falkenburger et al., 2010; Alberdi et al., 2015). Current recovery for channels containing variants were fit poorly using this squared exponential but could be better fit using another sigmoidal equation, the 4-parameter logistic: y=ymax+(y0−ymax(1+tymax−ymin2b). A calculated liquid junction potential (−3.62 mV) was not corrected.