Physiol Rev 89: 847– 885, 2009;
doi:10.1152/physrev.00029.2008.
Hyperpolarization-Activated Cation Channels: From Genes
to Function
MARTIN BIEL, CHRISTIAN WAHL-SCHOTT, STYLIANOS MICHALAKIS, AND XIANGANG ZONG
Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie,
Pharmakologie für Naturwissenschaften, Ludwig-Maximilians-Universität München, Munich, Germany
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I. Introduction
II. A Short Overview on Basic Biophysical Properties of Ih
A. Channel gating by membrane hyperpolarization
B. Modulation by cyclic nucleotides
C. Ion selectivity
D. Pharmacological profile
III. The HCN Channel Family
A. Transmembrane segments and voltage sensor
B. Pore loop and selectivity filter
C. Cyclic nucleotide-binding domain and C-linker
D. Dual channel gating
E. Functional differences between HCN channel types
IV. Regulation of HCN Channels
A. Regulation by acidic lipids
B. Regulation by protons
C. Regulation by chloride
D. Regulation by Src kinase-mediated tyrosine phosphorylation
E. Regulation by p38-mitogen-activated protein kinase
F. Transmembrane and cytosolic proteins interacting with HCN channels
V. Tissue Expression of HCN Channels
VI. Physiological Roles of HCN Channels in Neurons
A. Principles
B. Role of Ih in dendritic integration
C. Role of Ih in working memory
D. Role of Ih in constraining hippocampal LTP
E. Role of Ih in motor learning
F. Role of Ih in synaptic transmission
G. Role of Ih in resonance and oscillations
H. Role of Ih in the generation of thalamic rhythms
VII. Role of Ih Channels in Cardiac Rhythmicity
A. HCN4
B. HCN2
C. Conclusions and open questions
VIII. Role of Ih in Disease
A. Inherited channelopathies
B. Transcriptional channelopathies
IX. HCN Channels as Novel Drug Targets
A. Heart rate-reducing agents
B. Blockers of neuronal Ih
X. Conclusions and Future Directions
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Biel M, Wahl-Schott C, Michalakis S, Zong X. Hyperpolarization-Activated Cation Channels: From Genes to
Function. Physiol Rev 89: 847– 885, 2009; doi:10.1152/physrev.00029.2008.—Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation
channels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart and
nervous system. The current produced by HCN channels has been known as Ih (or If or Iq). Ih has also been
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0031-9333/09 $18.00 Copyright © 2009 the American Physiological Society
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designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemaker
cells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidence
that Ih is also involved in a number of basic physiological processes that are not directly associated with rhythmicity.
Examples for these non-pacemaking functions of Ih are the determination of the resting membrane potential,
dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into the
structure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCN
channel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channel
types arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of the
impact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCN
channels as drug targets.
I. INTRODUCTION
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II. A SHORT OVERVIEW ON BASIC
BIOPHYSICAL PROPERTIES OF Ih
Native Ih as well as the currents induced by heterologously expressed HCN channels are characterized by
four major hallmark properties: 1) channel activation by
membrane hyperpolarization, 2) facilitation of channel
activation by direct interaction with cAMP, 3) permeation
of Na⫹ and K⫹, and 4) a specific pharmacological profile
that includes sensitivity to external Cs⫹ concurred with
relative insensitivity to Ba2⫹ (Fig. 1).
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Since their first discovery in sinoatrial node cells (49,
51, 113, 116, 430) and neurons (169, 234) in the late 1970s
and early 1980s, hyperpolarization-activated currents (Ih)
have sparked continuing interest of physiologists and researchers working in biomedical disciplines. The exceptional position of Ih among ionic currents mainly arises
from its unique ion selectivity and gating properties. Ih is
a mixed cationic current carried by Na⫹ and K⫹. Since its
reversal potential is around ⫺20 mV at physiological ionic
conditions (114), Ih is inwardly directed at rest and,
hence, depolarizes the membrane potential. However, unlike the vast majority of cellular conductances that are
activated upon membrane depolarization, Ih is activated
by hyperpolarizing voltage steps to potentials negative to
⫺55 mV, near the resting potential of cells. In addition,
activation of Ih is facilitated by cAMP in a direct, protein
kinase A (i.e., phosphorylation)-independent fashion. Because of its quite unusual biophysical profile, the hyperpolarization-activated current was designated “funny”
current (If) in heart (49), while other researchers used the
term Iq (for “queer”) instead of Ih for the neuronal current
(169). In this review, we will generally use the term Ih.
Many physiological roles have been attributed to Ih.
First and foremost, the properties of Ih suggested that the
current plays an important role in the initiation and regulation (49, 117) of the heart beat (“pacemaker current”)(51, 113, 430). However, even now, 30 years after
this hypothesis has been raised, strong controversy over
the exact role of Ih in cardiac pacemaking exists. Genetic
studies in men and mice (see sect. VII) will be very instrumental in solving this physiological conundrum (171, 174,
226, 370). Ih is also involved in the control of rhythmic
activity in neuronal circuits (128, 221, 222, 258, 261, 355)
(e.g., in thalamus) and contributes to several other basic
neuronal processes, including determination of resting
membrane potential (98, 125, 226, 230, 266, 291–293, 300),
dendritic integration (239 –241, 423), synaptic transmission (32, 33), and the temporal processing of visual signals
in the retina (105, 106, 209, 247).
The ion channels underlying Ih have been discovered
about a decade ago (153, 192, 211, 227, 228, 251, 332, 333,
341, 344, 400). With reference to their complex dual gating
mode (7, 93, 112), these proteins were termed hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.
In mammals, the HCN channel family comprises four
distinct members (HCN1-4). Currents obtained after heterologous expression of the cDNAs of HCN1-4 channels
reveal the principal features of native Ih, confirming that
HCN channels indeed represent the molecular correlate
of Ih.
In the first three sections of this review, we give a
brief overview on the biophysical properties of Ih/HCN
currents and correlate these properties with structural
domains of HCN channels. Readers who are interested in
getting a more detailed and complete overview on the
mentioned topics are referred to a number of excellent
reviews that extensively deal with these issues and also
describe the discovery of Ih and the advance in the Ih field
from a more historical point of view (29, 40, 82, 93, 152,
226, 300, 324, 334). Section IV is a first important focus of
this review, presenting recent data on the cellular regulation of HCN channels. Section V briefly summarizes the
data available on the tissue expression of HCN channels.
The major focus of this review is on the roles of HCN
channels in neuronal function and cardiac rhythmicity
(sects. VI and VII). We discuss these issues in the context of
recent results from the analysis of HCN channel knockout
mice. Finally, we give an overview on the role of HCN
channels in the pathologies of human diseases (sect. VIII)
and discuss pharmacological agents interacting with
these proteins (sect. IX).
FUNCTION OF HCN CHANNELS
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A. Channel Gating by Membrane Hyperpolarization
In general, Ih currents activate with hyperpolarizing
steps to potentials negative to ⫺50 to ⫺60 mV (Fig. 1A).
Unlike most other voltage-gated currents, Ih does not
display voltage-dependent inactivation. Typically, two kinetic components can be distinguished upon activation of
Ih: a minor instantaneous current (IINS) (236, 237, 312),
which is fully activated within a few milliseconds, and the
major slowly developing component (ISS) that reaches its
steady-state level within a range of tens of milliseconds to
several seconds under fully activating conditions. While
there is no doubt that ISS is generated by cations passing
the well-characterized pore of HCN channels, the ionic
nature of IINS is a matter of current dispute. IINS is not
consistently observed in all measurements of Ih and, if so,
its amplitude is usually small.
However, in some neurons, a large IINS lacking timedependent ISS has been reported (12, 100, 326). Speculations on the nature of IINS reach from models where this
current represents a leak conductance or an experimental
artifact to models in which IINS is caused by a second pore
that is found within the same HCN channel that produces
ISS or a second channel population associated with HCN
channels (236, 237, 312). Depending on the cell type, the
activation of ISS can be empirically described by either a
single (120, 136, 200, 261, 264, 355, 393, 425) or double
exponential function (17, 104, 131, 175, 248, 402). As
mentioned above, kinetics of ISS are quite variable (111,
118). While most Ih channels activate quite slowly with
time constants (␶act) ranging between hundreds of milliseconds and seconds (131) (Fig. 1B, inset), there are also
currents with profoundly faster activation (17, 169, 175,
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234, 248, 264), for example, in hippocampal CA1 neurons,
where ␶act values in the range of 30 –50 ms have been
found (146, 300, 324). The diversity of ␶act probably results
from an interplay of several factors. First of all, the differences reflect the diverse intrinsic activation properties
of distinct HCN channel isoforms underlying the Ih of a
given cell type. Second, there is growing evidence that the
cellular microenvironment that fine-tunes HCN channel
activity (e.g., auxiliary subunits, concentration of cellular
factors, etc.) can profoundly vary from cell type to cell
type. Third, Ih measurements are highly sensitive to experimental conditions (e.g., pH, temperature, patch configuration, expression system, ionic composition of solutions, etc.), a circumstance that may explain that even for
the same channel different kinetics have been found by
different laboratories. As mentioned above, Ih activates at
around resting membrane potential. The voltage dependence of activation shows a typical S-shaped dependence
that can be fit using Boltzmann functions (20, 25, 131, 175,
200, 216, 234, 253, 261, 354, 357, 393) (Fig. 1B). Such fits
reveal half-maximal activation (“midpoint”) potentials
(V0.5) of around ⫺70 to ⫺100 mV in most cell types.
However, like for ␶act, V0.5 values can vary profoundly
in vivo (28, 111, 118). For example, V0.5 of Ih recorded in
sinoatrial node cells is in the range of ⫺60 to ⫺70 mV
(3–5, 121, 124, 167) while currents found in ventricular
cardiomyocytes activate at much more hyperpolarized
voltages with V0.5 being negative to ⫺90 to ⫺140 mV (138,
319, 346). Like for ␶act, the strikingly large range of V0.5
values probably results from the combination of intrinsic
parameters such as the HCN channel isoform and modulatory factors present in a given cell type, and extrinsic
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FIG. 1. A: voltage dependence of
HCN2 activation. Family of current
traces (bottom panel) and pulse protocol
(top panel). B: activation curve of HCN2
currents in the absence of cyclic nucleotides (open circles) or after intracellular
perfusion with 1 mM cAMP (solid circles)
or 1 mM cGMP (triangles), respectively.
Inset: time course of HCN2 activation in
the absence (black line) and the presence
of cAMP (red line). C: dose-response relationship for the shift in the HCN2 activation curve as a function of cAMP concentration. D: current-voltage relationship for the fully activated HCN2 channel
determined at 5.4 mM (solid circles) and
30 mM (open circles) extracellular K⫹,
respectively. E: extracellular block of
whole cell current by 2 mM Cs⫹, 2 mM
Ba2⫹, 20 mM tetraethylammonium (TEA),
and 1 mM 4-aminopyridine (4-AP), respectively. F: percentage inhibition of steady-state
current at ⫺140 mV by 20 mM TEA, 1 mM
4-AP, 2 mM Ba2⫹, and 2 mM Cs⫹.
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factors such as the experimental settings during Ih measurements.
B. Modulation by Cyclic Nucleotides
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C. Ion Selectivity
Ih is a mixed cation current that is carried by both K⫹
and Na⫹ under normal physiological conditions (110, 116,
145, 227, 333, 424). The ratio of the Na⫹ to K⫹ permeability of the channel (PNa:PK) ranges from 1:3 to 1:5, yielding
values for the reversal potential between ⫺25 and ⫺40
mV (Fig. 1D) (20, 25, 31, 60, 95, 103, 113, 131, 175, 180, 200,
203, 227, 253, 261, 264, 301, 333, 341, 354, 357, 383, 391,
393, 402, 424). As a consequence, activation of Ih at resting membrane potentials results in an inward current
carried mainly by Na⫹, which depolarizes the membrane
toward threshold for firing of action potentials. Ih is almost impermeable to Li⫹ (180, 424) and is blocked by Cs⫹
(109, 137, 227). K⫹ is not only a permeating cation, it also
affects the permeation of Na⫹ (20, 95, 103, 109, 132, 145,
175, 200, 227, 234, 248, 250, 261, 354, 357, 383, 385, 424).
Both the current amplitude and the PNa:PK ratio of Ih
channels depend on the extracellular K⫹ concentration
(Fig. 1D) (227). An increase in extracellular K⫹ concentration results in a strongly increased current amplitude
and in a slightly reduced selectivity for K⫹ over Na⫹ (145,
424). The interdependence of Na⫹ and K⫹ permeation of
Ih channels is illustrated by the finding that the channels
conduct little, if any, Na⫹ in the absence of K⫹. In contrast, current modifications upon reduction of extracellular Na⫹ levels are merely the result of the altered driving
force (116, 300). Although Ih channels do not conduct
anions, their conductance is sensitive to external Cl⫺
levels (144, 411) (see sect. IVC for further discussion). For
a long time, only monovalent cations were expected to
permeate through the Ih channels. However, there is recent evidence for a small but significant Ca2⫹ permeability of these channels (440, 441). At 2.5 mM external Ca2⫹,
the fractional Ca2⫹ current of native Ih as well as that of
heterologously expressed HCN2 and HCN4 channels is
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The second key feature of Ih is its regulation by cyclic
nucleotides (122; Fig. 1B). Hormones and neurotransmitters that elevate cAMP levels facilitate activation of Ih by
shifting V0.5 values to more positive values and by accelerating the opening kinetics (25, 41, 49, 50, 122, 136, 151,
203, 216 –218, 260, 262, 303, 391). It has been shown that
the acceleration of the opening kinetics with cAMP can be
attributed to the shift in voltage dependence of activation
(412). Thus, in the presence of high cAMP concentrations,
Ih channel opening is faster and more complete than at
low cAMP levels (Fig. 1B, inset). Conversely, neurotransmitters that downregulate cAMP inhibit Ih activation by
shifting its activation curve to more hyperpolarized voltages (117, 123, 124, 147, 190, 299, 315, 318). The range of
V0.5 shift induced by saturating cAMP concentrations is
quite large (0 –20 mV), depending on the cell type (25, 41,
49, 50, 122, 136, 151, 203, 216 –218, 260, 262, 303, 384, 391)
and the expressed Ih channel isoform (192, 227, 228, 333,
341). The regulation by cellular cAMP level is of key
importance for the specific role that Ih channels fulfill in
physiological settings, since it enables these proteins to
operate as transmembrane integrators of electrical (membrane potential) and chemical (hormones and neurotransmitters) inputs. Most notably, the cAMP-mediated modulation of Ih channel activity is considered to play a major
role in the up- or downregulation of the heart rate during
sympathetic stimulation and muscarinic regulation of
heart rate at low vagal tone (49, 50, 117, 122–124).
