Molecular Pathophysiology of Congenital Long QT Syndrome

Physiol Rev 97: 89 –134, 2017
Published November 2, 2016; doi:10.1152/physrev.00008.2016
MOLECULAR PATHOPHYSIOLOGY OF CONGENITAL

LONG QT SYNDROME
M. S. Bohnen, G. Peng, S. H. Robey, C. Terrenoire, V. Iyer, K. J. Sampson, and R. S. Kass
Department of Pharmacology, Columbia University Medical Center, New York, New York; and The New York
Stem Cell Foundation Research Institute, New York, New York
Bohnen MS, Peng G, Robey SH, Terrenoire C, Iyer V, Sampson KJ, Kass RS.
L
Molecular Pathophysiology of Congenital Long QT Syndrome. Physiol Rev 97: 89 –134,
2017. Published November 2, 2016; doi:10.1152/physrev.00008.2016.—Ion
channels represent the molecular entities that give rise to the cardiac action potential,
the fundamental cellular electrical event in the heart. The concerted function of these
Downloaded from http://physrev.physiology.org/ by 10.220.33.3 on November 5, 2016

channels leads to normal cyclical excitation and resultant contraction of cardiac muscle. Research
into cardiac ion channel regulation and mutations that underlie disease pathogenesis has greatly
enhanced our knowledge of the causes and clinical management of cardiac arrhythmia. Here we
review the molecular determinants, pathogenesis, and pharmacology of congenital Long QT Syn-
drome. We examine mechanisms of dysfunction associated with three critical cardiac currents
that comprise the majority of congenital Long QT Syndrome cases: 1) IKs, the slow delayed
rectifier current; 2) IKr, the rapid delayed rectifier current; and 3) INa, the voltage-dependent
sodium current. Less common subtypes of congenital Long QT Syndrome affect other cardiac
ionic currents that contribute to the dynamic nature of cardiac electrophysiology. Through the
study of mutations that cause congenital Long QT Syndrome, the scientific community has ad-
vanced understanding of ion channel structure-function relationships, physiology, and pharmaco-
logical response to clinically employed and experimental pharmacological agents. Our understand-
ing of congenital Long QT Syndrome continues to evolve rapidly and with great benefits: genotype-
driven clinical management of the disease has improved patient care as precision medicine
becomes even more a reality.
I. INTRODUCTION 89 yielded much insight into the pathophysiology of congenital

II. IKs DYSFUNCTION IN CONGENITAL... 91 LQTS.
III. IKr DYSFUNCTION IN CONGENITAL... 99
IV. INa DYSFUNCTION IN CONGENITAL... 106 Clinically, congenital LQTS patients often first present after
V. OTHER SUBTYPES OF CONGENITAL LQTS 114 episodes of syncope and/or seizure, and the ECG reveals a
VI. GENOTYPE-DRIVEN CLINICAL... 116 prolonged QT interval. The ECG measures electrical activ-
VII. CONCLUSIONS 120 ity of the heart over time, at the patient’s body surface. The
primary electrical signals observed include the P wave,
which signifies atrial depolarization; the QRS complex,
I. INTRODUCTION
which arises from ventricular depolarization; and the T
wave, due to ventricular repolarization (FIGURE 1A). The
A genetic disorder disrupting electrical activity in the heart,
QT interval, therefore, reflects the time elapsed from the
congenital Long QT Syndrome (LQTS) can lead to life-
initiation of ventricular depolarization to the end of ventric-
threatening arrhythmias and sudden cardiac death. In the
ular repolarization. The QT interval shortens with increas-
first cases of congenital LQTS, described in 1957, several
children in one family presented with prolongation of the ing heart rate thus requiring a normalization, or “correc-
QT interval on the electrocardiogram (ECG) and congenital tion,” for heart rate. For a diagnosis of LQTS, this rate-
deafness (189). This came to be known as Jervell and corrected QT (QTc) interval prolongation on a 12-lead
Lange-Nielsen syndrome (JLNS), the autosomal recessive ECG generally is referenced as ⬎470 ms for males and
form of LQTS. The more common, autosomal dominant ⬎480 ms for females. QTc also varies with age, and thus, an
form of congenital LQTS that presents without deafness age-appropriate prolonged QTc interval in a patient aids
was first described in 1963 and 1964 in two separate cases the diagnosis of LQTS (209). However, diagnosis of LQTS
and became known as Romano-Ward syndrome (346, based on absolute QT interval cutoffs can be challenging,
459). Since these initial patient descriptions, advances in since there is considerable overlap in the QTc distribution
our understanding of the mechanisms of cardiac electrical of affected patients and otherwise healthy individuals
excitability at the tissue, cellular, and molecular level have (193). Asymptomatic patients can have intervals beyond
0031-9333/17 Copyright © 2017 the American Physiological Society 89

BOHNEN ET AL.
A The ventricular cellular action potential results from the

summation of a large number of ion channel currents and
QRS
electrogenic pumps that control the cellular membrane po-
tential (see Nerbonne and Kass review, Ref. 299), but in this
ECG
review we will focus on three key ion channels that are
P-Wave T-Wave
well-established to be linked to LQTS and that are illus-
trated in FIGURE 1B. The cellular resting membrane poten-
QT Interval tial is approximately ⫺85 mV, determined largely by in-
wardly rectifying K⫹ channels. Inward rectification is a
B property that hinders the outward flow of K⫹ as membrane
potentials become positive, but passes K⫹ more efficiently
at potentials negative to the K⫹ equilibrium potential where
the flow of K⫹ would be inward. Hence, the term “inward
Ventricular AP rectification” is used to describe these channels (304). Ac-
tivation of Nav1.5, the primary voltage-gated sodium chan-

nel in the heart, leads to sodium influx (INa) and membrane
depolarization. As the cell reaches approximately ⫺40 mV,
voltage-gated L-type calcium channels begin to open, lead-
ing to calcium influx (ICa) (299). Concurrently during the
IKs
upstroke of the action potential, potassium channels, in-
cluding those carrying the IKs and IKr delayed rectifier cur-
IKr
rents, begin to activate slowly. As the cell reaches ⫹30 mV,
INa
INa inactivates almost completely. At this time, a brief and
small repolarization of the membrane potential occurs via
fast activation of the transient outward K⫹ current, Ito. In
FIGURE 1. ECG to cellular ionic currents. A: membrane depolar-
the plateau phase that follows, influx of Ca2⫹ through volt-
ization and the rapid upstroke of the ventricular action potential give age-gated calcium channels is balanced mainly by IKs and
rise to the QRS complex. The duration of the QT-interval corre- IKr. The plateau phase ceases as calcium channels inactivate
sponds to the time to ventricular repolarization. The relatively stable and outward potassium efflux persists, leading to a net out-
membrane potential during the plateau phase of the action potential ward membrane current, and cell repolarization back to the
gives rise to a brief isoelectric period. Ventricular repolarization
gives rise to the T-wave. B: time course of several ionic currents that
cellular resting potential. During this process, the Na⫹-K⫹-
underlie ventricular action potential morphology (currents not to ATPase helps maintain intracellular concentrations of these
scale). The rapidly activating and inactivating INa drives membrane key ions (304).
depolarization. Two K⫹ currents, IKs and IKr, contribute most to the
repolarizing current necessary to drive membrane potential back to In LQTS patients, the QT is prolonged presumably due to
rest.
prolongation of underlying action potential durations
these cutoff and develop no arrhythmias; similarly, QTc

intervals below this cutoff can be seen in patients with es- K+ Na+
tablished LQTS (with clinical arrhythmias and positive ge-
netic testing) (16, 17, 338). Clinical scoring systems (368),
Extracellular
as well as genetic testing, can be helpful to assist with the
diagnosis of congenital LQTS (193), particularly when QT
intervals are on the borderline (within 20 ms of these cut-
offs) or when clinical history is equivocal. This review will
focus primarily on the various forms of congenital LQTS.
Cytosol
Ion channels are the molecular entities underlying most
ionic currents in the heart, allowing passive diffusion of
ions across the cell membrane’s electrochemical gradients K+ Na+
(FIGURE 2). A selectivity filter in the channel pore, deter-
mined by distinct atomic components (152, 315), endows FIGURE 2. Schematic of a generic K⫹ and Na⫹ ion channel. Ion
selective permeation of ions, such as Na⫹, K⫹, and Ca2⫹ channels allow for selective permeation of ions through the plasma
membrane down their electrochemical gradient. The classic K⫹
(246). Some ion channels exhibit voltage-dependent gating, channel consists of four identical pore-forming subunits, whereas
where voltage-sensing domains respond to changes in mem- each Na⫹ channel is formed by a single polypeptide with four homol-
brane potential to cause channel opening or closing (247). ogous domains.
90 Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org

CONGENITAL LONG QT SYNDROME
(APDs), most often caused by decreased repolarizing IKs or encodes hERG), respectively, while LQT3 is associated
IKr activity, or persistent sodium influx that extends with mutations in SCN5A, the gene coding for the Nav1.5
through the plateau phase. A loss of IKs or IKr function, or a sodium channel alpha subunit. Disease association for vari-
gain of INa function, predisposes ventricular myocytes to ants in these three proteins is supported by genome-wide
early afterdepolarizations (EADs), and in some cases to de- association studies (300) and functional electrophysiologi-
layed afterdepolarizations (DADs) which may underlie de- cal characterization of mutant channels. In addition, LQTS-
generation into a characteristic sinusoidal wave pattern on associated mutations exist less frequently in other ion chan-
the ECG, referred to as torsades de pointes, which may nels, modulatory channel subunits, and signaling- or cyto-
further regress into ventricular fibrillation and sudden car- skeleton-associated proteins. Understanding the molecular
diac death. EADs are driven in large part by calcium entry mechanisms that cause LQTS allows for optimization of
via L-type calcium channels during prolonged action poten- genotype-specific treatments. In this review, we discuss the
tial plateau phases, whereas DADs, which occur over the molecular physiology, biology, and pathophysiology un-
diastolic range of potentials after action potential repolar- derlying congenital LQTS, and the cellular and molecular
ization, are caused by intracellular calcium overload, also a underpinnings of genotype-driven clinical management of
consequence of action potential prolongation (126). Addi- LQTS.

tionally, arrhythmic activity may result from altered refrac-
toriness and impulse block, also putative consequences of
II. IKs DYSFUNCTION IN CONGENITAL
prior APD prolongation. Thus treatment in LQTS aims to
LQTS
prevent malignant ventricular arrhythmia by shortening the
QTc interval to minimize cardiac event rates.
A. Introduction
Ion channels may interact with a variety of molecular enti-
ties that contribute to their trafficking, stabilization, signal- Of the different subtypes of inherited LQTS, subtypes 1, 5,
ing, and function (299). Coordination among different ion and 11 are associated with mutations in proteins that par-
channel types facilitates the ionic balance necessary for the ticipate in the macromolecular complex which generates
generation of an action potential and normal electrical and modulates the slow delayed rectifier potassium current
propagation through the heart. LQTS mutations may cause (IKs), which plays a critical role in the repolarization of the
an increase or decrease in ion channel function, disrupting cardiac action potential. Among all LQTS subtypes, LQT1
normal ionic balance leading to pathological electrical ac- is the most common, representing 30-35% of all congenital
tivity in the heart. LQTS (8). Upregulation of IKs during ␤-adrenergic stimu-
lation is critical to normal physiology by shortening ventric-
There are 15 subtypes of congenital LQTS, each associated ular APD and allowing for adequate diastolic filling in the
with mutations on a different gene (15) (TABLE 1). The most context of an elevated heart rate (354). Cardiac events in
common subtypes, LQT1, LQT2, and LQT3, account for patients with IKs-associated LQTS are often triggered by
the vast majority of congenital LQTS. LQT1 and LQT2 are stress and exercise, consistent with the role of adrenergic
associated with mutations in KCNQ1 and KCNH2 (which stimulation in the regulation of IKs (350, 371). Insight into
Table 1. Subtypes of congenital LQTS and their associated genes, proteins, and effects on cardiac currents
LQT Subtype Gene Protein Current
LQT1 KCNQ1 KCNQ1 (Kv7.1) 2IKs

LQT2 KCNH2 hERG (Kv11.1) 2IKr
LQT3 SCN5A Nav1.5 1INa
LQT4 (ankyrin-B syndrome) ANK2 Ankyrin-B Multichannel interactions
LQT5 KCNE1 KCNE1 (minK) 2IKs
LQT6 KCNE2 KCNE2 (MiRP1) 2IKr
LQT7 (Andersen-Tawil syndrome type 1) KCNJ2 Kir2.1 2IK1
LQT8 (Timothy syndrome) CACNA1C Cav1.2 1ICa
LQT9 CAV3 Caveolin 3 1INa
LQT10 SCN4B Nav1.5 ␤4 1INa
LQT11 AKAP9 AKAP-9 (yotiao) 2IKs
LQT12 SNTA1 ␣1-Syntrophin 1INa
LQT13 KCNJ5 Kir3.4 (GIRK4) 2IKACh
LQT14 CALM1 Calmodulin Multichannel interactions
LQT15 CALM2 Calmodulin Multichannel interactions
Physiol Rev • VOL 97 • JANUARY 2017 • www.prv.org 91

BOHNEN ET AL.
the molecular mechanisms of disease mutations have (minK) generates IKs (36, 358). While KCNE1 had previ-
greatly improved our understanding of LQTS pathophysi- ously been thought to be an independent potassium channel
ology and helped provide a first step to the future develop- (167, 412), it was confirmed that KCNQ1 is actually the ␣-
ment of targeted therapies. or pore-forming subunit of IKs while KCNE1 is a critical ␤-
or modulatory subunit. Coexpression of KCNQ1 and
KCNE1 generates the hallmark IKs current with slow acti-
B. Physiology vation. KCNQ1 and KCNE1 have been shown to be ex-
pressed in all four chambers in the heart (45), as well as the
IKs is an outward potassium current with unique kinetics inner ear (301, 351), where IKs is thought to play a role in
and voltage dependence and plays a key role in the repolar- K⫹ secretion into the endolymph (68). This explains the
ization of the cardiac action potential (299). In 1969, the observation that congenital deafness is a key feature of
delayed rectifier potassium current in sheep Purkinje fibers JLNS. In addition, KCNQ1 and KCNE1 are expressed else-
was studied and shown to comprise two kinetically distinct where in the body, including the pancreas, the kidneys, and
components (305). These currents were later subjected to the brain (1).
pharmacological dissection in guinea pig ventricular myo-

cytes and identified as IKs and IKr (360). 1. KCNQ1, the pore-forming subunit
IKs is slowly activating and most prominent during the pla-

KCNQ1, like most voltage-gated potassium channels,
teau and repolarizing phases of the cardiac action potential,
consists of six transmembrane helices (451) (FIGURE 3A).
where it contributes to counterbalancing calcium influx and
Four subunits of KCNQ1 come together to form a chan-
repolarization (FIGURE 1B). Expression of IKs has been dem-
nel that is capable of voltage-dependent gating (FIGURE
onstrated in both human atrial and ventricular myocytes
3B). On each subunit, the helices S1–S4 comprise the
(198, 235, 457). In addition, it has also been measured in
voltage-sensing domain, where the S4 helix, with its pos-
cardiomyocytes from a variety of non-human mammalian
itively charged arginine residues, senses changes in mem-
species including dogs (241, 406, 437, 442, 493) and rab-
brane potential (310). Following the voltage-sensing do-
bits (352). On the other hand, IKs is expressed at very low
main is the pore domain, which comprises the pore-loop,
levels or absent in mouse hearts (478), most likely because
an extracellular segment containing the selectivity filter,
at very high heart rates in mouse heart (⬃500 beats/min)
and helices that line the pore, S5 and S6. Furthermore,
this channel would have little or no time to activate and not
the cytoplasmic loop between S4 and S5 plays important
affect cardiac electrophysiology in the mouse.
roles in the voltage sensor-to-pore coupling and in volt-
age-dependent gating (82, 229, 498), which has been
Importantly, IKs is subject to upregulation by ␤-adrenergic
demonstrated in other voltage-gated channels as well
stimulation to control APD in the face of sympathetic nerve
(75, 122, 355). The cytoplasmic loop between S2 and S3
activity (210, 444). During sympathetic activation, adren-
also plays a role in channel gating (498). The COOH-
ergic stimulation increases the outward IKs current, which
terminal domain (CTD) of KCNQ1 is large and contains
counterbalances the concomitant increase in inward cal-
four intracellular ␣-helices referred to as A-D. A wide
cium current, prevents prolongation of the cardiac APD,
range of functions has been attributed to the CTD includ-
and allows for adequate diastolic filling times between heart
ing calmodulin binding, interaction with ␤-subunits and
beats (386). However, insufficient IKs activation such as
scaffolding proteins, as well as channel assembly and
that seen in LQT1 results in failure to counterbalance the
trafficking (159, 469).
calcium influx, prolonging the action potential and increas-
ing susceptibility to arrhythmia. This is consistent with ex-
ercise being a key trigger of cardiac events seen in LQT1 2. KCNE1, the ␤-subunit
patients (371).
Coexpression of KCNE1 with KCNQ1 leads to a drastic
change in channel function to generate IKs. Most promi-
C. Molecular Biology nently, assembly with KCNE1 leads to a delay in the onset
of activation, an increase in channel amplitude (FIGURE 3C),
The first known subtypes of inherited LQTS, LQT1-3, were as well as a depolarizing shift in the current-voltage rela-
distinguished by mapping to distinct chromosomes, with tionship (not illustrated) (36, 358). This results in a channel
LQT1 mapping to chromosome 11 (191, 212, 213). Even- that, compared with most other voltage-gated potassium
tually it was found that the KCNQ1 (KvLQT1) gene is channels, activates at more positive voltages and with
responsible for LQT1 and encodes a potassium channel slower kinetics. KCNE1 is a 129-amino acid protein that
(451). The current conducted by this channel was rapidly consists of a single transmembrane helix, with an extracel-
activating and minimally inactivating, unlike any previ- lular NH2 terminus and intracellular COOH terminus
ously known current in the heart, but soon it was shown (412). It is thought to have extensive contact with KCNQ1
that KCNQ1 together with the accessory protein KCNE1 including the voltage-sensing domain (37, 84, 203, 309,

