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).