REVIEWS
Exploring HCN channels as novel
Pacemaker cells
Cardiac cells that display
automaticity. The primary
pacemaker cells of the heart
are localized in the sinoatrial
node region.
Sinoatrial node
Tissue located in the right
atrium of the heart that
generates cardiac sinus
rhythm.
*Center for Drug Research,
Department of Pharmacy,
Ludwig-Maximilians-
Universität München,
Butenandtstr. 7, 81377
Munich, Germany.
‡
Center for Integrated Protein
Science Munich, Ludwig-
Maximilians-Universität
München, Butenandtstr.
5-13, 81377 Munich,
Germany.
Correspondence to M.B.
e-mail: martin.biel@cup.
uni-muenchen.de
doi:10.1038/nrd3576
Published online
18 November 2011
NATURE REVIEWS | DRUG DISCOVERY
drug targets
Otilia Postea* and Martin Biel‡*
Abstract | Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key
role in the control of heart rate and neuronal excitability. Ivabradine is the first compound
acting on HCN channels to be clinically approved for the treatment of angina pectoris. HCN
channels may offer excellent opportunities for the development of novel anticonvulsant,
anaesthetic and analgesic drugs. In support of this idea, some well-established drugs that act
on the central nervous system — including lamotrigine, gabapentin and propofol — have
been found to modulate HCN channel function. This Review gives an up-to-date summary of
compounds acting on HCN channels, and discusses strategies to further explore the potential
of these channels for therapeutic intervention.
Hyperpolarization-activated cyclic nucleotide-gated HCN channels are members of the pore loop cation
(HCN) channels were discovered over ten years ago, yet channel superfamily 10. In mammals the HCN channel
many aspects of their activation mechanism, their modu- family comprises four homologous members (HCN1–
lation in vivo, their cellular and subcellular distribution, HCN4) that share about 60% sequence identity with
and their interaction with agonists or antagonists remain each other and 25% sequence identity with their closest
unclear 1–3. HCN channels allow the passage of a depolar- relatives — the cyclic nucleotide-gated channels (FIG. 1a).
izing mixed Na+ and K+ current — the hyperpolarization- HCN channels are present in all vertebrates and also in
activated current (termed Ih) or the funny current (termed several invertebrates11–20.
If) — which has a key role in controlling the rhythmic HCN channels are tetrameric complexes consisting of
activity (also known as the pacemaker current) in cardiac either identical (homomeric) or different (heteromeric)
pacemaker cells and spontaneously firing neurons. Ih is also types of subunits. The topology of a HCN channel sub­
involved in several non-pacemaking functions, including unit is shown in FIG. 1. The transmembrane core includes
determination of resting membrane potential, dendritic six α-helices (S1–S6) and a loop domain between the
integration, synaptic transmission and learning. Thus, S5 and S6 helix that forms the ion selectivity filter.
HCN channels are interesting targets not only for the Like other voltage-operated cation channels, HCN chan-
development of drugs to lower heart rate but also for the nels contain a positively charged S4 helix that functions
treatment of diseases related to impaired neuronal activity. as a voltage sensor 21. HCN channels contain a cyclic
In this Review we briefly outline the current knowl- nucleotide-binding domain (CNBD) in the carboxyl
edge on the structure–function relationships, expression terminus that is connected to the channel core via an
patterns and basic biophysical profiles of HCN channels. 80‑amino-acid peptide called the C‑linker. The struc-
This is followed by a summary of the role of HCN chan- ture of the CNBD–C-linker has been determined at
nels in disease. A more detailed overview of these topics atomic resolution22–23. Following the binding of cyclic
is provided in several recent publications1–6. This Review AMP or cyclic GMP to the channel, the CNBD–C-linker
focuses on the potential of HCN channels as targets for facilitates HCN channel activation by inducing a right-
bradycardic, anticonvulsant, analgesic and anaesthetic ward shift in the voltage-dependence of activation14,24,25
compounds, and discusses the challenges and future (FIG. 2a). Elucidation of the crystal structure of the HCN
directions in the development of novel drugs targeting channel core should shed further light on the gating
HCN channels. machinery that couples the movement of the S4 helix
in the electrical field with the conformational change in
The HCN channel family the CNBD–C-linker domain following cAMP binding.
HCN channels represent the molecular correlate of Ih When they are expressed in heterologous systems the
or If, which was identified in the late 1970s in sinoatrial HCN channel subunits give rise to currents that display
node cells and neurons1,7–9. the typical hallmarks of native Ih: activation with sigmoidal
VOLUME 10 | DECEMBER 2011 | 903
© 2011 Macmillan Publishers Limited. All rights reserved
REVIEWS
a TRP b Pore
TPCN
Extracellular
Kir
KCa
Kv1–9
K2P Intracellular
Kv10 Ca2+
Kv11 K+
Kv12
Na+
CNGB
CNGA + muscle33 and most parts of the CNS, except for the olfac-
S1 S2 S3 S4 S5 S6
+ tory bulb and some hypothalamic nuclei, which display
HCN2 +
HCN4
Ih(If)
HCN1 C-linker
HCN3
N C
Nav CNBD
Cav cAMP
Figure 1 | HCN channels: phylogenetic tree and transmembrane topology.
a | A schematic phylogenetic tree representing the superfamily of pore loop cation
channels is shown, including the hyperpolarization-activated cyclic nucleotide-gated
Nature Reviews
(HCN) channels. b | The diagram illustrates the transmembrane | Drug
