Dysfunctional Nav1.5 channels due to SCN5A mutations

Short abstract

The voltage-gated sodium channel 1.5 (Nav1.5), encoded by the SCN5A gene, is responsible for the rising phase of the action potential of cardiomyocytes. The sodium current mediated by Nav1.5 consists of peak and late components (I_Na-P and I_Na-L). Mutant Nav1.5 causes alterations in the peak and late sodium current and is associated with an increasingly wide range of congenital arrhythmias. More than 400 mutations have been identified in the SCN5A gene. Although the mechanisms of SCN5A mutations leading to a variety of arrhythmias can be classified according to the alteration of I_Na-P and I_Na-L as gain-of-function, loss-of-function and both, few researchers have summarized the mechanisms in this way before. In this review article, we aim to review the mechanisms underlying dysfunctional Nav1.5 due to SCN5A mutations and to provide some new insights into further approaches in the treatment of arrhythmias.

Impact statement

The field of ion channelopathy caused by dysfunctional Nav1.5 due to SCN5A mutations is rapidly evolving as novel technologies of electrophysiology are introduced and our understanding of the mechanisms of various arrhythmias develops. In this review, we focus on the dysfunctional Nav1.5 related to arrhythmias and the underlying mechanisms. We update SCN5A mutations in a precise way since 2013 and presents novel classifications of SCN5A mutations responsible for the dysfunction of the peak (I_Na-P) and late (I_Na-L) sodium channels based on their phenotypes, including loss-, gain-, and coexistence of gain- and loss-of function mutations in I_Na-P, I_Na-L, respectively. We hope this review will provide a new comprehensive way to better understand the electrophysiological mechanisms underlying arrhythmias from cell to bedside, promoting the management of various arrhythmias in practice.

Introduction

The α-subunit (Nav1.5) encoded by the SCN5A gene is the predominant element in heart tissue and plays a critical role in the excitability of cardiomyocytes. Nav1.5 channels mediate the inward sodium current (I_Na) and induce fast depolarization, thereby initiating the excitation–contraction coupling cascades in the cells. I_Na mediated by Nav1.5 can be classified into peak and late sodium currents (I_Na-P and I_Na-L). Mutations of SCN5A can impair Nav1.5 function and change the magnitude and duration of I_Na-P and I_Na-L, consequently leading to different types of fatal arrhythmias. Gain- or loss-of-function mutations are responsible for most of the pathogenesis of SCN5A mutation-induced cardiac disorders. More than 400 mutations have been identified in the SCN5A gene (updated SCN5A mutations from 2013 to 2018 are depicted in Figure 1). In this review, we will firstly introduce the biology of the Nav1.5 channel and then focus on the mechanisms underlying gain or loss of function and summarize arrhythmic consequences of mutant Nav1.5 and their clinical implications.

Figure 1.

Updated SCN5A mutations identified from 2013 to present. The red represents gain of I_Na-P, blue represents gain of I_Na-L, green represents loss of I_Na-P, yellow represents loss of I_Na-L, the mixed colors represent coexistence of gain- and loss-of-function mutations. (A color version of this figure is available in the online journal.)

Biology of the Nav1.5 channel

Sodium channels are hetero-multimeric proteins composed of a pore-forming α subunit and auxiliary β subunits. The α subunit consists of four homologous domains (DI–DIV). Each domain contains six transmembrane-spanning segments (S1–S6), of which the S4 segment functions as a voltage sensor and the S5 and S6 regions form the pore with the intermembrane P-loop.¹ The α-subunit (Nav1.5) encoded by the SCN5A gene is the predominant element in the heart and plays a critical role in the excitability of cardiomyocytes. In terms of its biophysical properties, Nav1.5 channels can be observed at three states: closed at resting membrane potential (approximately −85 mV), activated during depolarization, and inactivated. Transition between these states depends primarily on the transmembrane potential, time, temperature, and pH value. Recovery from inactivation occurs within the repolarization phase during diastole under physiological conditions. The upstroke speed of the AP and conduction is determined by the numbers of Nav1.5 channels that are available for opening. The inactivation process is rapid and stable for most ion channels. However, sodium channels may inactivate incompletely, therefore generating a so-called I_Na-L throughout the plateau phase of the AP.² In addition, some channels may reactivate during the repolarizing phase of the AP at a range of potentials in which inactivation is not complete and exhibits overlap with activation, resulting in the “window current.”³ Both I_Na-L and the window current can play critical roles in genetic and acquired cardiac diseases, as discussed below.

