2.1 Cell culture and expression of recombinant sodium channels
Cloning of the wild type and F1579A mutant rNaV1.4 channel constructs was performed as described previously (Lukacs et al., 2018).
Recombinant sodium channel-expressing cell lines were generated by transfection of wild type and F1579A mutant NaV1.4 BAC DNA constructs into HEK 293 cells (ATCC CRL-1573) by Fugene HD (Promega, Fitchburg, WI) transfection reagent according to the manufacturer’s recommendations. Cell clones with stable vector DNA integration were selected by the addition of Geneticin (Life Technologies, Carlsbad, CA) antibiotic to the culture media (400 mg/ml) for 14 days. HEK293 cells were maintained in Dulbecco’s Modified Eagle Medium, high glucose supplemented with 10% v/v fetal bovine serum, 100 U/ml of penicillin/streptomycin and 0.4 mg/mL Geneticin (Life Technologies, Carlsbad, CA). For experiments cells were plated onto 35-mm Petri dishes or T75 flasks, and cultured for 24–36 h. For manual patch-clamp experiments, Petri dishes were transferred to the recording chamber, where cells were kept under continuous flow of extracellular solution. For Port-a-Patch experiments, cells were dissociated from the dish with trypsin-EDTA, centrifuged, and suspended into the extracellular solution.