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).