This Review is part of a thematic series on Inherited Arrhythmogenic Syndromes: The Molecular Revolution, which
includes the following articles:
The Fifteen Years That Shaped Molecular Electrophysiology: Time for Appraisal [Circ Res. 2010;107:451– 456]
Defining a New Paradigm for Human Arrhythmia Syndromes: Phenotypic Manifestations of Gene Mutations in Ion
Channel– and Transporter-Associated Proteins [Circ Res. 2010;107:457– 465]
The Cardiac Desmosome and Arrhythmogenic Cardiomyopathies: From Gene to Disease [Circ Res. 2010;107:700 –714]
Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Voltage–Dependent L-Type
Calcium Channel [Circ Res. 2010;107:607– 618]
Inherited Dysfunction of Sarcoplasmic Reticulum Ca2⫹ Handling and Arrhythmogenesis [Circ Res. 2010;107:871– 883]
Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac Sodium Channel
Phenotypical Manifestations of Mutations in Genes Encoding Subunits of Cardiac Potassium Channels
Silvia Priori, Editor
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Phenotypical Manifestations of Mutations in the Genes
Encoding Subunits of the Cardiac Sodium Channel
Arthur A.M. Wilde, Ramon Brugada
Abstract: Variations in the gene encoding for the major sodium channel (Nav1.5) in the heart, SCN5A, has been
shown to cause a number of arrhythmia syndromes (with or without structural changes in the myocardium),
including the long-QT syndrome (type 3), Brugada syndrome, (progressive) cardiac conduction disease, sinus
node dysfunction, atrial fibrillation, atrial standstill, and dilated cardiomyopathy. Of equal importance are
variations in genes encoding for various subunits and regulatory proteins interacting with the ␣-subunit Nav1.5
and modifying its function. Based on detailed studies of genotype–phenotype relationships in these disease
entities, on detailed studies of the basic electrophysiological phenotypes (heterologous expressed wild-type and
mutant sodium channels and their interacting proteins), and on attempts to integrate the obtained knowledge, the
past 15 years has witnessed an explosion of knowledge about these disease entities. (Circ Res. 2011;108:884-897.)
Key Words: sodium channel 䡲 arrhythmias 䡲 sudden cardiac death
I
cardiomyopathy.8 Because many of these diseases associate
with sudden death in young individuals with a structurally
normal heart, potentially causal SCN5A variants have also
been identified in sudden infant or sudden unexplained death
syndrome (SIDS/SUDS).9,10 Detailed descriptions of the clinical (patients) and basic electrophysiological phenotypes
(heterologous expressed wild-type and mutant sodium channels) have profoundly increased our knowledge about these
disease entities and about the role of the sodium channel and
its interaction with various subunits. Indeed, besides
nitially based on genetic studies in the mid-1990s, the
SCN5A gene, encoding the major sodium channel (Nav1.5)
in the heart, has been unmasked as an important player in a
number of primary arrhythmia syndromes. Indeed, sodium
channel dysfunction resulting from mutation-based alterations in channel function is associated with the long-QT
syndrome (LQTS) (type 3),1 the syndrome of right precordial
ST elevation, ie, Brugada syndrome (BrS),2 (progressive)
cardiac conduction disease (CCD),2,3 sinus node dysfunction,4 atrial fibrillation (AF),5 atrial standstill,6,7 and dilated
Original received September 2, 2010; resubmission received December 8, 2010; revised resubmission received January 13, 2011; accepted January 28, 2011.
In December 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.5 days.
From the Heart Research Centre (A.A.M.W.), Department of Clinical and Experimental Cardiology, Academic Medical Center, University Medical
Center, University of Amsterdam, The Netherlands; and the Institut d’Investigació Biomèdica Girona-IdIBGi (R.B.), Universitat de Girona, Giona Spain.
Correspondence to Arthur A.M. Wilde, Department of Clinical and Experimental Cardiology, Academic Medical Center, PO Box 22700, University
Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands. E-mail [email protected]
© 2011 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.110.238469
884
Wilde and Brugada
Cardiac Disease and Subunits of the Sodium Channel
␤-subunits (␤1 to ␤4), the ␣-subunit Nav1.5 also interacts
with several regulatory proteins, of which, more recently,
malfunction resulting from disease-causing variants in their
encoding genes has been shown to underlie comparable
phenotypes.
This article aims to review the existing knowledge of the
basic and clinical aspects of sodium channel related diseases.
It begins with a review of sodium channel function in normal
and abnormal conditions that is followed by a description of
the diverse clinical phenotypes.
The Cardiac Sodium Channel
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The ␣-subunit of the cardiac isoform of the sodium channel is
known as NaV1.5 and is encoded by the SCN5A gene.
SCN5A maps to the short arm of chromosome 3 (3p21),
contains 28 exons spanning ⬇80 kb,11,12 and encodes the
␣-subunit of the voltage-gated cardiac sodium channel (designated Nav1.5), conducting the cardiac sodium current INa.
Sodium channels are expressed in several tissues and share a
common structure.13–16 Hence, the cardiac sodium channel is
a member of the voltage-dependent family of sodium channels, which consist of heteromeric assemblies of the poreforming ␣-subunit. NaV1.5 contains 2016 residues and has an
approximate molecular mass of 227 kDa. It consists of 4
homologous domains, known as DI to DIV, joined by
so-called linkers. These 3 linkers, as well as the N terminus
and the C terminus of the protein, are cytoplasmic. Each
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Non-standard Abbreviations and Acronyms
AF
CCD
LQTS
Nav1.5
nNOS
PK
SCD
SCN5A
SCN1B
SCN2B
SCN3B
SCN4B
SIDS
SUDS
atrial fibrillation
cardiac conduction disease
long QT syndrome
voltage-gated cardiac sodium channel
neuronal nitric oxide synthase
protein kinase
sudden cardiac death
␣-subunit of the cardiac sodium channel
␤1-subunit of the cardiac sodium channel
␤2-subunit of the cardiac sodium channel
␤3-subunit of the cardiac sodium channel
␤4-subunit of the cardiac sodium channel
sudden infant death syndrome
sudden unexplained death syndrome
domain, DI to DIV, contains 6 transmembrane helices
(namely S1 to S6) linked by intracellular or extracellular
loops. S5 and S6 in each of the domains form the pore-lining
helices (Figure 1).
Opening of the channel allows a rapid influx of positively
charged Na ions (INa) that will depolarize within tenths of a
millisecond the membrane potential of cardiac cells. As such,
Figure 1. Schematic representation of the ␣-subunits of cardiac sodium channel, consisting of 4 serially linked homologous
domains (DI–DIV), each containing 6 transmembrane segments (S1–S6) (A). In B, the interacting ␤-subunits and other regulatory
proteins are depicted.
886
Table 1.
