Recording of sodium currents
The recording of sodium currents was performed as previously reported (T. Yang et al., 2014). The intracellular solution contained (in mmol/L): 5 NaF, 110 CsF, 20 CsCl, 10 EGTA and 10 Hepes. Adjusted the pH to 7.4 with CsOH. The external solution contained (in mmol/L): 135 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 C6H12O6 and 10 Hepes. The pH was adjusted to 7.4 with NaOH. In addition, 0.005 mmol/L Nifedipine (Sigma, USA, Lot # MKCB9232) and 0.5 mmol/L 4-aminopyride (MedChemExpress, USA, Lot # 27027) were used to eliminate L-type calcium current and outward potassium currents.
Sodium currents were measured at room temperature (22-24℃). Recording microelectrodes were made of hard glass and their tip resistances were between 2~4 MΩ. The protocol for recording sodium current and sodium channel dynamics is as follows: (1) PeakI Na, current-voltage curve and activation were measured from a holding potential of -120 mV, and were elicited with steps of 5 mV from -90 mV to +60 mV with a cycle length of 500 ms. (2) Steady state inactivation was measured with a double pulse. The conditioning pulse was held at -120 mV, with steps of 5 mV from -90 mV to +60 mV with a cycle length of 500 ms, and followed a single 50 ms test pulse at -30 mV. (3) Recovery from inactivation was measured by double pulse as well. The conditioning pulse was held at -120 mV, and the first test pulse was kept at -30 mV for 50 ms, then recovered to -120 mV, with the interval of 2, 4, 6, 8, ······ 49, 51 ms, and this was followed by a single 50 ms test pulse at -30 mV. (4) LateI Na was elicited from -120 mV to -30 mV with a cycle length of 500 ms.
Voltage clamp protocols are inset in figures. EPC10 patch clamp amplifier and PatchMaster version v2x73.2 software (HEKA, Germany) are used for data acquisition. The stimulation frequency was 1 Hz and the sampling frequency was 20 kHz. To increase data accuracy, ~70% of the series resistance was corrected and automatic leakage compensation was carried out. Origin 2017 software (OriginLab, USA) was used to analyze data and prepare figures. Data were presented as the current amplitude / cell capacitance to reduce intercellular error. Activation and inactivation curves were fitted with Boltzmann function (y={1+exp[(V -V 1/2)/k ]}-1), and recovery from inactivation curve was fitted with single exponential function (y = A1*exp (-x/t1)+y0). The averageI NaL from 50 to150 ms after the test pulse was used (Glynn et al., 2015).