The SCN5A gene, which is expressed in a circadian pattern, is also expressed in extracardiac cells such as the excitable cells of the cerebral limbic system, and diverse subtypes of non-excitable cells, including microglia, astrocytes, T-lymphocytes, macrophages, fibroblasts, endothelial cells, and different type of cancer cells. Mounting evidence has demonstrated that sodium channels can take part in various effector functions and lead to non-classical effects in non-excitable cells. For instance, in cancer cells, Nav1.5 is related to enhanced invasiveness and metastasis. Nav1.5 affects Na⁺/H⁺ exchanger activity in breast cancer cells and causes local extracellular acidification, which results in activated cathepsin and consequently leads to the breakdown of the extracellular matrix. Na⁺ inflow is equally important in this process, as blocking Nav1.5 channels decreases the invasion of cancer cells. In addition, Nav1.5 in endosomes of macrophages from individuals with multiple sclerosis contributes to phagocytosis and pH regulation. It is suggested that targeting Nav1.5 could be a putative therapeutic approach in this disease.⁴

The Nav1.5 channel mediates the rapid entry of the sodium current (I_Na), a current that mainly contributes to the depolarization of the action potential (AP) in cardiac myocytes and the His-Purkinje system.⁵ I_Na mediated by Nav1.5 can be classified into peak and late sodium currents (I_Na-P and I_Na-L). I_Na-P occurs during phase 0 of the AP with a density of approximately 391 uA/uF and is quickly inactivated within 1–2 ms. The I_Na-L amplitude is much smaller than the I_Na-P amplitude in many species (approximately 0.1%–1%) and is inactivated more slowly during the plateau of the AP⁶ with the time constant ranging from 75 to 450 ms.^7,8 The I_Na-P is mainly associated with the initiation of cardiac excitability and electrical conduction. The I_Na-P drives the rapid AP upstroke, resulting in further channel activation. This transient increase in intracellular sodium leads to calcium current (ICa) influx via L-type voltage-gated channels when the voltage upstroke reaches approximately −25 mV. The depolarization-activated ICa induces Ca²⁺ release from intracellular sarcoplasmic reticular Ca²⁺ stores and initiates myocardial mechanical activity.⁹

Pathogenesis of SCN5A mutations

SCN5A gene mutations impair Nav1.5 function and consequently change the magnitude and duration of I_Na-P and I_Na-L, which lead to different types of fatal arrhythmias.¹⁰ SCN5A mutations are responsible for various types of cardiac disorders, including Brudaga syndrome (BrS),¹¹ long QT syndrome 3 (LQT3),¹² cardiac conduction disease (CCD),¹³ sick sinus syndrome (SSS),¹⁴ atrial fibrillation (AF),^15,16 progressive cardiac conduction defect (PCCD), dilated cardiomyopathy (DCM),¹⁷ multifocal ectopic Purkinje-related premature contraction (MEPPC),¹⁸ and the onset of a variety of non-cardiac diseases, including bowel syndrome,¹⁹ myotonic dystrophy,²⁰ epilepsy,²¹ pain,²² and ataxia.²³

SCN5A mutations result in the dysfunction of Nav1.5 due to defective protein trafficking, targeting, fixation to specific cellular compartments, post-translational protein processing, the modulation of biophysical properties and many unclear mechanisms.²⁴ Genotype and phenotype vary significantly, as the phenotypic characterization ranges from asymptomatic phenotypes to sudden cardiac death (SCD) in individuals that carry the same mutations. In addition, specific SCN5A mutations cause an individual phenotype or compound phenotypes, indicating that a complex pathogenesis underlies SCN5A mutations.