Gene
SCN1B
Circulation Research
April 1, 2011
Cardiac Sodium Channel Regulatory Proteins Linked to Arrhythmic Phenotypes
Locus
Protein
Phenotype(s)
Effect of Mutation(s) on INa
19q13.1
␤1-subunit
Brugada syndrome
Decrease in peak INa
48, 74
Reference
Cardiac conduction disease
Atrial fibrillation
SCN2B
11q23
␤2-subunit
Atrial fibrillation
Decrease in peak INa
48
SCN3B
11q23.3
␤3-subunit
Brugada syndrome
Decrease in peak INa
49, 79, 82
Idiopathic ventricular fibrillation
Increase in persistent INa
Sudden infant death syndrome
Atrial fibrillation
SCN4B
11q23.3
␤4-subunit
GPD1L
3p22.3
Glycerol 3 phosphate dehydrogenase 1-like (GPDL1)
Long QT syndrome type 10
Increase in persistent INa
84, 85
Decrease in peak INa
90, 93
Increase in persistent INa
96, 100
Increase in (persistent) INa
107, 108
Sudden infant death syndrome
Brugada syndrome
Sudden infant death syndrome
CAV3
3p25
Caveolin-3
Long QT syndrome type 9
Sudden infant death syndrome
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SNTA1
20q11.2
␣-1 syntrophin
Long QT syndrome type 12
Sudden infant death syndrome
sodium channel activity plays a central role in cardiac
excitability and conduction of the cardiac impulse. The S4
segments in each domain, containing a positively charged
amino acid at every third position, are held responsible for
voltage-dependent activation. At the end of the action potential upstroke (ie, phase 0), most channels are inactivated and
do not further allow passage of ions. Furthermore, this
inactivation process is voltage-dependent. It is generally
accepted that inactivation is mediated mainly by the inactivation gate (DIII to DIV linker), which blocks the inside of
the channel shortly after it has been activated. Furthermore,
the C-terminal cytoplasmic domain plays an important role in
inactivation. Channels can only be reactivated after recovery
from inactivation during phase 4. However, a small percentage of channels remain available to conduct and may reopen
during the plateau phase of the action potential (phases 2 and
3). This fraction of channels, less than 1% of total available
Na⫹ channels, contributes a small “late” Na⫹ current (INaL) to
total membrane current. Under normal conditions, INaL is very
small and has little impact on action potential morphology but
can be very important in pathological conditions involving
sodium channel mutations, among others (see below).
The cardiac Na⫹ channel is a multiprotein complex in
which auxiliary proteins interact with the ␣-subunit (Nav1.5)
to regulate its gating, cellular localization, intracellular transport, and degradation. By performing screening experiments
such as coimmunoprecipitation and yeast 2-hybrid, interactions with Nav1.5 have been demonstrated for several proteins. In addition, mapping techniques have been used to
discover the sites of interactions and the regulatory role of
these auxiliary proteins have been studied with coexpression
experiments in heterologous expression systems such as
human embryonic kidney cells (HEK-293), Chinese hamster
ovary cells (CHO), or Xenopus oocytes. Recently, genetic
screening studies have linked mutations in genes encoding
several of these subunits of the cardiac Na⫹ channel to
phenotypes similar to those previously associated with mu-
tations in SCN5A. Auxiliary proteins that interact with Nav1.5
may be classified as enzymes, adapter proteins, and regulatory proteins. Enzymes may directly interact with Nav1.5 and
control its cellular localization by ubiquitylation and internalization (ubiquitin-protein ligases). Enzymes may also alter
gating properties by phosphorylation (kinases) or dephosphorylation (phosphatase). Adapter proteins anchor Nav1.5 to
the cytoskeleton and play a role in the intracellular transport
and targeting of Nav1.5 to the sarcolemma. Regulatory
proteins modulate gating properties of Nav1.5 on binding.16
As indicated mutations in SCN5A lead to various phenotypes. In addition, mutations in 7 genes that encode for
auxiliary proteins of the cardiac Na⫹ channel have been
linked to arrhythmic phenotypes previously associated with
these phenotypes (Table 1). Four of these genes (SCN1B,
SCN2B, SCN3B, and SCN4B) encode for proteins with a
uniform molecular structure (␤1, ␤2, ␤3, and ␤4, respectively; Figure 1). These so-called ␤-subunits of the cardiac
Na⫹ channel contain an extracellular amino terminus, a single
transmembrane segment, and an intracellular carboxyl terminus. All 4 ␤-subunits are expressed in the heart, and are
particularly localized at the T-tubules/Z-lines and intercalated
disks in cardiac myocytes.17 The ␤-subunits play critical roles
in the regulation of sarcolemmal expression and gating of
Nav1.5, in the recruitment of cytoskeletal adapter proteins,
enzymes, and signaling molecules to the channel and in the
adhesion of Nav1.5 to the cytoskeletal framework and extracellular matrix.
For many of the identified putative causal variants in both
SCN5A, as well as in all its subunits (see below), one should
realize that almost always sound segregation data, linking the
genetic variant to the clinical phenotype lack. Other evidence
that the identified variants are disease-causing is based on
“weaker criteria” that include absence in control alleles,
conservation throughout species and functional data. Indeed,
in a study designed to identify the presence of rare variants in
the 3 main genes associated with LQTS, including SCN5A, in
Wilde and Brugada
Cardiac Disease and Subunits of the Sodium Channel
control individuals, these variants were particularly identified
in the intra domain linker and the C terminus of SCN5A.18 All
of these variants were altered amino acids considered wellconserved among species. This has led to the notion that the
pretest probability of pathogenicity of SCN5A variants identified in these regions to cause LQTS3 (see below) is far less
than 50%.19 With this caveat in mind, one should examine the
existing literature and be very cautious in calling a rare
genetic variant a novel arrhythmia syndrome–susceptibility
mutation.20 This holds in particular for variants identified in
single patients only once. Clearly, detailed functional studies
demonstrating clear aberration from normal behavior increase
the likelihood that certain variants are disease-causing.
Clinical Phenotypes Related to
SCN5A Mutations
Brugada Syndrome
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BrS is a hereditary disease responsible for ventricular fibrillation and SCD in the young. The disease is characterized by
the presence of right ventricular conduction abnormalities
and coved-type ST-segment elevation in the anterior precordial leads (V1 to V3).21 The prevalence of BrS is estimated
at approximately 3 to 5 in 10 000 people. Although the mean
age of onset of events is approximately 40 years, SCD can
affect individuals of any age, particularly men (75%). Of
those, 20% to 50% have a family history of SCD. The disease
exhibits an autosomal dominant pattern of transmission and
variable penetrance. Most mutations occur in genes related to
Na⫹ channels, although other channels may also be implicated in BrS.22,23
Between 20% and 25% of patients affected by BrS have
mutations in the SCN5A gene. To date, there are more than
200 known SCN5A BrS-related mutations described.24 These
mutations lead to a loss of Na⫹ channel function through
several mechanisms, including trafficking defects, generation
of truncated proteins, faster channel inactivation, shift of
voltage dependence of steady-state activation toward more
depolarized membrane potentials (Figure 2), or slower recovery from inactivation. Recently, it has been shown that in
several large SCN5A-related BrS families (5 of 13 families
with 4 or more phenotypically affected) affected individuals
did not carry the familial disease-causing mutation. These
observations could be explained by phenocopies but could
also explained by the presence of an anatomic substrate (an
area with slow conduction in the RV outflow track) with a
strong modifying role of SCN5A loss-of-function mutations.
Apparently, in some individuals, the substrate is sufficient to
provide the Brugada pattern spontaneously, ie, in the absence
of an SCN5A mutation. In any case, these observations
question a direct causal role of SCN5A mutations,25 and it
stresses once more how careful one should be when investigating mutations.
