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.