As mentioned above, it was this specific physiological function that earned Ih the name “pacemaker” current.
There is also good evidence that cAMP-dependent modulation of Ih is of crucial significance in some neuronal
circuits, e.g., in sleep-related thalamocortical circuits
(231). There are a few studies showing that Ih can also be
regulated by nitric oxide (NO)-mediated increase of
cGMP levels in brain (302) and heart (283). However, so
far the physiological role of this kind of modulation remains unclear. Like cAMP, cGMP shifts the voltage dependence of channel activation to more positive values
(Fig. 1B) (153, 227). While the extent of the shift is similar
for both cyclic nucleotides (i.e., both cyclic nucleotides
have the same efficacy at least in mammalian Ih), the
apparent affinities of Ih are ⬃10- to 100-fold higher for
cAMP (range of Ka ⫽ 60 –500 nM; Fig. 1C; Ref. 227) than
for cGMP (Ka ⫽ 6 ␮M for HCN2; Ref. 227). The principal
mechanism by which cyclic nucleotides regulate Ih channel gating was uncovered by DiFrancesco and Tortora
(122) in the early 1990s using current recordings in excised patches of sinoatrial node cells. It came as a big
surprise that in contrast to many other ion channels that
are regulated by cAMP via protein kinase A (PKA)-mediated serine or threonine phosphorylation (304, 337, 433),
Ih channels are activated by cAMP independent of phosphorylation (122). Like in the structurally related cyclic
nucleotide-gated (CNG) channels (39, 93, 201), Ih channels are activated by cAMP binding to a cyclic nucleotidebinding domain (CNBD) on the COOH terminus of the
channel (443). The molecular details underlying the modulation of Ih channels by cAMP have been extensively
studied when cloned Ih(HCN) channels were available
and will be discussed later. There also are some reports
on PKA-mediated phosphorylation of Ih channels (79,
403); however, phosphorylation could not yet be confirmed on the molecular level in HCN channels. Moreover,
the physiological relevance of PKA-induced Ih phosphorylation remains elusive.
FUNCTION OF HCN CHANNELS
333, 341, 344, 400) but are missing in Caenorhabditis
elegans, yeast, and prokaryotes.
In all mammals investigated so far, four homologous
HCN channel subunits (HCN1-4) exist. HCN1-4 are also
present in the genome of fishes (e.g., Tetraodon nigroviridis and Takifugu rubripes). Analysis of genomic sequences suggests that HCN2– 4 genes underwent duplications in the fish lineage increasing the number of potential
HCN species (194). HCN homologs have also been cloned
from several invertebrates including arthropods [e.g.,
spiny lobster (157), insects (158, 211, 251)] and sea urchins (149, 153). The overall characteristics of the Ih
currents from these species are strikingly similar to Ih
currents from mammals. Notably, HCN channels are not
present in C. elegans and yeast and also have not been
found in a prokaryotic genome.
Like other pore-loop channels, HCN channels are
complexes consisting of four subunits that are arranged
around the centrally located pore (Fig. 2). These subunits
form four different homotetramers with distinct biophysical properties (192, 227, 228, 333, 341). There is evidence
that the number of potential HCN channel types is increased in vivo by the formation of heterotetramers (8, 85,
135, 278, 397, 420, 428). So far, splicing variants of vertebrate HCN channels were not identified. In contrast, alternative splicing of HCN channel transcripts was reported for some invertebrates, including spiny lobster,
D. Pharmacological Profile
Ih channels are almost completely blocked by low
millimolar concentrations of Cs⫹ (Fig. 1, E and F) (109,
137, 227). Inwardly rectifying K⫹ currents that activate
over a similar voltage range as Ih are also blocked by this
cation (99, 288, 289). However, in contrast to these currents, Ih is insensitive to millimolar concentrations of
external Ba2⫹ and tetraethylammonium (TEA) (227). Ih is
also insensitive to 4-aminopyridine, a blocker of voltagegated K⫹ channels (227). A number of organic blockers
have been described to block Ih channels. Among these,
the most specific ones are bradycardic agents such as
ivabradine that block the Ih channels in the low micromolar range (see sect. IX).
C-linker
III. THE HCN CHANNEL FAMILY
HCN channels represent the molecular correlate of
Ih. Together with CNG channels and the Eag-like K⫹
channels (39, 93, 201), HCN channels form the subgroup
of cyclic nucleotide-regulated cation channels within the
large superfamily of the pore-loop cation channels (437).
HCN channels have been cloned from vertebrates and
several invertebrates (153, 192, 211, 227, 228, 251, 332,
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FIG. 2. Structure of HCN channels. HCN channels are tetramers.
One monomer is composed of six transmembrane segments including
the voltage sensor (S4) and the pore region between S5 and S6. The pore
region contains the selectivity filter carrying the GYG motif. The COOHterminal channel domain is composed of the C-linker and the cyclic
nucleotide-binding domain (CNBD). The C-linker consists of six ␣-helices, designated A⬘ to F⬘. The CNBD follows the C-linker domain and
consists of ␣-helices A-C with a ␤-roll between the A- and B-helices
(thick gray line). Human mutations involved in Ih channelopathies are
indicated as gray circles. The N-glycosylation site between S5 and the
pore loop is indicated as a Y. For details, see text.
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⬃0.5% (440, 441). The functional relevance of Ca2⫹ entry
through Ih channels is not clear at the moment. It was
assumed that in dorsal root ganglion neurons, Ca2⫹ influx
through Ih channels at negative potentials contributes to
activity-evoked secretion (441).
There is an ongoing controversy on the size of the
single-channel conductance of Ih. Originally, single-channel conductance was found to be very low, in the range of
⬃1 pS (110). This estimate is in good agreement with very
recent data (210). However, single-channel conductances
that are 10 –30 times higher have been reported for cloned
HCN channels (267) as well as for native cardiac (267)
and neuronal Ih (350). At this point, it is unclear how this
major discrepancy can be explained.
Furthermore, single-channel conductance as well as
other biophysical parameters may be dynamically regulated by modulatory factors and proteins assembled with
HCN channels in vivo. However, extreme care must be
taken before a final conclusion should be drawn on this
issue. It will be necessary to rigorously prove that the
recorded currents are indeed HCN channel currents. For
example, one problem with single-channel recording reported by Michels and co-workers (267, 268) is that the
ensemble records of single-channel activity poorly match
known kinetic properties of HCN channel current based
on whole cell recordings. This issue needs to be clarified
in the future.
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these two modes also affects the kinetics of the channel
activation and deactivation. The fact that these channels
are differentially sensitive to the direction of the voltage
change and that the activation curves in mode I and mode
II are separated by up to 50 mV is defined as “voltage
hysteresis.” This interesting gating behavior of spHCN is
probably the result of a slow conformational change attributable to lateral movement of S4 that stabilizes the
inward position of S4 upon hyperpolarization (52, 53, 133,
245, 246, 405). HCN1 channels behave similarly as spHCN
channels with respect to hysteresis behavior and mode
shift (246). In contrast, for HCN2, the voltage hysteresis is
less pronounced, but the effects of the mode shift on the
deactivation kinetics are present. For HCN4, only the
changes in deactivation kinetics are observed under certain conditions using high K⫹ concentrations in the extracellular recording solution. It has been suggested that the
mode shift is important for short-term, activity-dependent
memory in HCN channels (53).
A. Transmembrane Segments and Voltage Sensor
B. Pore Loop and Selectivity Filter
The transmembrane channel core of HCN channels
consists of six ␣-helical segments (S1-S6) and an ionconducting pore loop between S5 and S6 (Fig. 2). A highly
conserved asparagine residue in the extracellular loop
between S5 and the pore loop is glycosylated (N380 in
murine HCN2; Fig. 2). This posttranslational channel
modification was shown to be crucial for normal cell
surface expression (278). The voltage sensor of HCN
channels is formed by a charged S4-helix carrying nine
arginine or lysine residues regularly spaced at every third
position (81, 399). Positively charged S4 segments are
found in all voltage-dependent members of the pore-loop
cation channel superfamily (436). However, inward movement of S4 charges through the plane of the cell membrane leads to opening of HCN channels while it triggers
the closure of depolarization-activated channels such as
the Kv channels (245). The molecular determinants underlying the different polarity of the gating mechanism of
HCN and depolarization-gated channels remain to be clarified. However, there is initial evidence that the loop
connecting the S4 with the S5 segment plays a crucial role
in conferring the differential response to voltage (102,
224, 314) (Fig. 2).
Recently, it was shown that the voltage-dependent
activation of the HCN channel cloned from sea urchin
sperm (spHCN) can shift between two modes depending
on the previous activity (53, 133, 246). In mode I, gating
charge movement and channel opening occur at very
negative potentials, while in mode II, both processes are
shifted to ⬎50 mV more positive potentials (133). The
transition from mode I to mode II is favored in the open
state, while the transition from mode II to mode I preferentially occurs in the closed state. The shift between
Physiol Rev • VOL
As pointed out before, Ih channels slightly select K⫹
over Na⫹ and carry an inward Na⫹ current under physiological conditions (Fig. 2). Given this particular ion selectivity profile, it came as a big surprise that the pore
loop sequence of HCN channels is closely related to that
of highly selective K⫹ channels (436). Notably, the pore
contains the glycine-tyrosine-glycine (GYG) motif that in
K⫹ channels forms the selectivity filter for K⫹ (129, 448)
(Fig. 2). Thus, based on sequence analysis, HCN channels
are expected to be selective for K⫹ and to exclude Na⫹
from permeation. Also, conduction of divalent cations is
not consistent with current models, since a ring of acidic
residues (glutamate or aspartate) that is present in the
pore of Ca2⫹-permeable channels is missing in HCN channels (343, 431, 442). Several attempts have been made,
mainly using site-directed mutagenesis approaches, to
identify residues in the pore of HCN channels that confer
the unique permeation properties of these channels (19,
135, 237, 278, 428). However, these efforts were quite
unsuccessful. A major problem is that the pore region
turned out to be extremely sensitive to mutagenesis and
that even subtle mutations can lead to nonconducting
channels. Clearly, a high-solution crystal structure will be
required to establish a conclusive model of ion permeation in HCN channels.
C. Cyclic Nucleotide-Binding Domain and C-Linker
Regulation of HCN channels by cAMP is mediated by
the proximal portion of the cytosolic COOH terminus
(443) (Fig. 2). This part of the channel contains a cyclic
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Drosophila melanogaster, and Apis mellifera (156, 159,
298). Each HCN channel subunit consists of three principal structural modules: the transmembrane core and the
cytosolic NH2-terminal and COOH-terminal domains (Fig.
2). The transmembrane core harbors the gating machinery and the ion-conducting pore while the proximal part
of the cytosolic COOH-terminal domain consisting of the
CNBD and the peptide that connects the CNBD with the
transmembrane core (the “C-linker”) confers modulation
by cyclic nucleotides (93) (Fig. 2). The transmembrane
core and the proximal COOH terminus allosterically interact with each other during channel gating and reveal a
high degree of sequence conservation within the HCN
channel family (sequence identity of ⬃80 –90% between
HCN1-4) (28, 202). In contrast, cytosolic NH2 termini and
the sequence downstream of the CNBD vary considerably
in their length and share only modest to low homology
between various HCN channels (28).
FUNCTION OF HCN CHANNELS
mains of HCN2 were virtually identical, with the only
difference that cAMP binds in its anti conformation while
cGMP binds in the syn conformation (443). The CNBD
shows a highly conserved fold consisting of an initial
␣-helix (A-helix), followed by an eight-stranded antiparallel ␤-roll (␤1-␤8), a short B-helix, and a long C-helix
(Figs. 2 and 3). The C-linker consists of six ␣-helices
(A⬘–F⬘) (Figs. 2 and 3). Four C-linker CNBDs are assembled to a complex with a fourfold axis of symmetry that is
consistent with the tetrameric architecture of pore-loop
channels. Most of the subunit-subunit interactions in the
tetramer are mediated by amino acid residues in the
C-linker. The binding pocket for cAMP is formed by a
number of residues at the interface between the ␤-roll and
the C-helix (142, 443, 446). Within the binding pocket of
HCN2, a group of seven amino acids interacts with ligand
(446) (Fig. 3).
Three of these residues are located in the ␤-roll
(R591, T592, and E582 in HCN2) and four in the C-helix
(R632, R635, I636, and K638 in HCN2). Among these, only
one key residue of the C-helix (R632) was identified to
control the efficacy by which cyclic nucleotides enhance
channel opening (446). Four residues in the C-helix (e.g.,
R632, R635, I636, and K638) contribute to the higher
selectivity of HCN2 channels for cAMP than for cGMP
(446). However, it is not clear how these residues generate cAMP selectivity. It was suggested that cAMP selectivity at least partially is not due to its preferential contacts with the protein, but rather reflects the greater
hydration energy of cGMP relative to cAMP. This could
result in a greater energetic cost for cGMP binding to the
channel (447).
The CNBD is not only an important modulatory domain in HCN channels, like in the structurally related
Eag-like K⫹ channels (18), it may also be important for
normal cell surface expression of these proteins (6, 313).