Genotype–phenotype correlation studies in BrS have been
limited because of the low prevalence of mutation carriers. A
recently published study has proposed that the type of
SCN5A mutation may be useful for risk stratification. In this
study, a genotype-phenotype correlation was performed according to the type of SCN5A mutation (missense, truncated).
887
Patients and relatives with a truncated protein had a more
severe phenotype and more severe conduction disorders.26 At
this point in time, such data are too preliminary to use in
clinical decision making.
Lev-Lenègre Syndrome
Lev-Lenègre syndrome is a progressive cardiac conduction
disease (characterized by either prolongation of the P wave,
PR or QRS segment duration, complete AV block, and
bundle branch blocks) probably associated with gradually
developed ventricular fibrosis of the conduction system. The
first locus for the disease was reported in 1995, on chromosome 19q13.2-13.3.27 In 1999, the first mutations causing the
disease were described in SCN5A.3 Mutations in SCN5A lead
to a reduction in Na⫹ current, reducing the velocity of the
impulse conduction.28
Long QT Syndrome
LQTS is a cardiac channelopathy characterized by a QT
interval prolongation, responsible for ventricular
tachyarrhythmias with associated episodes of syncope and
SCD.29,30 LQTS is one of the leading causes of SCD among
young people. It can be congenital or acquired, generally in
association with drugs and electrolyte imbalance (hypokalemia, hypocalcemia and hypomagnesemia). The clinical presentation can be variable, ranging from asymptomatic patients to episodes of syncope and SCD caused by ventricular
tachyarrhythmias (torsades de pointes) in a structurally normal heart. Prolongation of the QT interval may arise because
of a decrease in the K⫹ repolarization currents, an increase in
Ca2⫹ entry or to a sustained entry of Na⫹ into the myocyte
(ie, increase in INaL). An increase in INaL is caused by either
impaired inactivation, ie, failure to inactivate completely
(Figure 3A) or an increase in “window current.” The latter is
generally attributable to a shift of voltage dependence of
steady-state inactivation toward more depolarized membrane
potentials leaving an increased “window” of inward sodium
current (Figure 3B).
Until now, 13 different types of LQTS have been described, most of them associated to K⫹ channel disorders.31
However, a 10% to 15% of the LQTS cases are related to
mutations in SCN5A (type 3 LQTS) or in Na current–
associated proteins. Patients with type 3 LQTS present
arrhythmias associated with bradycardia and symptoms at
rest, especially during sleep.32 Whether ␤-blockers are as
important in type 3 LQT patients as they are in LQT type 1
and 2 patients has been questioned. Data to support nonresponsiveness lack but, preliminary data on a large cohort of
LQTS type 3 patients suggest a beneficial effect (A Wilde et
al, unpublished data). The best therapy for type 3 LQT
patients is currently unknown, but high risk patients are
probably those who are symptomatic and those with the
longest QT intervals (⬎500ms). In the latter category a
prophylactic ICD might be considered. Gene-specific therapies include sodium channel blockers like flecainide and
mexiletine33–35 although detrimental attempts, with pathophysiological explanations based on sound experimental
studies, have been published.36 Furthermore, ranolazine, a
888
Circulation Research
April 1, 2011
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Figure 2. Two mechanisms leading to reduced peak inward
current (A). B, The voltage dependence of normal activation is
depicted by the yellow squares and activation at more depolarized potentials is depicted by the red squares. Shift of voltage
dependence of steady-state activation toward more depolarized
membrane potentials will lead to a decrease in the peak sodium
current. Furthermore, a hyperpolarizing shift of the voltage
dependence of steady-state inactivation curve will lead to a
decrease in the peak sodium current, because, for a given resting membrane potential, fewer sodium channels are available.
Both mechanisms, in addition to slower recovery from inactivation (not shown), have been described for BrS-associated
SCN5A mutations that alter channel characteristics.
new drug with late sodium current blocking efficacy, has
been shown to be able to shorten the QTc interval significantly.37 As many different mutations lead to an overlap
syndrome, ie, families with both gain-of-function and lossof-function phenotype38 the use of flecainide should be
Figure 3. The 2 mechanisms through which sodium channel
mutations lead to an increase in INaL. A, Either impaired inactivation, ie, failure to inactivate completely (A) or an increase in
“window current” caused by a depolarizing shift in inactivation
(“increased availability”), leaving an increased “window” inward
sodium current (B) causes a late sodium inward current. The
latter current flows through already activated but not yet inactivated sodium channels (B).
accompanied with serial ECGs because a BrS type ECG (with
the proarrhythmogenic substrate) may be the result.33 For all
of these drugs, long-term follow-up is not available.
Dilated Cardiomyopathy
Dilated cardiomyopathy is a cardiac structural disease characterized by decreased systolic function and ventricular
dilatation. An average 20% of cases are thought to be
familial, most associated with mutations in the cytoskeletal
Wilde and Brugada
Cardiac Disease and Subunits of the Sodium Channel
proteins. The identification of a mutation in SCN5A in a
family with dilated cardiomyopathy provided the first evidence of the disease being caused by an alteration in a cardiac
ion channel.8 Since then, other publications have confirmed
this association.39 The phenotype is usually associated with
arrhythmias (atrial and ventricular arrhythmias and conduction disease).
Atrial Fibrillation
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AF is defined as an unpredictable activation of the atria,
characterized by irregular fibrillatory ECG waves causing an
irregular ventricular response, which manifests clinically as
an irregular pulse. AF is one of the most common and yet
challenging arrhythmias encountered in clinical practice. The
prevalence of 1% among the general population increases to
10% among individuals older than 80 years, and it is
responsible for more than one-third of all cardioembolic
episodes.40
Although familial forms had remained mostly unknown,
the identification in 1997 of a genetic locus causing familial
AF, which defined AF in a subset of patients as a genetic
disease with an autosomal dominant pattern of inheritance,
initiated further research into the understanding the pathophysiology of this inherited form of the arrhythmia.41 To date,
most of the genes associated to AF encode K⫹ channel
proteins (KCNQ1, KCNE2, KCNE3, KCNA5, KCNJ2, and
KCNH2).42 The role of Na⫹ channel proteins in AF has
become evident in these last few years. In 2008, several
genetic variants in SCN5A, causing both loss and gain of INa,
have been associated with lone AF.5,43 Most of these phenotypes do not show alterations in the QT interval or in the ST
segment elevation.44,45 A family with AF with a combined
phenotype of LQTS type 3 has been recently described.46 In
addition, mutations in the ␤-subunits SCN1B, SCN2B, and
SCN3B have been associated with AF by decreasing INa.47– 49
It is hypothesized that loss of INa function leads to altered
atrial conduction parameters, increased fibrosis, and associated structural abnormalities (including increased diastolic
ventricular pressure in the setting of dilated cardiomyopathy
with subsequent enlargement of the atria), that all act together
to cause AF.