topology Discovery
of HCN
channels. The channels are tetramers (as shown in the upper panel). Each subunit
consists of six transmembrane α-helices (numbered S1–S6 on the figure). The fourth
α-helix is positively charged and forms the putative voltage sensor of the channel.
In the carboxyl terminus the subunits contain a cyclic nucleotide-binding domain
(CNBD) that is connected with the sixth transmembrane α-helix via the C‑linker.
HCN channels allow the passage of Na+ and K+. There is also evidence of minor
permeation of Ca2+. Cav, voltage-gated calcium channel; CNGA, cyclic nucleotide-gated
cation channel A subunit; CNGB, cyclic nucleotide-gated channel B subunit; If, funny
current; Ih, hyperpolarization-activated current; K2P, two-pore potassium channel;
KCa, calcium-activated potassium channel; Kir, inwardly rectifying potassium channel;
Kv1, voltage-gated potassium channel subunit Kv1; Nav, voltage-gated sodium
channel; TRP, transient receptor potential channel; TPCN, two-pore calcium channel.
kinetics following membrane hyperpolarization (FIG. 2b);
lack of voltage-dependent inactivation; conduction of Na+
and K+; a shift in the activation curve as a result of direct
interaction with cAMP; and inhibition by millimolar con-
centrations of external Cs+ (REFS 1,2). Currents mediated
by the four HCN channels differ from each other with
regard to their activation kinetics, which are fast in HCN1,
intermediate in HCN2, and slow in HCN3 and HCN4
(REFS 12,15,17,26). The speed of channel activation is also
strongly voltage-dependent, and increases as activation
voltages become more hyperpolarized. HCN channels
also differ from each other in their response to cAMP.
The activation curves of HCN2 and HCN4 are shifted
by about +15 mV, whereas HCN1 and HCN3 are only
weakly modulated by cAMP, if at all. As mentioned
above, HCN channels can be assembled into hetero­tetra­
mers. The biophysical properties of these heteromeric
channels are a mixture of those determined for pure
homomeric channels1,27. It is likely that the formation of
heteromeric channels is an important process underlying
the diversity of the native Ih observed in different types
of neurons1,9.
Expression pattern of HCN channel subtypes. HCN
channels are predominantly expressed in the brain and
heart. An overview of the different expression sites, as
well as the implicated functions and/or phenotypes of
904 | DECEMBER 2011 | VOLUME 10
© 2011 Macmillan Publishers Limited. All rights reserved
the individual channel isoforms, is provided in TABLE 1.
Expression of HCN channels in the central nervous
system (CNS) has been extensively studied at both the
protein and transcript level28–32 (reviewed in REF. 1).
Among the four subtypes, HCN2 is expressed most
extensively and is found in most parts of the brain. By
contrast, expression of HCN1 and HCN4 is spatially more
restricted. HCN1 is enriched in the neocortex, hippocam-
pus, cerebellar cortex and brainstem, whereas HCN4 is
mainly present in thalamic nuclei, basal ganglia and the
olfactory bulb. HCN3 is weakly expressed in heart
higher HCN3 expression levels26,31. HCN channels are also
found in retinal cells32,34 and in the peripheral nervous
system, including dorsal root ganglion neurons32,35,36.
In all mammals studied so far, including humans
and mice, HCN4 represents the principal HCN channel
isoform present in the sinoatrial node12,20,32,37,38. The expres-
sion of other isoforms in the sinoatrial node is much
weaker and also seems to vary between species20,32. The key
importance of HCN4 for the generation of sinoatrial If is
corroborated by electrophysiological studies that revealed
a 70–80% reduction in the total If in HCN4‑deficient
sinoatrial node cells compared with wild-type cells39,40.
By contrast, deletion of HCN2 diminished total If only
by about 20% in murine sinoatrial node cells41. HCN
channels are also present in non-pacemaking cardio-
myocytes of the atrium and ventricle14–15,20. The func-
tion of HCN channels in these cell types remains to be
determined. HCN channel transcripts have also been
identified in other organs including the testis, smooth
muscle and kidney 1. However, in most of these organs
the presence of the HCN channel protein has not yet been
detected. Similarly, the functional roles of HCN channels
in these organs are unclear. It should also be noted that
the expression pattern of HCN channels displays an age-
dependence and may change during development42–44.
Regulation of HCN channel function
Like most ion channels, HCN channels form macro­
mol­ec­ular complexes that consist of the principal ion-
conducting channel core and auxiliary subunits (for
example, scaffolding proteins and modulator proteins)
that are either permanently assembled with the channel
core or that can bind and unbind in a regulated fashion.
Affinity purification, combined with high-resolution
mass spectrometry, has revealed that HCN channels
in the brain are tightly co-assembled with peroxisomal
biogenesis factor 5‑like protein (PEX5L; also known
as TRIP8b) in a 1:1 ratio45. Functional studies suggest that
TRIP8b has an antagonistic effect on the cyclic nucleotide
gating of HCN channels45–47. TRIP8b is also an important
modulator of cell-surface expression and intraneuronal
trafficking of HCN channels48. There are more than ten
alternative amino‑terminal splice forms of TRIP8b in the
brain. Interestingly, some of these variants enhance HCN
channel densities, whereas others strongly downregulate
the levels of HCN channels. These findings suggest that
TRIP8b acts as a gatekeeper of neuronal HCN channel
activity (reviewed in REF. 49).
www.nature.com/reviews/drugdisc
 REVIEWS