A KCNE1 KCNQ1
Extracellular
S1 S2 S3 S4 S5 S6
+
+
+
FIGURE 3. Molecular biology of IKs and
Cytosol regulation by PKA-mediated signaling.
A: the IKs macromolecular complex, in-
cAMP cluding KCNQ1, KCNE1, and associated
–

+ + scaffolding and signaling proteins. B and
C + C: single pulse voltage-clamp recordings
S27 P PKA AC9 PDE4D3 of KCNQ1 expressed alone or coex-
pressed with KCNE1 in Xenopus oocytes.
– AKAP-9 D: dialysis with 200 ␮M cAMP and 0.2
␮M okadaic acid (OA) increases IKs am-
PP1 plitude and slows deactivation when het-
N C
erologously expressed in CHO cells.
[From Chen et al. (78).]
B C D 60mV
–40mV
KCNQ1+KCNE1
–80mV
+cAMP/OA
Control
KCNQ1
40pA/pF
10µA
1s 2s 1s
378, 479), the pore domain (84, 262, 311), as well as the 1988 it was shown that stimulation of PKA activity by a
CTD (161, 510). Previous crosslinking studies suggest that cAMP analog can upregulate the delayed rectifier current
KCNE1 is located in a cleft between the voltage-sensing (444). Later it was shown that the scaffolding protein
domain and pore domain of different KCNQ1 subunits (84, A-kinase anchoring protein 9 (AKAP-9), also known as
479), underlying its ability to modulate KCNQ1 gating. yotiao, plays a central role in adrenergic regulation of IKs
With respect to the stoichiometry of KCNE1 to KCNQ1, by compartmentalizing key elements of the PKA signal-
some studies suggest a fixed 2:4 ratio (278, 326), while ing pathway, allowing for spatiotemporal control.
others suggest a flexibility in stoichiometry (293, 456) that
AKAP-9 binds to the CTD of KCNQ1 and recruits sig-
allows for modulation of kinetics of assembled channels
naling proteins including protein kinase A (PKA), protein
to provide another level of flexibility in channel function.
phosphatase 1 (PP1) (255), adenylyl cyclase 9 (AC9)
Although three other members of the KCNE family,
KCNE2-KCNE4, also are expressed in the heart (45) and (238), and the phosphodiesterase PDE4D3 (419) (FIGURE
are capable of modulating KCNQ1 activity (45, 154, 3A). Together these proteins form the IKs macromolecu-
367, 422), whether they associate with KCNQ1 in vivo lar complex that can tightly control the phosphorylation
to contribute to potassium currents in the heart remains state of the channel in response to adrenergic stimula-
to be explored. tion. PKA phosphorylates KCNQ1 at the S27 residue,
adding a phosphate group and hence a change in charge
3. Molecular components of adrenergic stimulation to this residue, which leads to increased channel activa-
tion and slower deactivation (226, 255) (FIGURE 3D). In
There have been considerable efforts to elucidate the mo- addition, phosphorylation of AKAP-9 itself contributes
lecular pathway for the ␤-adrenergic regulation of IKs. In to the PKA-mediated upregulation of IKs (78).

BOHNEN ET AL.
4. Role of PIP2 and calmodulin in IKs function D. Molecular Pathophysiology
The lipid molecule phosphatidylinositol 4,5-bisphosphate There are more than 530 disease-causing mutations associ-
(PIP2) is critical for KCNQ1 function. PIP2 is found in the ated with the IKs complex (15). While most of these are
inner leaflet of plasma membranes (260) and can regulate a missense mutations, they also include nonsense mutations,
variety of ion channels (51, 83, 172, 179, 250, 475). PIP2 splice site mutations, frameshifts, as well as deletions. The
mainly binds to KCNQ1 at its cytoplasmic loops and the vast majority are mutations in KCNQ1, which cause LQT1,
COOH-terminal region near S6 (80, 208, 498), which are but a number of LQTS-causing mutations have also been
thought to form the interface between the voltage-sensing found in KCNE1 and AKAP-9, which are classified as
domain and the pore domain (82, 497). PIP2 binding is LQT5 and LQT11, respectively. JLNS, an autosomal reces-
sive form of LQTS with severe bilateral sensorineural deaf-
mediated by electrostatic interactions between the an-
ness, has so far only been associated with mutations in
ionic lipid headgroup and positively charged channel res-
KCNQ1 or KCNE1 (423). Nonetheless, the majority of
idues (418). Rundown of PIP2 in the membrane leads to a
LQTS arising from mutations in IKs are autosomal domi-
drastically reduced IKs amplitude and accelerated deacti-
nant in a form known as Romano-Ward syndrome, which is
vation, suggesting that PIP2 stabilizes the open state of

not associated with deafness.
IKs (250). Furthermore, PIP2 rundown reduces the cur-
rent amplitude of KCNQ1 in the absence of KCNE1
Congenital LQTS patients with dysfunction in IKs present
(239). In addition, PIP2 plays a critical role in the cou- with prolongation of the QT interval on ECG. An ECG
pling between voltage sensor movement and pore open- characteristic associated with LQT1 patients more specifi-
ing/closing (498). Interestingly, one modulatory effect of cally is a broad based T wave (FIGURE 4A). Dysfunction of
KCNE1 on KCNQ1 is a large (100-fold) increase in chan- IKs reduces repolarizing current during the plateau phase,
nel sensitivity for PIP2, which contributes to augmenta- thereby leading to APD prolongation (FIGURE 4B). With a
tion of KCNQ1 current amplitude (239). Whether PIP2 prolonged APD, the cardiomyocyte is vulnerable to EADs
level is under control by physiological mechanisms to (441, 500), where a second depolarization occurs prior to
modulate IKs in cardiomyocytes remains to be demon- the complete repolarization of the first due to recovery of
strated. voltage-gated Na⫹ or Ca2⫹ channels from inactivation,
triggering life-threatening arrhythmia such as torsades de
Calmodulin is a calcium-binding protein that serves in myr- pointes (26, 464). As previously mentioned, during adren-
iad calcium signaling pathways, some of which regulate ion ergic stimulation IKs plays an especially important role in
channels (399). Calmodulin has been linked to IKs function controlling the cardiac APD. This manifests clinically in
(141, 379). Multiple studies have shown that calmodulin LQT1 patients as a susceptibility to arrhythmia during ex-
binding to the CTD of KCNQ1 is important for normal ercise (371).
channel trafficking and folding (141, 379).
1. LQT1
5. Channel trafficking
Missense mutations are responsible for the majority of
LQT1 cases and can cause channel loss of function through
A variety of molecular entities are involved in the traf- a variety of molecular mechanisms, including defects in ion
ficking of KCNQ1KCNE1. The small GTPase-RAB11 permeation (altering the pathway through which ions flow
plays an important role in the exocytosis of KCNQ1- through open channels), channel gating (mechanisms that
KCNE1 to the plasma membrane, while RAB5 mediates regulate the opening and/or closing or channels), traffick-
endocytosis of KCNQ1-KCNE1 from the plasma mem- ing, KCNQ1-KCNE1 interaction, PKA-mediated signaling
brane into endosomes (374). In a physiological stress pathway, PIP2 binding, and calmodulin binding (FIGURE 4,
response, serum and glucocorticoid-regulated kinase 1 C AND D) (TABLE 2). Non-missense mutations can also cause
(SGK1) upregulates KCNQ1-KCNE1 expression by in- LQT1. Mutations belonging to certain groups may bear
creasing RAB-11-dependent channel exocytosis (374). implications on patient phenotype, severity of arrhythmia,
There is also evidence to suggest that KCNQ1-KCNE1 as well as response to therapy.
can be internalized from the plasma membrane via clath-
rin-mediated endocytosis, a process facilitated by A) PERMEATION DEFECT. A number of LQT1 mutations that
KCNE1 (480). Furthermore, KCNQ1 expression is reg- lead to defects in permeation are found in the pore region of
ulated by ubiquitination, which can label membrane pro- KCNQ1. Pore mutations are thought in general to carry
teins for internalization and degradation. For example, greater risk of cardiac events than other mutations. One
the ubiquitin ligase Nedd42 can ubiquitinate KCNQ1 study shows that mutations in highly conserved residues,
(190), while ubiquitin-specific protease 2 (USP2) can pre- many of which are located in the pore-loop, are associated
vent channel ubiquitination (221). Both processes allow with higher risk of cardiac events (61, 197). Three LQT1
for control of KCNQ1 expression. mutations near the selectivity filter, T322M, T322A, and

A LQT1 ECG C
40mV
–40mV
–80mV
KCNQ1 + KCNE1
IKs
LQTS IKs
B WT
LQT1 FIGURE 4. IKs dysfunction leading to con-
genital LQTS. A: ECG from a LQT1 patient
demonstrates a characteristic broad-
based T wave (unpublished data). B: simu-
AP lated action potential (top) and IKs (bottom)

in WT (black) and heterozygous LQT1
(gray) conditions, demonstrating the effect
5μA of 50% reduction in IKs. C: single pulse
voltage-clamp IKs recording in Xenopus
IKs oocytes demonstrating loss of channel
2s
function in an IKs-associated LQTS muta-
tion. D: topology of KCNQ1 and KCNE1 in
D KCNE1 KCNQ1 the plasma membrane. Several examples
of LQT1- and LQT5-associated mutations
N are highlighted in blue and teal, respec-
S225L tively. These mutations represent a variety
of mechanisms of loss-of-function including
Extracellular
disruption of permeation (yellow-filled
square), gating (yellow-filled circle), traffick-
ing (yellow-filled triangle), PKA-mediated
L51H + signaling (yellow-filled star), KCNQ1-
+ G314S KCNE1 interaction (white-filled square),
D220H + PIP2 affinity (white-filled circle), and cal-
modulin affinity (white-filled triangle).
A341V
A344V
Cytosol D76N
L114P S373P
G189R V254M
P127T R539W
C S546L
T587M
N C
G325R, have been shown to abolish channel conductance B) GATING DEFECT. LQT1 mutations that cause defects in
(13, 61). They exert dominant negative effects on wild-type KCNQ1 gating can be found in the pore domain (S5–S6).
(WT) IKs current in heterologous expression systems (61), Similar to other voltage-gated potassium channels, the
suggesting that mutant subunits coassemble with WT sub- COOH-terminal region of the S6 helix plays a key role in
units to form disrupted channels. Molecular dynamic sim- KCNQ1 gating (55, 157, 247). Scanning mutagenesis and
ulations suggest that these mutations disrupt the conforma- heterologous expression studies show that a number of
tion of the selectivity filter, leading to diminished K⫹ per- LQT1-linked residues in this region, such as F351 and
meation. A pair of adjacent LQT1 pore mutations, G314S L353, control KCNQ1 gating properties. For example,
and Y315C, located in the selectivity filter, dramatically F351A leads to a drastic slowing of channel activation and a
reduce IKs current amplitude and exert dominant negative depolarizing shift in voltage dependence of activation, while
effects on WT currents (52, 237). Immunofluorescence L353K leads to a constitutively open channel (55). To further
studies show that Y315C traffics to the membrane nor- elucidate the mechanism of F351A, a technique known as
mally, suggesting that the mutation results in trafficking of voltage clamp fluorometry (VCF), which utilizes fluorophore
non-conducting channels. labeling to allow simultaneous measurement of voltage sensor

BOHNEN ET AL.
is made constitutively active (41). Structural and muta-

Table 2. Representative LQT1-associated mutations classified tional studies suggest that R231 forms a salt-bridge in-
by mechanism teraction with E160 in S2 that stabilizes the channel in its
Reference closed state (340, 393, 472). Mutating R231 may disrupt
Mechanism Mutations Nos. this interaction and cause a defect in channel gating,
resulting in constitutive activation. One tool that can be
K⫹ permeation G314S, Y315C 52, 237
used to elucidate the effects of these S4 mutations on
T322A, T322M, G325R 13, 61
voltage sensor movement is VCF. The technique is capa-
Gating D202H 113, 114
ble of providing insights on IKs channel gating not possi-
S225L 52, 170
ble with current measurement alone.
R231C 41
A344V 391
LQT1 gating mutations are also present in other transmem-
Trafficking Y111C, L114P, P117L 98, 375
T587M, R591H, R594Q 205
brane regions (S1–S3) of the voltage-sensing domain. For
PKA-mediated G189R, R190Q, R243C, 39
example, the mutation D202H leads to biophysical defects
in IKs, shifting the current-voltage relationship to more pos-

signaling V254M
A341V 169 itive potentials, slowing activation, and accelerating deac-
G589D 255 tivation, all leading to reduced channel opening (114). Sin-
KCNQ1-KCNE1 gle-channel recordings of IKs utilized as a tool to study
interaction S546L, K557E 110 effects of select mutations (113) shows that D202H causes
PIP2 affinity R539W, R555C 312
a decrease in channel open probability, a decrease in open
S546L, K557E 110
states dwell time, and an increase in closed states dwell
Calmodulin affinity S373P, W392R 379
time, while maintaining single-channel conductance.
Large-scale defects
Nonsense R518X, Q530X 181
C) TRAFFICKING DEFECT. In addition to altering channel gating,
Deletion-insertion,
frameshift ⌬544 81 LQT1 mutations can also disrupt channel trafficking. Sev-
Splice site 1032G⬎A 286 eral LQT1 mutations in helix D of the CTD, including
T587M, R591H, and R594Q, have been found to diminish
channel trafficking to the membrane (205). These muta-
movement and channel current, has been used (37, 292, 308, tions are thought to disrupt a coiled-coil motif in helix D
309, 348, 498). It was shown using VCF that F351A alters the that plays an important role in channel trafficking (205).
coupling between voltage sensor and the pore (309), resulting Yet trafficking mutations are not limited to the CTD of
in a slowly activating channel that partially resembles IKs. In KCNQ1. LQT1 mutations in the NH2-terminal region of
addition, a LQT1 mutation in on the S6 helix, A344V, also KCNQ1, including Y111C, L114P, and P117L, have also
affects channel gating by shifting the current-voltage relation- been found to reduce surface expression of KCNQ1 and
ship of IKs in the depolarizing direction by 30 mV, thereby increase retention in the endoplasmic reticulum (98). This
destabilizing channel opening (391). region appears to be a conserved trafficking determinant
across the KCNQ family. In particular, Y111C and L114P
In addition to those in the pore domain, a number of LQT1 disrupt SKG1’s ability to increase channel trafficking to the
mutations that alter channel gating are found on the volt-
plasma membrane, suggesting that the NH2 terminus may
age-sensing S4 helix of KCNQ1. For example, the mutation
be important in RAB-mediated exocytosis of channels
S225L exerts dominant negative suppression of WT IKs
(375).
current (52). This mutation alters channel gating, shift-
ing the current-voltage relationship of IKs toward more
D) DEFECT IN PKA-MEDIATED SIGNALING. Given the critical im-
depolarized membrane potentials (170). The effect of this
mutation on voltage sensor movement remains to be de- portance of adrenergic stimulation and PKA-mediated sig-
termined, but given its location it is possible that it dis- naling in the regulation of IKs and repolarization of the
rupts the movement of the S4 helix in response to changes cardiac action potential during stress, disruption of this
in voltage. Another LQT1-associated mutation on S4, stimulation is expected to prolong the QT interval. For
R231C, decreases peak IKs amplitude, but it also leads to example, the mutation G589D is thought to disrupt a leu-
constitutive activation at the same time (41). Interest- cine zipper motif to which AKAP-9 binds, resulting in fail-
ingly, in one family, this mutation causes familial atrial ure of IKs to be stimulated by cAMP (255). A mutation on
fibrillation, which is more consistent with action poten- S6, A341V, exhibits dominant suppression of IKs that fails
tial shortening than prolongation (41, 296). To explain to respond to adrenergic stimulation with cAMP (169).
this finding, the study has used a computational model to This effect is mediated by a reduction in the phosphoryla-
show that the atrial action potential is more susceptible tion S27 on KCNQ1 and is not due to disruption in AKAP-
to shortening than ventricular action potential when IKs 9’s interaction with KCNQ1. This result implicates a role of