Sick Sinus Syndrome
Diseases of the sinoatrial node (brady-tachy syndrome, sinus
bradycardia, sinus arrest) have been associated with loss-offunction mutations in SCN5A, especially severe in patients
with compound heterozygote mutations.4 Similar with other
loss-of-function phenotypes, families may show important
overlapping features, with family members displaying sick
sinus syndrome, whereas others may display BrS or progressive cardiac conduction disease.45,50,51
Atrial standstill has also been described in SCN5A families. A combined loss-of-function mutation with a loss-offunction connexin 43 polymorphism also leads to atrial
standstill.52
Genetic Modulators in Na Current and Sudden
Cardiac Death
Modulation of the phenotype may be related to the presence
of environmental factors (ie, inducers like fever in BrS or
889
medications in LQTS) or additional genetic factors. Among
these genetic factors, it is well accepted that the presence of
compound heterozygotes will confer a worse prognosis in
family members. As such, compound heterozygotes involving genes associated with Na current have been described in
LQTS,53 BrS,54 and sick sinus syndrome.4
Polymorphisms have recently acquired more importance in
the explanation of certain phenotypes of genetic diseases.
Genetic variants in the SCN5A promoter region may have a
pathophysiologic role in BrS. A haplotype of 6 polymorphisms in the SCN5A promoter has been identified and
functionally linked to a reduced expression of the sodium
current. This variant was found among patients of Asian
origin, and it could play a role in modulating the expression
of BrS in far eastern countries.55 In addition, the common
H558R polymorphism has been shown to partially restore the
sodium current impaired by other simultaneous mutations
causing either cardiac conduction disturbances (T512I)56 or
BrS (R282H).57 The common variant H558R seems to be a
genetic modulator of BrS among carriers of an SCN5A
mutation, in whom the presence of the less common allele
makes BrS less severe (shorter QRS complex duration in lead
II, lower J-point elevation in lead V2, and a trend toward
fewer symptoms).58
Finally, the presence of the genetic variant S1103Y, which
accelerates sodium channel activation, has been associated
with arrhythmias and sudden cardiac death in blacks.59,60 Of
interest in experimental settings, the late sodium current was
exacerbated by lowering intracellular pH, making it an
important mechanism predisposing a Y1103 carrier to sudden
infant death syndrome (SIDS). Indeed, this variant has been
identified in black SIDS cases.61
Proteins Associated With Arrhythmias
Mutations in all 4 ␤-subunit– encoding genes are found in
individuals with various arrhythmic phenotypes, reflecting
their importance in normal cardiac electric activity. Interestingly, most such mutations are located in the extracellular
amino terminus of the ␤-subunits (Table 2 and Figure 4),
suggesting this domain may play an important role in the
interaction and regulation of Nav1.5 by the ␤-subunits.
␤1-Subunit
Two splice variant transcripts of SCN1B, generating ␤1 and
␤1 B proteins, are expressed in the heart.62 The ␤1-subunit
coimmunoprecipitates with Nav1.563 and is found to colocalize with Nav1.5 already within the endoplasmic reticulum,
suggesting that they mediate the transport of the channel
complex to the sarcolemma.64 The ␤1-subunit is believed to
interact with Nav1.5 at multiple sites. An interaction between
the extracellular domain of the ␤1-subunit with the extracellular loops between segments 5 and 6 of domains III and IV
(S5-S6 loops of DIII and DIV) of Nav1.5 is required for the
regulation of channel gating.65,66 In addition, an interaction
may also exist between the carboxyl terminus of these 2
subunits.67
Accordingly, the p.D1790G mutation in the carboxyl
terminus of Nav1.5, discovered in a large family with LQT-3
has been shown to affect the inactivation of the cardiac Na⫹
890
Table 2.
Circulation Research
April 1, 2011
Mutations in Cardiac Sodium Channel ␤-Subunits Linked to Arrhythmic Phenotypes
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␤-Subunit
Nucleotide
Change
Variant
Mutation
Type
Location
Phenotype(s)
Effect of INa
␤1
536G⬎A
W179X
Nonsense
C terminus
Brugada syndrome and cardiac
conduction disease
Decrease in peak INa and positive
shift of activation
␤1
537G⬎A
W179X
Nonsense
C terminus
Cardiac conduction disease
Decrease in peak INa
74
␤1
259G⬎C
E87Q
Missense
N terminus
Cardiac conduction disease
Decrease in peak INa and positive
shift of activation
74
␤1
254G⬎A
R85H
Missense
N terminus
Atrial fibrillation
Decrease in peak INa and positive
shift of activation and inactivation
48
␤1
457G⬎A
D153N
Missense
N terminus
Atrial fibrillation
Decrease in peak INa
48
␤2
82C⬎T
R28W
Missense
N terminus
Atrial fibrillation
Decrease in peak INa and positive
shift of activation
48
␤2
83G⬎A
R28Q
Missense
N terminus
Atrial fibrillation
Decrease in peak INa and positive
shift of activation and inactivation
48
␤3
17G⬎A
R6K
Missense
N terminus
Lone atrial fibrillation
Decrease in peak INa
␤3
29T⬎C
L10P
Missense
N terminus
Brugada syndrome
Decrease in peak INa and
negative shift of inactivation
47, 82
Decrease in peak INa and positive
shift of activation and inactivation
79, 84
Lone atrial Fibrillation
␤3
161T⬎G
V54G
Missense
N terminus
Idiopathic ventricular fibrillation
Sudden infant death syndrome
Reference
74
47
␤3
106G⬎A
V36M
Missense
N terminus
Sudden infant death syndrome
Decrease in peak INa and
increase in persistent INa
84
␤3
389C⬎T
A130V
Missense
N terminus
Atrial fibrillation
Decrease in peak INa
49
␤3
482T⬎C
L161T
Missense
Transmembrane
segment
Lone atrial Fibrillation
Decrease in peak INa
47
␤4
535C⬎T
L179F
Missense
Transmembrane
segment
Long QT syndrome type 10
Increase in persistent INa and
positive shift of inactivation
85
␤4
671G⬎A
S206L
Missense
C terminus
Sudden infant death syndrome
Increase in persistent INa and
positive shift of inactivation
84
channel by disrupting the interaction between Nav1.5 and
␤1-subunit.68 In heterologous expression systems, coexpression of SCN5A with SCN1B have demonstrated variable
effects of ␤1-subunit on gating, depending on the expression
Figure 4. Schematic representation of the ␤-subunits of cardiac sodium channel (␤1 [A], ␤2 [B], ␤3 [C], and ␤4 [D]), each
containing 1 transmembrane segment. For each ␤-subunit, identified variants that have been linked to either syndrome (see color
code below the schematic illustrations) are depicted.
