Horishita T , Yanagihara N , Ueno S , Okura D , Horishita R , Minami T , Ogata Y , Sudo Y , Uezono Y , Kawasaki T .

Fig. 1. Effects of APAS on peak sodium inward currents in Xenopus oocytes expressing Nav1.3 with β1 and β3 subunits at two holding potentials. (A) Representative traces are shown. Sodium currents were evoked by 50-ms depolarizing pulses to −10 mV from Vmax or V1/2 in both the absence and presence of 10 μmol/L of APAS. APAS were perfused for 3 min to reach equilibrium. (B) Percentage inhibition of sodium currents of APAS were calculated (n = 6). APAS inhibited the peak INa induced by Nav1.3 co-expressed with β1 by 26 ± 4% and 27 ± 4% at Vmax and V1/2, and reduced that by Nav1.3 co-expressed with β3 by 17 ± 1% and 34 ± 4% at Vmax and V1/2, respectively. Open columns represent the effect at Vmax holding potential and closed columns indicate the effect at V1/2. Data are presented as means ± SEM. **P < 0.01; ***P < 0.001, compared to the control, based on paired t-test (two-tailed). (C) Concentration-response curves for inhibitory effects of APAS on sodium currents elicited by 50-ms depolarizing pulses to −10 mV from V1/2 holding potential (n = 6). The peak current amplitude in the presence of APAS was normalized to that of the control and the effects are expressed as percentages of the control. Data are presented as means ± SEM. IC50 values and Hill coefficients were calculated using GraphPad Prism. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, one-way analysis of variance (ANOVA) followed by Dunnet pot hoc test. APAS = allopregnanolone sulfate; IC50 = half maximal inhibitory concentration; Nav = voltage-gated sodium channel; Vmax holding potential = holding potential causing maximal current; V1/2 holding potential = holding potential causing half-maximal current.

Fig. 2. (A) Effects of APAS on activation of sodium currents in oocytes expressing Nav1.3 with β1 (upper panel) and β3 subunits (lower panel) (n = 6). Currents were elicited using 50-ms depolarizing steps between −80 and 60 mV in 10 mV increments from a Vmax holding potential. Representative INa traces from oocytes expressing Nav1.3 with the β1 and β3 subunits in both the absence and presence of 100 μmol/L of APAS at Vmax holding potential are shown (left panel). APAS were perfused for 3 min to reach equilibrium. The effects of APAS on normalized I–V curves elicited from Vmax holding potential are shown (closed circles, control; open circles, APAS) (middle panel). Peak currents were normalized to the maximal currents observed at −10 mV. The effects of APAS on normalized activation curves elicited from Vmax holding potential are shown (closed circles, control; open circles, APAS) (right panel). Activation curves were fitted to the Boltzmann equation; V1/2 is shown in Table 1. (B) Effects of APAS on inactivation curves in oocytes expressing Nav1.3 with β1 and β3 subunits (n = 6). Currents were elicited by a 50-ms test pulse to −10 mV after 200-ms prepulses ranging from −140 mV to 0 mV in 10 mV increments from a Vmax holding potential. Representative INa traces from oocytes expressing Nav1.3 with the β1 and β3 subunits in both the absence and presence of 100 μmol/L of APAS are shown (left panel). Effects of APAS on inactivation curves (closed circles, control; open circles, APAS) are shown (middle and right panel). Steady-state inactivation curves were fitted to the Boltzmann equation and the V1/2 values are shown in Table 1. Data are expressed as means ± SEM. (C) Use-dependent block of sodium channels on Nav1.3 with β1 and β3 subunits (n = 6). Currents were elicited at 10 Hz by a 20-ms depolarizing pulse of −10 mV from a V1/2 holding potential in both the absence and presence of 100 μmol/L of APAS. Representative INa traces from oocytes expressing Nav1.3 with the β1 and β3 subunits in both the absence and presence of 100 μmol/L of APAS are shown (left panel). Peak currents were measured and normalized to the first pulse and plotted against the pulse number. Closed circles and open circles represent control and the effect of APAS, respectively (middle panel). Data were fitted to the monoexponential equation and values for fractional blockage of the plateau of normalized INa are shown (right panel). Data are expressed as means ± SEM. **P < 0.01, and ***P < 0.001, compared to the control, based on paired t-test (two-tailed). APAS = allopregnanolone sulfate; Nav = voltage-gated sodium channel; Vmax holding potential = holding potential causing maximal current.