S6 in the regulation of the phosphorylation state of brane. These results are consistent with the role calmodulin
KCNQ1. plays in channel assembly and trafficking (141, 379).
Mutations that lead to defective adrenergic stimulation of H) NON-MISSENSE MUTATIONS. Non-missense mutations can
IKs in LQT1 patients are suggested to be associated with also cause LQT1. For example, nonsense LQT1 mutations
higher risk of cardiac events during exercise and greater such as R518X and Q530X introduce a stop codon, leading
response to ␤-blocker therapy. One study has identified to early termination of channel transcription and loss of IKs
missense mutations in the cytoplasmic loops between S2/S3 function (181). These mutations are mostly associated with
and S4/S5 of KCNQ1 to be associated with an elevated risk autosomal recessive LQTS, although autosomal dominant
of aborted cardiac arrest and sudden cardiac death (39). cases have also been reported for R518X. One study shows
Four mutations in these cytoplasmic loops, G189R, that these mutant channels only mildly affect WT IKs cur-
R190Q, R243C, and V254M, all diminish IKs upregulation rent, consistent with their mostly recessive mode of inheri-
in response to forskolin, an activator of the PKA signaling tance (349). It is thought that the nonsense transcripts are
pathway, suggesting that patients with these mutations are selectively degraded and do not interfere with WT channel
expected to be especially susceptible to arrhythmic events production. Interestingly, the same study suggests that

during stress. This study bears implications on better risk- LQT1 patients with nonsense mutations have reduced risk
stratification for LQT1 patients and predicting response to of cardiac events compared with patients with missense
␤-blocker therapy. In addition, it suggests that the cytoplas- noncytoplasmic loop mutations, although the explanation
mic loops are involved in adrenergic stimulation of remains to be determined.
KCNQ1.
Another non-missense LQT1 mutation is the deletion-inser-
E) DISRUPTED KCNQ1-KCNE1 INTERACTION. A number of LQT1 tion at residue 544, denoted ⌬544, which leads to a frame-
mutations disrupt the KCNQ1-KCNE1 interaction. Two shift that alters subsequent 107 amino acids and introduces
such mutations, S546L and K557E, are located in the helix an early stop codon (81). It is an autosomal recessive mu-
C of the CTD of KCNQ1 and disrupt its interaction with tation occurring in the CTD. The mutation has been shown
the COOH terminus of KCNE1, as demonstrated by GST to disrupt channel assembly in vitro (364), underscoring the
pulldown assays (110). The same mutations also decrease role of the CTD of KCNQ1 in channel assembly.
channel affinity for PIP2, leading to decreased current am-
plitude and a depolarizing shift in the current-voltage rela- LQT1 mutations can also disrupt transcript splicing. A base
tionship. The decreased PIP2 affinity may be due to disrup- substitution at a consensus splice donor site the end of exon
tion of a potential PIP2 binding site on helix C. 7, 1032G⬎A, can lead to a dropped exon 7 or exons 7 and
8 (286). In terms of channel regions affected, exon 7 en-
F) DECREASED PIP2 AFFINITY. Some LQT1 mutations have been
codes parts of the pore-loop and S6, while exon 8 encodes
shown to decrease channel PIP2 affinity. In addition to the the rest of S6 and part of the CTD. Transcripts lacking one
mutations described above, two LQT1 mutations on helix or both of these exons therefore are not expected to produce
C of the CTD, R539W and R555C, both lead to decreased functioning channels. A study shows that this splicing mu-
PIP2 affinity, decreased IKs amplitude, and a depolarizing tation exerts a dominant-negative suppression of WT IKs,
shift in the current-voltage relationship, underscoring the likely through a direct interaction between mutant and WT
importance of the CTD in PIP2 binding (312). The cytoplas- channels preventing trafficking to the membrane (428). In
mic loops of KCNQ1 between S2/S3 and S4/S5 also play addition to splice donor sites, splice acceptor sites can also
important roles in PIP2 binding and channel gating (208, be mutated in LQT1, such as the base substitution 922-1
498). Several LQT1-linked residues in these cytoplasmic
G¡C at the end of intron 6, which leads to the loss of exons
loops, including residues 195, 258, and 259, are thought to
7 and 8 (286).
form a binding site for PIP2. Disease mutations in this bind-
ing site may result in decreased PIP2 affinity, leading to
altered channel function (80, 498). 2. LQT5
G) DECREASED CALMODULIN AFFINITY. A number of LQT1 mu- Coassembly of KCNQ1 with the KCNE1 subunit is key to
tations have been found to weaken calmodulin binding to the generation of the IKs current. Thus it follows that
KCNQ1 as the underlying mechanism of disease. For ex- KCNE1 mutations can alter the physiologically critical cur-
ample, the mutations S373P and W392R, located in the rent of the assembled channel and lead to LQT5. Similar to
CTD, reduce calmodulin binding both in the absence and LQT1, the mode of inheritance for LQT5 can be autosomal
presence of KCNE1 (379). Both of these mutations cause dominant (RW) or recessive (JLNS) (107, 430). Mutations
decrease in surface channel expression and dramatic reduc- in KCNE1 can lead to defects in gating, trafficking,
tion in IKs amplitude. Overexpression of calmodulin is able KCNQ1-KCNE1 interaction, as well as adrenergic stimu-
to increase S373P mutant channel expression in the mem- lation (FIGURE 4D) (TABLE 3).

BOHNEN ET AL.
A) GATING DEFECT. One LQT5 mutation that affects channel interaction (166). Furthermore, the mutation P127T, lo-
gating is D76N. It is an autosomal dominant mutation in cated in the COOH-terminal region of KCNE1, appears to
the COOH terminus of KCNE1 that has been shown to disrupt the interaction of KCNE1 with helix C in the CTD
drastically suppress IKs amplitude, accelerate deactivation, of KCNQ1 (110). Interestingly, the mutation was also
and cause a depolarizing shift in the voltage dependence of found to diminish PKA-stimulated upregulation of IKs by
IKs activation when expressed in Xenopus oocytes and decreasing phosphorylation at the S27 residue. Since PKA
CHO cells (76, 405). Overexpression of the mutant KCNE1 phosphorylation at this site was previously shown to be
in guinea pig ventricular myocytes leads to APD prolonga- independent of KCNE1 (226), it is possible that the disrup-
tion and early afterdepolarizations (176). The mutant ap- tion in adrenergic stimulation by P127T is independent of
pears to traffic normally to the cell surface (53) and does not the mutation’s disruption of KCNQ1-KCNE1 interaction
disrupt KCNE1 binding to the COOH terminus of KCNQ1 (110).
(510). However, it reduces IKs upregulation secondary to
stimulation of the PKA signaling pathway, suggesting a role 3. LQT11
for the COOH terminus of KCNE1 in adrenergic stimula-
tion of IKs (226). AKAP-9 is a scaffolding protein and part of the IKs macro-

molecular complex that plays a critical role in the compart-
B) TRAFFICKING DEFECT. In addition to channel gating, LQT5 mentalization of adrenergic-stimulated PKA signaling path-
mutations can lead to defects in channel trafficking. The way leading to IKs upregulation. That a mutation in
JLNS mutation L51H, located in the transmembrane helix AKAP-9, S1570L, can cause LQTS is a testament to the
of KCNE1, results in diminished KCNE1 trafficking to the critical role of PKA signaling in the regulation of IKs mac-
cell surface (53). Furthermore, when coexpressed with romolecular complex (79). This mutation is located near
KCNQ1 in HEK cells, the mutant KCNE1 decreases the COOH-terminal binding domain of AKAP-9, disrupt-
KCNQ1 trafficking to the surface (53, 220). In addition, ing its interaction with KCNQ1, reducing cAMP-stimu-
coexpression of the mutant KCNE1 with KCNQ1 in CHO lated phosphorylation of KCNQ1, and abolishing IKs up-
cells leads to a diminished current amplitude and channel regulation in response to cAMP-mediated stimulation.
biophysical properties that resemble KCNQ1 alone rather Computational modeling suggests that a disruption in the
than IKs, consistent with a drastic reduction of functional basal phosphorylation state of IKs alone can alter IKs func-
KCNE1 in the membrane. These results together suggest tion sufficiently to prolong APD (79).
that the mutant KCNE1 interacts with KCNQ1 to disrupt
the trafficking of both proteins to the membrane surface,
leading to a reduction in current. The transmembrane re- E. Molecular Pharmacology
gion of KCNE1 may therefore be important to channel
trafficking. 1. ␤-Blockers
C) DISRUPTED KCNQ1-KCNE1 INTERACTION. LQT5 mutations ␤-Blockers have been demonstrated as a particularly effec-
can disrupt the interaction between KCNE1 and KCNQ1 tive form of therapy for LQT1 patients, who are more sen-
required to generate IKs. For example, the double mutation sitive to stress- and exercise-induced arrhythmia than other
T58P/L59P, located in the transmembrane region of LQT subtypes (282, 371). Insufficient upregulation of IKs
KCNE1, results in near-complete loss of IKs amplitude but by adrenergic stimulation to counterbalance concomitant
has minimal effect when coexpressed with WT IKs in Xeno- rise in inward calcium current is thought to underlie APD
pus oocytes (181). The mutation leads to a diminished abil- prolongation in LQT1 during sympathetic activation (386).
ity for KCNE1 to associate with KCNQ1 in coimmunopre- ␤-Blockers antagonize adrenergic receptors and helps pre-
cipitation studies, suggesting that the transmembrane re- vent this imbalance between potassium and calcium cur-
gion of KCNE1 is important for the KCNQ1-KCNE1 rents, decreasing predisposition for cardiac arrhythmic
events.
2. Channel activators
Table 3. Representative LQT5-associated mutations classified
by mechanism
While IKs plays a critical role in cardiac repolarization, it is
Reference not a direct target of drugs currently used to treat LQTS.
Mechanism Mutations Nos.
However, conceptually, IKs activators could allow for more
precise rescue of disease phenotype. In this section we
Gating D76N 76, 405
briefly review IKs activators and refer readers to other stud-
Trafficking L51H 53
ies in which activators of the ATP-sensitive K⫹ channel,
KCNQ1-KCNE1 interaction T58P/L59P 166, 180
such as nicorandil, have been used in LQTS patients and
P127T 110
model systems (14, 388). Currently there are no IKs activa-
PKA-mediated signaling D76N, P127T 110, 226
tors being used in clinical trials or therapy, but a number of

small molecules that activate IKs have been identified at the 1), underlies congenital Long QT (LQT) syndromes type 2
benchside and may guide future development of therapeutic and 6, which arise from mutations in the KCNH2 and
agents. For example, the compounds DIDS and mefenamic KCNE2 genes, respectively. LQT2 is the second most com-
acid both increase IKs current amplitude (5). In addition, mon cause of congenital LQTS. KCNH2 mutations lead to
DIDS has been shown to drastically slow IKs deactivation. defective hERG protein, resulting in a decrease in IKr. Mu-
Interestingly, the effects of these drugs appear to be de- tations in KCNE2 cause defects in the KCNE2 (or MiRP1)
pendent on the presence of KCNE1. Compared with protein leading to LQT6, which also results in a decrease in
KCNQ1 alone, current augmentation by these com- IKr. Irrespective of the underlying cause, a decrease in IKr
pounds is much greater in the presence of KCNE1, sug- delays repolarization of the cardiac action potential pro-
gesting their effects are mediated by the ␤-subunit. In- longs the QT interval on the ECG, and predisposes patients
deed, deletion of the residues 39 – 43 of KCNE1 leads to to lethal arrhythmia. This section reviews IKr dysfunction
a diminished response to these compounds. To demon- leading to congenital and IKr-mediated drug-induced
strate the potential for activators as a class of therapeutic LQTS. [For a detailed summary of hERG channel structure,
agents for LQTS in in vitro studies, DIDS and mefenamic molecular biology, and basic electrophysiology, see Van-
acid have been shown to rescue IKs function in a LQT5 denberg et al. (436).]

mutation, D76N. Future studies will be required to better
understand their mechanisms of action and to develop
IKs-specific activators that can be effective and used B. Physiology
safely in patients.
Heterologous expression of hERG reveals a strong, near
Another KCNQ1 activator, ML277, has been shown to identical resemblance to IKr in cardiomyocytes (359, 427).
augment current for KCNQ1 alone more effectively than IKr is distinguished based on its relatively slow activation
KCNQ1 with KCNE1 (492). In fact, its activating effect and deactivation kinetics, combined with rapid inactivation
diminishes with progressive increase in KCNE1:KCNQ1 and recovery from inactivation (FIGURE 5). Inactivation re-
stoichiometry, suggesting that the ␤-subunit may act to pre- fers to a conformation of the channel protein in which the
clude the drug from binding to the channel (491). While a channels cannot conduct ions even if the activating machin-
drug with such properties is expected to have minimal ef- ery is in a conformation that would promote conduction of
fectiveness in human cardiomyocytes, surprisingly the same ions. Channels will not conduct ions when in an inacti-
study showed that the drug can augment IKs and shorten vated state, but will conduct ions after the channels re-
action potential in human iPSC-derived cardiomyocytes cover from the inactivated state, a recovery that takes
from a healthy control. The mechanism underlying this ef- place at negative voltages (174). Deactivation refers to
fect requires further elucidation. the transition to a conformation in which channels return
to a closed, nonconducting state, a transition that also
3. Channel blockers occurs at negative (diastolic) voltages. hERG channels
undergo voltage-dependent and C-type inactivation. Be-
cause channel activation is slow relative to the rapidly
IKs-specific blockers are not useful as therapeutic agents for
occurring inactivation process, the hERG I–V curve takes
LQT1, but serve as useful tools for research by allowing for
on a bell-shaped relationship, as shown in FIGURE 5C.
pharmacological dissection of IKs currents. Chromanol
Upon repolarization of the membrane, channels recover
293B is the first known IKs-specific blocker, with an IC50 of
from inactivation much faster than they deactivate. This
6.9 ␮M in Xenopus oocytes (62, 139). A more effective
crucial channel property results in a marked outward K⫹
blocker, HMR1556, with an IC50 of 0.12 ␮M, was de-
veloped using Chromanol 293B as the lead compound current during the repolarization phase of the cardiomy-
(139). These drugs inhibit KCNQ1 with KCNE1 more ocyte action potential. By this time, a large percentage of
effectively than KCNQ1 alone. Blockers of IKs are spec- hERG channels have recovered from inactivation, such
ulated to serve useful roles in the treatment of disease that outward potassium efflux helps return the cell to its
conditions resulting from IKs gain of function, such as resting potential, despite the fact that the electrochemical
familial atrial fibrillation. gradient for potassium efflux decreases as repolarization
progresses (FIGURE 1B).
III. IKr DYSFUNCTION IN CONGENITAL Since hERG inactivation and recovery from inactivation
LQTS proceed more rapidly than activation or deactivation of
hERG (365, 394, 401), IKr contributes to prolongation of
the plateau phase duration and thus cardiomyocyte con-
A. Introduction traction, in addition to cardiac repolarization (361, 394).
As hERG recovers from inactivation during cell repolariza-
Alterations in IKr, the rapid component of the delayed rec- tion, the repolarization itself promotes greater hERG recov-
tifier current in the cardiomyocyte action potential (FIGURE ery from inactivation due to the voltage dependence of

BOHNEN ET AL.
A KCNE2 hERG
Extracellular
S1 S2 S3 S4 S5 S6
+
+
+
+
FIGURE 5. hERG structure and electro-
Cytosol physiology. A: schematic of the IKr channel
complex. Four hERG1 subunits tetramer-
ize to comprise the pore-forming alpha

subunit of IKr. hERG1 contains a voltage-
cNBD sensing domain (purple), including the S4
helix which contains positively charged gat-
PASD
C ing residues, and a pore domain (gray).
KCNE2, an accessory subunit of the IKr
channel complex, consists of a single
N C transmembrane helix (blue). B: voltage-
clamp protocol (top panel) and heterolo-
gously expressed hERG1 ionic currents
B +50 mV C (bottom panel) recorded from a Xenopus
–70 mV oocyte. Currents were recorded at poten-
–80
tials that ranged from ⫺70 to ⫹50 mV;
deactivating (“tail”) currents were mea-
1.0
sured at ⫺70 mV. C: current-voltage (I-V)
relationship for hERG1 currents measured
0.8
at the end of test pulses, as indicated by
Relative current
red circle in B. D: voltage dependence of

0.6 hERG1 current activation. The peak of tail
currents measured at ⫺70 mV (indicated
0.4 by blue square in B) were normalized to the
largest value and plotted as a function of
0.5 μA 0.2 the test potential. E: voltage dependence
of hERG1 inactivation. Channel availability
0.0 is decreased at positive potentials, result-
–80 –60 –40 –20 0 20 40 ing in a decreased magnitude of peak out-
1 sec mV ward currents and the bell-shaped I-V rela-
tionship depicted in C. [B–E from Sangui-
D E netti (356), with permission of Springer.]
1.0 1.0
0.8 0.8
Channel availability
Relative current
0.6 0.6
0.4 0.4
0.2 0.2
0.0 0.0
–80 –60 –40 –20 0 20 40 –120 –80 –40 0 40
mV mV
hERG inactivation gating (359, 394, 427). As the cell con- URE 5E) acts to oppose cell depolarization (242, 394),
tinues to repolarize and return to its resting membrane po- which helps prevent premature heart beats from leading to
tential, the slower process of channel deactivation pro- an early action potential and a tachyarrhythmia. Loss of
gresses, leading to closure of hERG (436). The fraction of hERG function thus predisposes to arrhythmia in the set-
channels remaining open near the resting potential (see FIG- ting of premature beats(46).

C. Molecular Biology hERG subunit (see below) (94). Furthermore, the PAS do-
main and cNBD appear to bind, working in concert to
The KCNH2 gene, located on chromosome 7q35-36, en- modulate hERG gating by positioning the NH2-terminal
coding the human ether a go-go-related K⫹ channel pro- residues in close proximity to the cytosolic side of S6 (23,
tein (hERG), was first discovered in 1994 (460). Muta- 100, 160, 287).
tions in KCNH2 associated with congenital LQTS were
discovered a year later in 1995 (97), and soon after, it hERG can coassemble with two different ␤-subunits in het-
was determined that hERG represents the ␣-subunits of erologous systems: KCNE1, encoded for by the KCNE1
the K⫹ channel responsible for IKr (359, 427). hERG may gene; and KCNE2 (or the MiRP1 protein), encoded for by
be referred to as “hERG1,” since other hERG proteins the KCNE2 gene (FIGURE 5A) (2, 259). KCNE1 and
(hERG2 and hERG3) have since been discovered (385). KCNE2 are single-pass transmembrane subunits that can
hERG1a, the main hERG isoform present in cardiomyo- interact with the hERG channel (2, 3, 20, 259). While the
cytes, is 1,159 amino acids in length, with a predicted molec- precise physiological role of the KCNEs in regulating hERG
ular weight of 127 kDa. As with most voltage-gated K⫹ chan- and IKr remains unclear (21, 257, 462), mutations in
nels, functional hERG channels are composed of four hERG KCNE1 and KCNE2 lead to LQT5 and LQT6, respectively,