Gain-of-function mutations and arrhythmias

Long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization, which predisposes individuals to develop torsades de Pointes (TdP) and SCD. LQTS3 is caused by gain-of-function mutations of SCN5A. Approximately 8–10% of patients with SCN5A mutations are positively phenotypic as having LQTS.^25,26 The first SCN5A mutation related to LQT3, the deletion of amino acids 1505–1507 (ΔKPQ), was identified by Wang et al.²⁷ According to previous reports, cardiac events primarily occurred during sleep in LQT3 patients, and 18% died suddenly.²⁸ The gain-of-function SCN5A mutation leads to enhanced I_Na-P and I_Na-L, which finally triggers life-threating arrhythmias primarily in LQT3 patients.

Gain-of-function mutations of I_Na-P

The underlying mechanisms of SCN5A mutations that lead to the gain-of-function of I_Na-P are mainly due to abnormalities in mutation-induced kinetic properties, including augmented I_Na-P amplitudes, negative shifts in the voltage-dependence of activation, and an increased speed of recovery from inactivation. The most recently identified SCN5A mutations over the last five years (from 2013 to 2018) are shown in Table 1.^29–34

Table 1.

The newest identified gain of INa-P function of SCN5A mutations from 2013 to 2018 and their reported electrophysiological properties.

Mutation	Protein domain	Biophysical properties of mutant protein	Cardiomyopathy and accompanied features	References
I141V	DI/S1	Increased Iwindow: negative shift of activation	PVC, tachycardia	33
G213D	DI/S3-S4	Increased INaP: negative shift of act, positive shift of inactivation	AA, VA, DCM	34
Q1476R	DIII-DIV	Increased INaP: positive shift of inactivation; increased INaL	LQT3	32
G1748D	N-terminus DIV S6	Increased INaP: positive shift of inactivation, accelerated recovery from inactivation	LQT3	29
I1768V	DIV/S6	Increased INaP, Iwindow: negative shift of the activation, faster recovery from inactivation	LQT3, SCD	31
H1849R	C-terminus	Increased INaP and INaL: negative shift of inactivation, slower inactivation	LQT, AF, VT, SCD	30

AA: atrial arrhythmia; AF: atrial fibrillation; DCM: dilated cardiomyopathy; LQT: long QT syndrome; PVC: polymorphic ventricular complexes; SCD: sudden cardiac death; VA: ventricular arrhythmia; VT: ventricular tachycardia.

First, gains of channel function can be caused by variants that lead to augmented I_Na-P amplitudes. LQT3 mutations, such as I1748V³¹ and G1748D,²⁹ exhibited greater I_Na-P than wild type, whereas variants A572D³⁵ and G615E³⁶ also showed a significant gain of function of I_Na-P, but with an unclear clinical phenotype. However, some SCN5A mutations identified in clinical LQTS patients, such as F1250L³⁷ and N406K³⁸ variants, showed no significant changes in I_Na-P amplitudes. These phenomena suggested that although altered I_Na-P amplitudes affect phenotypes of the diseases directly, there may be other unknown mechanisms that are related to certain environmental factors or unknown gene mutations that contribute to genotype–phenotype interactions.

Second, the gain of function of I_Na-P could be generated by a negative shift in voltage-dependent activation potentials. It was reported that Nav1.5 reached its maximal current at −20 mV, while the variants G1748D,²⁹ H1849R,³⁰ S216L,³⁹ G983D, and F816Y⁴⁰ showed peak inward currents at a more negative voltage. Patients with G1748D and H1849R showed typical LQT3 features. However, patients that carried the variants S216L, G983D, and F816Y showed an unclear phenotype. These cases indicate that not all negative shifts of activation result in a gain of function of SCN5A and manifest the LQT3 phenotype; there must be other unidentified mechanisms that underlie genotype–phenotype interactions.

