Discussion
Q1491H and G1481V are SCN5A heterozygous mutations identified in two infants who died from long QT (Q1491H 700 ms AVB 2:1; G1481V 600 ms AVB 2:1) at 5 (G1481V) and 12 (Q1491H) weeks of age, respectively. The mutations were located in the Nav1.5 intracellular loop between domain III and IV near the IFM motif (Fig 1) , which is involved in fast inactivation16. Such mutations in the inactivation gate are often associated with changes in inactivation of voltage-gated Na+ channels. Both mutations were identified in clinical practice. SCN5A gene is known as a clinically actionable gene (https://search.clinicalgenome.org). The variants pathogenic status is thus critical to inform appropriately the family and their use in pre-symptomatic diagnosis.
The Q1491H mutation was located four amino acids downstream from the IFM motif. Glutamine has a polar uncharged side chain whereas histidine has electrically charged side chains. A biophysical characterization revealed that this mutation has a marked effect on the normal function of the NaV1.5 channel. We observed a loss-of-function for peak current density that could be due to a trafficking defect. We also observed a loss-of-function for recovery time (a slow time constant: 56.01 ± 8.73%, Table 1 ), which led to a decreased inward Na+ current and a longer recovery from inactivation time. An increase in the window current may lead to an increase in Na+ influx, which can increase the risk of fatal ventricular arrhythmias. This was correlated with the depolarized shift of steady state inactivation (+20.27 mV) and activation (+9.36 mV) that drove channel activity to more positive potentials. We also showed that the Q1491H mutant channel displays a persistent current that is inhibited by 25 𝛍M TTX (Fig. 6) and ranolazine (Fig. 7) , which is characteristic of LQT3. This mutation also displayed pronounced frequency-dependent inactivation at 10 Hz, suggesting that the Nav1.5/Q1491H channel has an unstable inactivated state. These results provide a rationale for linking this mutation and the clinical phenotype, especially for the long QTc interval (700 ms) measured in the infant prior to its sudden death.
The G1481V mutation was located four amino acids upstream from the IFM motif. Since valine and glycine are both uncharged, this may explain the more modest effect on the function of the Nav1.5/G1481V channel. The G1481V mutation resulted in an increase in the current density(Fig. 2) as well as a +7.03-mV depolarized shift in inactivation, which increased by 1.4-fold the window current(Fig. 3) . This mutation did result in a significant increase in persistent current; however the use of other expression system such as iPSCs is warranted. Furthermore, the recovery from the inactivation was also faster indicating that sodium channel will recover faster from inactivation All these changes represent a gain of function of G1481V mutant sodium channels.
The biophysical characterization of both Q1491H and G1481V SCN5Amutations thus revealed a functional defect. The manual patch-clamp is not validated as a well-established method in a variant classification perspective17,18. Considering the clear biophysical defects, a “Pathogenic Strong 3” (PS3) criterion in the ACMG classification process. Interestingly, both variants were re-classified as either likely pathogenic (class 4) or pathogenic (class 5), indicating that with the support of the biophysical characterization, both SNC5A variants can now be used for pre-symptomatic diagnosis.
Interestingly, although the patient with the G1481V mutation presented a long QTc interval of 600 ms, the increase in the persistent current was not statistically significant. In addition, there was no significant difference in the time constants of inactivation for all conditions (data not shown).
The III-IV linker acts as a lid in which the inactivation particle isoleucine-phelynalanine-methionine (IFM) amino acid trio occludes the inner pore by binding to the docking site like a latch. We used the recently crystalized rat Nav1.5 to visualize the position of the two residues and their interacting sites19. The IFM motif is highly conserved in all Na+ channels. The sites of the Q and G mutations investigated in the present study were located up- and downstream, respectively, from the IFM. They are highly conserved in several Na+ channels, underlining their importance during the process of inactivation. The structure revealed that Q1493 residue interacts with N1661 (N1659 in hNav1.5) and is one of the key residues for optimum IFM docking. It has been shown earlier that mutating N1661 to alanine abolishes fast inactivation20. The recently published rat Nav1.5 structure showed that Q1493 at the C-terminal of the DIII-DIV linker (Q1491 in hNav1.5) forms a strong hydrogen bond with N1661 (Fig. 8) . Q1491H mutation may lead to a weaker interaction with N1659 because of histidine shorter imidazole side chain. This will impair fast inactivation and lead to a gain of function effect. This may explain the appearance of persistent current and the rightward shift of the steady-state inactivation curve of Q1491H mutant. This could impede this interaction and contribute to change in pore closure and the slowing of slow inactivation. On the other hand, G1481 which is located at the tight turn of DIII S6 and the beginning DIII-DIV linker is important for keeping the flexibility of movement of the IFM motif. A valine substitution, with its bulky side chain, at this position will lead to less flexibility of the IFM motif movement. This structural insight explain the mild slow fast inactivation kinetics of G1481V mutant compared to wild type channel. However, the effects of this mutation is expected to be less drastic compared to Q1491H mutation. This is manifested by the little effects of G1481V mutation on channel function
Many SCN5A mutations, especially in the III-IV linker and the voltage sensor domain, have been linked to BrS and LQT3. The difference between BrS and LQT3 is that BrS appears with age21. The first mutation to be associated with LQT3 was a three-amino-acid deletion (Lys1505-Pro1506-Gln1507, ∆KPQ). This mutation shares the same deletion (Q1507) as the delQKP deletion at position 1507-1509. Both are located in the III-IV linker region and are associated with a gain-of-function and a late persistent Na+ current22,23. This region is a hotspot of mutations that induce inactivation disturbances, including a depolarized shift in inactivation that enhances the window current, as is the case for Q1491H and G1481V. This same effect was observed with F1486del, which involves a deletion of a phenylalanine on the IFM motif24. This mutation alters lidocaine sensitivity, which is a local anesthetic that blocks late currents associated with LQT3 mutations. Although IFM/QQQ mutants also display an alteration in lidocaine sensitivity25, this effect is not seen if the phenylalanine (F) is replaced by a glutamine (Q)26. Other mutations have been shown to induce a long QTc interval, including N1774H, which is located in the C-terminal27. Like the Q1491H mutations, N1774H results in a loss-of-function in peak current density and an increase in the late current. The N1774H mutation was detected in a 19-year-old woman. The S1333Y mutation on the S4-5 linker results in an enhanced window current and a persistent current. The S1333Y mutation was detected in a 25-day-old infant28. Although all these mutations lead to LQT3, they are located in different parts of the Nav1.5 channel that all play a role in inactivation. The ineffectiveness of mexiletine and flecainide in case 1 and propranolol in case 2, is not very clear especially that these drugs all have some sodium channels blocking properties. The possibility of Inter-individual pharmacokinetic and pharmacodynamic differences could explain the non-uniform clinical response to these drugs.