Gating Pore Currents in Mutant Na⨅1.4 Channels

Gating pore currents through mutant Na_V1.4 channels were studied using two-electrode voltage clamp in Xenopus oocytes. All procedures were carried out as described previously (Männikkö et al., 2018 (link)). Briefly, oocytes for Na_V1.4 expression were isolated from Xenopus laevis in accordance with the UK Animal (Scientific Procedures) Act 1986. The oocytes were injected with rat SCN4A encoding R669G mutant channel (analogous to human R675G mutation) and SCN1B messenger RNA transcribed in vitro (mMESSAGE mMACHINE kit, Thermo Fisher Scientific) at a 1:1 mass ratio using Nanoject (Drummond). GeneClamp 500B, Digidata 1200, and pCLAMP programs (Molecular Devices) were used to collect data. The bath solution contained 60-mM sodium methanesulfonate, 60-mM guanidine sulfate, 1.8-mM CaSO₄, and 10-mM Hepes (pH 7.4), and the oocytes were perfused with 1–2-μM tetrodotoxin (TTX) to block the main pore. To measure gating pore currents, the mean current during the last 100 ms of a 300-ms step to voltages ranging from −140 mV to +50 mV in 5-mV increments was plotted against the voltage. Holding voltage was -100 mV. Data were analyzed and presented using pCLAMP (Molecular Devices) and Origin (OriginLab) software. All data are presented as mean ± SEM.