Third, gain of function of I_Na-P can be induced by a faster recovery from inactivation. According to this underlying mechanism, variants A572D and G983D showed a faster resumption of inactivation due to a fast component of the recovery. Moreover, the dedication of the fast component to the recovery from inactivation was relatively augmented in G983D. The A572D variant manifested as atria tachycardia, and G983D manifested as an abnormal T-wave on the electrocardiography (ECG).^41–43 The configuration of the T-wave pointed out the differences in the time course of ventricular repolarization. Morphologic changes in the T wave are sometimes more immediately remarkable than the mere prolongation of the QT interval; in some cases, the morphology of the T-wave is the only sensitive sign of ventricular repolarization disturbances.^44,45

Gain-of-function mutations of I_Na-L

I_Na-L has also been called steady-state I_Na, slow inactivation, persistent current, and late current. Under physiological conditions, the amplitude of I_Na-L is larger in mid-myocardial cells (M cells) and Purkinje fibers than epicardial and endocardial cells.⁴⁶ Although the magnitude of persistent I_Na-L is negligible compared to I_Na-P (0.1%–1%), the delay in inactivation breaks the delicate equilibrium of inward and outward currents, resulting in a prolongation of the action potential duration (APD), which manifests as a prolonged QT interval on ECG.⁷

An increase in I_Na-L due to acquired conditions or inherited SCN5A mutations in favor of intracellular Ca²⁺ loading,^47,48 the occurrence of early and delayed after depolarization (EAD and DAD),^49,50 triggered activities,⁵¹ and spontaneous diastolic depolarization⁵² that promotes the spatial and temporal dispersion of ventricular repolarization can lead to reentrant arrhythmias (Figure 2).

Figure 2.

The ion mechanism of gain of function of I_Na-L leading to arrhythmia.

The detrimental effects of a pathological persistent I_Na-L contribute to the development of arrhythmic disorders. The mechanisms are as follows:

(i) During phase 2 of the AP plateau, membrane resistance is high while the ionic conductance is low,⁵³ which caused marked APD prolongation. A prolonged APD helps L-type Ca²⁺ channels recover from inactivation and reactivate to form the upstroke of an EAD during the AP plateau.⁵⁴ (ii) An increase in I_Na-L due to delayed inactivation increased intracellular Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX, 3 Na⁺ out, 1 Ca²⁺ in)^55,56 and interacted with calmodulin in a protein kinase II (CaMKII)-dependent manner,^51,57 which had a positive feedback on Na+ loading. Additionally, it increased sarcoplasmic reticulum (SR) Ca²⁺ loading-induced Ca²⁺ release.^47,58 The increases in I_Na-L, the activity of CaMKII and SR Ca²⁺ release contributed to a substrate precipitating DAD.⁵⁹ (iii) Diastolic depolarization during phase 4 of the AP usually occurred in spontaneous pace-making cells of the sinoatrial and atrioventricular nodes.⁶⁰ However, spontaneous diastolic depolarizations were often observed in Purkinje fibers and atrial tissue isolated from a diseased heart with a persistent I_Na-L. According to reports, the gain of function of SCN5A may lead to spontaneous AP firing and abnormal automaticity, especially in myocytes that were relatively depolarized and had low resting K⁺ conductance.⁶¹ After all, the formation of EAD and DAD due to the gain of function of I_Na-L occurred more frequently in M cells than endo and epi and increased the transmural dispersion of ventricular repolarization, which finally caused reentrant arrhythmic events, which manifested as TdP and ventricular fibrillation (VF).

Previous studies revealed several gain-of-function mechanisms of I_Na-L with an alteration in channel kinetics, including a slower speed of inactivation or a positive shift in the voltage-dependence of inactivation (Table 2).^62–70

Table 2.

The newest identified gain of INa-L function of SCN5A mutations from 2013 to 2018 and their reported electrophysiological properties.

