2.2 Automated whole-cell patch-clamp electrophysiology

Na_V channel currents from HEK293 cells stably expressing Na_V subtypes and the β1 auxiliary subunit were recorded using an automated whole-cell patch clamp system QPatch 16X (Sophion Bioscience A/S, Ballerup, Denmark). As per the manufacturer’s guidelines, the cells were cultured for 48 h to achieve ∼80% confluency, detached using Detachin (Genlantis) and resuspended to 5 × 10⁶ cells/mL in serum free media [CHO-cell SFM (Life Technologies), 25 mM HEPES and 100 U/mL penicillin/streptomycin]. The extracellular solution comprised (in mM) 1 CaCl₂, 1 MgCl₂, 5 HEPES, 3 KCl, 140 NaCl and 20 TEA-Cl, with the pH adjusted to 7.3 with NaOH. The intracellular solution comprised (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES and 10 NaCl, with the pH adjusted to 7.3 with CsOH. The osmolarity of both solutions were adjusted to 320 mOsm with sucrose. Compounds were prepared in extracellular solution containing 0.1% bovine serum albumin (Sigma-Aldrich). To obtain the dose-response curves, cells were maintained at a holding potential −80 mV and Na⁺ currents were elicited by 20 m voltage steps to 0 mV from a −120 mV conditioning pulse applied for 200 m. Increasing concentrations of the peptide were incubated with the cells at the holding potential for 2 min before the voltage protocol was applied.