sO8
C 
D sO8

 sO8
UGEQPFU


++OCZ






s s s

8O O8 P#
sE#/2 E#/2 UGEQPFU

Figure 2 | Functional properties of HCN channel currents. a | The graph shows the voltage-dependence of
hyperpolarization-activated cyclic nucleotide-gated channel 2 (HCN2) activation in the absence or presence of 10 μM
cyclic AMP. cAMP induces a positive shift in the activation curve. The inset shows the accelerating effect of cAMP on
0CVWTG4GXKGYU^&TWI&KUEQXGT[
HCN2 channel activation. In this experiment, currents were evoked in an inside-out patch in the absence or presence of
10 μM cAMP by stepping from a holding potential of –40 mV to –120 mV (data were compiled from REF. 15). b | A family
of HCN2 current traces are shown that are activated following hyperpolarizing steps from a holding potential of
–40 mV to various test potentials, ranging between –140 mV and –50 mV. The current is activated with sigmoidal kinetics
and does not display voltage-dependent inactivation. I/Imax, ratio between current at a given membrane potential (I) and
the maximal current (Imax); nA, nanoampere; Vm, membrane potential.

Diastolic depolarization
Slow membrane
depolarization that occurs
between action potentials
(that is, in the diastole of
the heart) in pacemaker
cells of the cardiac system.
The functional significance of several other proteins
that have been proposed to be assembled with HCN
channels is less clear. Such proteins include: potassium
voltage-gated channel, Isk-related family member 2
(also known as MIRP1)50; potassium channel regulator 1
(REF. 51) ; filamin A 52; the complex formed by GRP1
(general receptor for phosphoinositides 1)-associated
scaffold protein (also known as tamalin) and amyloid-β
A4 precursor protein-binding family A member 2 (also
known as MINT2)53; as well as caveolin 3 (REF. 54). Further
studies will be required to validate the specific roles of
these proteins with regard to HCN channel function.
In addition to auxiliary subunits, HCN channels are
regulated by several low-molecular-weight factors. The
best characterized of these factors is phosphatidylinositol
4,5‑bisphosphate, which was shown to shift the activation
curve of HCN channels by about +20 mV55,56. This regu-
lation is of crucial importance because it is needed to
activate HCN channels in the physiological voltage range.
In addition to phosphatidylinositol 4,5‑bisphosphate,
other acidic lipids — like phosphatidic acid and arachi-
donic acid, which are products of diacylglycerol kinase–
phospholipase A2 signalling pathways — directly facilitate
HCN channel gating57. HCN channel activity is also regu-
lated by extracellular concentrations of K+ (REF. 58), extra-
and intracellular concentrations of H+ (REFS 59,60), and
external concentrations of Cl– (REFS 58,62).
A third group of HCN channel regulators comprises
protein kinases and phosphatases. SRC kinase directly
interacts with HCN1, HCN2 and HCN4, and regulates
Ih by phosphorylating tyrosine residues. The regulation
of HCN channels by SRC kinases has been demonstrated
under physiological conditions in sinoatrial pacemaker
cells in murine and rat hearts as well as in neurons19,63,64.
HCN channel currents are upregulated in hippocampal
pyramidal neurons by p38 mitogen-activated protein
kinase65, whereas the phosphatase calcineurin inhibits
HCN channels in hippocampal pyramidal neurons by
inducing a hyperpolarizing shift in the activation curve66.
It remains to be determined whether the bidirectional reg-
ulation exerted by p38 MAPK and calcineurin is caused
by direct phosphorylation and dephosphorylation of the
channel or by a mechanism involving other proteins.
cGMP-dependent protein kinase 2 has been identified as
another regulator of HCN channels in the brain; it binds
to the C terminus of HCN2 and phosphorylates a specific
serine residue in the CNBD67. This modification induces a
hyperpolarizing shift in the activation curve. Thus, cGMP
regulates HCN channel activity in two ways: it enhances
HCN currents by directly binding to the CNBD and it
inhibits gating by phosphorylating the CNBD.
HCN channels in human disease
HCN channels have an important role in the generation
of cardiac pacemaker activity and the control of neuronal
excitability. Impairment of HCN channel function (for
example, resulting from inadequate expression levels or
mutations in the primary sequence) has been implicated
in the pathology of diseases of the heart and the nervous
system. Here, we give a brief overview of pathologies
involving HCN channels (TABLE 1; for further details see
REFS 1,68–72).
HCN channels in arrhythmogenic diseases. Cardiac pace­
maker cells generate a specialized kind of action potential
(known as a pacemaker potential) that is characterized
by the presence of a progressive diastolic depolarization in
the voltage range between –65 mV and –45 mV (FIG. 3).

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REVIEWS

Although the diastolic depolarization results from the are heterozygous for the respective HCN4 mutations.
Sinus bradycardia
Heart rhythm that originates
concerted action of several currents, If is generally con- A possible explanation for this finding might be that,
from the sinus node with a sidered to have a key role because it is activated at nega- like in mice, homozygous deletion of HCN4 function39
resting heart rate of 60 beats tive potentials and can therefore potentially serve as a or complete loss of cAMP-dependent modulation79 may
per minute or less. primary initiator of diastolic depolarization. Importantly, result in embryonic lethality.
up- and downregulation of If by an increase or decrease The first HCN4 channelopathy was identified in a
Syncope
Partial or complete loss
in sinoatrial cAMP levels, respectively, is believed to single patient who developed an idiopathic sinus node
of consciousness, with have a pivotal role in the autonomic control of heart rate. dysfunction that was characterized by a marked sinus
interruption of awareness of As described above, HCN4 is the predominant compo- bradycardia, episodes of syncope, intermittent atrial
oneself and one’s surroundings. nent of sinoatrial If in all mammals. The intensive search fibrillation and chronotropic incompetence even under
Syncope often occurs as a
result of an irregular heartbeat.
for genetically determined cardiac channelopathies has maximal workload73. Molecular genetic analysis revealed
resulted in the identification of six different mutations the presence of a single base deletion in exon 5 of the
Chronotropic incompetence in the HCN4 gene. All six mutations are associated HCN4 gene, which leads to the generation of a truncated
The inability to increase heart with the induction of sinus bradycardia73–78. In addition, protein lacking the CNBD and subsequent downstream
rate commensurately with one mutation (D553N) is associated with a complex sequence (referred to as L573X). In heterologous expres-
increased activity or demand.
arrhythmia syndrome74. All patients identified so far sion systems the truncated HCN channels are normally
targeted to the plasma membrane but — as expected —
they do not respond to cAMP.
In 2004 a missense mutation (D553N) in the HCN4
Table 1 | Expression pattern and involvement of HCN channels in disease gene was identified in a Japanese family 74. The patients
Isoform Expression sites Phenotypes had severe bradycardia combined with a complex pat-
tern of rhythmic disturbances that included recurrent
Nervous system
syncopes, QT prolongation, polymorphic ventricular tachy­
HCN1 Neocortex, • Mouse knockout: impaired motor learning, cardia and torsade de pointes. In vitro studies indicate that
hippocampus, enhanced hippocampal-dependent learning
brain stem19,28,30,31 and memory, less cold allodynia, epilepsy and
the D553N mutation is associated with impairment of
altered vision116,126,141 the cell-surface expression of HCN4. Downregulation
of HCN4 expression could explain the bradycardia but
HCN2 Ubiquitous28,30,31 • Mouse knockout: ataxia, absence
epilepsy41, as well as reduced inflammatory it does not readily explain the other complex symptoms
and neuro­pathic pain127 of these patients.
HCN3 Olfactory bulb, • Mouse knockout: not reported Another point mutation (S672R) in the HCN4 gene,
hypothalamic localized at a highly conserved position in the CNBD
nuclei, retinal cone domain, was identified in an Italian family with asymp-
pedicles2,26,28,31,34 tomatic sinus bradycardia75. Heterologous co-expression
HCN4 Thalamic nuclei, • Mouse knockout: not reported of mutant and wild-type HCN4 channels to mimic
basal ganglia, heterozygosis as present in patients revealed that the
olfactory bulb28,30,31 S672R mutation does not impair the cAMP-dependent
Heart modulation of the channel; rather, it induces a 5 mV
HCN1 Sinoatrial and • Not reported hyperpolarizing shift in the If activation curve. This
atrioventricular shift would explain the basal bradycardia combined with
node20, 142,143 normal autonomic control of heart rate that is observed
HCN2 Ubiquitous20,32 • Mouse knockout: sinus arrhythmia41 in patients with this mutation. Unexpectedly, autonomic
up- and downregulation of heart rate is completely
HCN3 Heart muscle 33
• Mouse knockout: increased T wave amplitude
in the electrocardiogram at basal heart rate33 preserved in patients with bradycardia who express an
HCN4 variant with a truncated and hence non-functional
HCN4 Sinoatrial and • Mouse knockout: embryonic lethal phenotype,
atrioventricular node, bradycardia, nonfunctional pacemaker cells39 CNBD77 (HCN4‑695X). This finding indicates that
Purkinje fibres20,32,39,145 • Conditional mouse knockout: repetitive sinus HCN4 is crucial for determining the basal heart rate but
pauses80,81, deep bradycardia, heart block40 challenges the notion that it is required for autonomic
(a disease in the electrical system of the heart) control of cardiac pacemaking.
• Human mutation: bradycardia, QT prolongation,
Although all four HCN4 mutations described above
syncope and conduction disturbances in
the sinoatrial and atrioventricular node73–76 occur in the C terminus of HCN4, a fifth mutation
(G480R) associated with asymptomatic bradycardia
Other tissues
was discovered in the pore region of this channel76. This
HCN1 Not reported • Not reported mutation involves the GYG signature that forms the
HCN2 Not reported • Not reported ion selectivity filter of potassium and HCN channels.
HCN3 Mouse liver, lung • Not reported Surprisingly, heterologous expression of this channel in
and kidney18,144 Xenopus laevis oocytes and human embryonic kidney
HCN4 Lung, skeletal • Not reported
293 (HEK293) cells showed that the ion selectivity of
muscle18, human HCN4 is not affected by the G480R mutation. Rather,
testis17 (but not the mutation shifts the activation curve to values that
mouse testis18) are more hyperpolarized and leads to a pronounced
HCN, hyperpolarization-activated cyclic nucleotide-gated channel. decrease in the cell-surface expression of HCN4 (REF. 76).