Recently, a four-amino acid motif (EEYP) in the B-helix of
HCN2 was identified that strongly promotes channel export from the endoplasmatic reticulum and targeting to
the cell membrane (287).
D. Dual Channel Gating
FIG. 3. Sequence alignment of HCN2 and spHCN COOH terminus.
Residues that have been reported by Zhou and Siegelbaum (446) to
interact with cAMP are indicated in red. Residues that have been identified by Flynn et al. (142) to control cGMP efficacy in spHCN are
indicated by arrows. Tyrosine residues that are phosphorylated by cSrc
kinase are shown in blue (Y453 in HCN2 corresponds to Y531 in HCN4).
␣-Helices are indicated as green boxes, and the eight ␤-sheets are
indicated as green arrows and numbered from 1– 8.
Physiol Rev • VOL
Unlike CNG channels, which obligatorily require
binding of cAMP to open (39, 201), HCN channels are
principally operated by voltage, i.e., membrane hyperpolarization is necessary and sufficient to activate these
channels. cAMP and cGMP can be considered as stimulatory modulators (or coagonists) of HCN channels that
do not open the channels in the absence of membrane
hyperpolarization but rather facilitate voltage-dependent
activation by shifting the voltage dependence of activation to more positive voltages (122). Electrophysiological
studies in excised patches from sinoatrial node cells in-
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nucleotide-binding domain composed of ⬃120 amino acids (CNBD) and an 80-amino acid-long C-linker region
that connects the CNBD with the S6 segment of the
channel core (Figs. 2 and 3). Evolutionarily, the CNBD is
an ancient protein domain that has been conserved across
a wide range of proteins, including bacterial transcription
factors (e.g., CAP; Ref. 419), cAMP- and cGMP-dependent
protein kinases (307, 387), cAMP-dependent guanine nucleotide exchange factors (Epac; Ref. 321), and CNG
channels (39, 201). The determination of the crystal structure of the C-linker-CNBD of HCN2 in its cAMP- and
cGMP-bound form (443) and of spHCN in its cAMP-bound
form (142) has provided important insights into the
molecular determinants of cyclic nucleotide-dependent
channel modulation. So far, high-resolution structures of
unliganded CNBDs from HCN channels are not available.
The structures of the two liganded C-linker-CNBD do-
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Physiol Rev • VOL
clic nucleotide-dependent gating (for a recent overview,
see Refs. 28, 93). Among these, cyclic allosteric models
derived from the classic Monod-Wyman-Changeux
(MWC) model developed for hemoglobin (274), currently
give the best approximation to HCN channel behavior (7,
112). In its basic form, the MWC model assumes that each
of the four subunits of the tetrameric channel is independently gated by voltage. Every time a voltage sensor
switches to the activated state, the probability for channel
opening increases. The opening/closing reactions occur
allosterically and involve concerted transitions of all four
subunits. This transition occurs if the channel is unliganded, partially liganded, or fully liganded and is energetically stabilized by a constant amount for each cAMP
bound. The model further assumes that cAMP has a
higher binding affinity to open than to closed channels (7,
112, 413). The model reproduces the characteristic features of HCN channel gating like the voltage dependence
and the activation by cyclic nucleotides. In addition, several kinetic features are well described by the model,
including the delay in current activation and deactivation.
However, in its original form, the model also has several
limitations. For example, there is evidence (86) that the
final allosteric open/close transition in HCN2 channels is
not voltage dependent as suggested previously (7). In
addition, the mode shift and the voltage hysteresis of HCN
channels cannot be explained by simple MWC models. To
solve these issues, several extensions of the cyclic allosteric model have been proposed among which the dimerof-dimers model (396), the circular four-state model by
Männikkö et al. (246), and an extension of the MWC
model made by Zhou and Siegelbaum (446) are the most
recent ones. Despite the progress that clearly has been
made in mathematically modeling HCN channel gating, it
is obvious that these approaches have to be accompanied
by new high-resolution structures of HCN channels in
closed and open state(s) to achieve a full understanding
of channel behavior.
E. Functional Differences Between HCN
Channel Types
All four mammalian HCN channel subtypes have
been expressed in heterologous expression systems and
shown to induce currents that display the principal biophysical properties of native Ih (153, 192, 211, 227, 228,
333, 341, 400). However, the individual HCN currents
quantitatively differ from each other with respect to their
respective activation time constants (␶), their steady-state
voltage dependence, and the extent of cAMP-dependent
modulation. Before we will discuss these issues in more
detail, it is important to recall that at least some of the
reported differences may not reflect the intrinsic biophysical diversity of individual HCN channel types. Instead,
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dicated that the modulatory effect of cAMP is due to its
direct binding to the channel rather than being conferred
by protein phosphorylation (122). The identification of
CNBDs in the primary sequence of HCN channels strongly
supported this model and suggested that mechanistically
there were probably similarities between the action of
cyclic nucleotides in HCN and CNG channels. As discussed in the previous section, crystal structure data
along with site-directed mutagenesis and molecular dynamics simulations allowed to identify structural determinants controlling cAMP and cGMP binding (142, 412,
414, 443, 446, 447).
There is now good evidence that “disinhibition” is the
fundamental principle underlying the cAMP-dependent
modulation of HCN channels. The C-linker-CNBD is an
autoinhibitory domain that in the absence of cAMP lowers
open probability. Binding of cAMP increases channel activity by removing tonic channel inhibition that is conferred by this domain (412, 414). The exact sequence of
molecular events leading from initial cyclic nucleotide
binding to facilitation of voltage-gated channel opening is
still an unsolved issue. Addressing this problem is complicated by the fact that in the available crystal structures
the C-linker seems to be caught in the resting conformation (i.e., the conformation that is occupied in the absence
of cAMP) while the CNBD is in its active (i.e., cAMPbound) conformation (93, 94, 198). According to a recent
model (93), in the absence of cAMP, the C-linker is
thought to be in a “compact” conformation that produces
an inhibitory effect on channel opening. Binding of cAMP
in turn induces a conformational change in the CNBD
involving the C-helix. The conformational change in the
C-helix is then coupled to the C-linker that occupies a
more “loose” conformation leading to an alteration of the
intersubunit interface between helices of neighboring
subunits. The resulting change in the quaternary conformation removes the inhibition of the COOH terminus and
destabilizes the closed state, thus promoting the opening
of the channel. One would expect that the described
activation process comes along with large translocations
of subdomains in the C-linker-CNBD. However, recent
data suggest that this may not be necessarily the case.
Fluorescent resonance energy transfer (FRET) measurements in a CNG channel suggested that movements within
the C-linker are probably subtle, involving only limited
rearrangements (386).
The finding that cAMP facilitates activation of HCN
channels by affecting their voltage dependence suggests
that the gating mechanism operated by hyperpolarization
and the one operated by cAMP are closely related to each
other, if not identical. In other words, HCN channels
behave in the presence of a given cAMP concentration
simply as if they were experiencing a stronger voltage
drop. Several kinetic models have been proposed to describe the tight interconnection between voltage- and cy-
FUNCTION OF HCN CHANNELS
Physiol Rev • VOL
unpublished observations). V0.5 of HCN3 ranges between
⫺80 and ⫺95 mV. Activation constants of 250 – 400 ms at
⫺140 mV place the kinetics of HCN3 in between those of
HCN2 and HCN4 (271, 372). Remarkably and unlike in all
other HCN channels, cyclic nucleotides do not induce a
positive shift of V0.5 in HCN3. Human HCN3 was found to
be insensitive to cAMP or cGMP (372), while the murine
channel seems to be even slightly inhibited by cyclic
nucleotides (shift of V0.5 by ⫺5 mV) (271). The structural
determinants underlying this unexpected behavior are
unclear. Interestingly, when the CNBD of HCN4 is replaced by the corresponding domain of HCN3, cAMP
sensitivity is fully maintained, suggesting that the CNBD
of HCN3 is principally able to bind cAMP and to mediate
cAMP-dependent gating (372). Thus, within the HCN3
channel, the CNBD may be functionally silenced by a
structural change in channel domains that communicate
cAMP binding with channel gating. A similar mechanism
may explain the lack of cAMP sensitivity in members of
the Erg K⫹ channel family that also contain a CNBD (54,
184, 443).
The properties of an HCN channel cloned from sea
urchin sperm (spHCN) differ from mammalian HCN channels (153). In the absence of cAMP, the spHCN current
develops with a sigmoidal time course, and then decays to
a much lower degree. In contrast, in the presence of
saturating cAMP, hyperpolarizing voltage steps produce
large currents of sigmoidal waveform lacking inactivation, which are virtually identical to those of mammalian
HCN currents. The large augmentation of spHCN channels by cAMP is produced by removal of inactivation with
little or no shift in steady-state voltage dependence (142,
153, 347). Mutation of a conserved phenylalanine residue
in the S6-helix of spHCN to a leucine (F459L; F431 in
HCN2) is sufficient to convert spHCN to a channel that
behaves very much like HCN2, i.e., it shows no inactivation and its voltage dependence is positively shifted by
cAMP (347). The ability to accomplish this conversion
with a single point mutation in S6 argues strongly that the
two channel types use the same fundamental mechanisms
for gating and that the different phenotypes represent
differences in the precise relationship between the energetics and kinetics of the different gating processes.
There is one other property in which spHCN differs
from its mammalian homologs. In HCN1-4, saturating concentration of either cAMP or cGMP induces the same
maximal current, i.e., both cyclic nucleotides are full agonists of these channels (202). In contrast, cGMP behaves
as a partial agonist on the spHCN channel, activating only
⬃50% of the maximal current obtained at saturating cAMP
concentrations (202). Using a combination of X-ray crystallography and electrophysiology, Flynn et al. (142) revealed that the efficacy of cyclic nucleotides in channel
activation is controlled by complex interactions of these
ligands with residues in the ␤-roll and the C-helix of the
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these differences rather can be attributed to the fact that
measurements of Ih are very sensitive to experimental
parameters and that the same HCN channel may be embedded in quite diverse modulatory networks, depending
on the expression system or cell type (111, 118). The
intrinsic sensitivity to external and internal factors also
makes it difficult to unambiguously match the Ih characterized in a native type with Ih currents induced by a heterologously expressed HCN channel isoform. Assembly of different HCN channel subtypes to heterotetrameric channel
complexes may further increase heterogeneity of Ih channels.
Within the HCN channel family, HCN1 is the subtype
with the fastest kinetics (191, 331, 372). Like in native Ih
activation kinetics of HCN1 is strongly voltage dependent,
with ␶act values ranging from 30 to 300 ms at ⫺140 to ⫺95
mV (191, 331, 372). HCN1 is also the HCN channel type
with the most positive V0.5 value (range ⫺70 to ⫺90 mV)
(8, 28, 372). On the other hand, compared with HCN2 and
HCN4, HCN1 reveals only a weak shift of the activation
curve in the presence of saturating cAMP concentrations
(⫹2 to ⫹7 mV) (8, 372, 409, 412, 414). Taken together,
these findings suggest that HCN1 activation is energetically favored compared with the other channels. One
reason for this finding is that the tonic channel inhibition
produced by the C-linker-CNBD is weaker in HCN1 than
in HCN2 or HCN4. Indeed, deletion of the CNBD in HCN1
or HCN2 which is equivalent with a total removal of tonic
inhibition resulted in channels with almost identical V0.5
values (412). However, HCN1 lacking a CNBD still activates five- to sixfold faster than HCN2 lacking this domain. This shows that gating differences between both
channels are not solely determined by the different degree
of tonic inhibition conferred by the CNBD but that the
opening of the core HCN2 channel is also inherently
slower than that of HCN1 (412).
While HCN1 is the fastest member within the HCN
channel family, HCN4 is by far the channel with the
slowest opening kinetics (192, 228, 341). ␶act values for
this channel range between a few hundred milliseconds at
strongly hyperpolarized voltages (⫺140 mV) up to many
seconds at normal resting potential (⫺70 mV) (192, 228,
341). HCN2 occupies an intermediate position with ␶act
ranging from 150 ms to 1 s (228, 372). V0.5 values between
⫺70 and ⫺100 mV have been reported for HCN2 and
HCN4 (8, 28, 372). Both channels also have in common
that their steady-state activation curves are very sensitive
to cAMP with shifts of V0.5 of 10 –25 mV (8, 192, 228, 277,
341, 409, 412, 414, 443).
So far, electrophysiological measurements of HCN3mediated currents have been only reported by a few
groups (80, 271, 372). A major obstacle to a full characterization of this particular channel type is that the HCN3
protein tends to accumulate in intracellular compartments and reveals only weak cell surface expression (170;
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IV. REGULATION OF HCN CHANNELS
HCN channels are tightly regulated by interacting
proteins as well as by low molecular factors (e.g., protons,
chloride ions) in the cytosol and the extracellular space
(Fig. 4). These molecules control the functional properties of the channels in the plasma membrane, regulate
their cell surface expression (i.e., the number of functional channels in the membrane), and control their targeting to defined cellular compartments. In the following
section, we give an overview on HCN channel regulators
that have been identified in the last couple of years.
FIG. 4. Regulation of HCN channels by interacting proteins and low
molecular factors. Only one subunit of the tetrameric channel complex
is shown. Proteins interacting with HCN channels and low molecular
factors regulating HCN channels are shown. KCR1, MiRP1, Filamin A,
TRIP8b, S-SCAM, Tamalin, Mint2, cSrc, Cl⫺, and H⫹ interact with different channel portions as indicated. The tyrosine phosphorylation site
in the C-linker is shown as a circle with a “P” in it.
Physiol Rev • VOL
A. Regulation by Acidic Lipids
It has been long known that native Ih as well as
heterologously expressed HCN currents display a rapid
rundown when measured in excised patches or during
prolonged whole cell recordings (43, 85, 118, 119, 122,
227). This effect is caused by an ⬃30 to 50 mV hyperpolarizing shift of the activation curve. It was speculated
that washout or depletion of cAMP may account for up to
20 mV of this shift; however, the mechanistic basis of the
rest of the voltage shift was unclear (85, 119). Recently,
two groups provided convincing evidence that phosphatidylinositol 4,5-bisphosphate (PIP2) is probably the missing factor that underlies the so far unexplained gap in the
voltage shift (308, 449).