system used. However, in most cases, an increase in peak INa
amplitude,69,70 a shift of steady-state inactivation toward
more positive potentials,63,71 and a decrease in persistent INa
amplitude has been observed.71,72 Surprisingly, ventricular
myocytes from SCN1B knockout mice showed increased
expression of Nav1.5 on mRNA and protein level, an increase
in peak INa amplitude, and no change in Nav1.5 gating.73
These findings are not in agreement with those observed in in
vitro experiments and suggest that heterologous expression
systems may not mimic the intracellular environment (in
particular the protein–protein interactions) present in cardiac
myocytes. However, the SCN1B knockout mice also displayed lower heart rates and repolarization delay (reflected by
prolonged QTc and action potential durations), and this may
result from an increase in persistent INa attributable to the
absence of the suppressive effects of ␤1-subunit on this
current. Accordingly, individuals with the previously mentioned D1790G mutation in Nav1.5, which disrupts the
interaction of Nav1.5 with the ␤1-subunit, also experienced
sinus bradycardia and QTc prolongation.68
Mutations in SCN1B have been found in individuals with
BrS, cardiac conduction disease (CCD), or AF. In a recent
study by Bezzina and colleagues, 282 probands with BrS and
44 with CCD were screened for mutations in SCN1B.74 A
nonsense mutation (c.536G⬎A resulting in p.W179X) was
identified in 4 related individuals, of whom 3 displayed signs
of BrS and CCD on their ECGs. This mutation is predicted to
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produce a prematurely truncated protein lacking the
membrane-spanning segment and the carboxyl terminus. A
different nonsense mutation (c.537G⬎A resulting in
p.W179X) was identified in a father and his daughter, of
whom only the daughter displayed signs of CCD (right
bundle branch block and prolonged PR interval). In both
individuals, BrS was unlikely, as established by a negative
sodium channel drug challenge test. Finally, a missense
mutation (c.259G⬎C resulting in p.E87Q), located in the
amino terminus of the ␤1-subunit, was found in 3 family
members, of whom 2 displayed signs of CCD on their ECGs
(complete left bundle branch block, right bundle branch
block, and left anterior hemiblock). It must be noted that the
families in this study were too small to perform linkage
analysis between the phenotypes and the mutations, and that
the mutations were also found in individuals with no phenotype. In another study, Roden and colleagues screened 842
AF patients for mutations in SCN1B to SCN4B and identified
a mutation in SCN1B in 2 unrelated individuals.48 The
mutations (c.254G⬎A resulting in p.R85H, and c.457G⬎A
resulting in p.D153N) were both located in the amino
terminus of the ␤1-subunit. Coexpression of all of these
mutant ␤1-subunits with Nav1.5 in CHO cells resulted in a
decrease of peak INa density compared with coexpression of
Nav1.5 with normal ␤1-subunits, suggesting that ␤1-subunits
increase the sarcolemmal expression of Nav1.5 in cardiac
myocytes.48,74 In addition, p.W179X, p.E87Q, and p.R85H
also caused a shift of steady-state activation toward more
positive potentials, indicating delayed activation of the channels. These findings are in line with the INa reduction found
for virtually all SCN5A mutations related to BrS and CCD,
and for few SCN5A mutations related to AF.13 INa reduction
is speculated to predispose to AF by slowing the conduction
velocity of electric stimuli through the atria, thereby facilitating the maintenance of reentrant excitation waves.75
␤2-Subunit
The ␤2-subunit, encoded by SCN2B, coimmunoprecipitates
with Nav1.5 and colocalizes at the intercalated disks in
cardiac myocytes.63,76 Coexpression of Nav1.5 with the ␤2subunit resulted in a shift of the steady-state activation toward
more positive potentials compared with Nav1.5 expression
alone.48 A similar effect was observed when Nav1.5 was
coexpressed with the intracellular domain of the ␤2-subunit,
suggesting that this portion of the protein may interact with
Nav1.5 to modulate the gating.77 A recent study revealed that
the effect of the ␤2-subunit on Nav1.5 activation depends on
the presence of sialic acid residues on the ␤2-subunit (ie, the
extent of ␤2-sialylation). When completely sialylated, ␤2subunit caused a reverse shift of the steady-state of activation,
ie, toward more negative potentials.78
In the above-mentioned study by Roden and colleagues,
mutations in SCN2B were identified in 2 unrelated individuals with AF.48 The mutations (c.82C⬎T resulting in p.R28W,
and c.83G⬎A resulting in p.R28Q) were located in the amino
terminus of the ␤2-subunit, and their coexpression with
Nav1.5 resulted in a decrease of peak INa density and a shift
of steady-state activation toward more positive potentials
compared with when Nav1.5 was coexpressed with normal
891
␤2-subunits.48 Both of these effects led to INa reduction, and,
accordingly, the individuals carrying the mutations were
reported to display electrocardiographic BrS-like changes.
Although ␤2-subunits were previously shown not to colocalize with Nav1.5 in the endoplasmic reticulum, indicating they
are transported separately to the sarcolemma,63 the decrease
in peak INa density by the mutant ␤2-subunits suggests that
␤2-subunits play a role in controlling the sarcolemma expression of Nav1.5.
␤3-Subunit
The ␤3-subunit, encoded by SCN3B, coimmunoprecipitates
with Nav1.5.79 An initial study showed that the ␤3-subunit is
highly expressed in the ventricles and Purkinje fibers but not
in atrial tissue of sheep hearts.80 However, recently, the
␤3-subunit expression was demonstrated in mouse atrial and
ventricular tissues.81 Compared with Nav1.5 alone, coexpression with the ␤3-subunit resulted in increase in peak INa
amplitude and a shift of steady-state inactivation toward more
positive potentials (in Xenopus oocytes),80 or no change in
peak INa amplitude and a shift of steady-state inactivation
toward more negative potentials (in CHO cells and in
HEK-293 cells).72,79 These findings illustrate again that the
regulatory effects of ␤-subunits on Nav1.5 may vary significantly between heterologous expression systems, probably
because of differences in intracellular environments of various expression systems and conditions in which the cells are
cultured (eg, lower culture temperature for Xenopus oocytes).
Interestingly, coexpression of Nav1.5 with ␤1- and ␤3subunits caused an additional shift of inactivation toward
more negative potentials, suggesting that ␤-subunits may act
synergistically to regulate gating.72 Compared with wild-type
mice, SCN3B knockout mice displayed slower heart rates, longer
P wave durations, and prolonged PR intervals on their ECGs,
indicating slowing of electric conduction in the heart.81 In
addition, electrophysiological studies demonstrated inducibility
of atrial tachycardia, AF, and ventricular tachycardia in hearts of
SCN3B knockout mice, indicating increased atrial and ventricular arrhythmogenesis.81 Ventricular myocytes isolated from
SCN3B knockout mice hearts showed a decrease in peak INa
density and a shift of steady-state inactivation toward more
negative potentials compared with ventricular myocytes isolated
from wild-type hearts.81 These data are in agreement with those
obtained from coexpression studies of ␤3-subunit with Nav1.5 in
Xenopus oocytes.80
To date, 4 mutations in SCN3B have been linked to
arrhythmic phenotypes in humans. Using a candidate gene
approach, Antzelevitch and colleagues identified a missense
mutation in SCN3B (c.29T⬎C resulting in p.L10P, located in
the amino terminus of ␤3-subunit) in a patient displaying the
ECG signs of BrS.82 By a similar approach, Makielski and
colleagues identified a missense mutation (c.161T⬎G resulting in p.V54G, also located in the amino terminus of
␤3-subunit) in a patient with aborted cardiac arrest, attributable to documented ventricular fibrillation, in the absence of
structural heart disease or any well-defined arrhythmic phenotype.79 Coexpression studies revealed that both these mutations cause a decrease in peak INa density and a shift in
steady-state inactivation compared with coexpression of
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Nav1.5 with normal ␤3-subunits. Additionally, immunocytochemistry revealed that the mutant ␤3-subunits decreased the
expression of Nav1.5 by disrupting its transport from the
endoplasmic reticulum to the sarcolemma.79,82 It should be
noted that in both cases, there is not much evidence for
loss-of-function of cardiac sodium channel activity as even in
the presence of procainamide conduction intervals are within
normal limits, whereas with SCN5A loss-of-function mutations, conduction is generally prolonged at different levels.26,83 Recently, the p.V54G mutation was also linked to
SIDS.84 Makielski and colleagues screened 292 SIDS cases
for mutations in ␤-subunit– encoding genes and found a
mutation in SCN3B in 2 cases (V54G, and c.106G⬎A
resulting in p.V36M, located in the amino terminus). Coexpression of Nav1.5 with p.V36M ␤3-subunit resulted in a
decrease in peak INa density but, interestingly, also in an
increase of the persistent INa, compared with coexpression
with normal ␤3-subunits, showing that mutations in ␤3subunits may also cause INa gain of function.84 Taken
together, findings from these translational studies are largely
in line with those obtained from in vitro experiments and
SCN3B knockout mice and indicate that ␤3-subunit plays an
important regulatory role in the sarcolemmal expression and
gating of Nav1.5. However, recently, Wang and colleagues
sequenced 477 patients with AF for mutations in SCN3B and
identified a missense mutation in SCN3B (c.389C⬎T resulting in p.A130V, located in the distal portion of the amino
terminus) in one patient.49 When coexpressed with Nav1.5,
the mutant ␤3-subunit resulted in a decrease in peak INa
density and no gating changes. Surprisingly, Western blot
analysis revealed that the mutation did not affect the surface
expression of Nav1.5. Based on these data, the authors
speculated that p.A30V may decrease INa by impairing the
conductance, but not the intracellular trafficking, of Nav1.5.