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QT prolongation
The QT interval represents
the time for electrical
activation and inactivation
of the ventricles — the lower
chambers of the heart.
Prolongation of the QT interval
can result in the potentially
lethal arrythmia known
as torsades de pointes.
Torsades de pointes
A form of polymorphous
ventricular tachycardia
that is associated with
prolongation of the cardiac
QT interval that can lead to
sudden cardiac death.
NATURE REVIEWS | DRUG DISCOVERY
Similarly, another mutation in the HCN4 gene (A485V)
downstream of the GYG signature has been identified
in three Moroccan families with sinus bradycardia, and
this mutation profoundly impairs HCN4 function
and synthesis78.
mV
The key role of HCN4 in cardiac pacemaking is
corroborated by genetic mouse models. Constitutive
cardiac deletion of HCN4 results in embryonic death
because of the failure to generate mature sinoatrial pace-
maker cells39. There is, however, an ongoing controversy
concerning the relevance of HCN4 in the adult murine
heart. Herrmann et al.80 reported that the conditional
deletion of HCN4 in mice does not induce bradycardia
at rest — rather, it leads to arrhythmia characterized by
recurrent sinus pauses81. By contrast, using a slightly dif-
ferent strategy to conditionally delete HCN4 in sinoatrial
node cells, Baruscotti and colleagues40 observed a pro-
gressive development of severe bradycardia and atrio-
ventricular block in mice, which led to cardiac arrest
and death. A cardiac phenotype was also observed in
HCN2‑knockout mice. HCN2 makes up about 20% of
total sinoatrial If 41. Deletion of the HCN2‑mediated cur-
rent fraction does not impair basal heart rate nor does
it interfere with autonomic regulation of the heartbeat.
Rather, HCN2‑deficient mice display a mild sinus dys-
rhythmia at rest. So far, HCN2 channelopathies have not
been reported in humans, and no naturally occurring
deletions or mutations in HCN1 have been described.
Besides the pathology resulting from inherited HCN
channel mutations, there is evidence that upregulation
of the levels of HCN channel expression may have a role
in the induction of ventricular and atrial arrhythmia in
heart failure (reviewed in REFS 82,83). Basal levels of
HCN channels are very low in healthy heart muscle cells
compared with sinoatrial pacemaker cells. However,
elevated mRNA and protein levels of HCN2 and HCN4
have been found in ventricular and atrial samples from
failing hearts explanted from patients with end-stage
ischaemic cardiomyopathy 84. It is therefore tempting to
assume that increased If densities that may result from
HCN channel upregulation could endow cardiomyo-
cytes with pacemaker-like activity, thereby promoting
ectopic pacemaker activity.
HCN channels in neurological diseases. HCN channels
are widely expressed in the CNS and peripheral nervous
system. At the cellular level several basic functions have
been attributed to these channels, including neuronal
pacemaking, control of the membrane resting potential
and dendritic integration. It was therefore hypothesized
that the dysfunction and/or inadequate expression of
HCN channels may be a disease-causing factor. This
assumption has been supported by the analysis of
HCN1- and HCN2‑deficient mice that has revealed
several phenotypes resembling human neurological dis­
orders, including epilepsy, ataxia and impaired learning.
Conversely, channelopathies for the two major neu-
ronal HCN channels, HCN1 and HCN2, have not been
reported so far in humans, and patients with mutations
in HCN4 do not have obvious defects other than cardiac
bradycardia or arrhythmia. Thus, our current knowledge
© 2011 Macmillan Publishers Limited. All rights reserved
REVIEWS
a
Iv
0
–20
Diastolic
depolarization
–60
If
b
Iv Na+ Extracellular
+ + – –
+ +
+ Iv +
+ +
+ +
+ Iv +
+ +
– – + +
Iv K+
Intracellular
Figure 3 | Mechanism of action of ivabradine on
sinoatrial HCN channels. a | Hyperpolarization-
activated cyclic nucleotide-gated (HCN) channels
Nature Reviews allow
| Drug Discovery
the passage of the funny current (If), which is a major
current producing slow diastolic depolarization of the
sinoatrial pacemaker potential. For clarity, other currents
contributing to diastolic depolarization are omitted.
Ivabradine (shown on the figure as ‘Iv’) reduces the
frequency of sinoatrial pacemaker potentials by blocking
HCN channels and hence reducing the slope of diastolic
depolarization. b | Current-dependent block of HCN
channels by ivabradine. Ivabradine enters the pore of
HCN channels from the intracellular side when the
channels are in the open state (left side of panel). When
the membrane potentials are more positive than about
–20 mV, HCN-mediated currents are mainly carried by K+
and are outwardly directed. The outward current drives
ivabradine into the pore of the channel, where it tightly
binds to its binding site (right side of panel). The polarity
of the plasma membrane is indicated by ‘+’ or ‘–’ signs.
The movement of the positively charged S4 helix of HCN
channels in the electrical field is also indicated.
on the pathophysiological importance of HCN channels
is by and large based on studies in animals (mostly mice
and rats) and has yet to be confirmed in humans.
HCN channels are known to be involved in two com-
plex groups of diseases: epilepsies and neuropathic pain
disorders1,4,71,72,85,86. Both groups of diseases are charac-
terized by enhanced firing of neurons and/or aberrant
neuronal firing patterns. HCN channels are key determi-
nants of neuronal network activity. Even subtle changes
in their expression levels, localization to subcellular
compartments, subunit composition or their cellular
regulation can have profound consequences on neu-
ronal network activity. Given the multifaceted functions
of HCN channels, effects resulting from HCN channel
dysfunction are intrinsically complex and may strongly
depend on the pathophysiological context. For exam-
ple, both upregulation87,88 and downregulation89 of HCN
channels have been associated with increased firing of
VOLUME 10 | DECEMBER 2011 | 907
REVIEWS
Inotropic effects
Effects pertaining to the force
of muscular contractions,
particularly those of the heart.
908 | DECEMBER 2011 | VOLUME 10
peripheral sensory neurons following nerve injury. The
involvement of HCN channels in the development of
epilepsies is even more complex. As in pain disorders,