Mutation	Protein domain	Biophysical properties of mutant protein	Cardiomyopathy and accompanied features	References
R225P	DI/S4	Increased INaL and Iwindow: slower inactivation, shallower activation curve slope	Multifocal Ventricular Ectopy-associated Cardiomyopathy, LQT	66
N406Ka	DI/S6	Decreased INaP: positive shift of activation; increased INaL	BrS, LQT	65
R800L	DII/S3-S4	Increased INaL, Iwindow: incomplete inactivation and slowed decay of currents	LQTS, in compound with A261V-SNTA1	64
A1180Va	DII-DIII	Decreased INaP: negative shift of inactivation; increased INaL	DCM, AVB	63
V1328M	DIV/S6	Increased INaL: positive shift of inactivation	drug-induced BrS	69
F1473C	DIII-DIV	Increased INaL, Iwindow: negative shift of the inactivation	LQT3, TdP, VT, AVB	62
F1486dela	DIII-DIV	Decreased INaP; increased INaL: positive shift of inactivation, negative shift of activation, slower Na+ current decay	BrS, LQT	65
N1774D	C-terminus	Increased INaL: negative shift of activation, slower Na+ current decay; increased INaP	LQT	65
S1787N	C-terminus	Increased INaL due to splice variant and environmental factors	LQT3	67
M1851V	C-terminus	Increased INaL: slower inactivation, faster recovery from inactivation, positive shift of inactivation	AF, VA	70
V2016Ma	C-terminus	Increased INaL: PKA activation; decreased INa	LQT, SND	68

^aBoth gain and loss of function mutations;

AF: atrial fibrillation; AVB: atrioventricular block; BrS: Brugada syndrome; DCM: dilated cardiomyopathy; LQT: long QT syndrome; SND: sudden nocturnal death; TdP: Torsade de pointes; VA: ventricular arrhythmia; VT: ventricular tachycardia

First, gain-of-function mutations of SCN5A resulted in slower inactivation kinetics and increased I_Na-L, which included the LQT3-causing variant A993T as well as A572D⁴³ and K480N⁴² in patients with an unclear phenotype. In a patient with a mutation of T1526P,⁷¹ the cardiac examination was nearly normal, but T1526P showed a fractional gain-of-function property by a reduced speed of inactivation.

Second, gain of function can be caused by a positive shift in the voltage-dependence of inactivation, which also increased the magnitude and duration of I_Na-L. For example, the LQT3 mutation F1486L,⁷² which results in a gain of function, showed a positive shift in the voltage-dependence of inactivation. This effect of the N1325S^73,74 variant with an unclear phenotype was also observed in patients.

Loss-of-function mutations and arrhythmias

SCN5A loss-of-function mutations often cause BrS which is characterized by ST-segment elevation in the right precordial leads (V1–V3). Over 300 SCN5A loss-of-function mutations have been identified in connection with BrS.^75,76 Misfolded channels, trafficking defects, and negatively shifted steady-state inactivation curves contribute to a reduced availability of functional Nav1.5 channels on the plasma membrane.⁷⁷

CCD mutations were also related to loss of function in Nav1.5.⁷⁸ SCN5A loss of function reduced the AP upstroke velocity, which further delayed the rapid conduction of the electrical impulse through the highly specialized conduction system.⁷⁹ Most SCN5A loss-of-function mutations prolonged the rising time of the AP and rendered it more difficult to reach the membrane potential, which is necessary for the fast AP upstroke. In addition, defects in channel gating kinetics, an inability to conduct sodium, and channel retention in the ER would further reduce the availability of channels. The SCN5A mutations that lead to a loss of channel functions can be classified as follows:

Loss-of-function mutations of I_Na-P

Mutations that lead to a loss of function of I_Na-P are related to an alteration in channel kinetics, including decreased I_Na-P amplitudes and retention in the ER, but the function of the channels was restored when they reached the membrane (Table 3).^80–107

Table 3.

The newest identified loss of INa-P function of SCN5A mutations from 2013 to 2018 and their reported electrophysiological properties.

