The mutant channel L1821fs/10 exhibited a loss of function biophysical phenotype resulting in a mixed clinical syndrome characterized by SSS, CCD and recurrent VT. This discovery provides the fifth SCN5A
mutation associated with multiple clinical phenotypes [7
]. Generally, mutations in SCN5A
that cause a decrease in peak INa
density appear to be associated with BrS1/CCD [3
]. This can occur by two general mechanisms: a decrease in channel expression or a change in channel kinetics that tends to decrease peak INa
How does a loss of function mutation in some patients cause BrS1, SSS, or CCD, or in some patients, mixtures of these syndromes? It has been proposed [18
] that a rightward shift in the voltage dependence of the mutant sodium channel activation curve, as found with L1821fs/10 () is a common feature of CCD [5
]. The slower time to peak INa
() in our study indicates that the mutant channels require a more positive membrane potential to fully activate and more time to reach the membrane potential at which the maximum current amplitude occurs than WT channels, which might manifest on the ECG in a widening of the QRS in CCD [5
]. However, the mechanisms by which the proband showed SSS and monomorphic VT but not BrS1, are unknown.
The mutation, L1821fs/10, also showed decreased INa with slow decay and a noninactivating component (late INa) in those cells that had measurable macroscopic currents ( and ), similar to the gain-of-functional molecular phenotype of LQT3. This introduces seemingly paradoxical terminology, how can a mutation cause both “loss of function” and “gain of function”. The key distinction is timing, “loss of function” generally refers to a loss of peak or early INa, and “gain of function” generally refers to late INa. For L1821fs/10 mutant channels, peak INa is decreased but late INa is increased relative to peak INa.
This combination of molecular phenotypes has been reported previously for ΔK1500, a deletion of a lysine in the III–IV linker of SCN5A
, which is associated with BrS1, LQT3 and CCD [10
] and for 1795insD, an insertion of aspartic acid in the C-terminal domain of SCN5A
, which causing both LQT3 and BrS1 [8
]. In heterologous expression system, L1821/fs10 also has very similar molecular phenotype of both loss-of-function and gain-of-functional sodium channel features with a previously reported mutation of a methionine to a leucine (M1766L) from a patient with LQT3 [20
]. When expressed, M1766L showed a profound trafficking defect, but had increased late INa
and the expression defect was “rescued” by mexiletine, or, as in the patient, the mutation was rescued when expressed in the context of a sodium channel containing the common polymorphism H558R [14
In this case, the L1821fs/10 proband did have a prolonged QTc, but it was in the presence of right bundle branch block, and his VT was monomorphic, not torsades. Although this mutation has a biophysical phenotype that could potentially cause LQT3, there is no evidence for LQT3 in the patient. The reduced current expression was not restored with mexiletine, quinidine, or cisapride, drugs previously reported to rescue expression defective SCN5A mutations [17
]. Co-expression with β subunits and low temperature of incubation, which increased some expression levels in M1766L [20
], did not increase expression level for L1821fs/10. Further, the cell surface staining with anti-Flag antibody (: in nonpermeabilized cells) and co-labeling with anti-Flag antibody and anti-calnexin antibody (: in permeabilized cells) indicated that the L1821fs/10 channel reached the plasma membrane. From this we conclude that the markedly decreased peak INa
of L1821fs/10 was mainly caused by biophysical abnormalities.
Recently, the Nav
1.5 C-terminal domain has attracted considerable attention for having a direct structural role in the control of channel inactivation in the studies by Kass and colleagues [11
]. They postulate that the proximal part of the C-terminal domain is a critical structure for stabilizing the inactivated state of the channel during prolonged depolarization. The naturally occurring C-terminal truncation mutation, L1821/fs10 (Q1832 stop) in our study not only showed results similar to the experimental mutation (S1885 stop) designed to test biophysical function of the C-terminus, namely increased late INa
resulting from channels failing to inactivate during prolonged depolarization and slows the channel’s recovery from inactivation, as well as shifts the steady state inactivation curve in the hyperpolarizing direction [23
It is interesting that selected family members preferentially presented the phenotype with different degrees of severity, the marked clinical phenotype suffered by the proband contrasts sharply with the lack of symptoms in four carriers in the preceding generations and the very mild clinical phenotypes observed in a sister and a cousin. In general, but with one exception, the asymptomatic carriers were females, which agrees with a male predominance for loss-of-function SCN5A mutations causing BrS1. The mechanisms for this variability in this instance and in general are incompletely understood. We previously reported that the two common polymorphisms (H558R and S524Y) caused a profound expression defect in the Q1077 background, but not the Q1077del background [16
], and that H558R can modify expression of an arrhythmia causing mutation M1766L [14
]. These and other polymorphisms were absent in these patients. Loss of function for the BrS1 mutation G1406R was more severe in the Q1077 background [17
]. Although mRNA for Q1077 and Q1077del are present in equal ratios in different individuals [26
], should the protein levels vary this could account for clinical variability. Other unknown genetic, developmental, or acquired abnormalities may also account for clinical variability. For example structural abnormalities such as increased fibrosis has been shown to interact with decreased INa
] to affect excitability and conduction. Understanding the mechanisms for variability of the clinical phenotype may lead to improved understanding of pathogenesis and of possible therapy.
It is important to emphasize that these studies in heterologous systems, like previous work in the field, may not reflect what occurs in the myocytes, which has additional subunits and interacting proteins. Also these clones do not contain the introns or promoters that might affect expression in the myocytes. These experiments in heterologous systems only suggest a possible biophysical phenotype that may lead to the clinical syndromes, but further studies in more integrated systems is necessary to describe the full pathogenetic pathway.