there is evidence that both up- and downregulation of
HCN channel subunits, particularly subunits of HCN1
and HCN2, are associated with epilepsies 90. There is
growing evidence that several mechanisms, including
transcriptional control, trafficking and channel modu-
lation (for example, alterations in subunit composition
that affect the biophysical properties of Ih), act at dif-
ferent temporal and spatial scales to modulate Ih in the
brain of patients with epilepsy. For a detailed overview of
the role of HCN channels in the development of different
types of epilepsies, see REFS 4,72,86.
HCN channels as drug targets
HCN channel blockers as heart rate-reducing agents.
Increased heart rate is an important risk factor in vari-
ous cardiac diseases, including heart failure, arterial
hypertension and coronary heart disease 91–93. The
reduction of an increased basal heart rate is beneficial
to patients and has been shown to prolong lifespan94.
Currently used bradycardic drugs (such as β‑adrenergic
receptor antagonists) and blockers of the cardiac L‑type
calcium channel efficiently reduce heart rate, but their
use is limited by adverse reactions (for example, negative
inotropic effects and interactions with vascular and
pulmonary smooth muscle)93,95. Inhibitors of HCN chan-
nels — particularly inhibitors of the major sinoatrial
isoform, HCN4 — are well suited for therapeutically
lowering heart rate. Importantly, as HCN channels are
specifically expressed in the heart and the CNS, drugs
interfering with HCN channel activity will not be associ-
ated with adverse effects on airway and vascular smooth
muscle tone.
Drugs identified as selective bradycardic agents
include alinidine (ST567), which is a derivative of cloni-
dine, ZD7288 and the ‘bradines’, which are a group of
compounds derived from the calcium channel blocker
verapamil96. The group of bradines comprises zatebradine
(UL‑FS49), cilobradine (DK‑AH269) and ivabradine
(Procoralan; Servier); chemical structures of these and
other drugs discussed in this article are provided in FIG. 4.
These substances block sinoatrial If and hence reduce the
slope of the slow diastolic depolarization phase as well as
the frequency of pacemaker potentials (FIG. 3a).
The mechanism of action of ivabradine has been
studied in detail in isolated rabbit sinoatrial node cells97
(FIG. 3b). Ivabradine can access HCN channels only from
the intracellular side when the channel pore is opened by
hyperpolarization and the current is inwardly directed.
During the following depolarization phase in the action
potential cycle, which is characterized by an outward ion
flow, ivabradine is driven into the pore where it binds
(to a site that has not yet been identified) and blocks
the HCN channels (FIG. 3b). This mechanism of action
is consistent with the observed use-dependency of
ivabradine: the more frequently the direction of ion flow
through the HCN channel changes, the more efficiently
the ivabradine molecule can access its binding site in the
inner pore.
© 2011 Macmillan Publishers Limited. All rights reserved
Use-dependency is a very useful clinical property,
as it specifically increases the efficiency of ivabradine
during tachycardia. Interestingly, the mode of action of
ivabradine seems to depend on the particular isoform
of HCN channels. Ivabradine displays pronounced
current-dependency in HCN4 and in native sinoatrial
If 98; by contrast, it is a closed-channel blocker of HCN1
channels. Zatebradine — which is structurally closely
related to ivabradine — blocked HCN channels in a
purely voltage-dependent fashion and did not display
current-dependency 97. Together, these findings suggest
that zatebradine and ivabradine, although structurally
closely related to each other, have slightly different bind-
ing sites on HCN channels. The interaction of both com-
pounds with HCN channels may be affected by residues
that are either distinct or spatially oriented in a different
way in the respective HCN channel isoforms.
Ivabradine was approved by the European Medicines
Agency in 2005, and is the first clinically approved
compound that specifically targets HCN channels. The
therapeutic indication of ivabradine is the symptomatic
treatment of chronic stable angina pectoris in patients
with coronary artery disease with a normal sinus
rhythm, who have contraindication or intolerance for
beta blockers. Ivabradine may also be effective in various
other cardiovascular conditions in which an exclusive
lowering of heart rate is beneficial (for example, chronic
heart failure, atherosclerosis and heart transplantation)99.
In particular, ivabradine and other If blockers may sup-
press the generation of ectopic action potentials that
are thought to be triggered by the upregulation of HCN
channels in congestive heart failure100,101. Importantly, in
contrast to β‑adrenergic receptor antagonists, ivabradine
does not induce depression of left ventricular function in
patients with left ventricular dysfunction102.
A large-scale multicenter clinical trial — BEAUTIFUL
(morbidity-mortality evaluation of the If inhibitor
Procoralan in patients with coronary disease and left ven-
tricular dysfunction) — enrolling almost 11,000 patients
evaluated the efficacy of ivabradine in reducing morbidity
and/or mortality in patients with coronary artery dis-
ease with left ventricular dysfunction103,104. This study
revealed a clear benefit for the subgroup of patients with
a basal heart rate ≥70 beats per minute with respect to the
occurrence of fatal and non-fatal myocardial infarction
and revascularization. In another clinical study — SHIFT
(systolic heart failure treatment with the I f inhibitor
ivabradine trial) — that included about 6,500 patients
with chronic heart failure, ivabradine lowered major
risks associated with heart failure when it was adminis-
tered in addition to guideline-based and evidence-based
treatment 105 (most patients received beta blockers and
antagonists of the renin–angiotensin–aldosterone sys-
tem). In agreement with the results of the BEAUTIFUL
study, the therapeutic benefit associated with ivabradine
directly correlated with pre-treatment heart rate (it was
significant in patients with a median baseline heart rate
that was higher than 77 beats per minute).
Given their promising therapeutic value, it is clearly
desirable to further optimize bradycardic drugs acting
on HCN channels. Ivabradine and the other compounds
www.nature.com/reviews/drugdisc
 REVIEWS