PIP2 is not a novel entry in the list of ion channel
regulators but has been found to control numerous ion
transporters and ion channels including many K⫹ channels, TRP channels, and Ca2⫹ channels (for recent reviews, see Refs. 150, 378). In HCN channels, PIP2 acts as
an allosteric activator from the intracellular site that facilitates channel activation by shifting V0.5 ⬃20 mV toward more positive potentials. Importantly, this action is
independent of the presence of cyclic nucleotides. As a
result, PIP2 adjusts HCN channel opening to a voltage
range relevant for the physiological role of Ih channels.
PIP2-mediated regulation of HCN channels may be of
physiological significance for the function in neuronal
circuits, as enzymatic degradation of phospholipids reduces channel activation and slows down firing frequency
of neurons. Different levels of PIP2 may also contribute to
the profound variations in the half-maximal activation
voltages of Ih in cardiac cells of different developmental
stage or distinct regional distribution (77, 316, 325). The
molecular determinants conferring the effect of PIP2 on
HCN channel gating are not yet known. Most likely, HCN
channels are activated by an electrostatic interaction between the negatively charged head groups of phosphoinositides and the channel protein. A similar mechanism for
PIP2 modulation of gating was identified in voltage-gated
and inwardly rectifying K⫹ channels (30, 186, 297, 349). It
was proposed that the interaction between PIP2 and Ih
channels relieves an inhibition of channel opening conferred by an inhibitory channel domain. This “PIP2 responsive” domain is clearly different from the CNBD because the PIP2 effect is still present in channels lacking
the CNBD.
There is recent evidence that in addition to PIP2
other acidic lipids may also serve as allosteric modulators
of HCN channels. Fogle et al. (143) showed that phosphatidic acid and arachidonic acid, which are products of
diacylglycerol kinase/phospholipase A2 signaling pathways, directly facilitate HCN channel gating by shifting
the V0.5 to more positive values (shift of 5–10 mV in
HCN2). The molecular mechanism of this facilitation re-
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CNBD. Replacement of a valine in the ␤-sheet to threonine (V621T) in conjunction with another replacement in
the C-helix (I665D) (Fig. 3) is sufficient to convert cGMP
into a full agonist of spHCN. The V621T mutation probably leads to binding of cGMP in the syn conformation
(like in HCN2), while the aspartate in the C-helix is necessary to stabilize the movement of the C-helix after
primary cGMP binding, a process which is coupled to the
opening conformational change in the channel pore. All
four mammalian HCN channels carry a threonine residue
at the position equvialent to V621 in spHCN, which may
explain why cGMP is a full agonist of these channels.
Interestingly, however, mammalian channels also harbor
an isoleucine at the position equivalent to I665 in spHCN.
Flynn et al. (142) speculated that in mammalian HCN
channels the opening allosteric conformational change
may be less energetically costly than in spHCN. Therefore, unlike in spHCN, in mammalian channels an interaction with an aspartic acid residue in the C-helix may not
be required to promote full activation.
FUNCTION OF HCN CHANNELS
mains to be elucidated, but there is initial evidence that it
involves direct binding of both metabolites to the channel
and is independent of cAMP pathways. It was speculated
that the regulation of the voltage dependence of Ih by
acidic lipids may be involved in the fine tuning of subthreshold properties of excitable cells (143).
B. Regulation by Protons
C. Regulation by Chloride
Although Ih is a pure cationic current, its conductance is affected by the concentration of small anions,
notably by Cl⫺ (144, 411). Frace et al. (144) showed that
Physiol Rev • VOL
the amplitude of rabbit sinoatrial Ih decreased when extracellular Cl⫺ was replaced by larger anions such as
aspartate. The molecular basis of the regulation by external Cl⫺ was studied in heterologously expressed HCN
channels (411). The effect of Cl⫺ was found to be pronounced for HCN2 and HCN4, while it is rather weak for
HCN1 (411). A single amino acid residue in the pore
region was identified as molecular determinant of extracellular Cl⫺ sensitivity (411) (Fig. 4). Channels with high
Cl⫺ sensitivity (HCN2 and HCN4) carry an arginine residue at this position (R405 in HCN2; R483 in HCN4), while
HCN1 carries an alanine (A352). The regulation of HCN
channels by Cl⫺ is probably relevant for heart (patho)
physiology. A reduction of the amplitude of sinoatrial Ih
could be involved in the generation of arrhythmias observed in hypochloremia.
Cl⫺ regulates HCN channel function also from the
intracellular site. In a recent study, it was shown that
intracellular Cl⫺ acts as a physiological suppressor of the
instantaneous component of Ih (IINS) (272). An increase
of intracellular Cl⫺ from physiological concentrations (10
mM) to high concentrations (140 mM) almost completely
abolished IINS while it had no significant effect on the
steady-state component of Ih. The physiological relevance
of this regulation remains to be determined.
D. Regulation by Src Kinase-Mediated
Tyrosine Phosphorylation
On the basis of experiments with pharmacological
blockers, it was assumed that cardiac Ih is regulated by
tyrosine kinases of the Src family (426). This hypothesis
was corroborated by yeast two-hybrid screens that provided evidence for direct interaction between Src and
HCN1 (332), as well as between Src and HCN2 (450).
Direct binding of Src to HCN2 (450) and to HCN4 (13) was
also shown by coimmunoprecipitation in heterologous
expression systems and native tissue.
Mapping experiments in HCN2 revealed that Src
binds via its SH3 domain to the C-linker-CNBD and phosphorylates the channel at this domain (450). As a consequence, the activation of the channel is accelerated. Conversely, inhibition of Src by pharmacological agents or
cotransfection of a dominant-negative Src mutant slows
down channel kinetics probably via dephosphorylation by
not yet defined cellular phosphatases. The residue conferring modulation by Src (Y476) has been identified in
HCN2 by mass spectrometry. The residue is localized in
the B⬘-helix of the C-linker (Figs. 3 and 4) and is conserved in all HCN channel isoforms, suggesting that regulation by Src may be a commonality of the HCN channel
class. In agreement with this notion, replacement of Y476
by phenylalanine in either HCN2 or HCN4 yields to channels that are no longer regulated by Src. A recent study
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The activity of HCN channels depends on both intracellular (281, 451) and extracellular (369) concentrations
of protons. Intracellular protons shift the voltage dependence of channel activation to more hyperpolarized potentials and slow down the speed of channel opening. In
the murine HCN2, a protonable histidine residue (His321)
localized at the boundary between the voltage-sensing
S4-helix and the cytoplasmatic S4-S5-linker has been identified to confer intracellular pH (pHi) sensitivity (451)
(Fig. 4). At acidic (pHi 6.0) and alkaline pHi (pHi 9.0), the
midpoint potential of HCN2 activation is shifted by ⬃10
mV to more hyperpolarized and depolarized potentials,
respectively, compared with physiological pHi (pH 7.4)
(451). The modulation of HCN channels by intracellular
protons may have an important physiological impact on
the modulation of HCN channel activity in the brain (281),
for example, for the regulation of thalamic oscillations
and the respiratory frequency. The protective action of
carbonic anhydrase inhibitors in generalized seizures has
been attributed to the high sensitivity of HCN channels to
pHi (282). Inhibition of the carbonic anhydrase causes an
increase in pHi and augments Ih in thalamocortical neurons. As a result, these neurons are depolarized, and their
engagement in synchronized paroxysmal discharges is
reduced. Inhibition of HCN channels by intracellular acidosis could also be pathophysiologically relevant during
cardiac ischemia and heart failure (70, 451).
Acidic extracellular pH (pHe ⬍5.0) was found to
activate Ih in a subset of rat taste cells. It was assumed
that this mechanism contributes to sour taste transduction in these cells (369). Heterologous expression of the
two HCN channel isoforms present in taste cells, HCN1
and HCN4, verified the findings from native cells. Acidification to pH 3.9 profoundly sped up channel kinetics,
shifted the threshold of activation by up to ⫹50 mV, and
induced a depolarizing shift of V0.5 by up to ⫹35 mV with
respect to neutral pH. The structural determinants in the
HCN1 or HCN4 protein underlying regulation by external
pH have not been reported so far.
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E. Regulation by p38-Mitogen-Activated
Protein Kinase
1. Regulation by MiRP1
The MinK-related protein MiRP1 (encoded by the
gene KCNE2) was reported to interact with several HCN
channel types (101, 317, 438). MiRP1 is a member of a
family of single transmembrane-spanning proteins and is
an established auxiliary subunit of the HERG delayed
rectifier K⫹ channel (2, 390, 445). MiRP1 was found to
interact with HCN2 in rat neonatal cardiomyocytes and
canine sinoatrial node tissue (317). Overexpression studies in Xenopus oocytes (438) and neonatal rat cardiomyoctes (317) showed that MiRP1 increases current densities and accelerates activation kinetics of HCN2. In
contrast, MiRP1 did not affect voltage dependence of activation. In overexpression systems, MiRP1 was also found
to interact with HCN1 (438) and HCN4 (101). MiRP1
increased current densities of HCN4, but unlike in coexpression experiments with HCN1 or HCN2, it slowed
down kinetics and induced a negative shift of the activation curve of this channel (101). Collectively, these results
may suggest that MiRP1 is an auxiliary subunit of HCN
channels. However, extreme care should be taken before
a final decision on this issue is made. It will be necessary
to verify the interaction between MiRP1 and HCN channels including its functional implications in native tissues.
Furthermore, the specificity of antibodies used for immunoprecipitations must be confirmed in adequate knockout
models.
2. Regulation by KCR1
In addition to tyrosine kinases, HCN channels are
also regulated by the serine/threonine kinase, p38-mitogen-activated protein (MAP) kinase (310). In hippocampal
pyramidal neurons, activation of p38-MAP kinase significantly shifts the voltage-dependent activation towards
more positive potentials. This regulation may functionally
affect temporal summation and neuronal excitability. It is
not clear whether p38 induces the observed effects by
direct phosphorylation of the HCN channel protein or by
phosphorylation of another protein interacting with these
channels.
F. Transmembrane and Cytosolic Proteins
Interacting With HCN Channels
There is growing evidence that ion channels usually
are macromolecular protein complexes that in addition to
the principal pore-forming subunit contain auxiliary proteins that are required for the fine-tuning of electrophysiological properties, the functional coupling to signaling
pathways, and trafficking to specific cellular compartments. HCN channels are no exception from this rule. In
the last couple of years, several proteins interacting with
HCN channels have been identified (Fig. 4).
Physiol Rev • VOL
Very recently, another transmembrane protein
(KCR1) was reported to interact with HCN2 and native
cardiac Ih channels (268). KCR1 is a plasma membraneassociated protein with 12 putative transmembrane regions that like MiRP1 can associate with the HERG K⫹
channel (183, 213). Upon overexpression in CHO cells,
KCR1 reduces HCN2 current densities and affects singlechannel current parameters of this channel. Overexpression in rat cardiomyocytes also reduced current densities
of native Ih and suppressed spontaneous action potential
activity of these cells. From these experiments, it was
concluded that KCR1 is an inhibitory auxiliary subunit of
HCN channels and serves as a regulator of cardiac automaticity. As discussed for MiRP1, further experiments
will be required to verify this hypothesis.
3. Regulation by neuronal scaffold proteins
Several scaffold proteins interact with the COOH
terminus of HCN channels. These proteins were mainly
identified in neurons and may regulate channel targeting
to distinct subcellular compartments (e.g., dendrites or
synapses). A brain-specific protein termed TRIP8b (TPRcontaining Rab8b interacting protein) interacts through a
conserved tripeptide sequence in the COOH terminus of
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identified an additional tyrosine residue in HCN4 (Y531 in
A⬘-helix of the C-linker) that may also be involved in
Src-mediated channel regulation (223) (Fig. 3). In agreement with findings of Arinburg et al. (13), this study also
found that Src not only speeds up channel kinetics of
HCN4 but also induces a ⫹10 to 15 mV shift of the V0.5. At
this point, one can only speculate on the mechanism by
which Src-mediated phosphorylation facilitates channel
opening. It is tempting to assume that the presence of the
bulky negatively charged phosphate group in the B⬘-helix
of the C-linker destabilizes the interaction between neighboring C-linkers within the HCN channel tetramer. As a
consequence, the C-linker tetramer would occupy a less
compact conformation and would impose a weaker inhibitory impact on the channel gate as if present in the more
compact dephosphorylated conformation.
Regulation of Ih by tyrosine phosphorylation through
Src kinase has been demonstrated under physiological
conditions in sinoatrial pacemaker cells in the murine
(450) and rat heart (13, 439) as well as in neurons (450).
These results support the notion that the control of the
phosphorylation status is indeed an important regulatory
mechanism to adjust the properties of Ih to the specific
requirements of different types of neurons and heart cells.
FUNCTION OF HCN CHANNELS
4. Regulation by filamin A
HCN1, but not HCN2 or HCN4, was found to bind
filamin A via a 22-amino acid sequence downstream of the
CNBD (161). Filamin A is a putative cytoplasmic scaffold
protein that binds actin and thereby links transmembrane
proteins, among those are the K⫹ channels Kv4.2 and Kir
2.1, to the actin cytoskeleton (305, 329). On the basis of
heterologous overexpression in filamin A-expressing and
filamin A-deficient cell lines, it was proposed that filamin
A causes clustering and slows down activation and deactivation kinetics of HCN1 (161).