Mutation	Protein domain	Biophysical properties of mutant protein	Cardiomyopathy and accompanied features	References
A226V	DI/S4	Decreased INaP	BrS, in compound with p; R1629X	102
C335R	DI/S5-S6	Decreased INaP	AF, BrS	107
D349N		No detected current	SSS	89
R367G		Decreased INaP: trafficking defect	CCD	40
R367H	DI/S5-S6	Decreased INaP: positive shift of activation, negative shift of inactivation, faster recovery from inactivation	BrS, SUNDS	106
R376C	DI/S5-S6	Decreased INaP: positive shift of activation	SSS, SCD	91
R526H	DI/DII	Decreased INaP: trafficking defect	BrS, SCD, RBBB	94
S528A	Phosphorylation site	Decreased INaP: trafficking defect	BrS, SCD, RBBB	94
R811H	DII/S4	Decreased INaP: negative shift of inactivation, slower recovery from inactivation	BrS	82
L812Q	DII/S4	Decreased INaP, Iwindow: trafficking defect, negative shift of inactivation	BrS	96
I890T	P-loop of DII	Decreased INaP: positive shift of the activation	BrS	80
S910L	DII/S5-S6	Decreased INaP: trafficking defect, positive shift of activation	BrS, DCM	20
R1023C	DII-DIII	No detected current	ERS, VF, structural myocardial alteration	84
A1055G	DI/S5-S6	Decreased INaP: negative shift of inactivation, degradation	ERS	100
W1095X		No detected current	BrS, epilepsy	87
S1218I	DIII/S1	Complete loss of INaP: trafficking defect	BrS	82
D1275V	DIII/S3	Decreased INaP: positive shift of activation, enhanced degradation, trafficking defect	CCD, DCM, SND, AT, VT	105
N1380del	DIII/S5-S6	No detected current	CCD, VT	103
R1390Ha	DIII/S4	Decreased INaP: positive shift of activation, negative shift of inactivation, slower recovery from inactivation; increased INaL: slower deactivation	BrS, LQT, AA, VA	99
A1428S	DIII/S5-S6	Decreased INaP	BrS	97
D1430N	DIII/S5-S6	Complete loss of INaP: blockade of ion permeation	BrS, SCD	86
N1472del	DIII-DIV	Decreased INaP: positive shift of the activation and inactivation, slower recovery from inactivation	BrS, LQT, syncope, SCD, 2:1 AV block	83
1493delKa	DIII-DIV	Decreased INaP: trafficking defect; enhanced recovery from inactivation	CCD, VA, SCD	85
R1629X	DIV/S4	No detected current	BrS, in compound with p; A226V	102
R1632C	DIV/S4	Decreased INaP: negative shift of inactivation, slower recovery from inactivation	BrS, sinus node dysfunction(SND)	98
D1690N	DIV/S5-S6	Decreased INaP: positive shift of activation, slower recovery from inactivation;	BrS	101
G1748D	N-terminus of DIV/S6	Decreased INaP: positive shift of activation curve, faster inactivation	BrS	29
F1775Lfs15a	DIV/S6	Decreased INaP	overlap syndrome of SSS and BrS	81
L1786Qa		Decreased INaP: positive shift of activation, negative shift of inactivation; increased INaL	overlap syndrome of LQT3 and BrS	92
D1790N		No detected current	SSS	89
D1816VfsX7a	Truncation of C-terminus	Decreased INaP: trafficking defect, positive shift of activation; increased INaL: positive shift of inactivation, accelerated activation, faster recovery from inactivation	BrS, VF, bradycardia, AF	90
Q1832E	C-terminus	Decreased INaP: trafficking defect	BrS, SIDS, in compound with R1944△	104
R1860Gfs12a	Truncation of C-terminus	Decreased INaP: negative shift of inactivation, degradation, positive shift of activation; increase INaL: delayed inactivation	SSS, AF, AVB	93
V2016M	SIV motif	Decreased INaP: trafficking defect, positive shift of activation	BrS	95
c; 4297G>C	DIII/S5-S6	Decreased INaP: prolonged recovery from inactivation, positive shift of activation, affect translation process or degradation of the mutant protein	ERS	88

^aBoth gain and loss of function mutations;

AA: atrial arrhythmia; AF: atrial fibrillation; AT: atrial tachycardia; AVB: atrioventricular block; BrS: Brugada syndrome; CCD: cardiac conduction disease; DCM: dilated cardiomyopathy; ERS: early repolarization syndrome; LQT: long QT syndrome; RBBB: right bundle branch block; SCD: sudden cardiac death; SIDS: sudden infant death syndrome; SND: sudden nocturnal death; SSS: sick sinus syndrome; SUNDS: sudden unexpected nocturnal death syndrome; TdP: Torsade de pointes; VA: ventricular arrhythmia; VT: ventricular tachycardia.

