Figure legends
Figure 1: (A) Standard ECG recorded soon after birth. Paper speed 25 mm/s; 10 mm/1 mV. The QT interval was 720 ms, and the QRS was 60 ms and 2:1 AVB developed due to an extremely prolonged QT interval. (B) Sequence analysis of the G1481V and Q1491H mutations. (C) The Q1491H mutation resulted from a G- to -T substitution at position 4473, leading to a glutamine (Q)-to-histidine (H) substitution at residue 1491. The G1481V mutation resulted from a G- to -T substitution at position 4442, leading to a glycine (G)-to-valine (V) substitution at residue 1481. (D) Schematic representation of the four domains of the α-subunit of the Nav1.5 channel showing the locations of the Q1491 and G1481V mutations. DEKA represents the selectivity filter of the channel. The Q1491H mutation caused a glutamine-to-histidine substitution four amino acids downstream from the IFM motif. The G1481V mutation caused a glycine-to-valine substitution four amino acids upstream from the IFM motif.
Figure 2 : Analysis of whole-cell Na+ currents recorded from HEK 293 cells expressing Nav1.5 WT, Q1491H, and G1481V. (A) Example of raw traces from Nav1.5/WT, Q1491H, and G1481V were obtained using depolarizing pulses from –100 mV to +30 mV in 5-mV increments. (B) Current-voltage relationship of WT ● (n=9), Q1491H ◼ (n=9), and G1481V ▲ (n=9). The current amplitude was normalized to the membrane capacitance to obtain the current density (pA/pF).
Figure 3: Gating properties of steady-state activation and inactivation and window currents. (A) Voltage-dependence of steady-state activation and inactivation in WT (●, activation, n=9, and inactivation, n=9), Q1491H (◼, activation, n=9, and inactivation, n=7), and G1481V (▲, activation, n=9, and inactivation, n=7). The inactivated currents were generated using the protocol described in the insets, and the activated currents were obtained from the IV recording. The resulting data were fitted to a standard Boltzmann function. (B) The predicted window current was obtained using equation 1 (see Methods).
Figure 4: The gating properties of slow inactivation, recovery from slow inactivation, and closed-state inactivation. (A) Slow inactivation in WT (●, n=6), Q1491H (◼, n=6), and G1481V (▲, n=6). The two-pulse protocol described in the inset was used to generate the currents. (B) Time courses of recovery from slow inactivation in WT (●, n=7), Q1491H (◼, n=8), and G1481V (▲, n=7). Closed-state inactivation in WT (●, n=7), Q1491H (◼, n=8), and G1481V (▲, n=7).
Figure 5: Frequency dependence of WT (●, n=8), Q1491H (◼, n=8), and G1481V (▲, n=8). Currents were evaluated at (A) 2, (B) 5, and (C) 10 Hz. Fifty pulses were applied at –40 mV from a holding potential of –140 mV. Peak currents were normalized to the first peak current and were plotted versus the pulse number.
Figure 6: Persistent Na+ currents in WT, Q1491H, and G1481V inhibited by TTX. Recordings were performed using a –30-mV depolarizing pulse (see protocol in inset). (A) Currents recorded without TTX (left) and with 25 𝛍M TTX (right). (B) Currents normalized to the maximum current, without TTX (left) and with 25 𝛍M TTX (right). (C) Histograms of persistent Na+ currents. The persistent currents accounted for 0.58 ± 0.10 of the peak current amplitude for WT without TTX (n=6), 2.66 ± 0.29 for Q1491H without TTX (n=13), 1.25 ± 0.22 for Q1491H with 25 𝛍M TTX (n=13), and 1.33 ± 0.35 for G1481V without TTX (n=11) at –30 mV (**P<0.01, ***P<0.001).
Figure 7: Effects of ranolazine on persistent sodium current. (A) Persistent Na+ current traces using the same protocol as shown in Fig. 6 showing the effect of 100 µM ranolazine. (B) Bar graph of persistent Na+ currents inhibition by ranolazine (*P<0.05).