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.