Biophysical characteristics of Nav1.5/Q1491H and Nav1.5/G1481V
Currents were elicited by sequential depolarizing steps of the cell membrane from –100 mV to +30 mV in 5-mV increments (Fig. 2A)with the Low Na+ external solution (see Methods). The I-V curves were constructed by measuring the peak of each Na+ current and were normalized to the membrane capacitance to obtain current densities (pA/pF). The current density of the Q1491H mutant channel was lower than that of the WT channel while the current density of the G1481V mutant channel was higher than that of the WT channel. These results suggest that the expression levels of the Q1491H and G1481V mutant channels were different than that of the WT channel. The potential of the maximum peak current amplitude of the Q1491H channel was shifted toward more positive potentials compared to the WT channel (Fig. 2B) . To study the activation and inactivation parameters, we first calculated the G-V curves (steady-state activation), which were fitted with a standard Boltzmann function. The V1/2 of the Q1491H mutant channel was positively shifted by 9 mV, with significant differences in k values. There was no difference between the V1/2 values of the WT channel and the G1481V mutant channel (Fig. 3A and Table 1).
Voltage-dependent steady-state inactivation was assessed by applying 500-ms pre-pulses ranging from –140 mV to –30 mV to allow the channels to enter the inactivated state followed by a test pulse at –30 mV to assess the number of functional channels. The current amplitude of the test pulse was normalized to the maximum current recorded during the pre-pulse and was plotted versus the voltage of the pre-pulse to obtain the voltage-dependent inactivation curve, which was then fitted to a standard Boltzmann function. V1/2 and k were generated by fitting each data set with a standard Boltzmann function (see values in Table 1 ). A +20-mV shift for Q1491H and a +7-mV shift for G1481V were observed, but k was not significantly affected (Fig. 3A) .
Window currents were determined and were used to assess the open probability. The total areas were 1.4 and 11-fold larger, respectively, for G1481V and Q1491H than for the WT channel and were shifted to more positive voltages (Fig. 3B) .
Slow inactivation was measured using a two-pulse protocol, with an initial conditioning pulse (pre-pulse) and a final test pulse. The current was normalized to the current amplitude during the pre-pulse. There was no difference between the G1481V and WT channels in terms of the kinetics of entry into slow inactivation. However, the Q1491H mutant channel entered into slow activation faster than the WT channel(Fig. 4A) . Recovery from slow inactivation was measured using a two-pulse protocol. The channels were first inactivated by a 500-ms pre-pulse and were then allowed to recover from inactivation. The peak currents were normalized to the maximum peak current. The Q1491H mutant exhibited a complex recovery with two (fast and slow) time constants(Table 1) . The G1481V mutant recovered from slow activation more quickly than the WT channel (Fig. 4B) . The currents of the mutant channels (Q1491H and G1481V) did not decrease during closed-state inactivation. Since the WT channel only exhibited a\(\tau\)fast time constant, \(\tau\)slowis not present in Table 1 . Closed-state inactivation was assessed using a two-pulse protocol. The currents were normalized to the first pulse. Neither mutated channel was affected, unlike the WT channel, which showed closed-state inactivation (Fig. 4C) .
We recorded the frequency-dependence of the Na+currents in order to determine the channels that enter the inactivated state by applying a series of 50 depolarizing pulses at –40 mV. To evaluate current inhibition during rapid pulsing, the channels were pulsed 50 times at 2 Hz, 5 Hz, and 10 Hz. There was a significant difference between the WT channel and the Q1491H mutant channel at 10 Hz, with the mutant channel displaying a reduced current during rapid pulsing. There was no difference between the G1481V mutant channel and the WT channel (Fig. 5) .