␣-subunits, forming either a hERG1a homomeric channel, or and mutation of either KCNE1 or KCNE2 can predispose
a hERG1a/hERG1b heteromeric channel that contributes patients to drug-induced LQT syndrome, possibly via mod-
to native cardiac IKr activity. The heteromeric assembly of ulation of hERG activity (2, 9, 53, 295, 376, 402).
hERG isoforms 1a and 1b has distinctively altered kinetics
compared with hERG1a monomeric channels, including KCNE1 and hERG associate in heterologous systems and
faster activation and deactivation. hERG1b is expressed in may contribute to regulation of IKr; however, KCNE1 is
smaller amounts at the mRNA level in the heart compared better characterized physiologically as the ␤-subunit of the
with hERG1a, but nevertheless adds to the complexity of KCNQ1 complex that produces IKs (36, 53, 259, 358).
hERG channel regulation and potential therapeutic modal- KCNE2 was reported to alter gating properties and drug
ities (195, 196, 231, 323, 353, 426). responses of hERG channels(2), and mutations in KCNE2
leading to LQT6 likely result from changes in hERG and
Each of the four hERG ␣-subunits consists of six transmem- thus IKr current activity, producing a pro-arrhythmic
brane helices, S1–S6, as shown in FIGURE 5A. The voltage- state, further supported by the KCNE2 T10M mutation
sensing domain spans from S1 to S4; the S4 transmembrane that confers an arrhythmogenic substrate to auditory
segment of hERG contains the primary positively charged stimuli, a known trigger of LQT2-associated arrhythmia
amino acids required for voltage-sensing and opening of the (see FIGURE 7) (2, 151, 182, 251). It is possible that
activation gate (325, 408, 505). The pore domain of the physiologically relevant KCNE2 expression exists only in
channel, spanning from S5–S6, harbors the K⫹ permeation the Purkinje fibers and pacemaker cells of the human
pathway necessary for K⫹ conduction (FIGURE 5A) (105). heart (297, 330), which further confounds the exact role
The long cytoplasmic NH2 terminus contains the PAS do- of KCNE2 as a ␤-subunit of hERG in vivo. Moreover,
main with a PAS-cap, and together these sequences make up KCNE2 coassembles with KCNQ1, which decreases IKs
the “EAG” domain that is conserved among EAG-related current(192), adding to the complexity and diversity of
voltage-gated potassium channels(274). The long cytoplas- IKs and IKr channel subunit interactions.
mic COOH terminus of the channel contains the cyclic
nucleotide-binding domain (cNBD) (56), as well as a more
distal RXR endoplasmic reticulum retention signal (224), D. Molecular Pathophysiology
and a coiled-coil domain (188).
A decrease in IKr results in LQT2 via mutations in the
The NH2-terminal PAS domain accelerates deactivation of KCNH2 gene that encodes hERG, and LQT6 via mutations
hERG and plays a role in channel trafficking (77, 274, 436). in the KCNE2 gene that encodes KCNE2 (or MiRP1) pro-
Slow deactivation of hERG may involve interaction of the tein. The mechanisms underlying disease pathogenesis are
PAS domain in the NH2 terminus of the channel, with the described below.
S4 –S5 linker (450), which couples movement of the trans-
membrane voltage sensor to activation gate movement 1. LQT2
(247). Experimental hERG mutants with a deleted PAS do-
main have faster deactivation kinetics (274, 401). Proper Patients with congenital LQT2 often present clinically after
folding of the PAS domain also leads to trafficking of hERG syncope or seizures not explained by a known medical con-
from the endoplasmic reticulum to the plasma membrane dition. Prolongation of the QT interval on the ECG sup-
(314). The cNBD contributes to channel trafficking and ports the diagnosis, and in LQT2 in particular, the T-wave
gating as well, while cAMP binding to the cNBD domain may appear as a classic notched, or “bifid” T-wave (FIGURE
results in changes in the gating kinetics of the channel, 6A) (503). Syncope occurs following a common pathologi-
which are altered in the presence of KCNE2, an accessory cal process in the LQT syndromes, wherein delayed ventric-

BOHNEN ET AL.
A COOH terminus, including 8% in the c-NBD (207). In

LQT2 ECG terms of molecular mechanism, a small proportion of LQT2
mutations result in nonsense-mediated mRNA decay (150).
More commonly, decreased protein trafficking to the cell
surface leads to eventual endoplasmic reticulum-associated
degradation (ERAD) of mutant hERG (22, 149, 443, 512).
Several important general characteristics of KCNH2 mu-

tations associated with congenital LQT2 were recently
B highlighted in a large-scale functional study of 167 dif-
WT ferent LQT2-associated missense mutations (23). Func-
LQT2 tional analysis was performed by voltage clamp after
coexpression of WT and mutant subunits in HEK293
AP cells. Anderson et al. (23) found that 1) hERG trafficking
defects comprised the most common (88%) mechanism

of loss of function overall, derived from mutations in the
PAS domain, pore domain, and C-linker/cyclic nucleo-
tide-binding domain. Only the distal COOH-terminal re-
gion did not yield trafficking defects as the primary mech-
anism of loss-of-function. 2) Greater than 70% of pore
mutations led to dominant negative suppression of
IKr hERG, whereas the other intracellular domain mutation
locations did not yield dominant negative suppression of
current. 3) All mutant channels, regardless of mutation
location within the channel, were rescued pharmacolog-
ically by E4031, a hERG pore blocker that stabilizes
FIGURE 6. IKr dysfunction leading to congenital LQTS. A: ECG from channel structure and folding, thus promoting channel
a LQT2 patient demonstrates a characteristic “notched,” or bifid, T retention at the cell surface (148). Coexpression of WT
wave with QTc prolongation (unpublished data). B: simulated action with pore mutant channel rendered heteromeric pore mu-
potential (top) and IKr (bottom) in WT (black) and heterozygous LQT2 tant-WT channels especially responsive to pharmacolog-
(gray) conditions, demonstrating the effect of 50% reduction in IKr.
ical correction compared with coexpression of WT with
non-pore mutant channels. Overall, these data suggest
ular repolarization (and resultant QT interval prolongation that pharmacological recovery of hERG channel function
on the ECG) precipitates an EAD, in some cases DADs, and is feasible for a large proportion of LQT2 muta-
ventricular tachycardia. EADs trigger the torsades de tions(345) and raise the possibility of this approach as a
pointes sinusoidal waveform on the ECG, which may prog- therapeutic strategy for LQT-2 patients, but to our
ress to ventricular fibrillation and sudden cardiac death knowledge this has not been implemented in the clinic.
(356).
A) MUTATION SEVERITY: HAPLOTYPE INSUFFICIENCY VS. DOMINANT-
Homozygous mutations in KCNH2 leading to LQT2 are NEGATIVE LOSS OF FUNCTION. Heterozygous mutation result-
extremely rare in humans, resulting in either death in utero, ing in defective KCNH2 gene expression or hERG protein
or very severe prolongation of the QT interval upon birth that does not impact normal functioning of the WT
(175, 194). Heterozygous mutation leading to one defective hERG protein that remains yields haplotype insuffi-
hERG copy is far more common and may result in signifi- ciency. Only the gene product from the mutant KCNH2
cant prolongation of the cardiac action potential duration allele is negatively affected, while WT hERG subunits
(see FIGURE 6B). hERG mutations leading to LQT2 occur still homomerize to form functional channels. In con-
by a variety of genetic mechanisms. To date, ⬃500 muta- trast, some hERG mutations have increased pathogenic-
tions in KCNH2 have been identified in association with ity by exerting a dominant-negative loss of channel func-
LQT2 (23). A study analyzing 226 different LQT mutations tion, wherein the mutant and defective KCNH2 gene
in genotype-confirmed LQT2 patients reported that 62% of product reduces function of the WT hERG protein en-
mutations were missense, 24% were frameshift, while 14% coded in the patient’s genome via heteromerization of
were a combination of nonsense mutations, inframe inser- mutant with WT channel, rendering the healthy hERG
tions/deletions, or splice site mutants in the KCNH2 gene. subunits nonfunctional when combined with mutant
Of the combined 226 mutations, 32% resided in transmem- hERG, either by decreasing forward trafficking of mutant
brane and pore-pore domains; 29% in the NH2 terminus, WT channels, or decreasing function of the heteromers at
including 8% in the PAS/PAC domains; and 31% in the the cell surface. In a study of LQT2 patients with 44 unique

KCNE2 hERG
G626A/D/V/S
N Y611H
G584S
Extracellular T10M
S1 S2 S3 S4 S5 S6
+ FIGURE 7. Topology of hERG and KCNE1
+ G628S in the plasma membrane, representative
+ LQT2- and LQT6-associated mutations
highlighted. Different mechanisms of loss
V65M + of function in hERG or KCNE2, including
gating (yellow-filled circle), K⫹ permeation
(black-filled square), trafficking (white-filled
Cytosol
triangle), or combined defects (green-filled
star) are categorized.

V822M
cNBD
PASD M124R R835W
C
D16A
N C
mutations in different regions of the hERG channel, it was tions, categorized by mechanism of loss of function (see
discovered that those patients harboring mutations in the TABLE 4). Representative LQT-associated hERG mutations
pore region of the channel were more susceptible to cardiac are divided into four general classes: I) decreased hERG
events than patients with non-pore region hERG mutations, synthesis, II) trafficking defect, III) gating defect, and IV)
likely due to a greater dominant-negative suppression of IKr
decreased K⫹ permeability (436).
current exerted by pore vs. non-pore mutations (187, 283).
Perhaps counterintuitively, more harmful mutations in one I) Decreased hERG synthesis. Defective biogenesis of
KCNH2 allele, resulting in a premature stop codon for hERG may occur via mRNA processing abnormalities or
instance, prevents formation of a hERG protein product mRNA instability (150, 504). The hERG R1014X mu-
from the mutant KCNH2 allele, leading to haplotype insuf- tant mRNA transcript is degraded by “nonsense-medi-
ficiency and the possibility of a less severe phenotype com- ated mRNA decay,” a cellular damage-control mecha-
pared with some dominant-negative mutations, as WT nism that destroys mRNA transcripts harboring non-
hERG remains unaffected(356). sense mutations (premature stop codons), which prevents
translation of a shortened hERG peptide (150, 356).
Studies of specific hERG mutants have greatly enhanced our Other nonsense-mediated decay mutants include
understanding of the underlying mechanisms of loss of W1001X (150) and Y652X (409). These mutations would
function leading to LQT2. FIGURE 7 provides a schematic of result in haplotype insufficiency, as there would be 50%
representative LQT-associated hERG and KCNE2 muta- loss of function while the remaining WT hERG channels
Table 4. Representative LQT2- and LQT6-associated mutations classified by mechanism

Mechanism Mutations Reference Nos.
hERG LQT2 mutations

K⫹ permeation G628S 59, 116, 357
Gating D16A, G584S, T613A, I711V, R835W 23, 329, 509
Trafficking Y611H, V822M 512
Decreased hERG synthesis R1014X, W1001X, Y652X 50, 150, 356, 409
Multiple mechanisms (e.g., gating and trafficking) F29L, M124R 23, 206, 390
KCNE2 LQT6 mutations
Gating T10M, V65M 151, 182

BOHNEN ET AL.
function normally. Nevertheless, a 50% reduction in IKr with a milder loss-of-function phenotype directly and solely
can lead to clinically significant LQTS. attributed to its effect on inactivation. This finding adds
further to the complexity of hERG mutant phenotypes as
II) hERG trafficking defects. Defective hERG trafficking is most other pore mutations cause increased arrhythmic
the most common mechanism of loss of hERG function (22, events compared with non-pore hERG mutations (283).
23, 436), and most KCNH2 missense mutations cause traf- Recently, the hERG T613A mutation in the outer region of
ficking defects in hERG (22). Trafficking mutants may re- the pore helix, a regulatory site for C-type inactivation, was
sult in either dominant negative loss of hERG function (22, found to cause greater than 80% inhibition of maximal
201) or haploinsufficiency (131). If WT hERG associates hERG current via a hyperpolarizing shift in channel inacti-
with trafficking-defective mutants to form heteromeric vation when expressed in Xenopus oocytes alone; coexpres-
channels in the endoplasmic reticulum (ER) or Golgi appa- sion of WT and mutant channel resulted in an intermediate
ratus, dominant negative suppression of IKr results: 93% loss of channel function, without dominant-negative sup-
reduction in IKr was observed by this mechanism (123). pression of current (329).
Defects in trafficking may be further subdivided by the un- Gating defects can arise from mutations across a wide range

derlying mechanism, protein trafficking, or protein misfold- of locations in hERG, and predispose individuals to LQT2
ing (22, 124, 512); hERG protein becomes core glycosy- of varying severity dependent on the functional impact of
lated in the ER, with modifications made in the Golgi ap- the mutation.
paratus. When the mature hERG protein traffics to the
plasma membrane, it weighs 155 kDa, versus 135 kDa for IV) hERG K⫹ permeation defect. The potassium selectivity
the core-glycosylated-only hERG protein. This difference in filter in the pore of the hERG channel allows for potassium
molecular weight aids in determining whether hERG pos- permeation with great specificity. Conserved amino acid
sesses a trafficking and/or protein folding defect (512). The residues comprise the selectivity filter, and mutation of any
hERG Y611H and hERG V822M LQT2 associated muta- of these residues or nearby amino acids may greatly alter
tions were found to have a molecular weight of 135 kDa, potassium permeation, even if only one of four hERG
and thus had decreased trafficking to the plasma membrane ␣-subunits within the mature channel bears the mutation
due to protein misfolding. These mutant hERG channels (i.e., dominant negative suppression of IKr). The hERG
were retained in the ER, and subsequently ubiquitinated G628S mutation, for instance, traffics to the cell surface and
and degraded in proteasomes (512). gates normally, which was confirmed by voltage-clamp flu-
orometry experimentation, but the mutant hERG channels
III) hERG gating defects. Alteration in activation, deacti- do not conduct potassium under physiological conditions
vation, and/or inactivation kinetics can result in loss of due to blockade of potassium permeation by intracellular
function of hERG and a decrease in IKr at physiologically potassium (116). The G628S mutation was also found to be
relevant membrane potentials. Any hERG mutation that, cause lethal arrhythmia in transgenic rabbits with the mu-
for instance, enhances the speed of inactivation, or causes a tation, as these rabbits possessed greater than 50% inci-
depolarizing shift in channel activation, ultimately leads to dence of sudden cardiac death after one year (59).
loss of hERG function at voltages ranging from the resting
potential to the plateau phase of the action potential, de- B) COMBINED MECHANISMS OF LOSS-OF-FUNCTION IN LQT2. Many
pending on the nature of the gating defect (48, 77, 290, 357, mutations leading to LQT2 confer a loss-of-function hERG
363, 509). Several hERG gating defect mutations have been phenotype by multiple molecular mechanisms. Kanters
described in mammalian cell lines (48, 363, 509). et al. (206) recently reported the mechanistic basis of chan-
nel dysfunction of the hERG F29L mutation in a founder
Both the I711V and R835W mutations represent examples population of Danish families, which results in a malignant
of c-NBD region LQT2 mutations in the COOH terminus form of LQT2. The mutation causes clinically significant
of the channel, with altered channel gating. These hERG prolonged QT, with a penetrance of 73%. The F29L muta-
mutants deactivate faster at ⫺50 mV compared with WT tion was found to 1) reduce trafficking of hERG to the cell
hERG. In addition, the R835W mutation confers a right- surface, posited to occur based on decreased glycosylation
ward, depolarizing shift (⫹16 mV) in the V0.5 of activation, of the hERG F29L channel; and 2) reduce steady-state in-
while inactivation measured at 0 mV remains unchanged activation current density, positively shift the voltage de-
(23). Within the NH2 terminus of hERG, the LQT2 hERG pendence of inactivation, and increase the rate of deactiva-
D16A mutation exhibits a ⫹13 mV rightward shift in the tion. Similarly, the M124R mutation in the NH2 terminus
V0.5 of activation, with a minor slowing of inactivation at 0 residing close to the hydrophobic surface of hERG, results
mV and a slowing of deactivation at ⫺50 mV (23, 302). in faster channel deactivation, in addition to reduced traf-
ficking to the cell surface (23, 390). Furthermore, the exact
The hERG G584S pore mutation increases the rate of chan- amino acid substitution can impact the mechanism of loss
nel inactivation (509). G584S represents a pore mutation of function of the hERG channel: the LQT2-associated

G626A mutation in the conserved K⫹ selectivity filter in the E. Molecular Pharmacology

channel pore traffics to the cell surface normally, while the
LQT2-associated G626D, G626V, and G626S mutations hERG channel modulation by pharmacological agents has
had trafficking defects. The exact amino acid substitution become an increasingly studied research area because it is
has potential therapeutic implications: of the three G626 now well established that a wide variety of clinically used
trafficking mutants, only hERG G626S had its trafficking drugs block hERG and thus inhibit IKr, leading to drug-
defect corrected by E4031, an experimental pharmacologi- induced LQTS. The section below details mechanisms
cal agent that increases forward trafficking of hERG chan- and the biomedical relevance of drug-induced LQTS, fol-
nels in addition to its pore blocking effects (23). lowed by a discussion of various pharmacological tools
that hold promise as novel agents for the treatment of
2. LQT6 LQT2 (and LQT6) in the future.
KCNE2 (or MiRP1) protein, encoded for by the KCNE2 1. Drug-induced LQTS
gene, modulates hERG activity as an accessory ␤-subunit