Although this is an interesting hypothesis, it requires further
experimental investigation. Recently, 3 additional loss-offunction mutations in SCN3B have been associated with lone
AF (R6K, L10P, M161T).47 It must be noted that all SCN3B
mutations linked to arrhythmic phenotypes are located in the
amino terminus of the ␤3-subunit, suggesting this portion of
the protein is important for its regulatory role in the expression and gating of Nav1.5.
␤4-Subunit
The ␤4-subunit is encoded by SCN4B and coimmunoprecipitates with Nav1.5,85 and its expression in the heart has been
demonstrated on both mRNA and protein levels.86,87 Coexpression of Nav1.5 with the ␤4-subunit did not change peak
INa amplitude but shifted the steady-state inactivation toward
more negative potentials compared with expression of Nav1.5
alone.84,85 Recently, the ␤4-subunit has been proposed as a
potential genetic modifier of conduction slowing in individuals with cardiac Na⫹ channel loss of function.86 This study
aimed to assess the role of genetic modifiers on the variable
severity of conduction slowing in patients with the SCN5A
p.1795insD mutation. To investigate the effect of genetic
background without the confounding influences of environmental factors, 2 different mouse strains (FVB/N and 129P2),
both carrying the mouse homolog of the p.1795insD muta-
tion, were studied. Similar to the human phenotype, both
transgenic mice strains displayed bradycardia and conduction
slowing. However, 129P2 mice displayed more severe conduction slowing. Pan-genomic mRNA expression profiling
and subsequent Western blot analysis revealed a decrease in
SCN4B mRNA and ␤4-subunit protein levels, respectively, in
129P2 mice compared with FVB/N mice. In addition, measurements of INa in ventricular myocytes did not find differences in peak INa but uncovered a shift of steady-state
activation toward more positive potentials in 129P2 mice
compared with FVB/N, indicating delayed opening of the
Na⫹ channels. Finally, computer simulation models predicted
that this delay in activation may be responsible for the
differences in conduction slowing between the mouse
strains.86 This report first recognizes a ␤-subunit of the
cardiac Na⫹ channel as a genetic modifier of phenotype
severity in individuals with a mutation in SCN5A.
The importance of the ␤4-subunit is further reflected by
recent studies linking mutations in SCN4B to LQTS (LQT10)
and SIDS. Ackerman and colleagues identified a mutation
(c.535C⬎T resulting in p.L179F, located in the transmembrane segment of the ␤4-subunit) in a LQTS family and a
history of unexpected and unexplained sudden deaths.85
Coexpression of Nav1.5 with mutant ␤4-subunits resulted in
an increase of persistent INa and a shift of steady-state
inactivation toward more positive potentials compared with
coexpression of Nav1.5 with normal ␤4-subunits. In the
above-mentioned study on 272 SIDS cases, a mutation in
SCN4B (c.671G⬎A resulting in p.S206L, located in the
carboxyl terminus) was also identified.84 Coexpression experiments in HEK-293 cells showed that, similar to p.L179F, the
p.S206L mutation also resulted in an increase in persistent INa
and a shift of steady-state inactivation toward more positive
potentials. Accordingly, adult rat ventricular myocytes, adenovirally transduced with the p.S206L mutant ␤4-subunits,
displayed an increase in persistent INa and prolongation of
action potential duration compared with myocytes transduced
with wild-type ␤4-subunits.84 Taken together, effects of these
2 SCN4B mutations on the persistent INa and Nav1.5 gating
are in line with effects previously described for SCN5A
mutations related to LQTS.88
Glycerol 3 Phosphate Dehydrogenase
1-Like Protein
Approximately 20% of BrS cases have been linked to
mutation in SCN5A (see below), and only in a few cases have
mutations in ␤-subunit– encoding genes been found. In search
for other genetic causes, London and colleagues linked a
multigenerational BrS family to a locus at chromosome
3p22-24 and excluded a disease-causing mutation in SCN5A
by linkage and direct sequencing.89 In a further study, the
authors identified a missense mutation (c.899C⬎T resulting
in p.A280V) in GPD1-L, located within the previously found
locus, encoding the glycerol-3-phosphate dehydrogenase
1-like protein (GPD1-L).90 This protein displays 84% homology with the glycerol-3-phosphate dehydrogenase 1 (GPD1),
a cytoplasmic enzyme that catalyzes the dehydrogenation of
glycerol-3-phosphate (G3P) to dihydroxyacetone phosphate
(DHAP) in the glycolytic pathway and the reduction of
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DHAP to G3P in the synthesis of triglycerides.91 GPD1-L
was shown to be expressed abundantly in the heart. Compared with coexpression of Nav1.5 with wild-type GPD1-L,
its coexpression with p.A280V GPD1-L resulted in a marked
decrease of peak INa density and reduction of cell surface
expression of Nav1.5.90 In another study, Ackerman and
colleagues screened a large cohort of SIDS cases for mutations in GPD1-L.92 They found 3 novel missense mutations in
GPD1-L (c.307G⬎A resulting in p.E83K, c.430A⬎G resulting in p.I124V, and c.877C⬎T resulting in R273C). Similar
to the BrS-linked p.A280V mutation, coexpression of Nav1.5
with each of these mutations cells caused a decrease in peak
INa density. Accordingly, adenovirus-mediated transduction
of neonatal mouse myocytes with p.E83K GPD1-L reduced
INa compared with when myocytes were transduced with
wild-type GPD1-L.93 Based on these data, GPD1-L was
suggested to play a role in the intracellular trafficking of
Nav1.5 to the sarcolemma.90 –93
More recently, Makielski and colleagues demonstrated in
HEK-293 cells that, like GPD1, GPD1-L catalyzes the
reaction of G3P to DHAP, and that the BrS-linked p.A280V
mutation and the SIDS-linked p.E83K mutation reduce the
enzymatic activity of GPD1-L.92 Next, they showed that
reduced GPD1-L activity results in an increased phosphorylation of Nav1.5 at residue S1503, and this phosphorylation is
previously recognized to decrease INa.94 The authors hypothesized that GPD1L loss of function increases the substrate
G3P and feeds the protein kinase (PK)C-mediated phosphorylation of Nav1.5. Indeed, additional experiments, whereby
PKC activity was stimulated by G3P or inhibited by staurosporine, indicated that Nav1.5 phosphorylation and subsequent INa decrease occur through a G3P-dependent PKCmediated pathway.92 Although this is an interesting model,
future studies confirming these findings in a more native
environment are required. Finally, it is worth mentioning that,
by using a GST pull-down assay, Makielski and colleagues
also showed a previously unrecognized interaction between
Nav1.5 and GPD1-L, which was not disrupted by mutations in
GPD1-L. However, they did not determine the location of this
interaction or whether it was direct or mediated by channel
interacting proteins.
