Novel G1481V and Q1491H SCN5A Mutations Linked to Long QT Syndrome Destabilize the Nav1.5 Inactivation State

Figure 1. (A) A standard electrocardiogram was recorded soon after birth. Paper speed was 25 mm/s; 10 mm/1 mV. The QT interval was 720 ms; the QRS was 60 ms; and 2:1 atrioventricular block 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 4 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 4 amino acids downstream from the isoleucine, phenylalanine, and methionine motif. The G1481V mutation caused a glycine-to-valine substitution 4 amino acids upstream from the isoleucine, phenylalanine, and methionine motif.

Figure 2. Analysis of whole-cell Na+ currents recorded from human embryonic kidney (HEK) 293 cells expressing Nav1.5 wild type, 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 wild-type • (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 wild type (• activation, n = 9; inactivation, n = 9); Q1491H (▪ activation, n = 9; 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 wild type (•, n = 6), Q1491H (▪, n = 6), and G1481V (▲, n = 6). The 2-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). (C) Closed-state inactivation in WT (•, n = 8), Q1491H (▪, n = 10), and G1481V (▲, n = 7).

Figure 5. Frequency dependence of wild type (•, n = 8), Q1491H (▪, n = 8), and G1481V (▲, n = 8). Currents were evaluated at (A) 2 Hz, (B) 5 Hz, 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 vs the pulse number.

Figure 6. Persistent Na+ currents in wild type, Q1491H, and G1481V inhibited by tetrodotoxin (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 wild type 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 Figure 6 showing the effect of 100 μM ranolazine. (B) Bar graph of persistent Na+ currents inhibition by ranolazine (∗P < 0.05).

Figure 8. Open state structure of the human Nav1.5 sodium channel. (A) Top view of the cryogenic electron microscopy (Cryo-EM) structure of rat Nav1.5 channel showing the IFM motif in red spheres. (B) Bottom view of the Cryo-EM structure of the rat Nav1.5 channel showing the isoleucine, phenylalanine, and methionine (IFM) motif in red spheres. (C) Close-up view of the IFM motif flanked by G1481 on the N-terminal and Q1491 on the C-terminal. A strong hydrogen bond between N1661 and Q1491 (2.9A) ensures the proper docking of the IFM motif. DII, sodium channel domain 2; DIV, sodium channel domain 4.