(FIGURE 5A). Mutations in KCNE2 cause LQT6 due to a Most drugs that block IKr conduction bind to the open state
decrease in IKr (see FIGURE 7 AND TABLE 4) (2). The V65M of the hERG channel, binding the inner pore thereby pre-
mutation in KCNE2, for example, induces faster inactiva- venting permeation of potassium (67, 215, 267, 400, 483,
tion of colocalized hERG/mutant KCNE2 channel com- 513). The anti-arrhythmic pharmacological agent MK-499
plexes (182). The KCNE2 T10M was found linked to pa- interacts mostly with polar and/or aromatic residues within
tients with cardiac arrhythmia and LQTS, with particular the channel pore, including Thr623, Tyr652, and Phe656,
susceptibility to arrhythmia onset in the setting of auditory to induce hERG block (266). Different hERG-blocking
stimuli and hypokalemia, both common triggers of arrhyth- drugs may interact with different combinations of these
four residues (266, 319). For instance, the anti-arrhythmic
mia in LQT2 patients as well. Coexpression of KCNE2
agent dofetilide interacts with Phe656 to mediate hERG
T10M with hERG decreases tail currents and causes a hy-
binding (233). While the aromatic and polar residues above
perpolarizing shift in steady-state inactivation, along with
are integral to hERG’s supreme sensitivity to block by phar-
slowed recovery from inactivation compared with WT
macological agents, C-type inactivation may increase affin-
channels in CHO cells. The impact of the KCNE2 T10M
ity of drugs to hERG’s pore residues and enhance drug
mutation in the setting of known LQT2 arrhythmia triggers block (125, 171).
supports the hypothesis that KCNE2 associates with hERG
in the human heart to regulate IKr (151). As KCNE2 is Some drugs, including pentamidine (90, 228) and probucol
known to interact with other K⫹ ␣-subunits in addition to (156), cause a reduction in IKr by reducing trafficking of
hERG, defective KCNE2 may lead to varying phenotypes. hERG to the plasma membrane, leading to prolongation of
Indeed, the KCNE2 M54T mutation leads clinically to the QT interval. The widely clinically employed drugs flu-
LQT6 in addition to sinus bradycardia, the latter clinical oxetine and ketoconazole block hERG directly and de-
finding mediated by reduction of the HCN4 pacemaker crease hERG channel density at the cell surface (337, 411).
channel activity (297). The complexity of interactions of Several other pharmacological agents precipitate drug-in-
KCNE2 with a variety of potassium channels may inform duced LQTS in susceptible patients, via hERG blockade or
differential phenotypes observed clinically in those harbor- modulation of other important ionic currents (e.g., INa, IKs)
ing a KCNE2 mutation. (439, 482).
3. Autoimmune-associated LQT syndrome KCR1, a 12-pass transmembrane segment regulatory pro-

tein, interacts with hERG and mitigates the proarrhythmic
effects of hERG-blocking agents (178, 223). In heterolo-
Recently, Yue et al. (494) discovered that anti-Ro antibod- gous conditions, KCR1 decreases the sensitivity of hERG to
ies block the hERG channel, resulting in an autoimmune- a variety of well-established hERG channel blockers, such
associated LQTS. The anti-Ro antibodies, present in the as sotalol, quinidine, and dofetilide (223). Loss of function
serum of patients with a variety of common autoimmune of KCR1 is associated with greater risk of arrhythmia: the
illnesses, were found to inhibit IKr in HEK293 cells, and a KCR1 E33D mutant found in a patient with ventricular
guinea pig autoimmune model with circulating anti-Ro an- fibrillation and QT-interval prolongation conferred re-
tibodies had a prolonged QTc interval, caused by inhibition duced protection against drug-induced hERG blockade
of native IKr. Anti-Ro likely blocks hERG by binding within (168). The KCR1 I447V variant is associated with lower
the pore region of the channel. Patients with anti-Ro-posi- frequency of drug-induced arrhythmia, and reduced sensi-
tive autoimmune illness may be well advised to take partic- tivity hERG block by dofetilide when coexpressed heterolo-
ular caution to avoid medications that prolong the QT in- gously (322). These results suggest that functional KCR1
terval (494). provides protection against a drug-induced decrease in IKr,

BOHNEN ET AL.
with a mechanism of action thought to entail modulation of have been found, each having specific binding residues that
KCR1 ␣-1,2-glucosyltransferase activity (291). mediate their mechanism of hERG activation within the
“type 2” paradigm.
2. Drug screening for hERG toxicity
Other compounds have predominating mechanisms of
hERG activation that do not fall into the “type 1” or
Given the propensity for clinically relevant pharmacologi-
“type 2” classes. For instance, mallotoxin and KB130015
cal agents to block hERG, leading to drug-induced arrhyth-
primarily enhance hERG activation while shifting the
mia, it is now a required practice for drugs early in devel-
voltage dependence of activation to more negative mem-
opment to undergo hERG toxicity screens. High-through-
brane voltages, likely by binding within the channel pore
put screening techniques, including radioligand binding,
(140, 499). Some compounds, such as A935142 (407),
ion flux and fluorescence assays, and hERG-Lite which
have both “type 1” and “type 2” characteristics. As ac-
measures cell surface density of hERG, detect candidate
tivators bind hERG at various locations within the chan-
drugs with strong hERG-blocking and/or traffic-altering
nel, efforts to correlate binding location and channel stoi-
properties (436). The development of automated patch-
chiometry with mechanism of hERG activation by small
clamp electrophysiology enhances the throughput of data

molecule compounds will aid the design of more selective
acquisition tremendously, allowing for numerous hERG re-
and potent hERG channel activators by a variety of
cordings from different cells, applying different drugs and
mechanisms (476).
at different concentrations, to be collected simultaneously.
Automated patch-clamp technology is the best initial high-
throughput hERG toxicity drug screening method to date IV. INa DYSFUNCTION IN CONGENITAL
(163). A focus on hERG toxicity screening early in the drug LQTS
development process may save time, and prevent or mini-
mize adverse clinical outcomes due to inadvertent IKr block.
A. Introduction
3. Pharmacological treatment in LQT2
LQT3, LQT9, LQT10, and LQT12 account for 5–10% of
congenital LQTS (7) and are associated with gain-of-func-
␤-Blocker treatment represents first-line pharmacological tion mutations in the cardiac voltage-gated Na⫹ channel
therapy in LQT2 patients. For a more detailed discussion of complex of proteins. As such, patients diagnosed with Na⫹
␤-blocker treatment in congenital LQTS, refer to section VI channel-associated LQT often, though not exclusively,
of this review. have a unique risk profile for arrhythmia triggers and re-
quire different pharmacological approaches to disease man-
A growing interest in hERG activators has developed. Such agement than patients with LQT1 or LQT2 (370). Electro-
agents may be especially useful in the setting of congenital physiological experiments have elucidated the connection
LQT2 and/or drug-induced LQT syndromes. hERG activa- between aberrant function of single ion channels and the
tors may be subdivided into type 1 activators type 2 activa- unique clinical presentation of this set of diseases. In each
tors, based on mechanism of hERG activation (436). case, congenital mutations result in a failure of the cardiac
Na⫹ current (INa) to properly inactivate. However, even
Type 1 hERG activators include RPR260243, an experi- within the subset of Na⫹ channel-associated LQT, differ-
mental small molecule compound that slows hERG deacti- ences in mechanisms of pathological INa generation and risk
vation in a temperature- and voltage-dependent fashion as stratification in some patients makes LQT3 an important
its primary means of channel activation (204, 321). case study in the value of patient-specific medicine. Below
RPR260243 may slow deactivation via binding to the we will discuss the role of INa in the conduction of electrical
S4 –S5 linker and the cytoplasmic regions of the S5 and S6 impulses in the heart, mechanisms by which congenital mu-
hERG domains (321). tations may alter these currents, and advances in the discov-
ery of potent and selective pharmacological agents to treat
Type 2 hERG activators act primarily by slowing channel disease.
inactivation, and/or shifting the voltage dependence of in-
activation to more positive membrane voltages (69, 138,
153, 164). Type 2 activators may also have other relatively B. Physiology
minor effects on deactivation kinetics and voltage depen-
dence of activation, as well as single-channel open proba- In the heart, the majority of voltage-gated Na⫹ current is
bility (69, 138, 164, 165, 407, 511). Several type 2 hERG carried by the cardiac isoform of the voltage-gated Na⫹
activators have been described, including experimental channel, Nav1.5 (137). Upon membrane depolarization to a
compounds PD118057 (320), ICA105574 (28, 134, 263), threshold around ⫺50 mV, channels activate and allow the
and NS1643 (153, 155, 318), and key insights into their influx of Na⫹ down its electrochemical gradient. The rapid
mechanism of action alongside their hERG binding location influx of positive charge across the cell membrane drives the

A B C
0nA 1.0
C
Normalized Current
O 0.8
0.6
30pA
0.4
2pA
40ms –10 50ms

10ms 0.2 500ms
1nA
–100 –100
0
10ms –140 –120 –100 –80 –60 –40 –20 0 20 40
Membrane Potential (mV)

D SCN4B SCN5A
N I II III IV
Extracellular
+ + + +
+ + + +
+ + + +
+ + + +
Cytosol
C IFM motif
Syntrophin-α PDZ binding
N domain
C
Dystrophin
FIGURE 8. Physiology and molecular biology of INa. A: consecutive recordings of single Na⫹ channels
recorded under cell-attached conditions in HEK293 cells transfected with Nav1.5 cDNA (unpublished data).
B: WT Na⫹ currents recorded under whole cell patch-clamp conditions and obtained by depolarizations from
⫺120 to ⫺30 mV (unpublished data). At high gain (inset), whole cell currents return nearly to 0 nA. C: voltage
dependence of activation (red-filled square) and inactivation (blue-filled circle) (unpublished data). D: topology of
Nav1.5 in the plasma membrane with accessory proteins implicated in LQTS.
membrane potential from its resting voltage to a peak of highly voltage dependent. The voltage dependence of inac-
⫹50 mV, near the Na⫹ equilibrium potential (FIGURE 1). tivation is independent of the voltage dependence of chan-
The rapid activation of Nav1.5 is therefore critical to main- nel activation, as inactivation may occur from either closed
taining the speed of cardiac conduction. This rapid depo- or opened states (66). Closed-state inactivation can be mea-
larization coordinates the stimulation of voltage-gated sured with subactivation threshold depolarizing pulses.
Ca2⫹ and K⫹ channels necessary for excitation-contraction When these pulses are sufficiently long to approach equilib-
coupling and, eventually, repolarization (299). rium, these experiments are referred to as “steady-state in-
activation” experiments. As the holding potential becomes
In a healthy human heart, the Na⫹ current is rapidly atten- more depolarized, channels enter a closed-inactivated state
uated in milliseconds as channels inactivate, a process that and are unavailable to open upon supra-activation thresh-
was first observed and defined in Hodgkin and Huxley’s old depolarization with a midpoint near ⫺70 mV (FIGURE
giant squid axon experiments (173). Inactivation is respon- 8C). Because this midpoint is in the range of the resting
sible for the refractory period in neurons and myocytes membrane potential in a ventricular myocyte (⫺85 mV),
during which the firing of new impulses is not possible. small changes in the V0.5 of steady-state inactivation caused
Similar properties are observed in Nav1.5, with inactivation by mutations or drugs, or small changes in resting mem-
progressing with a time to half peak current on the order of brane potential, can have a large impact on channel avail-
1 ms and a mean open channel duration of roughly 0.5 ms ability, Na⫹ current density, and conduction velocity (86).
(49) (FIGURE 8, A AND B).
The voltage dependence of inactivation from the open state
Na⫹ channel inactivation is a complex multistep process has been more difficult to dissect, as channel fast-inactiva-
that involves multiple channel components and is itself tion is a nonequilibrium process. The rate of decay of Na⫹

BOHNEN ET AL.
currents increases with increasing voltage over the physio- nature of eukaryotic voltage-gated Na⫹ channels allows the
logically relevant range, reaching half-inactivation in 1.5 voltage sensors to modulate different aspects of channel
ms at ⫺40 mV and 0.5 ms at ⫹20 mV (34). Recovery from gating. Mutagenesis (66, 384), cysteine accessibility (380,
inactivation has the opposite voltage dependence of the on- 381, 383), and voltage-clamp fluorometry (71, 72) have
set of inactivation: channels recover more rapidly at hyper- revealed distinct properties of each voltage sensor. Move-
polarized potentials, and very slowly at potentials more ments of the DIV voltage sensor have been shown to corre-
positive than activation threshold (approximately ⫺45 mV) late with the voltage dependence and kinetics of inactiva-
(508). The presence of multiple inactivation states is evident tion (71, 147), and DIV-S4 activation is necessary and suf-
in the time course of recovery from inactivation. Following ficient for inactivation gating (66). The putative docking
an initial depolarizing pulse to inactivate channels, channel site for the inactivation gate resides in the intracellular
recovery during a period at resting membrane potential fol- NH2-terminal loops of S4 in DIII and DIV, potentially
lows a biexponential time course, suggesting the existence bridging our understanding of voltage-sensing domains and
of two distinct populations of channels (508): one in the voltage-dependent gating transitions (268, 415).
fast-inactivated state and one in the slow-inactivated state.
1. Accessory proteins

C. Molecular Biology
In the heart, Nav1.5 is associated with several accessory
Nav1.5 is encoded by the gene SCN5A (452). Unlike the proteins that modulate its trafficking and biophysical prop-
canonical voltage-gated K⫹ channels, Nav1.5 is composed erties. Four genes that encode ␤-subunits of the voltage-
of a single polypeptide chain (137). The protein folds into gated Na⫹ channel have been identified, SCN1B through
four homologous asymmetric domains, each containing six 4B, and all are expressed in the heart (253, 342). These
transmembrane helices (S1–S6) (70) (FIGURE 8D). S1 ␤-subunits have an Ig (immunoglobulin)-like NH2-terminal
through S4 form a voltage-sensing unit in each domain. S4 domain, a single transmembrane helix, and a short cyto-
spans the membrane and contains several charged arginine plasmic COOH-terminal domain. While they are similar in
residues capable of “sensing” changes in membrane poten- structure, the ␤-subunits interact with the ␣-subunit
tial. Depolarization drives a conformational change in S4 through different mechanisms. ␤1 and ␤3 interact nonco-
that is transmitted to the pore-forming helices, S5 and S6. valently with the ␣-subunit, and ␤2 and ␤4 interact via
Along with the extracellular reentrant loop that connects covalent disulfide bond (490).
them, S5 and S6 from each domain form the central ion
conduction pathway (70). Immunohistochemical studies suggest that ␤-subunits play an
important role in subcellular localization of different channel
The intracellular segments of Nav1.5 have been shown to be isoforms (253). Nav1.5 predominantly colocalizes with the ␤2
critical to proper channel function. The linker between do- and ␤4 subunits, encoded by the gene SCN2B and SCN4B, in
mains III and IV contains the three amino acid isoleucine, the intercalated disks. ␤4 has no significant effect on Nav1.5
phenylalanine, methionine (“IFM”) motif, shown to be a activation or inactivation gating (490). Small amounts of ␤1
key molecular determinant of fast inactivation (313, 465). subunit were also found to colocalize with Nav1.5 (101, 211,
Upon channel opening, this motif has been proposed to 253). Unlike ␤4, ␤1 displays a dramatic functional effect on
bind to a hydrophobic cluster in the intracellular linkers inactivation of Nav1.5 currents expressed in tsA cells. ␤1, but
between S4 and S5 of domains III and IV (268, 415). Con- not ␤2, caused a significant positive shift in the V0.5 of steady-
sistent with these findings, disruption of the structure of the state inactivation (101, 211, 342).
DIII/DIV linker by proteolytic cleavage (434) or mutation
(47) can impair inactivation, and coexpression of a cytoso- Nav1.5 is also associated with several other structural and
lic peptide containing the IFM motif can potentiate inacti- signaling complexes in the cardiomyocyte. Caveolin and
vation (111). syntrophin are two such proteins that have been implicated
in functional perturbation of INa and Na⫹ channel-associ-
The intracellular COOH-terminal domain has been shown to ated LQT. The six known caveolin proteins are encoded by
play an important role in channel function. The COOH ter- three caveolin genes: CAV1, CAV2, and CAV3 (30). Cav-3
minus has a structured proximal domain and unstructured is the most abundant isoform in cardiac tissue where it is
distal region (91). It has been proposed that interactions be- associated with cytoplasmic signaling factors (396), is re-
tween the COOH terminus and DIII/DIV linker are critical in sponsible for the formation of lipid raft microdomains and
the stabilization of the inactivated state (285), and further- caveolae (218), and potentially plays a role in t-tubule or-
more that this interaction may be facilitated by calmodulin ganization (133). Cav-3 null mice lacked caveolae and had
(435). In fact, several LQT3-causing mutations have been abnormal t-tubule development in skeletal muscle. Bio-
identified in the COOH terminus (34, 343). chemical and microscopic techniques have revealed that
Nav1.5 colocalizes with Cav-3 in lipid rafts, which facilitate
Multiple techniques have been used to study the role of the increase in peak INa density in response to ␤-adrenergic
voltage-sensing units in channel function. The asymmetric stimulation (486).

Caveolin and Nav1.5 also both associate with the cytoskel- underlie tissue-specific phenotypes (185), as well as ar-
etal dystrophin complex (106). Syntrophin-␣1 is a member rhythmia risk stratification (32). Two mechanisms have
of the dystrophin-associated protein complex and is the been established for the generation of persistent INaL, and
most abundantly expressed syntrophin in the heart. Syntro- an additional set of mechanisms has been proposed for
phins mediate the interaction of dystrophin with the PDZ- aberrant Na⫹ conduction in the absence of INaL. TABLE 5
binding domain on the COOH terminus of Nav1.5 (135). lists a set of Nav1.5 mutations known to perturb channel
Isolated cardiomyocytes from dystrophin knockout mice function by specific mechanisms.
showed markedly decreased INa density, and ECGs showed
significant QRS prolongation, consistent with an interac- A) CHANNEL BURSTING. Channel bursting is best exemplified
tion between the dystrophin-associated protein complex by the ⌬KPQ mutation, a deletion of amino acids 1505–
and ion channels in the heart. 1507 in the DIII/DIV linker near the IFM motif. ⌬KPQ was
first identified in 1995 (453) and characterized that same
year (47). Like WT currents, currents passed by single
D. Molecular Pathophysiology
⌬KPQ channels typically activate and inactivate quickly.
However, during a small fraction of depolarizing pulses,