Caveolin-3
Caveolae are small narrow-necked invaginations of the
plasma membrane of various types that are enriched with
lipids (cholesterol and sphingolipids) and proteins (ion channels, receptors, transporters, and signaling molecules).
Caveolins are responsible for the formation of caveolae and
the maintenance of their structure. Caveolin-3 is the main
isoform expressed in the heart. Coimmunoprecipitation of
Nav1.5 with caveolin-3 has been shown in both rat cardiac
myocytes and HEK-293 cells.95,96 Immunocytochemistry
showed that Nav1.5 colocalizes with caveolin-3,95 particularly in the caveolae at the sarcolemma but not intracellularly.97 Interestingly, Shibata and colleagues have shown that
␤-adrenergic stimulation of rat ventricular myocytes with
isoproterenol, in the presence of a PKA inhibitor, causes an
increase in peak INa. Importantly, this effect could be mimicked by the application of the stimulatory G-protein
893
␣-subunit, G␣s, and abolished by the addition of antibodies
against G␣s or caveolin-3.95,98 These findings suggest that
G␣s and caveolin-3 act in conjunction to increase peak INa
through a cAMP-independent pathway. The authors speculated that a reservoir of Nav1.5 may be located in caveolar
invaginations, where the proteins are not exposed to the
extracellular side of the myocyte. By ␤-adrenergic stimulation and through a G␣s- and caveolin-3– dependent pathway,
the caveolar neck will open and allow Nav1.5 to become
functional. In this line of research, it has also been demonstrated that a histidine residue at position 41 of G␣s is critical
in the regulation of cAMP-independent increase in INa.99 The
exact molecular mechanisms by which G␣s or caveolin-3
directly or indirectly (eg, through adaptor proteins such as
dystrophin and ankyrin that are also present in the caveolae)
interact with Nav1.5 and cause an increase in functional INa
remain to be unraveled.
Based on the preceding evidence, Vatta et al hypothesized
that mutations in caveolin-3 may underlie LQTS.96 They
screened 905 unrelated patients with LQTS for mutations in
CAV3, the gene encoding caveolin-3, and identified 4 novel
missense mutations (c.233C⬎T resulting in p.T78M in 3
patients, c.253G⬎A resulting in p.A85T in 1 patient,
c.290T⬎G resulting in p.F97C in 1 patient, and c.423C⬎G
resulting in p.S141R in 1 patient, LQT9). Three mutations are
located in the intramembrane domain, and the p.S141R
mutation is located in the cytoplasmic carboxyl terminus of
caveolin-3. Compared with the coexpression of Nav1.5 with
wild-type caveolin-3, its coexpression with mutant p.F97C or
p.S141R caveolin-3 resulted in an increase in persistent INa
with no changes in peak INa density and gating.96 This is in
agreement with the effect of SCN5A mutations linked to
LQTS type 3 on Nav1.5.13 Importantly, Vatta et al also
displayed that both p.F97C and p.S141R caveolin-3 coimmunoprecipitate with Nav1.5, indicating that the increase in
persistent INa is not attributable to loss of interaction between
mutant caveolin-3 and Nav1.5. More recently, Ackerman and
colleagues screened 134 SIDS cases for mutation in CAV3
and identified 3 distinct mutations in 3 cases (p.T78M
previously linked to LQT9, c.40G⬎C resulting in p.V14L,
located in the amino terminus, and c.236T⬎G resulting in
p.L79R, located in the intramembrane domain).100 Similar to
the LQTS-linked mutations in caveolin-3, coexpression of
Nav1.5 with each of these 3 mutations resulted in a marked
increase in persistent INa compared with coexpression with
wild-type caevolin-3.
Finally, it is noteworthy that the LQTS patient with the
p.F97C mutation in caveolin-3 showed QT prolongation only
but reproducibly during ␤-agonist inhaler therapy for
asthma.96 This phenomenon is in line with the previous
experimental data demonstrating that caveolin-3 plays a role
in the functional increase in INa on ␤-adrenergic stimulation95,98,101 and suggests that the mutation-induced persistent
INa may also increase during such conditions.
␣1-Syntrophin
The SNTA1-encoded ␣1-syntrophin (SNTA1) is a member of
the dystrophin-associated multiprotein complex that interacts
directly with the PDZ domain– binding motif formed by the
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last 3 residues (serine-isoleucine-valine) of the Nav1.5 carboxyl terminus.102 SNTA1 contains 4 domains, including 2
pleckstrin homology domains (PH1 and PH2), a PDZ domain
within the PH1 domain that interacts with Nav1.5, and a
syntrophin unique (SU) domain. Previously, SNTA1 had
been shown to interact also with the neuronal nitric oxide
synthase (nNOS), which is constitutively expressed the heart,
and the cardiac isoform of plasma membrane Ca2⫹/calmodulin-dependent ATPase (PMCA4b).103,104 Of note, in the
presence of SNTA1, PMCA4b acts as a potent inhibitor of
NO production by nNOS. Importantly, NO has been demonstrated to increase the amplitude of persistent INa in ventricular myocytes.105 Based on these findings, Makielski and
colleagues hypothesized that mutations in SNTA1 may disrupt its interaction with PMCA4b, thereby nullifying the
ability of PMCA4b to inhibit nNOS, resulting in increased
NO synthesis and increased persistent INa.106 They screened
50 unrelated individuals with LQTS for mutations in SNTA1
and identified one missense mutation (p.A390V) in one single
individual (LQT12). GST-pull-down assay revealed an interaction between Nav1.5 carboxyl terminus, nNOS, PMCA4b,
and wild-type SNTA1. The p.A390V mutation selectively
disrupted the interaction of PMCA4b with Nav1.5 and nNOS,
and this was associated with increased nitrosylation of Nav1.5
as detected by S-nitrosylation biotin switch assay. Moreover,
coexpression of Nav1.5, nNOS, PMCA4b, and p.A390VSNTA1 in HEK293 cells increased the peak and persistent INa
and shifted the steady-state inactivation toward more positive
potentials resulting in larger inward window current compared with coexpression with wild-type SNTA1. These gainof-function effects were partially abolished when cells were
cultured in the presence of NG-monomethyl-L-arginine (LNMMA), an NOS inhibitor. Moreover, adenoviral transduction of neonatal mouse myocytes with p.A390V-SNTA1 also
increased the persistent INa compared with wild-type SNTA1.106
Taken together, these data indicate that, by acting as a scaffolding protein for Nav1.5, nNOS, and PMCA4b, SNTA1 plays a
key role in regulating the level of Nav1.5 nitrosylation, and
thereby the amplitude of peak and persistent INa.