Decreased current amplitudes cause loss of function of Nav1.5. As shown in the variants R222stop and R2012H,⁴² patients diagnosed with BrS matched the loss of function with a reduction of I_Na-P. In addition, there were a number of SCN5A mutations, such as E161K¹⁰⁸ and P336L,¹⁰⁹ that exhibited a dramatic reduction in I_Na-P density, while the kinetics and gating properties of the mutant channels were unaffected. These findings demonstrated that the decrease in I_Na-P density of the mutant channels was primarily caused by their retention in the ER, but the function of the channels was restored when they reached the membrane. Thus, we hypothesized that amino acid mutations might affect the protein structure, which would lead to misfolding and ER retention.

Loss-of-function mutations of I_Na-L

Loss of SCN5A channel function manifesting as ECG ST-segment elevation in right precordial leads was thought to contribute to an early repolarization of the right ventricular sub-epicardial myocardium that differentially altered the AP morphology of epicardial versus endocardial cells.^11,110 Loss-of-function of SCN5A results in a reduction in I_Na-L; this alteration increases the relative amplitude of the fast, transient outward K⁺ current (I_to), which is the most prominent in epicardial cells of the right ventricle.^111,112 Under normal conditions, I_to inhibited the depolarizing effect of the I_Na-L during the AP plateau, resulting in a marked AP notch in association with depolarizing Ca²⁺ currents in a “spike-and-dome” morphology.¹¹³ Consequently, the loss of I_Na-L and increase in I_to leads to a negative shift in the membrane potential, resulting in an “all-or-none” repolarization and causing an enhanced dispersion of repolarization in epicardial cells.¹¹⁴ Finally, this results in premature repolarization, phase 2 reentry, and significant AP shortening. In contrast, endocardial cells show a much smaller I_to and I_Na-L reduction, which do not significantly affect AP morphology and duration. The transmural heterogeneity of the cellular membrane voltage ultimately causes ST-segment elevation, a J wave, and even severe “R on T,” which leads to fatal VF on an ECG (Figure 3).¹¹⁵ Previous studies showed that the alteration of channel kinetics, such as a faster speed of inactivation or a negative shift in the voltage-dependence of inactivation, causes a loss of function of I_Na-L (Table 4).¹¹⁶

Figure 3.

The ion mechanism of loss of function of I_Na-L leading to arrhythmia.

Table 4.

The newest identified loss of INa-L function of SCN5A mutations from 2013 to 2018 and their reported electrophysiological properties.

Mutation	Protien domain	Biophysical properties of mutant protein	Cardiomyopathy and accompanied features	References
R1629Q	DIV/S4	Decreased INaL: negative shift of inactivation, enhanced intermediate inactivation, prolonged recovery from inactivation	BrS, SCD	116

BrS: Brugada syndrome; SCD: sudden cardiac death.

First, faster inactivation kinetics contribute to a decreased I_Na-L. For example, V1591L was detected in a BrS patient with a decreased I_Na-L as a result of faster inactivation kinetics.¹¹⁷ However, an R568H variant identified in a patient⁴² diagnosed with QT prolongation also showed loss of function by faster inactivation kinetics. The mechanism of genotype-phenotype interactions remained unknown. Second, a negative shift of the voltage dependence of fast inactivation leading to a reduced I_Na-L caused loss of function of SCN5A. T1620K^118,119 mutant channels in CCD patients, for instance, inactivate rapidly at less depolarized potentials, which may result in a significant reduction in I_Na-L and consequently lead to delayed AP upstroke in the Purkinje system. Similarly, the R2012H variant in BrS patients also resulted in a loss of function due to a negative shift of the voltage-dependence of inactivation.⁴²

Loss-of-function mutations in both I_Na-P and I_Na-L

Studies pointed out that most of the mutant Nav1.5 associated with a conduction disorder displayed either a drastic current reduction or shifts of steady-state inactivation/activation or both,^120,121 which may involve the alteration of I_Na-P and I_Na-L at the same time. As seen in G514C^122,123 mutant channels, a positive shift of both steady-state activation and inactivation, leading to changes in both I_Na-P and I_Na-L, was observed and the shift of the activation curve predominated by only 3 mV, which was still sufficient to produce a reduced upstroke velocity. This type of mutation may cause overlap syndromes. It is reasonable to speculate that simultaneous alterations of inactivation/activation kinetics may lead to this type of phenotype.