1. LQT3
channels enter a slow gating mode, or “burst” mode, char-
acterized by a persistent noninactivating current.
Like other forms of LQT, the hallmark of LQT3 is a pro-
longation of the QT interval on the ECG arising from a Modal transitions have been shown to be highly dependent
prolongation of the ventricular action potential and delayed on inter-pulse duration and resting membrane potential.
ventricular repolarization. The discovery that mutations in INaL from ⌬KPQ channels in heterologous expression sys-
multiple genes can give rise to LQT has allowed for an
tems (289) is highly rate dependent, decreasing dramati-
improved understanding of the risk stratification for car-
cally with increased pulse frequency. In isolated transgenic
diac events in this set of patients (FIGURE 9, A–C). While
⌬KPQ mouse cardiomyocytes, changes in pacing rate
patients with K⫹ channel-associated LQT are generally at
caused pause-induced spontaneous depolarizations (127).
higher risk or cardiac events during exercise, patients with
This is consistent with a decrease in EAD frequency and
LQT3 have elevated risk at rest (370, 371). However, even
polymorphic VT during increased pacing in intact mouse
within the umbrella of LQT3 there is a range of triggers for
hearts (120), and explains the elevated risk of arrhythmia
arrhythmia that is not easily explained by a “one size fits
and sudden cardiac death at rest in human patients with
all” model, suggesting patient-specific differences in disease
LQT3 (370, 371).
mechanism (244).
LQT3 is a disease of impaired Nav1.5 inactivation. Failure The inverse dependence on interpulse duration suggests
of the channel to inactivate or remain inactivated during the that transitions between background and burst gating
plateau or repolarization phases of the ventricular action modes occur predominantly at rest. At higher resting poten-
potential allows for a depolarizing current sufficient to pro- tials, a larger fraction of channels exist in closed-inactivated
long the action potential, leaving patients susceptible to states, leaving fewer channels available to transition into
asynchronous EADs or DADs (184, 240). In most cases, bursting states. Indeed, INaL is smaller during test pulses
impaired inactivation is manifest as a sustained noninacti- from more depolarized holding potentials (185). Taken to-
vating inward current, or late current (INaL) (FIGURE 9D). gether, Clancy and Rudy (85) were able to develop a com-
Rather than inactivating completely, many disease-associ- putational model of ⌬KPQ function by allowing transitions
ated mutations cause a persistent INaL during prolonged between background and burst modes only when channels
depolarizations. It should be noted that INaL of smaller exist in the closed state. This model was able to reproduce
magnitude has also been detected in tissue isolated from single-channel and whole cell gating properties, as well as
normal hearts (432); thus in pathology some INaL may re- prolongation of the ventricular action potential and the
sult from the failure to control or modulate normal activity. occurrence of EADs.
Nevertheless, while currents through these channels still
provide the rapid influx of Na⫹ necessary for cardiac con- B) LATE REOPENING. Following the identification of modal
duction, at high gain one can observe INaL that amounts to transitions in ⌬KPQ channels, other SCN5a mutations
as little as 1% of the magnitude of the peak. Because the were identified in patients with LQT3 that cause INaL by a
membrane impedance during the plateau phase of the ac- different mechanism. At the whole cell level, the N1325S
tion potential is high, this small depolarizing current has a and R1644H mutations had markedly different inactiva-
substantial effect on AP duration. tion (109) and recovery from inactivation time courses
(448). Single-channel records revealed that these gating de-
The mechanisms by which gain-of-function Na⫹ currents fects were in fact not due to bursting channels, but to dis-
may arise have been studied in depth, and recent computa- persed channel reopening that occurred during prolonged
tional work suggests that these divergent mechanisms may depolarizations (109). Additional studies revealed that

BOHNEN ET AL.
A SCN5A
I II III E1295K IV
Extracellular
+ + + +
+ + + +
+ + + +
+ + + +
Cytosol I1768V
N1325S
Y1795C
ΔKPQ
N S1904L
C

B D
LQT3 ECG
WT
F1473C
1%
50ms
V5
2ms
C E
1.0
WT
F1473C
AP
Normalized Current
0.5
INa
–10
5s
–100
WT
0.0
LQT3
–90 –60 –30
Voltage (mV)
FIGURE 9. INa dysfunction leading to congenital LQTS. A: topology of Nav1.5 in the plasma membrane.
Several representative LQT3-associated mutations were selected because there is strong evidence that they
induce INaL by channel bursting (red-filled triangle) or by late reopening (red-filled circle), or prolong the action
potential without INaL (red-filled square). B: ECG from a LQT3 patient (unpublished data). C: simulated action
potential (top) and INa (bottom) in WT (green) and heterozygous LQT3 (purple) conditions. INa peak current was
truncated to enhance view of INaL. D: representative current recordings from WT (purple) and F1473C
(orange) Nav1.5 expressed in HEK293 cells at low and high gain (inset) elicited by depolarization to ⫺10 mV
(35). E: steady-state channel availability for WT (green triangle) and F1473C (purple square). [D and E from
Bankston et al. (35).]

Changes in steady-state properties of the cardiac sodium

Table 5. Representative LQT3-associated mutations classified current have been proposed as mechanisms for AP prolon-
by mechanism of gain of function gation and LQT, even in the absence of sustained INaL dur-
Reference ing prolonged depolarizing steps (FIGURE 9E) (6, 344, 395,
Mechanism Mutations Nos. 461). These mutations cause depolarizing shifts in channel
availability such that the window current is either larger, or
INaL: bursting ⌬KPQ (1505–1507) 47 exists at more depolarized voltages where rectification of
Y1795C 243 K⫹ currents increases the relative contribution of INa to
INaL: late reopening R1644H 109 total membrane current. These patients are therefore more
N1325S 109 susceptible to drug-induced LQT, most commonly arising
G1631D 447 from block of hERG channels, less commonly from block of
S1904L 34 other K⫹ channels and drugs that potentiate inward cur-
R1623Q 202 rents, including INa (439, 482). Additionally, several dis-
INaL: cAMP-dependent D1790G 416 ease-causing mutations have been shown to induce depolar-
Window current E1295K 6 izing shifts in channel availability in addition to canonical

G1631D 447 INaL during prolonged depolarization (35, 448, 463).
F1483del 397
Nonequilibrium gating I1768V 87 D) NONEQUILIBRIUM GATING DEFECTS. In addition to changes in
steady-state parameters of channel activity, time-dependent
changes in gating kinetics have also been posited as a mech-
these late reopenings contribute to a fraction of the INaL anism for AP prolongation. As membrane potential declines
from ⌬KPQ channels, as well (73). into the repolarization phase of the AP, WT channels re-
cover slowly from inactivation. As membrane repolariza-
An additional mutation, S1904L, was later shown to also tion is more rapid than recovery from inactivation, WT
induce late reopenings and INaL (34). Interestingly, this mu- channels never recover at potentials that facilitate channel
tation does not reside in the DIII/DIV linker, but rather activation. Some LQT3 mutant channels, such as the
exists in the distal COOH-terminal domain. Transition I1768V mutation, have enhanced recovery from inactiva-
metal ion FRET showed that this mutation destabilized a tion and are able to recover at voltages above the channel
critical intramolecular interaction within the COOH-termi- activation threshold (87). Importantly, this nonequilibrium
nal domain (144), contributing to our understanding of the reopening is different than window current because it oc-
inactivation gate as a macromolecular complex of different curs at potentials at which activation and steady-state chan-
channel components including the DIII/DIV linker and nel availability do not overlap.
COOH terminus (285).
E) PHOSPHORYLATION-DEPENDENT INaL.There exists one known
A computational model has been constructed to simulate mutation, D1790G, for which in vitro studies found INaL
late reopening currents at the single-channel and whole cell only arises after PKA-dependent phosphorylation (416).
level (34). Function of the S1904L mutation could be sim- D1790G channels showed marked INaL in response to
ulated by reducing the rate of transition into the slow inac- cAMP-dependent PKA stimulation and lacked INaL under
tivated states, allowing channels to reside for a prolonged basal conditions. Furthermore, the D1790E mutation
time in fast inactivated states from which they may reopen. showed no response to cAMP application, suggesting that
An interesting prediction of this model is that the amplitude the negative charge at residue 1790 is important in modu-
of INaL will not display the inverse rate dependence present lating PKA phosphorylation. Interestingly, alanine mu-
in bursting channels, and in fact the patient experienced tagenesis of consensus PKA phosphorylation sites showed
arrhythmic events that were not limited to periods of rest. that S38 and S525 are important in mediating INaL in
D1790G channels, despite residing in disparate parts of the
C) WINDOW CURRENT. In WT channels, the voltage depen- channel. These effects were found despite the fact that most
dence of channel availability (V0.5 ⫽ ⫺71 mV) and channel spontaneous arrhythmic events in LQT3 patients occur
during rest or sleep, suggesting perhaps a role of basal phos-
activation (V0.5 ⫽ ⫺25 mV) overlap only slightly. There-
phorylation of the Nav1.5 channel in D1790G patients
fore, there is a very small range, or “window,” of voltages in
(371).
which channels may activate but not completely inactivate.
This range may provide a small noninactivating current
known as “window current.” Typically, this window cur- 2. LQT associated with mutations to accessory
rent is very small and only exists at voltages at which repo- proteins
larizing currents through delayed rectifier K⫹ channels are
strong, and therefore contribute little to net membrane cur- In addition to mutations in SCN5A, mutations in the acces-
rent. sory proteins important in cardiac INa have been associated

BOHNEN ET AL.
with LQT (11), although there are few documented cases of E. Molecular Pharmacology
such diseases.
The pharmacology of Na⫹ channel-associated LQT has
3. LQT10 been difficult to navigate because the diversity in patient
phenotypes (392, 397) and risk of on- and off-target effects
of standard antiarrhythmic therapies (276) has limited their
Mutations in the ␤4 subunit give rise to LQT10. The L179F
use clinically. As a result, genetically identified LQT3 pa-
mutation was identified in a 21-mo-old girl with bradycar-
tients are the most likely to receive implantable cardio-
dia and severe QT prolongation (261). When coexpressed
verter-defibrillators (ICDs) as a precautionary measure de-
with Nav1.5 in HEK cells, this mutation caused both a
spite previously having been asymptomatic (372). There is
dramatic INaL and a depolarizing shift in the V0.5 of steady-
therefore a large unmet medical need for effective pharma-
state inactivation that increased the window current. Inter-
cological intervention, and our understanding of Na⫹ chan-
estingly, this ␤-subunit mutation induced a larger INaL than
nel blockade and ␤-adrenergic receptor blockade in LQT3
the ⌬KPQ deletion in the ␣-subunit. Mutations in SCN4B
continues to evolve.
have also been associated with Sudden Infant Death Syn-
drome (SIDS) (413). The SIDS-associated S206L caused a

1. Local anesthetics
depolarizing shift in stead-state inactivation and a dramatic
increase in INaL. Recently, a mutation was reported in the
The utility of local anesthetics in the treatment of LQT3 is
␤1b subunit, a splice variant of ␤1, that caused a speeding
rooted in their ability to inhibit the voltage-gated Na⫹
of recovery from inactivation, significant INaL, APD prolon-
channel with preference for INaL (FIGURE 10). Soon after the
gation in HL-1 cells, and QT prolongation and stress-in-
discovery of the ⌬KPQ mutation and its role in LQT3, it
duced syncope in the patient (342). This was the first report
was reported that the commonly prescribed local anesthetic
of a mutation in ␤1b associated with LQT.
and class 1b antiarrhythmic agent lidocaine inhibited INaL
more effectively than peak current (19). This phenomenon
4. LQT9 and LQT12 has been observed in block of several mutations by other
clinically relevant local anesthetic-like drugs including
LQT9 is a Na⫹ channel-associated arrhythmia arising from mexiletine (35, 258), flecainide (288), and ranolazine (127),
mutations in Cav3, the gene encoding Cav-3, a cardiac iso- as well as recent experimental drugs GS967 (43) and elecla-
form of caveolin (30). Four Cav-3 mutations were identified zine (128, 200), making them useful in normalizing the
in a sample of 905 LQTS patients (438). Sixty-seven percent QT-interval (130, 281, 284, 370) and preventing arrhyth-
of patients identified with mutations in Cav3 exhibited syn- mic events in some patients (347). This approach has re-
cope or cardiac arrest during periods of rest, consistent with cently been supported by a study of patients with a wide
the risk profile for patients with mutations directly in the range of LQT3 mutations in which a robust improvement in
␣-subunit, Nav1.5. Two mutations, F97C and S141R, were QT interval and a reduction in arrhythmic events was in-
studied further by coexpression in HEK-293 cells with duced by oral mexiletine (258). Because of the efficacy of
Nav1.5. The S141R mutation sped recovery from slow in- these drugs in treating LQT3 patients and because of a
activation, and both mutations caused a significant INaL. common underlying mechanism, preferential inhibition of
Consistent with the functional importance of the Cav-3/ late versus peak Na⫹ channel current, development of more
Nav1.5 interaction, three additional mutations in Cav3 selective and effective blockers of INaL is underway by the
have been shown to induce INaL and are associated with pharmaceutical community. Nevertheless, it is important to
SIDS (92). recognize and understand the mechanisms underlying inhi-
bition of this current.
In mouse skeletal muscle, knockout of Cav-3 caused a dis-
ruption of the dystrophin complex in lipid rafts (133). Per- Local anesthetic-like drugs bind to a conserved phenylala-
haps not surprisingly, mutations in syntrophin-␣, a member nine (127, 336) in the central cavity of the channel in a
of the dystrophin protein complex, are also associated with highly state-specific manner. This specificity is imparted
LQTS and give rise to variant 12 of the disease (473). Two both by state-dependent changes in drug access to the bind-
mutations in SNTA1, the gene encoding syntrophin-␣1 have ing site (243, 336) and by allosteric modulation of channel
been identified. The A257G mutation nearly doubled the gating (162). Clinically relevant channel blockers have been
peak current density, increased window current, and sped shown to bind preferentially either to inactivated states, as
recovery from fast inactivation, although it did not induce is the case for lidocaine, flecainide, and mexiletine, and, in
INaL relative to peak current density in isolated neonatal rat the case of ranolazine, to open channels (275).
cardiomyocytes (473). It is likely, however, that absolute
INaL was greater in A257G myocytes because of the increase The state dependence of drug binding has been assayed by
in channel expression. The A390V mutation does cause a electrophysiological as well as biochemical experiments. Li-
persistent INaL and a depolarizing shift in steady-state inac- docaine induces a hyperpolarizing shift in steady-state inac-
tivation in a nitric oxide synthase-dependent manner (431). tivation(202), slows recovery from inactivation, and stabi-

A B
F1473C
Mexiletine Ranolazine
1%
50ms
2ms

C D
* * *
Flecainide 80
60
Percent Block
40
20
0
Mexiletine Ranolazine Flecainide
FIGURE 10. Representative recordings at low and high (insets) gain of F1473C inhibition by 50 ␮M
mexiletine (A), 50 ␮M ranolazine (B), and 10 ␮M flecainide (C). D: mexiletine, ranolazine, and flecainide inhibit
INa with preference for INaL. [From Bankston et al. (35).]
lizes voltage-sensing units shown to provide the rate-limit- in patients suspected to have loss-of-function Na⫹ chan-
ing step in fast inactivation (27, 66, 382). Lidocaine has also nel mutations in Brugada Syndrome because of its ability
been studied at the single-channel level. In cell-attached to reduce peak INa and unmask latent disease phenotypes
patches, lidocaine reduced the propensity of ⌬KPQ chan- in a clinical setting (58). It has therefore been proposed
nels to burst, increased the number of depolarizing sweeps that an understanding of channel pathology and drug
with no channel openings, and had no effect on the mean properties may help guide a pharmacogenomic approach
open time of background or burst openings (31, 108). to disease management (88).
Taken together, these findings are consistent with stabiliza-
tion or promotion of inactivated channels. Ranolazine, on
2. ␤-Blockers
the other hand, has no effect on steady-state inactivation
and has therefore been interpreted as an open-channel
blocker (275). There remains some debate over the clinical utility of
␤-blockers in treatment of LQT3. A mainstay therapy for
It is therefore not surprising that mutations that alter patients with QT prolongation, clinical data suggest that
channel gating by different mechanisms will produce prophylactic ␤-blocker therapy in patients with LQT3 may
unique allosteric effects on drug binding. For example, be less effective at preventing cardiac events as it is in K⫹
flecainide (243) and mexiletine (347) have been shown to channel-associated LQT (282, 331). Interestingly, however,
more potently inhibit some mutations than others when ␤-blocker therapy did not show evidence of arrhythmia
expressed in heterologous systems. Mutation-specific induction, as might be expected from the heart rate-slowing
variability is therefore a major hurdle in disease manage- effects of ␤-blockade in LQT3 patients with elevated risk of
ment (88), as heightened sensitivity to Na⫹ channel arrhythmia at rest. This has prompted further investigation
blocking drugs can be pro-arrhythmic in certain settings into the mechanism of ␤-blocker activity in models of
(112, 276). In fact, flecainide is a useful diagnostic agent LQT3.

BOHNEN ET AL.
Experiments in animal models of LQT3 (64, 333) showed a ankyrin-B (271). These mutations in general lead to disrup-
protective effect of sympathetic stimulation against APD tion of normal targeting and regulation of transporters as
prolongation and torsades de pointes (387), suggesting a well as ion channels, leading to disrupted calcium handling
deleterious effect of ␤-blockade. However, experiments in that contributes to arrhythmia (272, 273). However, vari-
heterologous expression systems have shown that ␤-block- ation in severity of cardiac phenotypes exists between dif-
ers have a direct effect on Na⫹ channels (33). ␤-Blockers ferent mutations and can be correlated to the degree of loss
exhibit preferential inhibition of INaL and bind to the local of function observed in vitro (271).
anesthetic binding site, and at high doses have a similar
pharmaceutical profile as local anesthetics.
B. LQT7: Andersen-Tawil Syndrome Type 1
Ahrens-Nicklas et al. (12) constructed a computational
Andersen-Tawil syndrome type 1 arises from LQT7 muta-
model of ⌬KPQ-expressing ventricular myocytes stimu-
tions, which are loss-of-function mutations in the KCNJ2
lated by the ␤-agonist isoproterenol and inhibited by the
gene (327). This gene encodes Kir2.1, an inward rectifier
␤-blocker propranolol. Isoproterenol shortened APD and
potassium channel that underlies the cardiac IK1 current
EADs, consistent with the inverse-rate dependence of INaL
(104, 248, 303, 496), which contributes to setting the myo-

and animal models of LQT3. At low doses, propranolol
cardial resting potential and aids terminal repolarization of
mitigated this effect and prolonged APD, but at higher
the action potential (264). Because the membrane potential
doses where propranolol inhibits Na⫹ currents, it provided
of cardiomyocytes is more positive than the potassium equi-
a beneficial reduction in APD. Taken together, the experi-
librium potential, the channel actually conducts an outward
mental and computational data suggest a potential benefit
current in physiological conductions. Kir2.1 expression has
of ␤-blockade in treatment of LQT3, but more robust
been demonstrated in both human atria and ventricles
blinded studies must be performed to better understand the
(458). The protein consists of two transmembrane helices, a
clinical relevance.
pore-loop, as well as an NH2- and COOH-terminal region.
Four Kir2.1 subunits may come together to form a func-
V. OTHER SUBTYPES OF CONGENITAL tional channel. It has been shown that PIP2 binding to
LQTS Kir2.1 plays an important role in channel activation (179,
410).
A. LQT4: Ankyrin-B Syndrome
Andersen-Tawil syndrome is an autosomal dominant, mul-
tisystemic disorder characterized by periodic paralysis, ven-
The correct targeting of ion channels and transporters to
tricular arrhythmias, as well as dysmorphic features includ-
their respective subcellular domains is critical for normal
ing low-set ears, hypertelorism, and hypoplastic mandible
physiological function. It was discovered that autosomal
(362, 417). Early reports of patients with Andersen-Tawil
dominant loss-of-function mutations in ANK2, which en-
syndrome described QT prolongation on ECG; however,
codes ankyrin-B, can cause LQTS (273, 366). Ankyrin-B is
most ECGs of patients with this disorder are characterized
an adaptor protein that functions in the cytoskeletal net-
by prominent U waves that merge with the T wave, and a
work to ensure proper localization and stabilization of ion
characteristic prolonged “Q-T-U” interval. The physiolog-
channels and transporters. Although ankyrin-B was origi-
ical basis for the U wave is not well understood, but the
nally characterized as a 220-kDa protein, multiple splice
large U waves in Andersen-Tawil syndrome may be the
variants exist (474). The protein is composed of a mem-
result of delayed after depolarization or tissue-dependent
brane-binding domain, a spectrin-binding domain, and a
differences in repolarization including the mid myocardium
regulatory domain (271). It has been demonstrated to play
and His-Purkinje system (317, 328, 502). Andersen-Tawil
important roles in the normal localization of the Na⫹/Ca2⫹
syndrome type 1 results from KCNJ2 mutations, almost all
exchanger, the Na⫹-K⫹-ATPase, as well as the IP3 receptor
of which are dominant negative (424, 425). Many of these
(IP3R) which mediates intracellular calcium release (95,
mutations, such as R218W and G300D, located in the
269, 270, 273, 429). In addition, ankyrin-B has been shown
COOH-terminal region, either decrease PIP2 affinity or af-
to regulate voltage-gated Na⫹ channels (74), ATP-gated K⫹
fect residues involved in PIP2 binding (103, 249, 398, 501).
channels (236), as well as ryanodine receptors (65) in the
Some disease mutations, such as ⌬312–314, lead to dimin-
heart.
ished channel trafficking to the cell surface (44). Disease-
associated mutations can also be found in the NH2-terminal
While a loss-of-function ankyrin-B mutation was initially
region as well as the channel pore (424).
described as LQT4, it was later discovered that mutations in
ankyrin-B are not always associated with QT prolongation
and may present with a variety of cardiac phenotypes in- C. LQT8: Timothy Syndrome
cluding bradycardia, sinus arrhythmia, as well as atrial fi-
brillation (96, 232, 271). Most of these mutations, such as Timothy syndrome is caused by gain-of-function muta-
E1425G, are located in or near the regulatory domain of tions in the CACNA1C gene, which encodes Cav1.2, the