O
O
N O
O
O
N N
O O
O N
O
O  

O
O
N HN HN
H O
N HN N N N
N Cl Cl Cl Cl
N

N N

   
O O

Cl

Cl OH
N
H2N Cl
H2N F
N O
Br
N N F H
F
N
NH2
   

Cl F OH
NH
F
O
O F
F N Cl Cl
F
N O OH

   
Figure 4 | Structures of compounds interacting with hyperpolarization-activated cyclic nucleotide-gated
channels. Compound 1, ivabradine (Procoralan; Servier); compound 2, zatebradine; compound 3, cilobradine;
0CVWTG4GXKGYU^&TWI&KUEQXGT[
compound 4, ZD7288; compound 5, clonidine; compound 6, alinidine; compound 7, nicotine; compound 8, lamotrigine
(Lamictal; GlaxoSmithKline); compound 9, gabapentin (Gabapen; Pfizer); compound 10, halothane; compound 11,
isofluorane; compound 12, loperamide; compound 13, propofol; compound 14, ketamine.

currently available have only a modest affinity for HCN
channels. The IC50 value (the half-maximal inhibitory
concentration) of ivabradine is 1.5–4.5 μM under physio­
logical conditions97,106. Off-target effects of ivabradine
have not yet been reported 96,107,108. Nevertheless, to
further increase the specificity and long-term safety of
HCN channel-based therapy (as patients will have to
receive the treatment for many years), the development
of compounds acting in the nanomolar concentration
range would be a desirable goal. Achieving subtype
specificity among the four HCN channel subtypes is
another important goal. Ivabradine does not selectively
target specific HCN channel subtypes106,109.
As HCN4 is the principal HCN channel isoform in
the sinoatrial node (responsible for over 80% of total If), a
drug that specifically blocks this particular channel with-
out interfering with HCN1–HCN3 would be superior
to the currently available compounds. Interaction with
the HCN1 isoform expressed in retinal photoreceptors
and bipolar cells34,110 probably underlies the transient
changes in visual sensation that are observed in about
15% of patients following initial treatment with ivabra-
dine111. A recent study demonstrated that subtype speci-
ficity can be achieved by modifying the substituent on
the nitrogen atom in the benzazepin-4‑one backbone
of ivabradine106,112 (FIG. 5). Ivabradine does not cross the
blood–brain barrier — a property that is crucial for its
clinical use as a bradycardic agent113. Future development
of specific HCN channel blockers should also aim for
compounds that — unlike ivabradine — do not cross
the blood–retinal barrier.
Another strategy for developing cardiac-specific
HCN channel blockers could rely on using clonidine as
a lead structure. Clonidine is a well-known α2-adrenergic
receptor agonist, and is chemically related to the brady-
cardic agent alinidine. Studies in mice in which the
α2-adrenergic receptor was knocked out indicated that
inhibition of sinoatrial If contributes substantially to