Coexistence of gain- and loss-of function mutations

SCN5A mutations that present with an overlapped phenotype of LQT3 and BrS were also described.¹²⁴ In vitro studies suggested that these uncommon SCN5A mutations cause a mixed phenotype by altering the amplitude of I_Na-P and I_Na-L through enhanced sodium channel inactivation, a negative shift in steady‐state sodium channel inactivation, and enhanced tonic block in response to sodium channel blockers.^78,125

R1193Q,^126,127 E1784K,¹²⁸ and S216L variants in SCN5A are proposed to cause either BrS or LQTS. E1784K, originally described by Wei et al.¹²⁹ and subsequently explored by several other groups, is the most common mutation that causes both LQT3 and BrS phenotypes. I_Na-P amplitude is significantly reduced, whereas the steady‐state inactivation is shifted to much more positive potentials that lead to an enhanced I_Na-L. The R1193Q mutation reveals a slower inactivation with a persistent I_Na-L, which accounts for the gains of channel function and is in line with the LQT3 manifestations. Additionally, the steady-state inactivation of this mutation was shifted to a more negative voltage that lowered I_Na-L, which explains the loss of function of sodium current and BrS clinical characteristics. S216L is proposed to be an LQTS3-causing mutation because of a significant increase in the persistent I_Na-L, as well as an acceleration of the recovery from inactivation. It was also identified in a BrS patient due to a significant reduction in the I_Na-P. Therefore, S216L is considered to cause a mixed BrS/LQT phenotype. The mechanisms of the coexistence of gain and loss of channel functions remain unclear; studies speculate that some environmental factors may play a vital role in the formation of the phenotype, and there might be other gene mutations to identify.

Perspectives

Since the first-generation gene sequencing technology was invented by Sanger in 1977, it has made tremendous progress. The brand-new third-generation sequencing technology has made the identification of gene mutations and genotypes more convenient. Functional analysis, including automatic patch clamp to study ion currents, in silico model simulation, and cryo-electronic microscopy to observe protein structure are advanced approaches to investigate the function of ion channels. New mutant models, such as iPSC-CM and transgenic animals, are more practical methods to investigate genotype–phenotype interactions. In clinical practice, we should also take more advantage of the current methods, such as ECG. ECG reflects the immediate manifestation of cardiac electrical activities on the body surface, provides the most direct evidence, and is a convenient approach for diagnosis. The alteration of T wave morphology and duration on the ECG is now used to distinguish different types of gene mutations. These discoveries stand out as notable landmarks in the progression of modern medical science that will allow further interpretations of the related pathogenesis. Additionally, in reference to these underlying mechanisms, many targeting therapies are desperately needed. Therapies that target these specific mutations with gene therapy, including RNA interference (RNAi) and CRISPER/Cas9, are under exploration, in addition to other traditional medicine therapies that affect the physiologic derangements of the mutations. The present findings enhance the general concept that the in vitro characterization of mutant ion channel functions is a key component for the generation of specific therapeutic strategies for patient management. Late sodium channel blockers, including mexiletine, ranolazine, flecainide, and a new compound, GS‐6615, have been used in patients with LQT3 to restore the gain of channel function. Mexiletine also rescues the membrane expression of the Nav1.5 channel that expresses the BrS mutation. Mexiletine may be able to rescue the retention of Na1.5 in the ER to increase the sodium current, but it can also block the I_Na-L, which manifests as an inhibitory effect.

In this review, we summarized the gain and loss of channel functions in the SCN5A gene and discussed the underlying mechanisms of its genotype–phenotype relationship. The detailed mechanisms that underlie dysfunctional Nav1.5 due to SCN5A mutations are described herein, and we also provide some new evidence for additional approaches in the treatment of arrhythmias due to mutant Nav1.5 channels.

Authors’ contributions

Dan Han wrote the draft. Guoliang Li, Hui Tan and Chaofeng Sun revised it.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.