␣1C (pore-forming) subunit of a cardiac voltage-gated D. LQT13

calcium channel. The channel generates the L-type cal-
cium current, which allows calcium influx during the Mutations in the KCNJ5 gene can also cause LQTS. KCNJ5
plateau phase of the action potential, sustaining mem- encodes a G protein-coupled inwardly rectifying potassium
brane depolarization and leading to calcium release from channel, Kir3.4 (or GIRK4). Kir3.4 and another GIRK iso-
intracellular stores which triggers excitation-contraction form Kir3.1(GIRK1) may form homo- and heterotetramers
coupling in cardiomyocytes. The channel undergoes both to generate the IKACh current in the heart (99, 219, 222), a
calcium-dependent and voltage-dependent inactivation. potassium current that is stimulated by acetylcholine
Inactivation of calcium channels during the action poten- through a GPCR-mediated pathway which has been dem-
tial causes an imbalance between the inward calcium and onstrated to slow heart rate during parasympathetic stimu-
outward potassium currents, resulting in repolarization. lation (136, 467). Similar to other inward rectifier channels,
The ␣1C subunit consists of four homologous domains of Kir3.4 contains two transmembrane helices with a pore-
six transmembrane helices each (S1–S6), including a volt- loop in-between as well as an NH2- and COOH-terminal
age-sensing domain (S1–S4) and a pore domain (S5–S6), region. Activation of GPCR receptors by acetylcholine
similar to voltage-gated sodium channels. Different causes the G␤␥ subunit to activate Kir3.4, resulting in an

splice variants of the ␣1C subunit may be expressed in the outward K⫹ current and hyperpolarization of the mem-
heart (445). brane potential, which in pacemaker cells results in slowing
of heart rate (214, 341, 468).
Timothy syndrome is an extremely rare genetic multisys-
temic disorder that includes QT prolongation, syndactyly, The presence of IKACh has been best demonstrated in sino-
craniofacial abnormalities, congenital heart defects, as well atrial node, atrioventricular node, and atrial cells of the
as autism. All known Timothy syndrome mutations to date heart (225). While it has also been shown to be expressed in
are located at the COOH-terminal end of S6 helices in dif- the ventricles (42, 183, 217, 466, 485), its role there is not
ferent domains of ␣1C (54, 142, 403, 404). Most com- well understood. Identification of LQTS mutations on
monly, Timothy syndrome arises from the de novo missense KCNJ5, however, suggests a role for this current in control-
mutation G406R on exon 8a, which is present in a minor ling ventricular action potential (484). For example, the
cardiac splice variant of CACNA1C (404). This mutation disease mutation G387R, a conserved residue located at the
is located at the COOH-terminal end of the first S6 helix COOH-terminal region of Kir3.4, has been shown to cause
of the ␣1C subunit and dramatically disrupts voltage- autosomal dominant LQTS. The mutation has been shown
dependent inactivation of Cav1.2, leading to increased to reduce trafficking of both Kir3.4 as well as Kir3.1 to the
plasma membrane and decrease current amplitude. Interest-
calcium influx that can result in APD prolongation. Tim-
ingly, a number of these LQTS patients also have atrial
othy syndrome resulting from this mutation came to be
fibrillation, suggesting a role for Kir3.4 in electrical conduc-
known as the “classic” variant. Two patients were later
tion in both atria and ventricles.
found to have the mutations G406R and G402S, respec-
tively, on exon 8, the major splice variant in the heart
(403). Similar to the classic Timothy syndrome mutation, E. LQT14 and LQT15
these two “atypical” Timothy syndrome mutations also
disrupt channel inactivation. Interestingly, neither of the
Calmodulin is a ubiquitous calcium-binding protein that
two atypical Timothy syndrome patients had syndactyly,
critically mediates cellular calcium signaling. Calmodulin is
as opposed to 100% of patients with classic Timothy important not only for the calcium-dependent inactivation
syndrome, suggesting differential expression patterns of of L-type calcium channels and the inactivation of Nav1.5
the ␣1C splice variants. One Timothy syndrome muta- channels (435, 514), but also in the trafficking and assem-
tion, I1166T, shows minimal effect on Cav1.2 inactiva- bly of KCNQ1 (379). Three genes, CALM1-3, all encode
tion, but is thought to cause disease by increasing win- the identical calmodulin protein. For example, a recent
dow current, which occurs in voltage ranges where chan- study suggests that de novo mutations in CALM1 or
nels activate but do not completely inactivate (54). CALM2 are associated with severely prolonged QT inter-
Efforts are underway to develop mechanism-based ther- val, ventricular arrhythmias, as well as atrioventricular
apies. In a landmark study, it was found that roscovitine, block in infants (93). Located in calmodulin EF-motifs that
a cyclin-dependent kinase inhibitor, partially recovers directly bind calcium, these mutations result in a reduction
inactivation of mutant Cav1.2 channels (487, 488) and in calcium affinity that may lead to abnormalities in sodium
reduces APD in ventricular-like cardiomyocytes derived currents, potassium currents, as well as calcium dynamics in
from iPSC reprogrammed from the skin cells of a Timo- the heart. In addition to exhibiting prolonged QT, these
thy syndrome patient (489). The study provides a model patients show neurodevelopmental delay, consistent with
for future development of therapies using human iPSC- widespread calmodulin expression in different tissues
derived cardiomyocytes. (316).

BOHNEN ET AL.
VI. GENOTYPE-DRIVEN CLINICAL domains either directly or indirectly. Thus the location of
MANAGEMENT OF CONGENITAL LQTS the hERG mutation variably impacts susceptibility to com-
mon triggers in LQT2 patients.
A. Introduction In LQT3, rates of cardiac events are high, and risk is likely
related to the degree of channel dysfunction or late current.
Clinical management of congenital LQTS aims to minimize The canonical SCN5A ⌬KPQ mutation results in 2.4-fold
symptomatic arrhythmia and prevent life-threatening car- greater risk of experiencing cardiac events through age 40
diac events. As described in detail in the preceding sections, compared with the SCN5A D1790G mutation (244). Fur-
the three most common congenital Long QT syndromes thermore, while preliminary data suggest similar risk for
(LQT1 due to mutation in KCNQ1; LQT2 due to mutation mutations in the COOH-terminal versus transmembrane
in hERG; and LQT3 due to mutation in Nav1.5) result in region of SCN5A, a 2.5-fold risk of ACA and SCD is con-
lengthening of the APD in cardiomyocytes, leading to a ferred by mutations that cause both sodium late current and
prolonged QT interval on the ECG. window current, compared with mutations causing only
one such gain-of-function defect (38).

By consensus guidelines, management strategies in congen-
ital LQTS today are mostly uniform, regardless of geno- 2. Genotype-specific triggers of arrhythmia
type. However, given the remarkable strides made in under-
standing the molecular identity of LQTS subtypes, as well
The first recognition of the relevance of genotype in man-
as the specific impact of a wide range of LQT-associated
agement of LQTS occurred in the context of a seminal study
mutations, the ultimate hope is that understanding a pa-
linking molecular identity in LQTS with clinical features,
tient’s genotype and specific LQT mutation can lead to
establishing genotype-specific triggers of arrhythmic events
rational treatment considerations. This section reviews the
(371). Six hundred seventy symptomatic patients with
features of molecular physiology that may influence geno-
LQT1, LQT2, and LQT3 were followed, and strong differ-
type-specific strategies for clinical management.
ences arose in the trigger of arrhythmic events based on
patient genotype (371). LQT1 patients experienced 62% of
B. Risk Factors for Arrhythmogenesis their cardiac events during exercise, and swimming or div-
ing was found to be a particularly potent trigger of arrhyth-
1. Mutation location, type, and cardiac risk mia (10, 279). In contrast, LQT2 patients experienced the
highest percentage of arrhythmic events in the setting of
emotional arousal (43%), including sudden loud noises and
In LQT1, mutations in the transmembrane regions includ-
startle (e.g., alarm clock awaking the patient from sleep), or
ing cytoplasmic loop (C-loop) domains of KCNQ1 confers
other triggers of emotional stress and fear; 13% of LQT2
a twofold greater risk of cardiac events [defined as syncope,
patient triggers occurred in the setting of exercise. LQT3
aborted cardiac arrest (ACA), or sudden cardiac death
patients experienced the highest percentage of arrhythmic
(SCD)] compared with patients harboring mutations in the
events, 39%, at rest or during sleep (371). LQT1 patients,
COOH-terminal domain of the KCNQ1 protein (280). The
by extension, experience more cardiac events in the setting
C-loops are involved in sympathetic regulation of KCNQ1
of sympathetic nervous system stimulation induced by ex-
(256), and mutations in this region may underlie an in-
ercise than LQT2 or LQT3 patients. More generally, these
creased risk of cardiac events in these patients compared
findings highlight the varying pathophysiology of the LQT
with mutations in other regions of the channel due to the
subtypes, and the specific life-style modifications and treat-
substantial regulation of KCNQ1, and therefore IKs activ-
ment considerations dependent on genotype (described
ity, by sympathetic stimulation (39).
later in this section).
In LQT2, Shimizu et al. (389) reported that pore region
missense mutations, located in the S5-loop-S6 portion of 3. Role of heart rate in LQT variant susceptibility to
the hERG channel, confer greater risk of cardiac events than arrhythmia
non-pore mutations in hERG (389). Specific triggers of car-
diac events were further subcategorized in LQT2 based on The variable triggers of arrhythmia in congenital LQTS can
mutation location and age at the time of the cardiac event. be explained in part by the role of heart rate in arrhythmo-
A study of 634 LQT2 patients revealed that while patients genesis. Heart rate contributes greatly to action potential
with pore mutations in hERG confer a ⬎2-fold risk of car- and QT duration (298), and therefore impacts the most
diac events compared with individuals with non-pore mu- common forms of congenital LQT syndrome in a genotype-
tations overall, non-pore transmembrane domain muta- specific, and even mutation-specific manner.
tions specifically led to a 6.8-fold increase in exercise-in-
duced cardiac events (216), suggesting that adrenergic In LQT1, a higher basal heart rate increases risk of cardiac
pathways interact with hERG non-pore transmembrane events, providing some prognostic value, unlike in LQT2

patients (40, 57, 373). Functional IKs current is pivotal to Given the diagnostic utility of stress testing, attempts at
normal QT adaptation, or shortening with heart rate. At using this test to guide genotype and mutation-specific risk
increased heart rates (and/or during ␤-adrenergic stimula- stratification have been made. Laksman et al. (230) tested
tion of the heart), IKs facilitates cardiac repolarization and the hypothesis that exercise testing can predict QT interval
shortening of the QT interval: IKs has slow deactivation response and effect of ␤-blockers in LQT1 patients with
kinetics, rendering the channel open for a longer period of mutations in the C-loops (which, as described above, rep-
time. Residual IKs channel activation from previous cardiac resent the site of final sympathetic regulation of KCNQ1).
stimulation can enhance repolarization in the setting of In this retrospective analysis, C-loop KCNQ1 missense mu-
short diastolic intervals (119). Indeed, a hallmark of LQT1 tations were not associated with an increased QTc interval
is the failure of the QT interval to adapt to increases in heart during exercise stress testing or response to ␤-blocker ther-
rate, as often seen during diagnostic exercise stress testing apy (230). Despite the known limitations of retrospective
(29, 230, 470). This feature of IKs molecular physiology analysis, this study highlights the need for caution in the use
may also explain the predilection toward arrhythmias dur- of exercise stress tests for genotype-specific risk stratifica-
ing swimming and diving in LQT1, which can produce a tion.
complex autonomically-driven “diving reflex” with abrupt

changes in heart rate. 5. Female gender and pregnancy as risk factors for
arrhythmogenesis
In contrast to LQT1 and LQT2, LQT3 patients experience
greater risk of arrhythmic events at slower heart rates (332, Female gender is a risk factor for developing torsades de
370). Bradycardia can produce prime conditions for pointes in LQTS (186, 234), and the baseline QTc interval
SCN5A channel dysfunction, in a mutation-dependent in healthy females is prolonged compared with males. Fur-
manner. For example, the gain-of-function late sodium cur- thermore, women are at increased risk of developing drug-
rent generated by channel “burst” gating in the canonical induced torsades de pointes as a consequence of hERG-
⌬KPQ deletion mutation has been shown to be strongly blocking medications (254). In men, testosterone levels cor-
attenuated with increases in pacing rate (289). The fraction relate with QTc interval duration (507). In a large study of
of channels available to enter bursting channel states is 1,166 LQT2 syndrome patients, followed through age 40,
larger at slower pacing frequency (371). The observation of ACA and SCD occurred significantly more frequently in
strong association of arrhythmic events with bradycardia females (26%) than in males (14%). With regard to loca-
has led to concerns about the use of ␤-blockers, which slow tion of the mutation, males with pore mutations had a
heart rate, in the LQT3 subtype (33). However, it is impor- greater than twofold risk of a first ACA or SCD compared
tant to note that ␤-blockers have multiple intracellular tar- with males with non-pore mutations. Pore mutations did
gets that may be protective against arrhythmia, and direct not increase risk of these life-threatening cardiac events in
effects of ␤-blockers on sodium channel function have been females (265).
reported (33). Additionally, some LQT3 mutations can ex-
ert their effect via mechanisms other than channel bursting, Women with LQTS are particularly prone to developing
and therefore can have different features of rate dependence arrhythmias in the peripartum period. Seth et al. (377) dem-
(34). For example, the LQT3 SCN5A S1904L mutation onstrated that the postpartum state (defined as 9 mo post-
triggers arrhythmia more often at faster heart rates (185). delivery) confers a 2.7-fold increased risk of a cardiac event
Biophysical characterization alongside paired computa- and a 4.1-fold increased risk of a life-threatening cardiac
tional studies demonstrates increased late channel reopen- event. The majority of these events did not occur during
ings at faster pacing frequencies with the S1904L mutation, labor and delivery. This risk further appears to be genotype
in direct contrast to ⌬KPQ and F1473C mutations (35, specific; the LQT2 genotype, in particular, conferred
185). greater risk of cardiac events postpartum, compared with
females with LQT1 and LQT3 genotypes (377). Changes in
4. Genotype-guided exercise testing sex hormones associated with menopause may also affect
risk of arrhythmia. Within five years leading to the onset of
Since changes in heart rate can affect propensity for devel- menopause, the odds ratio for a syncopal event for LQT2
opment of arrhythmia, exercise testing in congenital LQTS females is 3.4 compared with LQT1 females, and after
has evolved into an essential tool in the diagnostic workup menopause, this increases to 8.1 (60). In LQT2, females
of affected patients. Almost 40% of LQTS patients do not older than 15 years of age have a 3.7-fold increased risk of
have a prolonged QTc interval on an ECG at rest. Exercise suffering cardiac events compared with males, while there is
stress testing can aid in diagnosis and risk stratification of no significant difference in risk of cardiac events between
congenital LQTS, with particular utility in uncovering cases males and females younger than 15 years of age (495). A
of concealed LQTS that have otherwise gone undiagnosed study of 634 LQT2 patients revealed that females ⬎13
(177, 339). Exercise ECG during stress and recovery is a years of age were nine times more susceptible specifically to
particularly helpful tool in uncovering concealed LQT1 “arousal” triggers compared with males ⬎13 years of age
syndrome (177). (216).