5. Regulation by caveolin-3
Ih channels localize to membrane lipid rafts in sinoatrial myocytes and in HEK293 cells expressing HCN4, the
major HCN channel isoform contributing to native sinoatrial Ih (26). Coimmunoprecipitation experiments indicate an interaction between HCN4 and caveolin-3, which
is a marker protein for so-called caveolae (27). Caveolae
represent a morphologically distinct type of lipid rafts. In
cardiomyocytes and pacemaker cells, several elements of
the ␤-adrenergic signaling pathway including ␤2-adrenoceptors that regulate HCN channel activity are localized in
caveolae. It was proposed that clustering of HCN4 in
caveolae is essential for normal function and regulation of
this channel. Indeed, disruption of lipid rafts by cholesterol depletion caused a redistribution of HCN channels
within the membrane and modified their kinetic properties (26).
V. TISSUE EXPRESSION OF HCN CHANNELS
Throughout the nervous system and the heart, the
hyperpolarization-activated current Ih as well as HCN
Physiol Rev • VOL
channel expression has been identified. In addition, Ih
was also found in some other tissues such as B cells of
pancreatic Langerhans islets (134), enteric (37, 179, 429),
lymphatic smooth muscle (254), uterine smooth muscle
(296), smooth muscle cells of the bladder (163) or portal
vein (164), testis (341), and the enteric nervous system
(427). However, HCN channel transcripts have only been
reported in some of these tissues or cell types (134, 164,
341, 427). Moreover, in most of these tissues, HCN channels are expressed at low levels, making it very difficult to
define the physiological role of Ih, if there is any. Another
problem is that in several cell types, HCN channels have
been detected only on the mRNA level, e.g., using RT-PCR
or in situ hybridization, while protein expression has not
been shown so far. Given these drawbacks, in this review
we will concentrate on HCN channels in heart and nervous system. We will also give only a rough outline of
tissue distribution of HCN1-4. Readers who are interested
in a more complete description of the expression pattern
are referred to a number of excellent studies that have
reported on this issue in great detail (126, 226, 275, 276,
294, 331, 332, 344, 345).
Briefly, in the brain, HCN1 is expressed in the neocortex, hippocampus, cerebellar cortex, and brain stem
(270, 275, 294, 331, 332). In addition, HCN1 expression
was reported in the spinal cord (270). HCN2 is distributed
nearly ubiquitously throughout most brain regions, with
the highest expression in the thalamus and brain stem
nuclei (275, 294, 331). In contrast, HCN3 is expressed at
very low levels in the central nervous system. Moderate to
high expression has only been detected in the olfactory
bulb and in some hypothalamic nuclei (275, 294). HCN4 is
strongly expressed in some parts of the brain, e.g., in
various thalamic nuclei and in the mitral cell layer of the
olfactory bulb (275, 294, 331). In other brain regions, the
expression of HCN4 is much lower. All four HCN channel
isoforms are expressed differentially in the retina (69, 193,
209, 276, 280). In the peripheral nervous system, all four
HCN subtypes have been reported in the dorsal root
ganglion, with HCN1 being the most abundant one (80).
All four HCN channel isoforms have been detected
in the heart. The expression levels of these isoforms strongly
depend on the cardiac region and, in addition, seem to
vary between species. In the sinoatrial node, in all species
analyzed so far (e.g., human, rabbit, guinea pig, mouse,
and dog), HCN4 is the major isoform accounting for ⬃80%
of Ih (276, 344, 388). The remaining fraction of this current
is species dependent. In rabbits, this fraction of Ih is
dominated by HCN1 (344), whereas in mice and humans,
HCN2 accounts for this fraction (226, 388). In addition, in
the mouse sinoatrial node, a significant amount of HCN1
mRNA was identified (249).
In other parts of the cardiac conduction system,
HCN4 is also the major isoform, with expression in the
atrioventricular node (126) and the Purkinje fibers (345).
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HCN channels (335). TRIP8b is thought to play an important role in the trafficking of vesicles to their final targets
(444). TRIP8b colocalizes with HCN1 in dendrites of cortical and hippocampal pyramidal cells. Functional coexpression of TRIP8b with HCN protein, either in native
cells or heterologous systems, results in a strong downregulation of HCN channels in the plasma membrane. On
the basis of these experiments and the analysis of HCN1
knockout mice, it was speculated that TRIP8 is involved
in the generation of somatodendritic HCN1 channel gradients in cortical layer V pyramidal neurons (335). HCN2,
but not other HCN channel types, interacts with the neuronal scaffold protein tamalin, mostly through a PDZ-like
binding domain (205). In addition, HCN2 interacts with
the scaffold proteins Mint2 and S-SCAM via distinct protein-binding domains at the COOH-terminal tail. In COS-7
cells, HCN2 levels were increased upon coexpression
with Mint2, suggesting that this protein is a positive regulator of cell surface expression of HCN channels (205).
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HCN1 expression has been reported in the atrioventricular node (249). For HCN3, either no expression (276) or
only very low expression levels have been reported in the
conduction system (249). HCN channels are also present
in atrial and ventricular myocytes. In these cells, HCN2 is
the dominant isoform displaying a rather ubiquitous distribution. Transcripts of HCN1, HCN3, and HCN4 have
also been detected in heart muscle (344, 375). In general,
expression levels of HCN channels are low in normal
heart muscle compared with cells of the conduction system. However, upregulation of HCN channels may occur
during heart diseases (72, 73, 139, 374) (see also sect. VIII).
A. Principles
Ih displays a set of unique biophysical features that is
essential to control excitability and electrical responsiveness of cells. With respect to these physiological roles,
two properties of Ih are of particular relevance. First, Ih
channels are partially open at rest. As a consequence, the
channels contribute to the setting of the membrane potential in many cells (98, 125, 226, 230, 266, 291–293, 300).
Second, Ih channels possess an inherent negative-feedback property because they can counteract both membrane hyperpolarization and depolarization by producing
a depolarizing inward current (due to Ih channel activation) or by facilitating hyperpolarization (resulting from Ih
channel deactivation), respectively. Thus Ih can actively
dampen both inhibitory and excitatory stimuli arriving at
the cell membrane and thus helps to stabilize the membrane potential (31, 253, 291, 354). We will now discuss in
some more detail these two key properties of Ih which
together provide the Leitmotiv for the specific functions
of this current in various physiological settings.
1. Activation of Ih at the resting membrane potential
In sinoatrial pacemaker cells of the heart as well as in
many neurons of the central nervous system, Ih channels
are constitutively open at voltages near the resting membrane potential (12, 226, 291–293, 326), pass a depolarizing noninactivating inward current, and hence set the
membrane potential to more depolarized voltages. In addition, constitutively open Ih channels per se stabilize the
resting membrane potential by lowering the membrane
resistance (Rm), which is defined as the ratio of a voltage
change and the required current (226, 291–293). Therefore, in the presence of Ih, any given input current evokes
a smaller change in membrane potential than in the absence of Ih. Constitutively open Ih channels seem to function as a slow “voltage clamp” (291), tending to stabilize
Physiol Rev • VOL
2. Negative-feedback properties of Ih
As mentioned above, voltage-dependent activation
and deactivation of Ih actively oppose deviations of the
membrane potential away from the resting membrane
potential (31, 253, 291, 354). This property is a consequence of the unusual relation between the activation
curve and the reversal potential of Ih. In contrast to
voltage-gated Ca2⫹ and Na⫹ channels (Fig. 5E), the reversal potential of Ih falls close to the base of its activation
curve (189) (Fig. 5E). If membrane hyperpolarization increases the fraction of open Ih channels, the depolarizing
inward current drives the membrane potential back towards the reversal potential of Ih and thereby to the initial
value close to the resting membrane potential (Fig. 5D).
This characteristic effect is called “depolarizing voltage
sag” (300, 324). Conversely, a depolarizing input causes
deactivation of the Ih that was active at rest. The loss of
a tonic depolarizing current causes a hyperpolarization
(“hyperpolarizing voltage sag”; Refs. 300, 324) again returning membrane potential toward rest (Fig. 5D, right).
Ih is involved in the control of a variety of basic and
complex neuronal functions, including dendritic integration, long-term potentiation, learning, and neuronal pacemaking, just to mention a few of them. In the following
sections we will review the role of Ih in these diverse
functions in more detail. Wherever possible, we will discuss the role of Ih in the light of recent findings obtained
from the analysis from HCN channel-deficient mouse
lines. These studies have allowed defining the specific
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VI. PHYSIOLOGICAL ROLES OF HCN
CHANNELS IN NEURONS
the membrane potential by opposing depolarizing or hyperpolarizing inputs (185). This effect suppresses lowfrequency fluctuations in membrane potential (291). In
dendrites, constitutively open Ih channels influence the
passive propagation of excitatory postsynaptic potentials
(EPSPs) (239 –241). In the absence of active voltage-dependent ion channels, the passive properties of a dendrite
(Fig. 5A) attenuate the amplitude and slow down the
kinetics of an EPSP (Fig. 5B) as it spreads from its site of
origin in the dendrites to the soma (Fig. 5, A–C). This
process is comparable to a wave traveling across water.
Just as a wave moving across water widens and diminishes in height over distance, EPSP signals can degrade
with distance along the neuronal membrane (Fig. 5B). The
presence of Ih in dendrites lowers Rm and thereby further
increases the amplitude attenuation of EPSPs. In addition, Ih counteracts kinetic filtering of propagating EPSPs
also by lowering Rm. Therefore, the EPSP upstroke and
decline are faster (Fig. 5C).
In the absence of Ih (e.g., after blockade of Ih by the
specific Ih blocker ZD7288 or after genetic deletion), somatic EPSPs rise and decay more slowly if they are
generated in distal dendrites compared with proximal
dendrites (239 –241, 423) (Fig. 5C).
FUNCTION OF HCN CHANNELS
861
contribution of distinct HCN channel subtypes to different physiological functions.
B. Role of Ih in Dendritic Integration
Dendritic integration is a process that is crucial for
signal processing in most neurons. Typical isolated EPSPs
are too small to bridge the gap between the resting membrane potential and the action potential threshold. Therefore, generation of action potentials at the soma usually
requires the integration of multiple synaptic inputs. To
ensure high fidelity of information processing, integration
of EPSP must be tightly controlled both in space and time.
Dendritic integration has been extensively studied in CA1
hippocampal and neocortical pyramidal neurons (241).
There is good evidence that in these neurons Ih and most
notably the Ih component encoded by HCN1 has an important role in regulating dendritic integration (239 –241,
394, 423). The way incoming EPSPs are summed up in
time largely depends on kinetic filtering by passive cable
properties of the dendrites. As mentioned above, denPhysiol Rev • VOL
dritic filtering slows down the time course of EPSPs
resulting in somatic EPSPs that rise and decay more
slowly if they are generated in distal dendrites compared
with proximal dendrites (Fig. 5, B and C). As a consequence of filtering, one would expect that repetitive
EPSPs arising from more distal synapses should summate
at the soma to a greater extent and over a longer time
course than EPSPs generated in more proximal dendrites.
However, in many neurons, including CA1 (239, 240) and
neocortical layer 5 pyramidal cells (38, 377, 423), this
localization dependence of temporal summation is not
observed. This discrepancy is probably explained by a
gradient of Ih whose density rises progressively by more
than sixfold with distance from the soma (225, 239 –241,
423) (Fig. 6). This gradient efficiently counteracts dendritic filtering. During the rising phase of an EPSP, Ih
channels rapidly deactivate. Turning off the inward current carried by Ih leaves an effective net outward current
that hyperpolarizes the plasma membrane and accelerates the decay of each EPSP (Fig. 5, B and C). Because the
density of Ih is higher in distal dendrites, distal EPSPs
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FIG. 5. Basic properties of Ih. A: equivalent electrical circuit of an idealized cylindrical segment of a dendrite. Three electrical components
are responsible for the passive features of a dendrite: the specific membrane resistance (Rm), the specific membrane capacitance (Cm), and the
intracellular resistance (Ri). For simplicity, the resistance of the extracellular space has been neglected. B: the passive properties of a dendrite
attenuate the amplitude (arrow) of an excitatory postsynaptic potential (EPSP) as it spreads from dendrites to the soma (EPSP at the distal synapse,
black line; the same EPSP after propagation to the soma, red line). C: EPSP recorded at the soma after propagation from a distal dendrite before
and after blockade of Ih with ZD 7288. In the presence of Ih (black line), the EPSP rise and decay faster than after blockade of Ih with ZD 7288 (red
line). D: Ih actively opposes changes in membrane voltage. Cartoon of a current-clamp experiment in a neuron. In the presence of Ih, a
hyperpolarizing current step (⫺200 pA) induces a depolarizing voltage sag (arrow, left panel) and a depolarizing current step (200 pA) induces a
hyperpolarizing voltage sag (arrow, right panel). Blockade of Ih by Cs⫹ eliminates the sag (red traces). E: the reversal potential (Vrev) of Ih falls near
the base of its activation curve. Therefore, Ih actively opposes changes in membrane voltage. Partial activation of the current drives the membrane
potential toward the reversal potential on the base of the activation curve (left, arrow in the activation curve) and thus is self limiting and stabilizing
for the membrane potential. In contrast, voltage-gated currents, whose reversal potential falls near the top of their activation curve (e.g., INa; right),
are destabilizing and self-regenerative. Partial activation drives the membrane potential towards the reversal potential at the top of the activation
curve (right, arrow in the activation curve).