According to this study, Vatta and colleagues identified a
missense mutation (p.A257G) in 3 of 39 individuals with
LQTS.107 Compared with wild-type SNTA1, p.A257GSNTA1 caused an increase in peak INa and a shift of
steady-state activation toward more negative potentials resulting in larger inward window current in HEK-293 cells and
neonatal rat myocytes. Although the p.A257G mutation did
not alter the persistent INa, its gain-of-function effects are in
line with those observed for LQTS type 3-related mutations
in SCN5A.88 Finally, mutations in SNTA1 have also been
linked to SIDS.108 Ackerman and colleagues screened 292
SIDS cases, and identified 6 distinct SNTA1 mutations
(p.G54R, p.P56S, p.T262P, p.S287R, p.T372M, and
p.G460S) in 8 cases. Interestingly, when coexpressed with
Nav1.5 in HEK-293 cells, the p.G54R and p.P56S mutations
did not affect INa compared with wild-type SNTA1. The
p.S287R mutation increased only the peak INa, whereas
p.S287R, p.T372M, and p.G460S caused similar gain-of-
function effects as the LQTS-linked p.A390V mutation (ie,
increase in peak and persistent INa and a shift of steady-state
inactivation toward more positive potentials). Because the
mutations causing an increase in both peak and persistent INa
(p.A390V, p.S287R, p.T372M, and p.G460S) were all located in or near to the PH2 and SU domains of SNTA1, the
authors speculated that these mutations disrupt the interaction
of SNTA1 with PMCA4b, thereby abolishing its inhibitory
effect on nNOS. In contrast, mutations located in the PH1
domain of SNTA1 (the LQTS-linked p.A257G and SIDSlinked p.G54R, p.P56S, p.T262P) may not possess the ability
to disrupt this interaction.
Proteins Not Associated With Arrhythmias
But With Specific Interactions With SCN5A
A number of other enzymes and regulatory proteins have
been shown to interact with the Nav1.5, but, as yet, no
disease-related variants in any of these proteins have been
identified (Table III and references in the Online Data
Supplement, available at http://circres.ahajournals.org). However, for a number of individual mutations in SCN5A, it has
been shown that the interaction between Nav1.5 and the
relevant protein is modified, giving rise to a distinct phenotype. Notable examples are the A1924T mutation in the IQ
motif of Nav1.5 that abolishes the enhanced entry of the
channels into slow inactivation induced by calmodulin, resulting in reduced cardiac excitability109; the BrS-linked
E1053K in Nav1.5 mutation that abolishes ankyrin G binding
to Nav1.5 and prevents normal channel trafficking to the
intercalated discs110,111; and the above-mentioned LQTS type
3–linked D1790G that abolishes binding of fibroblast growth
factor homologous factor 1B, thereby abolishing the negative
shift of inactivation.112
Conclusion
A short 2 decades of research have raised enormous insight
into the role of the cardiac sodium channels and their
regulatory subunits in arrhythmogenesis and have led to the
description of new disease entities. For some patients with
monogenic diseases, tailored therapy has ensued; for others,
nothing has changed or therapeutic choices have become
even more complicated. Basic insight into the role of all of
these proteins assembled in the multiprotein cardiac sodium
channel complex has been greatly advanced. Yet, many
details are still lacking, among which, for example, which ␤
subunits associate with which ␣ subunits. Is this only
Nav1.5? Furthermore, the spatial, temporal, and developmental distribution of these interaction are only partly known. In
addition, it is still incompletely understood why, eg, loss-offunction mutations in SCN5A lead to so many different
phenotypes. Gender, age, differential expression throughout
the heart, coexpression, eventual tissue specific, and expression of other genetic variants (eg, the connexin variant
discussed above) all may play a role, and the interaction of
SCN5A is complex but is being increasingly unraveled. In the
end, this may lead to a better prospect for all patients with
these rare diseases, as well as for those with far more
common disease entities, among which are heart failure– or
Wilde and Brugada
Cardiac Disease and Subunits of the Sodium Channel
ischemia-related atrial and ventricular arrhythmias, all with a
mutifactorial background.
Acknowledgments
We thank Dr Ahmad Amin for help with the manuscript and
preparation of the figures.
Sources of Funding
Support for A.A.M.W. is supported by the Leducq Foundation
(CVD05, “Alliance against Sudden cardiac Death”). R.B. is supported by Fondos Investigacion Sanitarias (FIS), Centro Nacional
Investigaciones Cardiologicas (CNIC), Leducq Foundation, Obra
Social La Caixa.
Disclosures
A.A.M.W. is a member of the scientific advisory committee of
PGxHealth. R.B. is a paid consultant for Gendia SL.
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Phenotypical Manifestations of Mutations in the Genes Encoding Subunits of the Cardiac
Sodium Channel
Arthur A.M. Wilde and Ramon Brugada
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Circ Res. 2011;108:884-897
doi: 10.1161/CIRCRESAHA.110.238469
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Online Table I: Proteins or protein families that have been reported to interact with and regulate Nav1.5, but
are not (yet) associated with arrhythmias. Protein
Type
Binding region on
Nav1.5
Effect on Nav1.5
Effect on INa
Ref
.
Nedd4-like
enzymes
(ubiquitin-protein
ligases)
Enzyme*
PY motif in C-terminus
Ubiquitylation and
internalization
Decrease in peak INa
1-3
14-3-3η
Regulator
y protein*
#
Cytoplasmic linker DI-II
Negative shift of inactivation
Decrease in INa
4
Fibroblast growth
factor homologous
factor 1B (FHF1B)
Regulator
y protein
Amino acids 1773-1832
in C-terminus
Negative shift of inactivation
Decrease in INa
5
Calmodulin
Regulator
y protein
IQ motif in C-terminus
Positive shift of inactivation
(only in Ca2+-free conditions)
Increase in INa
6-7
Ca2+/calmodulindependent protein
kinase IIδc
(CAMKIIδc)
Enzyme*
#
Not determined
Phosphorylation
Decrease in INa
8-9
Negative shift of inactivation
Increase in persistent
INa
Protein tyrosine
kinase Fyn
Enzyme*
Protein tyrosine
phosphatase H1
(PTPH1)
Enzyme
Telethonin
Delayed recovery from
inactivation
Cytoplasmic linker DIIIIV
Delayed recovery from
inactivation
Not determined
Phosphorylation
Increase in INa
10
Decrease in INa
11
Increase in INa
12
Positive shift of inactivation
PDZ domain binding
motif in C-terminus
Dephosphorylation
Regulator
y protein*
#
Not determined
Silencing induces:
Multicopy
suppressor of gsp 1
(MOG1)
Adapter
protein* #
Cytoplasmic linker DII-III
Increase cell surface expression
Increase in peak INa
13
Plakophilin-2
Adapter
and
regulatory
protein* #
Not determined
Silencing induces:
Increase in INa
14
Increase in peak INa
15
Negative shift of inactivation
Positive shift of activation
Decrease in INa
Negative shift of inactivation
Delayed recovery from
inactivation
Ankyrin G
Adapter
protein* #
Cytoplasmic linker DII-III
Transport to cell surface
* In vivo effect has been demonstrated in cardiac tissue.
# Co-localization with Nav1.5 (at the intercalated disk of myocytes): 14-3-3η
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