BOHNEN ET AL.
These clinical observations suggest that sex hormones may specifically swimming and possibly competitive athletics.
impact propensity for arrhythmia formation in LQTS in a LQT2 patients would be well advised to limit loud star-
genotype-specific manner. Sex hormones are known to play tling noises in their surroundings, including alarm clocks
a role in cardiac ion channel regulation and arrhythmia. and telephones (38).
Estrogen has been shown to decrease IKr activity and pro-
long the QTc interval (227), and ␤-estradiol inhibits hERG 2. ␤-Blockers and targeting of the sympathetic
channel expression in cultured HEK293 cells (24). Proges- nervous system
terone modulates both IKs and L-type calcium channels,
leading to a shortened QTc interval (294). According to consensus guidelines (335), ␤-blocker treat-
ment carries a class I recommendation in the setting of a
Animal models, including canine, mouse, rat, rabbit, and clinical diagnosis of LQTS. ␤-Blockers reduce overall risk of
guinea pig, have been extensively employed to better under- cardiac events in children and adults, and treatment repre-
stand gender differences in cardiac electrophysiology (186), sents a class IIa indication in all individuals confirmed as
and strongly support the role for male sex hormones short- carriers of LQTS mutations (118).
ening the QT interval via enhanced IKr activity (129, 245,

477). In a LQT2 rabbit model, estradiol enhances risk of However, the efficacy of ␤-blockers is strongly genotype-
sudden cardiac death, while progesterone is protective specific. ␤-Blockers produce greatest symptom relief and
(307). In canines, females have prolonged APD50, APD70, cardiac event reduction in LQT1 patients, followed by
and APD90 values compared with males. Dofetilide, a LQT2 patients, and are least effective in LQT3 patients
hERG channel blocker, prolonged the APD90 of Purkinje (334, 440). High-risk LQTS patient populations, including
fibers to a greater extent in female canines compared with LQT1 males and LQT2 females, experienced 67 and 71%
males with rate dependence, and the canine studies demon- risk reduction with ␤-blocker treatment, respectively (145).
strate consistency of sex-related differences in cardiac repo- Along with genotypic considerations, mutation-specific
larization between humans and canines (4). Further re- benefits exist based on location of the mutation in the ion
search is needed to elucidate the mechanisms underlying sex channel. For instance, ␤-blocker treatment resulted in 88%
hormone and arrhythmia formation, and to possibly un- risk reduction of life-threatening cardiac events in LQT1
cover therapeutic avenues through sex hormone sensitive patients harboring cytoplasmic-loop missense mutations
pathways. compared with LQT1 patients harboring missense muta-
tions in regions outside of the cytoplasmic loop (39).
␤-Blockers confer trigger-specific benefits as well, reduc-
C. Genotype-Specific Management of ing the risk of exercise-induced cardiac events by 78 and
Congenital LQTS 71% in LQT1 and LQT2 patients, respectively. How-
ever, ␤-blockers were not protective against events trig-
Congenital LQTS represents a potentially lethal condition, gered by arousal or rest/sleep in LQT1 and LQT2 (146,
yet advances in device therapy, medical options, and surgi- 216). These findings are in line with the molecular phys-
cal techniques have aided in the clinical management of the iology of these mutations. In nondiseased conditions, up-
disorder (421). Today, most clinical management of LQTS regulation of repolarizing currents by ␤-adrenergic stim-
is uniform across subtypes and consists of targeting of the ulation can enhance repolarization and favorably adapt
sympathetic nervous system, through ␤-blocker therapy QT interval for the concomitant increase in heart rate.
and left cervicothoracic sympathetic denervation (LCSD) in However, in the setting of increased heart rates and
selected refractory cases. Primary and secondary prevention LQT1 (and in some cases LQT2) disease, delayed rectifier
of cardiac arrest with an implantable cardioverter-defibril- current is not available for cardiac repolarization. One
lator (ICD), alongside antiarrhythmic therapy, is consid- possible mechanism for ␤-blocker effect could be preven-
ered after weighing risks and benefits for an individual pa- tion of sudden and sustained increase in heart rates,
tient. which in itself is associated with arrhythmia formation in
these subtypes.
As more is uncovered regarding the molecular physiology of
these conditions, there is growing potential for tailored In LQT2 events associated with sudden noises and startle,
therapy to a given patient’s genotype and specific LQT- both adrenergic and other neural pathways may be trig-
causing mutation. gered. As such, ␤-adrenergic blockade in LQT2 may not
fully mitigate the risks. Although ␤-blockers remain first-
1. Lifestyle modification line medical treatment in LQT2, a life-threatening cardiac
event rate of 6 –7% while on therapy remains a serious
Given the striking association of cardiac events with geno- concern (334).
type-specific triggers, counseling should be given regard-
ing lifestyle modification. As several examples, LQT1 It is important to note that ␤-adrenergic stimulation affects
patients are counseled about exercise limitations, most multiple intracellular targets, including the pathways for

intracellular calcium cycling that regulate L-type calcium settings and arrhythmic storm) and mexiletine (for suppres-
current, the ryanodine receptor, and sarcoplasmic reticu- sive therapy) are commonly clinically employed. Mexiletine
lum calcium uptake (18, 506). In LQTS, with impaired administration shortens the QT interval in LQT3 patients,
repolarizing currents (or enhanced depolarizing currents), and not in LQT2 patients (370). Mexiletine blocks mutant
␤-adrenergic stimulation can be pro-arrhythmic, regardless Nav1.5 channel function, diminishing the late sodium cur-
of the genotype producing the impaired repolarization. rent more than the peak sodium current by stabilizing the
␤-Blockers therefore are commonly prescribed to all LQTS inactivated state of the Nav1.5 channel (449). Ranolazine,
patients regardless of genotype. on the other hand, is thought to block Nav1.5 current via
open channel block (275). The utility of local anesthetic-
In selected cases, left cervicothoracic sympathetic denerva- like drugs is weighed against their off-target undesirable
tion (LCSD), which surgically interrupts adrenergic input to side effects. For example, flecainide inhibits peak INa and
the heart, can be considered. In a study of 147 high-risk has been reported to unmask Brugada syndrome-type pat-
LQTS patients, cardiac event rate dropped by 91% per terns on surface ECG (indeed, this class of agents is clini-
patient post-LCSD (369). When carried out at experienced cally utilized as a provocative challenge in the diagnosis of
centers, this treatment modality can be very effective in Brugada syndrome) (58). Various mechanisms of drug

refractory cases. block of Nav1.5 underlie, in part, the mutation-specific ef-
ficacy of LQT3 pharmacological agents (see sect. IV).
3. Other pharmacological treatments in LQT2
Pharmacological sodium channel blockade appears on the
⫹ surface to be a specific and targeted strategy against gain-
Treatment of LQT2 syndrome by increasing serum K con-
of-function mutations in the sodium channel, and in indi-
centration has been explored, as hERG conductance is
vidual cases does provide remarkable reduction in arrhyth-
modulated by changes in extracellular K⫹, and LQT2 pa-
mia burden. However, a mortality benefit for these agents in
tients can develop low body and/or serum potassium levels
LQT3 has yet to be established. Anti-arrhythmic therapy in
(436). IKr activity decreases in the setting of hypokalemia
LQT3, as with all LQT subtypes, must be considered care-
(359) and increases in the setting of hyperkalemia, due
fully after thorough review of all patient-specific factors,
mainly to extracellular K⫹ effects on inactivation gating
including mutation or genotype-specific risk, clinical vari-
(446, 454, 455).
ables (e.g., family history, age, gender), and overall treat-
ment plan (including role of ICD placement and/or LCSD)
As such, potassium supplementation and therapy with spi-
(143). Given the complexities of drug effect, these agents
ronolactone, a potassium-sparing diuretic, were tested as
are often employed empirically in LQTS, with close atten-
treatment options to enhance IKr activity, and each treat-
tion to clinical response.
ment significantly shortened the QTc interval (89, 117).
However, there was no evidence of a decrease in cardiac
events. Safety dilemmas have been raised, including hyper- D. Emerging Translational Strategies in
kalemia, with the use of spironolactone in LQT2 treatment Congenital LQTS
(38). Potential adverse effects of spironolactone may be
explained by the fact that its metabolism into canrenoic acid 1. Cellular models of LQTS
was actually shown to induce hERG blockade and reduce
channel activity (63).
The physiological significance of cardiac channelopathies
can be difficult to model at the cellular level because of the
4. Other pharmacological treatments in LQT3 complex interplay between several ionic currents in the car-
diomyocyte. While heterologous expression systems and
The efficacy of local anesthetic drugs in LQT3 treatment is animal models are invaluable tools in our understanding of
due to preferential inhibition of late sodium current (more disease phenotypes, they lack the genetic background and
than peak current). Limitation of the use of late sodium molecular biology of human cells. Induced pluripotent stem
current blockers is due, in part, to off-target drug effects as cell-derived cardiomyocytes (iPSC-CMs) represent a useful
well as to the relative selectivity of block of late versus peak platform for studying human cardiac ion channel function
sodium channel currents. The introduction of newer com- in health and disease in a cell system with human molecular
pounds with significantly higher selectivity for INaL block and physiological components, acquired from unaffected
offers great potential for more effective clinical manage- patients and/or those carrying mutations.
ment of LQT3. The need for improved pharmacotherapy is
supported by a high breakthrough cardiac event rate for In 2010, iPSC-CMs were generated from skin cells taken
LQT3 patients on ␤-blocker treatment, affecting as many as from two LQT1 patients with the autosomal dominant
15% of patients, generating a growing interest in INa-spe- KCNQ1 R190Q mutation (277). These cardiomyocytes
cific blocking agents (143). The use of the local anesthetic were spontaneously beating and classified as ventricular-,
class of sodium channel blockers lidocaine (during acute atrial-, or nodal-like based on cellular electrophysiology

BOHNEN ET AL.
and morphology. Compared with WT controls, LQT1 car- agents in the treatment of congenital LQTS. The experi-
diomyocytes demonstrate decreased IKs amplitude and pro- mental drug GS-967 preferentially inhibits late INa over
longed APD, consistent with LQT1 pathogenesis. More re- peak INa with considerable selectivity, and does so more
cently, Jouni et al. (199) derived iPSC-CMs from a LQT2 potently and efficaciously than flecainide and ranolazine,
patient harboring a hERG A561P mutation, to study the while also reducing arrhythmia burden in experimental rab-
electrophysiological consequences of hERG loss-of-func- bit cardiomyocytes (43). Computational modeling of GS-
tion in a patient-specific genetic background. Ventricular-, 967 suggests therapeutic benefit of late INa block by de-
atrial-, and nodal-like action potentials were recorded, and creasing arrhythmogenesis in the setting of LQTS (481).
a trafficking defect was characterized as the mechanism of Another experimental compound, F15845, selectively
loss of IKr, leading to a prolonged APD. Furthermore, a blocks late INa potently, preventing cardiac angina and ar-
study by Jones et al. (195) reported the use of mRNA silenc- rhythmia in animal models (324, 433). The benefits of any
ing in human iPSC-CMs to show that the hERG1b isoform selective late INa blocker must be weighed against the rela-
is a significant component of cardiac repolarization, and tive effects on peak INa, in addition to off-target effects that
that loss of hERG1b function provides a substrate for ar- include block of peak INa and IKr. Studies to date suggest
rhythmogenesis. Several additional studies have investi- that selective late INa blockers are not proarrhythmic; clin-

gated LQT3 in the context of iPSC-CMs (121, 252, 420). ical trials of these and related drugs are currently underway,
and it is expected that, in the near future, these studies will
In the context of drug development, iPSC-derived cardio- inform whether this drug class represents a viable treatment
myocytes provide a means for testing compounds in pa- option in congenital LQTS (25).
tient-specific genetic backgrounds, allowing for a targeted
approach in optimizing therapy. Terrenoire et al. (420) Along with more selective targeting of LQT-associated
studied human iPSC-CMs from a patient harboring a LQTS mutant ion channels, growing interest in both mutation-
mutation in SCN5A and a polymorphism in hERG. Upon specific and tissue-specific treatment strategies may be-
electrophysiological characterization of ionic currents, it come fruitful. For instance, ectopic rhythms leading to
was determined that the SCN5A F1473C mutation was ventricular fibrillation in LQT patients has been shown
responsible for the LQTS clinical phenotype via increased to originate 50% of the time from the Purkinje fiber cells
INaL, while the hERG K897T variant was a benign poly- in the cardiac specialized conduction system (158, 306,
morphism. Experiments in the iPSC-CMs revealed key rate- 414). The Purkinje system is especially prone to arrhyth-
dependent properties of INaL and its inhibition by mexile- mia formation, owing to differences in membrane current
tine; these results provided insight into control of arrhyth- density, APD and morphology, and calcium cycling (102,
mias in the proband. This study offers an example of how 132, 158). In a computational model of the His-Purkinje
iPSC-CMs can elucidate mechanisms of LQTS pathogenesis system, LQT3-associated mutations were shown to con-
to inform patient-specific therapy. fer a more severe phenotype in Purkinje fibers compared
with LQT1 and LQT2 mutations (185), predictions that
Use of iPSC-CMs is not without limitations. Discrepancies were confirmed in isolated murine Purkinje fiber cells
may still exist in the expression profile of cardiac ion chan- expressing ⌬KPQ mutant Na⫹ channels (175). Contin-
nels between iPSC-derived cardiomyocytes and native hu- ued research into the functional implications of muta-
man cardiac cells due in part to differences in cell maturity tions in different cell types may uncover further therapies
and growth environment. Cardiomyocyte differentiation directed at tissues most prone to arrhythmia formation.
can produce a mix of atrial-like and ventricular-like cells of For example, targeting the insertion of these Purkinje
varying morphology. Furthermore, recent publications us- fibers in the ventricular endocardium via catheter abla-
ing iPSC-CMs report a resting membrane potential depo- tion may prove to be an effective strategy in reducing
larized to near ⫺60 mV (252, 420), which can alter prop- arrhythmia burden (471).
erties of important cardiac channels such as Nav1.5 and
affect state-dependent pharmacological inhibition. Efforts
are underway to improve iPSC-CMs by using physiological VII. CONCLUSIONS
systems to promote cellular maturity such that they more
closely resemble native mature cardiomyocytes. As effi- The growing body of mechanistic insights into the patho-
ciency and specificity of cell differentiation improve, iPSC- physiology of channelopathies and other arrhythmia-caus-
CMs hold great promise as a model cell system for the study ing mutations has helped elucidate the molecular underpin-
of LQTS disease and patient-specific pharmacology (115). nings of congenital LQTS and genotype-phenotype correla-
tion in disease. Although insights from benchside to bedside
2. Pharmacological and medical approach to LQTS have facilitated progress toward better therapeutic strate-
gies, there remains a need for tailoring management toward
More specific targeting of the mechanisms underlying ion individuals in a genotype-driven and mechanism-specific
channel defects that predispose to arrhythmia guides the manner to optimize care. Techniques such as iPSC-derived
advancement and rational drug design of pharmacological cardiomyocytes and high-throughput patch clamping that

allow for screening of compounds in patient-specific con- Schulze-Bahr E, Semsarian C, Towbin JA, Watkins H, Wilde A, Wolpert C, Zipes DP,
Heart Rhythm S, European Heart Rhythm Association. HRS/EHRA expert consensus
texts may help pave the way for future development of statement on the state of genetic testing for the channelopathies and cardiomyopa-
therapeutics. In addition, continued progress toward fun- thies: this document was developed as a partnership between the Heart Rhythm
damental understanding of mechanisms of ion channel Society (HRS) and the European Heart Rhythm Association (EHRA). Europace 13:
function and drug-channel interaction will guide the devel- 1077–1109, 2011.
opment of more effective, mechanism-based molecular 9. Ackerman MJ, Tester DJ, Jones GS, Will ML, Burrow CR, Curran ME. Ethnic differ-
agents in the treatment of LQTS. ences in cardiac potassium channel variants: implications for genetic susceptibility to
sudden cardiac death and genetic testing for congenital long QT syndrome. Mayo Clin
Proc 78: 1479 –1487, 2003.
ACKNOWLEDGMENTS
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M. S. Bohnen, G. Peng, and S. H. Robey contributed ger for inherited long QT syndrome. Mayo Clin Proc 74: 1088 –1094, 1999.
equally to this work. 11. Adsit GS, Vaidyanathan R, Galler CM, Kyle JW, Makielski JC. Channelopathies from
mutations in the cardiac sodium channel protein complex. J Mol Cell Cardiol 61:
Present address of C. Terrenoire: The New York Stem Cell 34 – 43, 2013.
Foundation Research Institute, New York, NY 10032. 12. Ahrens-Nicklas RC, Clancy CE, Christini DJ. Re-evaluating the efficacy of ␤-adrener-

gic agonists and antagonists in long QT-3 syndrome through computational modelling.
Address for reprint requests and other correspondence: Cardiovasc Res 82: 439 – 447, 2009.
R. S. Kass, Dept. of Pharmacology, Columbia University 13. Aidery P, Kisselbach J, Schweizer PA, Becker R, Katus HA, Thomas D. Impaired ion
Medical Center, 630 W. 168th St., New York, NY 10032 channel function related to a common KCNQ1 mutation: implications for risk strati-
(e-mail: rsk20@cumc.columbia.edu). fication in long QT syndrome 1. Gene 511: 26 –33, 2012.
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Molecular Pathophysiology of Congenital Long QT Syndrome

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Physiol Rev 97: 89 –134, 2017

Published November 2, 2016; doi:10.1152/physrev.00008.2016

MOLECULAR PATHOPHYSIOLOGY OF CONGENITAL

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I. INTRODUCTION 89 yielded much insight into the pathophysiology of congenital

0031-9333/17 Copyright © 2017 the American Physiological Society 89

A The ventricular cellular action potential results from the

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these cutoff and develop no arrhythmias; similarly, QTc

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LQT1 KCNQ1 KCNQ1 (Kv7.1) 2IKs

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IKs is slowly activating and most prominent during the pla-

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4. Role of PIP2 and calmodulin in IKs function D. Molecular Pathophysiology

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is made constitutively active (41). Structural and muta-

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red circle in B. D: voltage dependence of

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A COOH terminus, including 8% in the c-NBD (207). In

Several important general characteristics of KCNH2 mu-

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Table 4. Representative LQT2- and LQT6-associated mutations classified by mechanism

hERG LQT2 mutations

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G626A mutation in the conserved K⫹ selectivity filter in the E. Molecular Pharmacology

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3. Autoimmune-associated LQT syndrome KCR1, a 12-pass transmembrane segment regulatory pro-

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40ms –10 50ms

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Changes in steady-state properties of the cardiac sodium