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FIG. 6. Proposed roles of Ih in various neuronal functions. A: the somato-dendritic gradient of Ih effectively counteracts kinetic filtering by
dendrites and normalizes the localization dependence of temporal summation. Summation of EPSPs from proximal (black trace) and distal (gray
trace) dendrites recorded at the soma after propagation. The voltage recordings are shown before (top panel) and after (bottom panel) inhibition
of Ih by the selective Ih blocker ZD 7288. B: hypothetical model of ␣2-adrenoceptor-cAMP-HCN regulation in prefrontal cortex neuron networks
involved in the control of spatial working memory. For details, see text. ␣2-R, ␣2-adrenoceptor; Gi, Gi protein. C: a model for the role of HCN1
channels in motor learning through history-independent integration of inputs by cerebellar Purkinje cells. Input-output function of wild-type (black;
top panel) and HCN1-KO Purkinje cells (gray; bottom panel). Presynaptic spike trains evoke synaptic currents in Purkinje cells that are integrated
to produce output spikes. The input-output function of wild-type Purkinje cells (black) is independent of the history of activity. In contrast, the
input-output function of Purkinje cells from HCN1-KO mice (gray) depends on whether the neuron is initially in an active (down arrow) or silent
(up arrow) state. D: HCN1 constrains LTP in perforant path synapses on distal dendrites of CA1 neurons (gray line, HCN1-deficient mice; black line,
WT mice), but not in Schaffer collateral synapses on proximal dendrites (not shown).
decay faster and are therefore shorter (Fig. 5C). As a
consequence, after propagation to the soma, the temporal
summation is more dampened for distal than for proximal
inputs. Therefore, the temporal summation of all inputs
reaching the soma is about equal (Fig. 6A). However, the
normalization of somatic EPSP time course by Ih comes
at a cost. As mentioned in section VIA, the activation of Ih
increases the dendrosomatic attenuation of EPSP amplitude and thus the dependence of somatic EPSP amplitude
on synapse location (Fig. 5B).
Collectively, these results suggest that the proposed
role of Ih in dendritic integration critically depends on an
increasing somatodendritic Ih gradient. However, this
may not apply to all types of neurons. For example, there
is a shallower Ih gradient or even a reversed Ih distribuPhysiol Rev • VOL
tion in a subset of CA1 pyramidal cells that show a similar
dendritic integration and temporal summation as pyramidal cells with a distal enrichment of Ih (61, 160). Moreover, location independence of EPSP summation is observed in cerebellar Purkinje cells, although Ih exhibits a
uniform dendritic density in these neurons (11). Thus
normalization of temporal summation may occur in a
variety of Ih distribution patterns. Using a modeling approach, Angelo et al. (11) concluded that it is the total
number of Ih channels, not their distribution, that governs
the degree of temporal summation of EPSPs. Finally,
other voltage-gated ion channels also have important
roles in dendritic integration. For example, a gradient of
A-type potassium channels rising from proximal to distal
dendrites (34, 181, 199, 377) may be involved in dendritic
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FUNCTION OF HCN CHANNELS
integration. In addition, it has been shown that the density
of AMPA glutamate receptors is increased in distal dendrites of several neurons, increasing the strength of distal
synapses (10, 242, 352). Thus the relative importance of
individual ion channels and receptors to the complex
process of dendritic integration may vary among different
neurons.
C. Role of Ih in Working Memory
Physiol Rev • VOL
ceptors giving rise to strongly increased cAMP levels (16).
In this situation, cAMP levels could significantly increase
the open probability of HCN channels and functionally
disconnect spinous inputs to the dendrites (15). Thus
connections of the prefrontal cortex would be functionally cut off.
An alternative modulation of HCN channels by ␣2adrenoceptors in prefrontal neurons has been proposed
by a different group (71). Carr et al. (71) suggested that
␣2-adrenoceptor activation suppresses HCN channels in
prefrontal neurons through a cAMP-independent signaling pathway that appears to be mediated by PLC-PKC
signaling.
D. Role of Ih in Constraining Hippocampal LTP
Mice in which the HCN1 gene has been selectively
deleted from forebrain show an improvement of hippocampal-dependent learning and memory formation,
compared with wild-type mice (292, 394) (Fig. 6D). Electrophysiological recordings indicated that this surprising
effect is concurred and probably also caused by a specific
enhancement of long-term synaptic plasticity (LTP) and
dendritic excitability in hippocampal CA1 neurons. CA1
neurons provide the major output of the hippocampus
and receive two major sources of excitatory synaptic
inputs (Fig. 7A). One set of inputs, the Schaffer collaterals, comes from hippocampal CA3 pyramidal neurons and
terminates on regions of CA1 neuron dendrites that are
relatively close to the cell body, an area with only moderate HCN1 expression. The second set of inputs, the
perforant path, represents a direct connection from layer
III neurons of the entorhinal cortex and terminates on the
distal dendritic regions of the CA1 neurons, an area where
HCN1 expression is very high. At the more proximal
Schaffer collateral pathway, both synaptic transmission
and long-term plasticity are relatively unaffected by the
deletion of HCN1, consistent with the relatively low expression of HCN1 in this dendritic region (292)(Fig. 6D).
In contrast, at distal perforant path synapses, a significant
enhancement in the integration of synaptic potentials and
an increase in LTP was observed, consistent with the
relatively high expression of HCN1 in this region (292)
(Fig. 6D). From these results, it was concluded that HCN1
channels impair spatial learning because they exert an
inhibitory constraint on dendritic integration and synaptic
long-term plasticity at the perforant path inputs to CA1
pyramidal neurons. It was proposed that high levels of
HCN1 may interfere with generation of Ca2⫹ spikes, a
process that is critical for the induction of LTP at dendritic synapses (394). These Ca2⫹ spikes are triggered by
synaptic activation of AMPA- and NMDA-receptors (394).
The activation of these glutamate receptors generates an
initial depolarization that activates most likely T-type
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A recently discovered role of Ih is the control of the
spatial working memory (415) (Fig. 6B). This form of
memory depends on prefrontal cortical networks that
have the unique ability to represent information that is no
longer present in the environment and to use this “representational knowledge” to guide behavior. In monkeys
performing a spatial working memory task, prefrontal
cortical neurons increase firing in response to visual stimuli presented in the preferred direction, while they decrease firing when stimuli are presented in the nonpreferred direction. This property is called spatial tuning. It
was proposed that spatial tuning is regulated by the
opposing effects of adrenoceptors and the D1-receptor
on intracellular cAMP signaling (15, 407). It has been
suggested that HCN channels are important downstream
targets for cAMP in this signaling pathway (415). HCN
channels colocalize and functionally interact with ␣2Aadrenoceptors on dendritic spines of prefrontal neurons. The
functional interaction was explained according to the
following model (Fig. 6B) (415): ␣2A-adrenoceptors are
activated by relatively low levels of norepinephrine released during alert and wakefulness (16). The activation
of ␣2A-adrenoceptors inhibits the production of cAMP via
Gi signaling and thus reduces the open probability of HCN
channels. As a result, Rm, the efficiency of synaptic input,
and the network activity of these neurons increase. On
the basis of this model, it was speculated that the reduced
HCN channel activity on spines receiving inputs from
neurons with similar spatial properties increases the firing to preferred spatial directions and thereby augments
relevant information (15). In contrast, activation of D1receptors increases the production of cAMP via Gs signaling. As a result, HCN channels open, reduce the membrane resistance, and, thereby, decrease inputs to the
spines (Fig. 6B). This could selectively disconnect spinous inputs to the dendrites. Thus activation of D1receptors on spines receiving inputs from neurons with
dissimilar spatial properties could suppress irrelevant information (“noise”) from the nonpreferred direction (15,
407, 422). So far, the regulation of HCN channels by
D1-receptors has not directly been demonstrated and
therefore remains hypothetical (15). During stress, higher
concentrations of norepinephrine may be present and
activate low-affinity ␣1- and very-low-affinity ␤1-adreno-
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F. Role of Ih in Synaptic Transmission
FIG. 7. Proposed roles of Ih in resonance and oscillation. A: anatomy of the hippocampal formation. For details, see text. EC, entorhinal
cortex; CA1, CA1 pyramidal neurons; PP, perforant path input; DG,
dentate gyrus; CA3, CA3 pyramidal neurons; SC, Schaffer collaterals.
B: resonance is formed by the interaction of active and passive properties in a neuron. The response to a ZAP current (current with linearily
increasing frequency) shows a prominent resonant peak (arrows). C: Ih
and passive membrane properties of the membrane act in concert to
produce resonance. The broken line shows the contribution of Ih, and
the black line shows the contribution of the passive membrane properties (␶m) to the total impedance. The bell-shaped curve is the resulting
impedance curve for the combined system (gray line).
(Cav3.3) and N-type (Cav2.2) voltage-dependent Ca2⫹
channels. Because HCN1 passes a depolarizing inward
current at rest, it will make the resting membrane potential more positive and, hence, shift a substantial fraction
of voltage-gated T- and N-type channel to their respective
inactivated states. Thus the availability of these channels
to participate in an action potential is decreased and Ca2⫹
influx is reduced. In other words, HCN1 can be considered as a partial brake that constrains dendritic Ca2⫹
spikes. Quantitatively, the inhibitory effect of HCN1 is a
function of the protein amount present in the membrane,
which may explain why it is only observed in distal synapses displaying high HCN1 expression levels.
Electrophysiological recordings have demonstrated
the presence of Ih in several presynaptic terminals, including the crustacean neuromuscular junction (33), avian
ciliary ganglion (141), cerebellar basket cells, and the
calyx of Held in the auditory brain stem (356). It was
suggested that an important role of these presynaptic Ih
channels consists in controlling synaptic transmission. In
support of this hypothesis, long-term facilitation of synaptic transmission in crustacean motor terminals was
shown to be conferred by cAMP-dependent upregulation
of Ih (32, 33). The downstream pathway coupling Ih activation to synaptic release is not yet known. However,
there is initial evidence that Ih channels may directly
interact with the release machinery, perhaps mediated by
the cytoskeleton (32, 33). In vertebrates, the functional
relevance of Ih in regulating synaptic transmission is currently disputed. Mellor et al. (263) reported that presynaptic Ih is involved in the generation of LTP in hippocampal mossy fiber synapses that terminate on CA3 pyramidal
cells. However, this finding was challenged by another
study that showed in the same system that LTP is independent of Ih (89).
G. Role of Ih in Resonance and Oscillations
1. Resonance
E. Role of Ih in Motor Learning
The role of Ih in motor learning has been recently
uncovered in mice with a global deletion of HCN1 (293)
(Fig. 6C). Deletion of HCN1 causes profound learning and
memory deficits in behavioral tests that require complex
and repeated coordination of motor output by the cerebellum (e.g., in visible platform, water maze, and rotarod
tasks). In contrast, the deletion of HCN1 does not modify
Physiol Rev • VOL
The term resonance describes the property of a neuron to selectively respond to inputs at a preferred frequency (189). In electrophysiological experiments, resonance can be induced by applying a current stimulus with
linearly increasing frequency (so-called ZAP current). In
many neurons, the voltage answer shows a prominent
resonant peak at the natural frequency or Eigenfrequency
of the neuron (Fig. 7B, arrow). To generate resonance, a
neuron needs to have properties of a low-pass filter com-
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acquisition or extinction of eyelid conditioning, a discrete
motor behavior that also involves cerebellar synaptic
plasticity. The actions of HCN1 were tested in cerebellar
Purkinje cells, the key component of the cerebellar circuit
required for learning of correctly timed movements (293).
In these spontaneously spiking neurons, HCN1 mediates a
depolarizing inward current that counteracts inputs that
hyperpolarize the membrane below the threshold for
spontaneous spiking. By stabilizing the integrative properties of Purkinje cells, HCN1 enables the Purkinje cells
to maintain an input-output relation that is independent of
the neuron’s previous history of activity (Fig. 6C). This
function of HCN1 is required for reliable encoding of
information and ensures accurate decoding of input patterns.
FUNCTION OF HCN CHANNELS
2. Oscillations
Sustained rhythmic oscillations are a hallmark feature of neuronal circuits in various brain regions (62, 65).
Oscillations arise from synchronized activity of neurons
and are thought to play an essential role in information
processing in neuronal networks. There are several types
of oscillations in different frequency bands, called delta,
theta, gamma, and fast “ripple” oscillations (62, 65). One
oscillation, the theta frequency oscillation (4 –12 Hz), is
prominent in all areas of the hippocampal formation.
These oscillations may be important for various cognitive
processes including processing, encoding, and storing of
spatial information (62) as well as for memory formation
and retrieval (63, 219). Hippocampal theta oscillations
have been proposed to originate from reciprocal interactions between rhythmic inputs from the medial septumdiagonal band of Broca, the entorhinal cortex, and other
subcortical structures (Fig. 7A; Refs. 66, 306, 406). The
inputs from septal neurons to the hippocampus seem to
be mainly responsible for the generation of the theta
rhythm (62, 306). In this process, the perforant path and
various other brain regions are also involved (155, 327).
In mice with a forebrain-restricted deletion of HCN1,
changes in hippocampal-dependent network oscillations
have been described (292). While low- and high-frequency
network oscillations appeared to be unchanged in knockout mice, there was a significant enhancement in the theta
frequency band (4 –9 Hz), during rapid-eye-movement
(REM) sleep and during wheel running (292). Consistent
with these results, in CA1 neurons responses to lowfrequency inputs at the theta range are preferentially inPhysiol Rev • VOL
creased in HCN1-deficient mice. HCN1 channels can
therefore be thought of as a partial brake that contributes
to resonance by preferentially dampening low-frequency
components of input waveforms at frequencies below the
theta range. Releasing the brake in knockout animals
causes a general enhancement in the voltage response to
low-frequency oscillatory currents.
In addition to the important role of Ih in the generation of theta oscillation, Ih is also involved in the generation of other types of oscillations. There are several studies that demonstrated the importance of Ih for ␥-oscillations in the hippocampus (97, 140), synchronized oscillations
in the inferior olive (21), and subthreshold oscillations in the
entorhinal cortex (108, 166). Moreover, Ih has been suggested to influence network oscillations that play an important role in learning and memory (140, 185, 230, 235).
H. Role of Ih in the Generation
of Thalamic Rhythms
Synchronized neuronal oscillations are produced in
thalamocortical networks during sleep (64, 255, 363, 364),
sensory processing (162), and seizures (256). The generation and synchronization of complex oscillations (e.g.,
spindle waves and slow waves) requires extensive synaptic interactions within the thalamocortical network (361)
(Fig. 8A). In contrast, there are other neuronal rhythms,
for example, the delta frequency rhythm that can be generated in single cells (255, 361, 367). We will first discuss
the role of Ih in the generation of characteristic firing
patterns that arise in single thalamocortical cells and then
discuss the role of Ih in oscillations that require more
extensive synaptic interactions. In our discussion, we
focus on data from in vitro slice preparations due to the
current paucity of in vivo data.
