Human embryonic kidney HEK293T cells were grown in a Dulbecco’s modified Eagle’s culture medium (DMEM/F-12, Invitrogen) containing 10% fetal bovine serum and maintained under standard conditions at 37°C in a humidified atmosphere containing 5% CO2. Cells were transfected using the jetPEI transfection reagent (Polyplus-transfection Inc.) with either wild-type or mutant NaV1.7 channel combined with β1 and β2 subunits (40:1 ratio). Recordings were made 48 to 72 h after transfection.
Whole-cell voltage clamp experiments were performed on transfected HEK293T cells exhibiting both red and green fluorescence in the expectation that such cells would also express NaV1.7, combined with β1 and β2 subunits. All the recordings were conducted at room temperature using an Axopatch 200B Amplifier, the Digidata 1550B Low Noise Data Acquisition System and the pClamp10.6 software (Molecular Devices). Data were filtered at 5kHz and digitized at 20kHz. Capacity transients were cancelled and series resistance compensated at 70%–90% in all experiments. The extracellular solutions contained (in mM): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.3 with NaOH (adjusted to 320 mOsm/L with glucose). Patch pipettes were filled with an internal solution containing (in mM) 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, pH 7.3 with CsOH (adjusted to 310 mOsm/L with glucose) and had a typical resistance of 2-3MΩ.
Whole-cell voltage clamp experiments were performed on transfected HEK293T cells exhibiting both red and green fluorescence in the expectation that such cells would also express NaV1.7, combined with β1 and β2 subunits. All the recordings were conducted at room temperature using an Axopatch 200B Amplifier, the Digidata 1550B Low Noise Data Acquisition System and the pClamp10.6 software (Molecular Devices). Data were filtered at 5kHz and digitized at 20kHz. Capacity transients were cancelled and series resistance compensated at 70%–90% in all experiments. The extracellular solutions contained (in mM): 140 NaCl, 3 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.3 with NaOH (adjusted to 320 mOsm/L with glucose). Patch pipettes were filled with an internal solution containing (in mM) 140 CsF, 10 NaCl, 1 EGTA, 10 HEPES, pH 7.3 with CsOH (adjusted to 310 mOsm/L with glucose) and had a typical resistance of 2-3MΩ.