Extended Data Fig. 2: Biophysical properties of the A1329D Nav1.2 channel mutation as determined by voltage clamp experiments.

A Peak sodium current (I_Na) density-voltage relationships. Currents were elicited from a holding potential of −120 mV by depolarizing voltage steps of 40 ms duration in 5 mV increment in the voltage range between −80 and +70 mV (voltage protocol shown in right inset in D). Left: representative I_Na traces in the voltage range between −80 and +20 mV; note that only the first 10-ms of the traces are shown. B Persistent inward I_Na-voltage relationships. Persistent current amplitude was determined as the inward current amplitude 40 ms after depolarization (arrows). Dotted lines indicate 0-pA level. C representative WT and A1329D I_Na traces elicited by −10 mV depolarizations. D Activation was determined from current-voltage relationships shown in A, whereas inactivation was determined from a holding potential of −120 mV, using 100-ms conditioning steps (between −120 and +10 mV) followed by a 20-ms test pulse to −5 mV to reveal the available current (arrow), at 0.1 Hz (voltage protocol shown in left inset). Voltage dependence of activation and inactivation, respectively, were obtained by measuring macroscopic currents and fitting the observed voltage dependence of the normalised conductance (G/Gmax) to a Boltzmann equation as follows: \(\frac{{\rm{G}}}{{{\rm{G}}}_{\max }}=\frac{1}{[1+{{\rm{e}}}^{({\rm{V}}-{{\rm{V}}}_{0.5})/{\rm{k}}}]}\), where V is the membrane voltage, V_0.5 represents the voltage for half-maximal activation or inactivation (V_0.5,act or V_0.5,inact, respectively), and k is the slope factor. Conductance (G) was calculated using the equation G = I/(V - V_rev), where V_rev represents the Na⁺ reversal potential. Normalized conductance values (G/G_max) were plotted against the membrane potential to generate activation curves. E Dependence of the time course of I_Na inactivation on the membrane potential. Left: Representative WT and A1329D I_Na traces elicited by −25 and −5 mV voltages. Note the slower time course of A1329D I_Na relative to WT. The fast time constants (τ_f) were obtained by fitting a double-exponential equation to the inactivating segment of individual I_Na traces as follows: \(\frac{{\rm{I}}}{{{\rm{I}}}_{\max }}={{\rm{A}}}_{{\rm{f}}}{{\rm{e}}}^{-{\rm{t}}/{{\rm{\tau }}}_{{\rm{f}}}}+{{\rm{A}}}_{{\rm{s}}}{{\rm{e}}}^{-{\rm{t}}/{{\rm{\tau }}}_{{\rm{s}}}}\), where t is time, A_f and A_s are the fractions of the fast and slow inactivation components, and τ_f and τ_s are the time constants of the fast and slow inactivating components, respectively. F Recovery from fast inactivation was evaluated using a paired-pulse voltage protocol from a holding potential of −120 mV (inset protocol). The first pulse inactivated the channels, followed by a second pulse to measure the fraction of current that recovered from inactivation after inter-pulse intervals of increasing duration. The time constants of recovery (τ) were determined by fitting the data with a single exponential function, as follows: \(\frac{{\rm{I}}}{{{\rm{I}}}_{\max }}=1-{{\rm{e}}}^{-{\rm{t}}/{\rm{\tau }}},\) where t is the time between the P1 and P2 test pulses. Data are represented as mean ± SEM; n, the number of experiments are shown between parentheses. Asterisks indicate statistically significant differences in the presence of A1329D relative to WT (*P < 0.05). The parameters of the fits and the results of the statistical evaluation are displayed in Supplementary Table 1.