1. Single cell oscillations in thalamic relay neurons
In vitro (22–24, 96, 231, 232, 259) and in vivo (9, 23,
107, 238, 328, 363) studies have shown that thalamocortical neurons fire in two distinct firing modes called transmission mode and burst mode (22–24, 96, 231, 232, 259)
(Fig. 8B). During wakefulness and REM sleep, thalamocortical neurons are depolarized by afferent inputs and
switch to the transmission or “single spike” mode (195,
196, 257) (Fig. 8, B and D). During this mode information
is gated through the thalamus and forwarded to the cortex
(255, 367). The transmission mode is characterized by the
generation of repetitive single Na⫹ spikes at depolarized
potentials where T-type Ca2⫹ channels are inactivated
(Fig. 8D). During this mode, sufficient excitation repetitively discharges thalamocortical neurons. The output frequency of these neurons increases with increasing depolarization induced by afferent excitatory inputs (165).
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bined with properties of a high-pass filter (65, 69, 187, 189)
(Fig. 7C). The low-pass filtering is caused by the membrane time constant ␶m. The high-pass filtering is caused
by the action of several voltage-gated currents including
Ih. To act as a high-pass filter, these currents need to be
able to actively oppose changes in membrane voltage
(Fig. 5, D and E). In addition, these currents have to
activate slowly relative to the membrane time constant.
Both requirements are met for Ih. At low frequencies, the
Ih channels have time to activate and actively oppose
changes in membrane potential. At high frequencies,
there is not enough time for the Ih channels to open. As a
result, high-frequency changes are not suppressed, rendering the neuron responsive for fast trains of spikes.
Resonance arises at intermediate frequencies where input-induced voltage changes are too high to be opposed
by Ih and too low to be filtered by ␶m. The importance of
Ih for the generation of membrane resonance has been
demonstrated for several neurons, for example, in neocortical pyramidal cells of the rat (398), in subicular pyramidal neurons of the rat (416), and in neurons from the
sensorimotor cortex of juvenile rats (188).
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During the burst mode, thalamic networks display a
monotonous repetitive firing pattern at hyperpolarized
membrane potentials (178, 195, 196, 368, 389). This firing
mode is characteristically seen during non-REM sleep or
epileptic discharges and has been considered to decrease
transfer of sensory information to the cortex (368).
In vitro studies have demonstrated that thalamic neurons
fire in the “burst mode” through the interaction of a
low-threshold Ca2⫹ current (IT) and Ih (Fig. 8, B and C)
(255, 300). The following model has been proposed: the
activation of Ih by hyperpolarization beyond ⫺65 mV
slowly depolarizes the membrane potential until a rebound low-threshold Ca2⫹ spike is generated by the activation of IT at more depolarized potentials. Because these
“slow spikes” are depolarizing and last for tens of milliseconds, typically a series of Na⫹ spikes rides on them.
The inactivation of IT terminates the low-threshold Ca2⫹
spike. During the spike, Ih is deactivated. As a result, the
termination of the spike is followed by a hyperpolarizing
“overshoot.” In turn, Ih is activated, the cycle repeats, and
continuous rhythmic burst firing is sustained. The intrinsic interplay of these currents promotes delta rhythmicity
in single thalamocortical neurons (128, 221, 222, 258, 261,
355). These single cell oscillations are synchronized in
large neuronal circuits and can be recorded in the EEG as
delta waves during non-REM sleep (128, 295, 366, 367).
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Electrophysiological recordings in thalamic slice
preparations of HCN2-deficient mice revealed that the
current produced by HCN2 constitutes the major component underlying thalamocortical Ih (226). Deletion of
HCN2 reduced the current amplitude of Ih by ⬃80% and
resulted in a 12 mV hyperpolarizing shift of the resting
membrane potential. Thalamocortical neurons of HCN2deficient mice displayed a higher susceptibility to fire in
the burst mode when experiencing excitatory inputs than
wild-type neurons (Fig. 9). Macroscopically, the altered
thalamic firing behavior of HCN2-deficient mice concurred with the presence of spike-and-wave discharges,
the clinical hallmark of absence epilepsy (Fig. 9A; see also
sect. VIII). The higher incidence of burst firing in HCN2deficient mice (Fig. 9B) may simply result from the fact
that the ratio of T-type channels present in the closed (but
activatable) versus inactivated state depends on the resting membrane potential. In HCN2-deficient neurons that
display a more hyperpolarized resting membrane potential, the fraction of T-type channels present in the closed
state will be higher than in wild-type cells where more
T-type channels will be in the inactivated state. Thus the
susceptibility of HCN2-deficient neurons to produce a
Ca2⫹ spike is higher than that of wild-type neurons because the T-type channels present in these cells are easier
to activate by excitatory inputs.
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FIG. 8. The role of Ih in the generation of thalamic oscillations. A, top: coronal section through the mouse brain. The cortex, the reticular
thalamic nuclei, and the thalamus are indicated. Bottom: schematic diagram of the thalamocortical loop: ⫹, excitatory glutamatergic synaptic
contacts; ⫺, inhibitory GABAergic contacts. For detailed information, see text. B: firing modes of thalamocortical neurons. C and D: higher temporal
resolution of sections shown in B. [Modified from McCormick and Bal (255).]
FUNCTION OF HCN CHANNELS
2. Network oscillations
Ih is involved in the generation of a number of rhythmic oscillations in thalamocortical networks. During the
early state of non-REM sleep, synchronized oscillations
are generated in the thalamocortical network that give
rise to spindle waves in the surface EEG (22, 363, 367).
Spindle waves are characterized by a crescendo-decrescendo type of oscillation at 6 –14 Hz that lasts for 1– 4 s
followed by a refractory period of 5–20 s that terminates
the oscillations (91, 204, 363, 367). These waves are assumed to have an essential functional role in synaptic
plasticity of cortical and thalamic neurons (368). Spindle
waves have been investigated in vivo (9, 92, 279, 362, 364,
365) and also in slice preparations (410). These studies
indicate that spindle waves are generated by a cyclic
interaction between excitatory thalamocortical cells
and inhibitory thalamic reticular neurons (23, 24, 363,
367, 410) (Fig. 8A). Thalamic reticular cells are the pacePhysiol Rev • VOL
makers for the generation of spindle wave oscillations
(148, 359). In these neurons, rhythmic bursts are generated by low-threshold Ca2⫹ spikes. Burst firing in thalamic reticular neurons induces rhythmic inhibitory
postsynaptic potentials (IPSPs) in thalamocortical cells.
These IPSPs hyperpolarize thalamocortical cells, remove
inactivation of the low-threshold Ca2⫹ current, and at the
same time activate Ih. The resulting depolarization triggers a rebound Ca2⫹ spike and a burst of action potentials
(Fig. 8C). These bursts reexcite the thalamic reticular
neurons and also stimulate cortical pyramidal cells. The
nearly simultaneous occurrence of spindle waves over
widespread cortical territories is produced by network
synchronization involving the cortex and the thalamus
(358). This synchronized activity underlies the presence
of spindle waves in the EEG.
The silent period between spindle waves is largely
determined by persistent Ih activation in thalamocortical
cells (22, 363, 367). This persistent activation of Ih results
from an increase in intracellular Ca2⫹ primarily triggered
by the rebound low-threshold Ca2⫹ bursts that occurred
during the spindle wave generation. The Ca2⫹ most likely
activate a Ca2⫹-sensitive isoform of adenylyl cyclase, producing an increase in cAMP synthesis that enhances Ih
(231). The persistent activation of Ih in turn suppresses
the next spindle wave until Ih is slowly decayed. Spindle
waves disappear during wakening or REM sleep. The
transition to these states is regulated by several neurotransmitters such as norepinephrine and serotonin that
both increase intracellular cAMP and lead to a consecutive upregulation of Ih (255).
VII. ROLE OF Ih CHANNELS
IN CARDIAC RHYTHMICITY
The heart beat originates from specialized pacemaker cells in the sinoatrial (SA) node region of the right
atrium. These cells generate a special kind of action potential (“pacemaker potential”) that is characterized by
the presence of a progressive diastolic depolarization
(DD) in the voltage range between ⫺65 to ⫺45 mV (Fig. 10;
see also Ref. 244 for an excellent recent overview on heart
automaticity). After the repolarization phase, it is the DD
that drives the membrane potential of a SA node cell back
toward threshold of Ca2⫹ channel activation, thereby
maintaining firing. The DD is generated by the concerted
action of several currents, among which Ih is considered
to play a prominent role because it is activated at negative
potentials and, therefore, potentially could serve as primary initiator of the DD. In addition to Ih, other inward
currents including Ca2⫹ currents (ICaT and ICaL) (168, 243,
244, 371), as well as the sustained inward current (Ist)
(273) whose molecular correlate is not yet known may
contribute to the DD. Furthermore, it was proposed that
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FIG. 9. Absence epilepsy and spike-and-wave discharges in
HCN2⫺/⫺ mice. A: comparison of spontaneous EEG patterns between
wild-type and HCN2⫺/⫺ mice. One-minute EEG recordings (L, left channel; R, right channel) are shown. Spike-and-wave discharges are indicated by asterisks. The horizontal bars demarcate the traces shown on
an expanded time scale below the 1-min recordings. B: different intrinsic
firing properties of wild-type and HCN2⫺/⫺ thalamocortical neurons.
The firing patterns elicited by 1-s depolarizing current pulses at resting
membrane potential of wild-type and HCN2⫺/⫺neurons are shown. The
amount of current injected is indicated above. Inset: traces elicited by
injection of 100 pA at an expanded time scale.
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the DD may be initiated by the decay of outward rectifier
K⫹ currents (IKr and IKs) or by mechanisms involving intracellular Ca2⫹ release through ryanodine (214, 215, 408) or
inositol 1,4,5-trisphosphate (IP3) receptors (265). Ih is not
only involved in principal rhythm generation, it also plays
a key role in heart rate regulation by the autonomic
nervous system. Sympathetic stimulation activates Ih and,
hence, accelerates heart rate via ␤-adrenoceptor-triggered
cAMP production (49, 50, 122) (Fig. 10, C and D) while
low vagal stimulation lowers heart rate via inhibition of
cAMP synthesis and an ensuing inhibition of Ih activity
(117, 123, 124). High vagal tone most likely lowers the
heart rate mainly via the activation of IKACh (117, 421).
A. HCN4
Recent genetic mouse models have made it possible
to evaluate the proposed roles of Ih in cardiac rhythmicity
under in vivo conditions. As mentioned above, HCN4
makes up ⬃80% of SA node Ih and has been considered to
be crucial for the generation of the heart beat (171, 172,
370). In support of this notion, mice with global- or heartspecific disruption of the HCN4 gene die in utero between
embryonic days 9.5 and 11.5 (370). Embryonic hearts of
HCN4-deficient mice analyzed before embryonic day 10
show a reduction of the beating frequency of ⬃40%. Importantly, these hearts do not respond to ␤-adrenergic
Physiol Rev • VOL
stimulation. Thus one may conclude that in the embryonic
heart HCN4 is not required for principal heart beat generation, at least not before embryonic day 11.5, but that
this channel is absolutely crucial for autonomous heart
rate regulation (370). In support of this conclusion, hearts
of mice carrying a mutation in the CNBD that abolishes
high-affinity cAMP binding (HCN4R669Q) also fail to respond to ␤-adrenergic stimulation (171). Further analysis
revealed that HCN4-deficient mice probably die from a
developmental defect of the SA node (370). These mice
develop normal embryonic pacemaker cells that produce
primitive pacemaker potentials but fail to generate adulttype pacemaker cells. Obviously, the latter cells are crucially required to drive the heart beat after embryonic day
11. Interestingly, HCN4R669Q mice are able to generate this
kind of pacemaker cells, but like HCN4-deficient mice
also die around embryonic day 11.5 (171). Together, the
mouse studies indicate that the HCN4 protein is required
1) for the formation of adult pacemaker cells during
embryonic heart development and 2) for conferring
cAMP-dependent upregulation of embryonic heart rate.
Both HCN4-mediated functions are dispensable in early
heart function and early embryonic heart development
but are needed for the transition to late embryonic stages.
Given its vital role in embryonic heart, it came as a
big surprise that mice in which HCN4 was deleted in adult
SA node using a temporally controlled knock-out ap-
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FIG. 10. Role of Ih in cardiac automaticity. A: anatomy and localization of the sinoatrial node. SAN, sinoatrial node (gray); CT, crista terminalis.
B: structure of the sinoatrioal node (left; arrow) and morphology of an isolated pacemaker cell (right). C, top: idealized pacemaker potentials in the
absence (black) and presence (red) of adrenergic stimulation. DD, diastolic depolarization. Bottom: proposed Ih time course during the pacemaker
potential. D, left: sinoatrial Ih activates faster in the presence of cAMP (red) than in the absence (black) of cAMP at maximal activation voltage (⫺140
mV). Right: activation curve of Ih in the absence (black) and the presence (red) of cAMP. In the presence of cAMP, the activation curve of Ih is
shifted to the right.
FUNCTION OF HCN CHANNELS
B. HCN2
With respect to the stabilizing function, HCN2 that
makes up ⬃20% of SA node Ih in mice may serve as a
complementary channel to HCN4 (226) (Fig. 11C). HCN2deficient mice also reveal a sinus dysrhythmia that is
characterized by varying peak-peak intervals in the electrocardiogram (226). Other parameters of the sinus rhythm
including autonomous heart rate regulation are normal in
these mice. Like for HCN4, the maximal diastolic potential (MDP) of HCN2-deficient SA node cells is slightly
hyperpolarized. It is important to note that HCN2 obviously is not crucial for the development of cardiac conduction system since HCN2-deficient mice do not display
increased embryonic lethality.