NATURE REVIEWS | DRUG DISCOVERY VOLUME 10 | DECEMBER 2011 | 909

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS
Absence epilepsy
A form of childhood epilepsy
that typically results from
abnormal transformation of
thalamocortical oscillations.
Temporal lobe epilepsy
A form of epilepsy in which
seizures typically involve the
hippocampus and adjacent
cortices. This form of epilepsy
is not thought to have a strong
genetic component.
Febrile seizure syndromes
Seizures that take place in
children at the onset of or
during fever. They are the most
common types of seizures in
humans, and usually short and
benign. However, when they
are long (especially longer than
30 minutes), these seizures
are associated with a high
probability of the development
of hippocampal epilepsy
(temporal lobe epilepsy) later in
life. There are both genetic and
environmental contributions to
febrile seizures.
910 | DECEMBER 2011 | VOLUME 10
the bradycardic action of clonidine114. Clonidine blocks
HCN4 with an IC50 of about 10 μM, but in contrast
to ivabradine it acts from the extracellular side of the
plasma membrane (M.B. and X. Zong, unpublished
observations) and shifts the voltage-dependence of the
channel by 10–20 mV to voltages that are more hyper-
polarized114. As clonidine has several neuronal effects
that are mediated by central α2-adrenergic receptors
(for example, analgesia and sedation), it cannot be used
as a bradycardic drug. However, compounds could be
designed — based on the clonidine backbone — that
do not act on α2-adrenergic receptors but preserve the
HCN-blocking effect.
HCN channels as targets for anticonvulsant drugs.
As discussed previously, dysregulation of HCN chan-
nel expression and aberrant HCN channel function
have been implicated in various types of idiopathic and
acquired epilepsies. HCN2-deficient mice have the typical
clinical hallmarks of absence epilepsy41. Similarly, deletion
of HCN1 in mice is associated with increased seizure
severity and risk of seizure-related death in different
limbic seizure induction models115,116. The results from
genetic mouse models suggest that suppression of Ih is
involved in the generation of neuronal hyperexcitability.
In support of this hypothesis, downregulation of HCN1
expression levels has been reported in experimental
temporal lobe epilepsy, which is the most common and
severe form of epilepsy in adults66,117,118. Surprisingly,
however, there is also growing evidence that not only
downregulation but also enhancement of HCN channel-
mediated currents may contribute to the pathology of
seizures. For example, an HCN2 variant (in which resi-
dues 719–721 are deleted) was identified in patients suf-
fering from febrile seizure syndromes; when heterologously
expressed, this variant leads to 35% larger currents than
controls119. Finally, in addition to the absolute expression
levels, the stoichiometry and subunit composition of het-
eromeric channels (for example, the HCN1:HCN2 ratio)120
as well as their targeting to neuronal compartments118 may
be crucial for inducing aberrant neuronal firing patterns.
The complexity and diversity of the mechanisms con-
necting impaired HCN channel activity with epilepsy
make it very challenging to develop a generally applica-
ble rationale for the design of anticonvulsant drugs based
on HCN channels. Indeed, there are reports that either
pharmacological block or activation may be beneficial
in the context of epilepsy, depending on the clinical cir-
cumstances. For example, the HCN channel inhibitor
ZD7288 was shown to reduce the generation of hipppo­
campal epileptic discharges in rabbits121. Conversely, two
well-established anticonvulsant drugs — lamotrigine
(Lamictal; GlaxoSmithKline) and gabapentin (Gabapen;
Pfizer) (FIG. 4) — that act by blocking voltage-gated sodium
and calcium channels, respectively, were shown to upregu-
late the activity of HCN channels122,123. The contribution
of HCN channel upregulation to the global clinical effect
of these compounds is currently unclear. However, it may
be speculated that the action of both drugs is directed
primarily at HCN1, which is the main HCN subtype in
the cortex and hippocampus.
© 2011 Macmillan Publishers Limited. All rights reserved
HCN channel blockers in the treatment of neuropathic
pain. There is growing evidence that HCN channels are
involved in the pathology of peripheral neuropathic pain
and in pain processing (reviewed in REFS 5,71). A hallmark
of neuropathic pain disorders is the generation of ectopic
discharges in primary sensory neurons and/or enhanced
nociceptive synaptic transmission in the spinal dorsal horn
that may be accompanied by changes in the descending
regulation of the supraspinal CNS. Several studies have
reported the upregulation of Ih densities following periph-
eral nerve injury, particularly in large and medium-sized
neurons in which most ectopic discharges are produced87,88.
Increased Ih densities might lead to a shift in the resting
membrane potential of neurons towards more positive
values and hence lower the threshold for the generation
of action potentials. By contrast, a study by Doan et al.89
suggests that the suppression of Ih by increasing neuronal
input resistance may also lead to enhanced peripheral
nerve firing. All four HCN channel subtypes have been
identified in peripheral sensory neurons, with HCN1 and
HCN2 being the most widely expressed isoforms32,124.
There is experimental evidence for increased HCN1 and
HCN2 expression in peripheral pain conditions35.
A recent study 125 showed that the anticancer agent
oxaliplatin induces the upregulation of HCN1 in cold-
sensitive primary afferent neurons. This process is of
clinical relevance because it may underlie the generation of
hypersensitivity to cold in patients receiving oxaliplatin-
based chemotherapy. The relevance of HCN1 in the
pathology of cold hyperalgesia is supported by the
findings of Momin and colleagues126. These authors
demonstrated that HCN1‑knockout mice display sub-
stantially less cold allodynia than their wild-type litter-
mates, but their responsiveness to inflammatory pain
stimuli is unaffected. There is evidence that HCN2 has
a central role in this particular pain modality 127. Genetic
deletion of HCN2 in a subfraction of nociceptive neurons
that are characterized by the expression of the voltage-
dependent sodium channel isoform Nav1.8 abolished
repetitive firing of these neurons in response to an eleva-
tion in cAMP. Inflammation did not cause hyperalgesia
to heat stimuli in these mice. Moreover, after a nerve
lesion, HCN2‑deficient mice displayed no neuropathic
pain in response to thermal or mechanical stimuli.
Together, these findings indicate that HCN channels
are promising targets for the treatment of peripheral pain
disorders. Indeed, low concentrations of the Ih blocker
ZD7288 reversed both pain behaviour and spontaneous
discharges in injured nerve fibres in rats87,128,129. Moreover,
intraperitoneal administration of ivabradine reversed
oxaliplatin-mediated cold hypersensitivity in mice125.
HCN1 seems to be a promising HCN channel subtype to
target in the context of peripheral pain disorders because
this channel is broadly expressed in sensory neurons and
it is transcriptionally upregulated in neuropathic pain.
In addition, a recent study 127 has indicated that HCN2 is
an attractive target for the development of analgesics to
combat both inflammatory and neuropathic pain.
The ideal analgesic compound should be strongly
selective for HCN1 and/or HCN2 over HCN4 to prevent
effects on cardiac rhythmicity. Moreover, it should not
www.nature.com/reviews/drugdisc
 REVIEWS

C D 
O
O
O O O

N 
R
O
N
O

'%=µ/?
N
/'.# 
O

O
N N
S O
O 
O
O
+XCDTCFKPG

O 
O /'.# +XCDTCFKPG '%
N   
O
O N %QORQWPF
O
'% *%0 *%0 *%0
Figure 5 | Development of derivatives of ivabradine with increased subtype specificity. a | Chemical structure
of ivabradine and two of its derivatives (MEL57A and EC18). b | EC50 (effector concentration for half-maximum
response) values for ivabradine, MEL57A and EC18 are shown, as determined for hyperpolarization-activated cyclic
nucleotide-gated channel 1 (HCN1), HCN2 and HCN4 expressed in human embryonic kidney 0CVWTG4GXKGYU^&TWI&KUEQXGT[
293 (HEK293) cells.
The ratios of the EC50 values of HCN4 versus HCN1 are given in parentheses for each compound. Ivabradine does not
differentiate between HCN1 and HCN4. By contrast, MEL57A is strongly selective for HCN1 over HCN4, whereas EC18
displays an approximately five times greater affinity for HCN4 than for HCN1 (data from REFS 106, 112).

