Figure 2

Gating properties of the channels. (A) The voltage dependence of the inactivation time constants of the WT (n = 9), R1451C (n = 8), and R1451L (n = 15) channels. The time course of the current decay elicited at depolarized voltages was best fitted to a single exponential function, and the resulting time constants (τ _h ) were plotted against voltage. The kinetics of fast inactivation were slower for the R1451C and R1451L channels, with R1451L being the slowest. The inset shows representative normalized Na⁺ currents elicited at −10 mV. The statistical significances of the differences between WT and R1451C/L (**P < 0.001, ***P < 0.001) and between R1451C and R1451L (^#P < 0.05, ^##P < 0.001, ^###P < 0.001) are shown. (B) Steady-state activation and inactivation of the WT and the mutants. No significant shifts in the V_1/2 of the R1451C/L channels were observed. Steady-state inactivation was determined using 10 ms test pulses to −10 mV after a 500-ms pre-pulse to potentials ranging from −140 mV to −30 mV (see protocol in the inset under the inactivation curves). The inactivation curves of R1451C and R1451L channels were left-shifted by −6 mV and −20 mV, respectively. The V_1/2, the k_v and the n values are given in Table 1. (C) Effect of R1451C/L on Na_V1.4 window current. The probability of being within this window was calculated through the following equation: (1/{1 + exp[(V_1/2act − V)/k_act]} × ((1 − C)/{1 + exp[(V − V_1/2inact)/k_inact]} + C). R1451C/L mutations produced a 5 and 7-fold increase in the window current, respectively (D) Recovery from inactivation of WT (n = 5), R1451C (n = 8) and R1451L (n = 6) channels. The cells were depolarized to −10 mV for 40 ms from a holding potential of −140 mV to inactivate the Na⁺ channels. Test pulses were then applied to −10 mV for 20 ms to measure current amplitudes, with an interval ranging from 0.1 to 4000 ms. The resulting curves were fitted to a single exponential equation. No significant effect was observed. The values are given in Table 1.