cross the blood–retina barrier to circumvent effects on
vision. An agent fulfilling these criteria is not yet available.
However, the study by Melchiorre et al.106 indicates that
HCN channel subtype-selective agents can be designed
based on the ivabradine backbone (FIG. 5). Interestingly,
the μ‑opioid receptor agonist loperamide (FIG. 4) was
also shown to block Ih in dorsal root ganglion neurons
in the low micromolar range36. It was speculated that this
block contributes to the analgesic action of loperamide.
Thus, loperamide may serve as another lead structure for
developing HCN channel blockers that are not structur-
ally related to the bradines. Similarly, clonidine (FIG. 4),
which blocks Ih in rat dorsal root ganglion neurons, may
serve as a starting point for the development of novel
analgesic compounds 130. Finally, the only clinically
approved HCN channel blocker — ivabradine — might
also be beneficial in the therapeutic management of neu-
ropathic pain. Although ivabradine is not selective for
any of the four HCN channel subtypes, it could be used
in a dose range that keeps heart rate under physiological
parameters125.
evidence that neuronal Ih is also inhibited by clinically
relevant concentrations of general anaesthetics133–135.
For halothane and isofluorane, the inhibitory effect
on Ih was shown to comprise at least two components:
namely, a hyperpolarizing shift in the activation curve
and a decrease in the peak current amplitude 135,136.
Studies in heterologous systems136 and HCN1‑knockout
mice137 indicate that HCN1 is the major channel con-
ferring the anaesthetic action of the drugs mentioned
above. Interestingly, despite being structurally unre-
lated, two other well-established intravenous anaes-
thetics — propofol and ketamine — also inhibit the
activity of HCN1 by shifting the voltage-dependence to
more hyperpolarized voltages and decreasing the peak
current 137,138 (FIG. 4). The importance of HCN1 as a tar-
get of anaesthetics is corroborated by the finding that
HCN1‑knockout mice are markedly less sensitive to the
hypnotic actions of propofol and ketamine137. A report
has also suggested that part of the mechanism of action
of local anaesthetics such as lidocaine may be caused by
HCN channel inhibition139.

Background potassium
channels
Potassium channels that are
constitutively open or possess
high basal activity (sometimes
also called ‘leaky’ channels).
HCN channel blockers in anaesthesia. General anaes-
thetics (for example, halothane and isofluorane; FIG. 4)
exert their pharmacological effects by modulating the
activity of ion channels in the CNS. Among the identi-
fied targets of these compounds are ionotropic recep-
tors (for example, the GABAA (γ-aminobutyric acid
type A) and NMDA (N-methyl-d-aspartate) receptors)
and several background potassium channels131,132. There is
New compounds: challenges and future directions
The successful therapeutic use of ivabradine in patients
with angina pectoris has served as a bona fide proof of
principle that interference with HCN channel function
can be beneficial in a clinical setting. However, to explore
the full spectrum of possible indications covered by HCN
channels, second-generation compounds will have to
be designed. Three crucial points should be considered

NATURE REVIEWS | DRUG DISCOVERY VOLUME 10 | DECEMBER 2011 | 911

© 2011 Macmillan Publishers Limited. All rights reserved

REVIEWS

in future studies. First, compounds with higher affinity
have to be developed. The currently available HCN
channel blockers (ivabradine) and agonists (lamotrigine
and gabapentin) discussed in this article display consid-
erably modest IC50 values in the low micromolar range.
Ideal compounds should operate in the nanomolar range
to lower the risk of off-target and toxic side effects. It has
been shown that nicotine blocks native neuronal HCN
channels at remarkably low concentrations 140 (with
an EC50 (effector concentration for half-maximum
response) value of around 60 nM). Although nicotine
itself is clearly not useful as a therapeutic HCN channel
blocker because it activates the nicotinic acetylcholine
receptor as well, analogues of this drug may interact with
HCN channels with greater selectivity.
The second issue to consider is the localization of the
drug binding side within HCN channels. Ivabradine,
like most other blockers, targets the intracellular side
of the HCN channel, which requires drug penetration
through the plasma membrane. Moreover, most blockers
are trapped within the ion-conducting pore, which
results in very slow off-kinetics and, in some cases, (for
example, with ZD7288) an almost irreversible block.
It would be desirable to design compounds that bind to
HCN channels in a reversible manner from the extracell­
ular side of the membrane. However, such compounds
are not yet available. Clonidine could serve as a template
for such a drug because it exerts its action by binding to
an external side of HCN channels (M.B. and X. Zong,
unpublished observations).
Probably the most important goal in the development
of drugs targeting HCN channels will be the design of
subtype-specific blockers. For example, a specific HCN4
blocker would effectively lower heart rate and probably
lack the side effects associated with vision that are typical
of nonspecific HCN channel blockers. Similarly, com-
pounds that selectively inhibit HCN1 would have no
major effects on cardiac pacemaker function but could
be used for the treatment of neuropathic pain or epi-
lepsy. Subtype specificity has not yet been achieved in a
satisfactory manner; however, a recent study 106 indicates
that it is in principle feasible to synthesize selective com-
pounds (FIG. 5). Recently, a series of novel HCN channel
blockers has been reported. The new compounds have
an indane backbone and display selectivity for HCN1
over HCN4 (REF. 146). Finally, it is important to note
that HCN channels also assemble into heterotetramers,
besides forming homomers. There is growing evidence
that an increase in the formation of HCN1–HCN2
heteromers has an important role in the pathology of
temporal lobe epilepsies4. It is tempting to speculate
whether altered hetero­merization of HCN subunits
could also be relevant in other types of epilepsies and
neuronal diseases. Thus, it will be necessary to search for
compounds that are selective for heteromeric channels
over homomeric channels.
An important drawback in the design of specific
HCN channel blockers is the lack of a high-resolution
structure of the transmembrane core. So far, only parts
of the C terminus of HCN channels have been crystal-
lized. As most ligands interact with the transmembrane
core (including the ion-conducting region) of the chan-
nel, structural information on this part of the channels
is urgently needed. Currently, several laboratories are
seeking to crystallize full-length HCN channels. Once
a three-dimensional structure is available, molecular
modelling approaches could be applied to identify drug
binding sites and optimize drugs targeting HCN channels
in terms of affinity, selectivity and mode of interaction
(for example, state-dependence of binding).

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Acknowledgements
This work was supported by the German Research Foundation
(the Deutsche Forschungsgemeinschaft).
Competing interests statement
The authors declare no competing financial interests.
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