C. Conclusions and Open Questions
In conclusion, genetic mouse studies indicate that
the two components of SA node Ih carried by HCN4 and
HCN2 are required for maintaining a stable cardiac
rhythm. However, neither HCN2 nor HCN4 is needed for
principal pacemaking and for autonomous rate regulation
in the adult heart. Further experiments (e.g., analysis of
HCN2/4 double knockout mice) will be required to solidify these conclusions. Importantly, it remains to be elucidated why heart rate regulation in early embryos requires
HCN4 but is independent of this channel in adult animals.
FIG. 11. Effect of deletion of HCN4 and HCN2 on cardiac pacemaking. A: HCN4 is not required for upregulation of the heart rate. Isoproterenol
(0.5 mg/kg) injected at time 0 increased the heart rate of control (n ⫽ 8; black curve) and knockout (n ⫽ 8; red curve) animals to similar maximum
levels. B: example ECG traces from control (top) and knockout mice (bottom) at 0.5 h (I) and 1.5 h (II) after isoproterenol injection. All traces are
displayed at the same scale. C: sinus dysrhythmia in HCN2-deficient mice. Telemetric ECG recordings (lead II) were obtained simultaneously from
wild-type (con) and HCN2-deficient mice (HCN-KO) at rest.
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proach were viable and displayed a rather mild cardiac
phenotype (172) (Fig. 11A). Like in embryonic pacemaker
cells of global HCN4-deficient mice, Ih in SA node cells of
the adult HCN4-deficient mice was reduced by ⬃80%
(172). However, the lack of this current component did
neither lead to a major impairment of pacemaker potential generation nor did it interfere with ␤-adrenergic regulation of heart rate (Fig. 11A). Unlike global knockout
mice, adult HCN4 knockout mice revealed a normal basal
heart rate and normal sympathetic and vagal heart rate
modulation. However, adult HCN4-deficient mice displayed a cardiac arrhythmia characterized by recurrent
sinus pauses (Fig. 11B). Pacemaker cells of adult HCN4
knockout mice were hyperpolarized by ⬃8 mV and in
most cases did not fire spontaneously under basal conditions. However, this functional impairment could be compensated by ␤-adrenergic stimulation. In conclusion,
these findings suggest that in the adult SA node, HCN4
may serve as a kind of a stabilizer of the pacemaker
potentials. In most situations, and particularly during
sympathetic stimulation, HCN4 does not seem to be required to promote stable pacemaking. However, after an
increase in repolarizing currents (e.g., vagal stimulation
or transition from activated to basal cardiac state), HCN4
is activated and provides a depolarizing current, keeping
the system well-balanced. The loss of this “depolarization
reserve” may explain the induction of recurrent sinus
pauses in HCN4 knockout mice.
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VIII. ROLE OF Ih IN DISEASE
Given the widespread expression and physiological
importance of HCN channels in nervous system and heart,
it is obvious to assume that impaired expression or malfunction of these channels is associated with the genesis
of human diseases. Indeed, evidence has accumulated
over the last couple of years that Ih is implicated in the
pathologies of at least three classes of diseases: epilepsies, neuropathic pain disorders, and cardiac arrhythmias.
A. Inherited Channelopathies
In a classical sense, the term channelopathy refers to
diseases that are caused by inherited genomic mutations
in an ion channel gene that lead to loss, impaired expression levels, or functional defects of the channel protein.
Interestingly, despite numerous efforts 10 years after the
discovery of the HCN channel genes, only four inherited
HCN channel mutations have been described (Fig. 2). All
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four are localized in the HCN4 gene and are associated
with the induction of sinus bradycardia (see sect. VIIC and
Ref. 174). All patients identified so far are heterozygous
for the respective HCN4 mutation. A likely explanation
for this finding may be that like in mice disruption (370) or
functional impairment (171) of both HCN4 alleles is associated with embryonic lethality due to the failure to develop a mature cardiac conduction system. Neuronal phenotypes have not been reported in the four groups of
patients. This is surprising given the expression of HCN4
in CNS. So far, disease-causing mutations in human
HCN1-3 genes have not been reported. On the basis of the
analysis of murine HCN knockout models, it is expected
that mutations in these genes would be implicated in
complex cardiac (HCN2) and/or neuronal (HCN1, HCN2)
phenotypes. However, one would also predict that even
the total loss of these individual channels does not lead to
lethality in mice. Thus one could assume that mutations in
HCN1-3 proteins have much more impact in humans than
in mice and are not detected because they are lethal.
Alternatively, the mutations exist but have escaped detection so far because they occur very rarely. More systematical screening and sequencing approaches in patients
will be required to clarify this issue.
B. Transcriptional Channelopathies
Transcriptional (or acquired) channelopathies result
from pathological alterations of the expression or localization of a normal (nonmutated) ion channel (417). Such
disorders occur more frequently than inherited monogenetic channelopathies, which are usually very rare. However, the analysis of transcriptional channelopathies is
also much more difficult and prone to misinterpretations
than the analysis of monogenetic diseases. The major
obstacle in the analysis is that expression and cellular
handling of ion channels usually is an extremely dynamical process that is regulated by numerous intracellular
and external factors. In general, this complexity makes it
difficult to decide whether an altered expression profile of
an ion channel 1) is causative to a disease, 2) is a compensatory process in response to a disease, or 3) represents a normal physiological variation that is neither causative nor compensatory to a disease. Having these limitations in mind, we will now summarize recent evidence
supporting a role of HCN channels in transcriptional diseases.
1. Epilepsy
First evidence connecting Ih and epileptic seizures
came from studies in a rat model of childhood febrile
seizures (hyperthermia model of febrile seizure) (83, 84).
At the age of 10 days, these rats are exposed to a single
period of hyperthermia lasting for ⬃30 min. Such animals
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In this context, it remains to be determined which current(s), if not Ih, are the downstream target(s) of cAMP
signaling in adult SA node cells. Finally, it should be
considered that the importance of HCN channels for cardiac rhythm initiation and frequency modulation may be
different between species.
The analysis of human HCN4 channelopathies supports this notion (269, 290, 338, 395). So far, four different
heterozygous HCN4 mutations have been identified in
humans (269, 290, 338, 395) (Fig. 2). The mutations lead to
loss of cAMP-dependent modulation (HCN4-573X) (338),
hyperpolarizing shift of the activation curve (S672R and
G480R) (269, 290), or a severe reduction of cell surface
expression (D553R) (395). Interestingly, all patients suffering from these mutations have in common that they
display a more or less severe bradycardia, a clinical phenotype that is not observed in HCN channel-deficient
mouse models. Another issue that is difficult to explain at
this moment is the effect of bradycardic agents, such as
cilobradine, ivabradine, and zatebradine (373), as well as
of clonidine (208). It was proposed that these agents
lower heart rate by blocking Ih channels and reducing the
firing frequency of SA node cells. In agreement with this
hypothesis, cilobradine lowers heart rate in wild-type
mice but does not do so in adult HCN4 knockouts (174).
On the other hand, given that HCN4 confers the heart-rate
lowering effect of cilobradine and related substances,
why then do HCN4-deficient mice (which corresponds to
a 100% pharmacological block) display no basal bradycardia? At present, there is no satisfying answer to this
conundrum; however, solving this issue will undoubtedly
profoundly advance our understanding of the molecular
basis of rhythmicity in normal and diseased heart.
FUNCTION OF HCN CHANNELS
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to cAMP that is consistent with an increase of HCN1
levels relative to HCN2. In the WAG/Rij model, but not in
GAERS, V0.5 of Ih was also shifted to more hyperpolarized
voltages. It was speculated that the hyperpolarizing shift
in combination with the reduced cAMP sensitivity locks
thalamocortical neurons in the burst firing mode (58).
Perhaps the strongest evidence linking Ih channels
with absences relates to HCN2-deficient mice (226) (Fig.
9). Like other absence models, HCN2-deficient mice display frequent bilaterally synchronous SWDs in the EEG
that are accompanied with brief episodes of immobility
(so-called behavioral arrest). In thalamocortical and thalamic reticular neurons of these mice, Ih was found to be
almost completely abolished (1, 226, 320, 435). The residual Ih (max. 20% of wild-type Ih) had a very slow kinetics
and a strongly hyperpolarized V0.5. As a consequence, the
resting membrane potential of thalamocortical neurons
from HCN2-deficient mice was shifted to hyperpolarized
potentials, which could well explain the increased burst
activity and network oscillations seen in these animals
(see also sect. VI).
Downregulation of Ih was also found in a pharmacological (kainic acid) rat model of temporal lobe epilepsy
(TLE) (342). Layer III pyramidal neurons of the entorhinal
cortex (EC) of these epileptic rats are hyperexcitable,
giving rise to profound synchronous network activity.
Electrophysiological recordings in EC neurons revealed a
significant reduction of dendritic Ih which was caused by
reduced HCN1 and HCN2 protein levels. Mechanistically,
hyperexcitability induced by downregulation of Ih could
be put down to the key role Ih plays in dendritic integration (see also sect. V). As predicted, reduction of Ih densities in dendrites leads to an increase of the dendritic
input resistance. This, in turn, enhances dendritic EPSP
summation and EPSP-spike coupling.
Rat and mouse models clearly indicated that impaired HCN channel function or expression is associated
with epileptiform activity. Such a clear correlation, although likely, is so far missing in humans. As mentioned,
HCN channelopathies involving epilepsy have not been
reported so far. However, changes in HCN channel expression have been found in the dentate gyrus from patients with temporal lobe epilepsy and severe hippocampal sclerosis (35). Since upregulation of HCN1 mRNA was
found only in cases of end-stage disease, long after the
onset of epilepsy, it was suggested that it is not causative
for the epilepsy but rather represents a compensatory
change of the brain.
In conclusion, the role of Ih in epilepsies seems to be
extremely complex and diverse. There is evidence that
1) both up- or downregulation can be associated with the
disease, 2) different HCN types contribute to different
extents to the disease, 3) the role of an HCN channel
strongly depends on its cellular localization, and 4) findings from rodents may not necessarily be applicable to
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reliably develop epileptic convulsions originating in the
hippocampus and reveal an increased tendency to develop seizures at adult stage. Electrophysiological recordings revealed an enhanced Ih (increase of Imax by 40% at
⫺70 mV) in hippocampal CA1 neurons of epileptic rats. In
addition, the Ih current showed a slight positive shift of
the V0.5 (⫹3 to ⫹5 mV) and slowed activation kinetics,
compared with wild-type rats. Functionally, the enhanced
Ih was coupled to an increased probability of rebound
depolarizations and action potential firing (83). Closer
inspection of the molecular basis of Ih observed after
febrile seizures revealed a downregulation of HCN1 in
hippocampal pyramidal neurons while HCN2 was upregulated, resulting in a decrease of the ratio of HCN1 vs.
HCN2 protein from 8:1 to ⬃4:1 (47, 48). The mechanism
underlying changes in HCN channel expression levels in
response to seizures is unclear. Reduction of HCN1 channel expression is probably a transcriptionally regulated
process that involves activation of calmodulin (CaM) kinase II and Ca2⫹ entry via AMPA receptors (322). In
contrast, upregulation of HCN2 was found to be independent of CaM kinase II (322). The consequences of up- and
downregulation of HCN channel subunits may be more
complex than originally expected. A recent coimmunoprecipitation study indicated that altered hippocampal
HCN1 and HCN2 expression levels are accompanied by a
long-lasting increase in the levels of heteromeric HCN1/2
channels (48). Thus the Ih of hippocampal neurons is
probably conferred by a complex mixture of HCN1 and
HCN2 homomers as well as by HCN1/2 heteromers.
Pathological deviations from the normal ratio of the individual HCN channel types may play a critical role in the
generation and the long-term maintenance of hippocampal hyperexcitability.
Altered HCN channel expression has also been found
in another class of seizures, the absence epilepsies. The
typical absence is clinically defined by a sudden, brief
impairment of consciousness and behavioral arrest. Immediately following the absence, there appears to be little
if any disruption of cognitive abilities. Absences mainly
originate in the cortex; however, the thalamus is also
involved in the pathology of the disease (256, 360). In
particular, the generation of spike-and-wave discharges
(SWDs), a diagnostic hallmark of absences, is correlated
with an increased prevalence of burst firing and synchronized oscillatory activity in thalamocortical circuits (256).
Given the key role of HCN channels in controlling the
firing behavior of thalamocortical neurons, abnormal
HCN channel expression may contribute to the generation of absences. In agreement with this hypothesis, in
two rat models for absence epilepsy [WAG/Rij (58, 376)
and GAERS (212)], increased levels of HCN1 were found
in thalamocortical neurons. In contrast, expression of
HCN2-4 was not altered. In both rat models, the Ih of
thalamocortical neurons displayed a reduced sensitivity
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human epilepsies. In most cases it remains unclear
whether an observed change in Ih produces hyperexcitability or represents a compensatory reaction to the state
of hyperexcitability. Thus the relevance of Ih in epileptogenesis cannot be addressed by a simplified general
model but has to be defined for various pathophysiological settings in a very specific and distinct manner.
2. Peripheral neuropathic pain
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Structural and functional remodeling of the heart
muscle is a clinical hallmark of a variety of cardiovascular
diseases including cardiomyopathies, infarction, chronic
hypertension, and inflammation (e.g., myocarditis) (14).
Initially, these adaptations are beneficial to maintain cardiac function (e.g., at pressure overload) but in later
stages of the diseases they contribute to contractile abnormalities and sudden death. On the cellular level, irregular expression of ion channels involved in the control of
cardiac contractility and automaticity plays an important
role in remodeling (176, 285, 286, 323, 392).
Upregulation of Ih has been described in a variety of
animal models of cardiac hypertrophy and heart failure as
well as in human patients suffering from these diseases
(for reviews, see Refs. 76, 173, 244). A common feature of
these diseases is the profound upregulation of Ih channels
in ventricular cardiomyocytes which normally express
only very low levels of these channels. In contrast, the
activation threshold of the Ih of diseased cardiomyocytes
is usually not shifted to more positive values compared
with normal ventricular Ih (76, 139, 177, 182). Increased Ih
densities strongly raise the tendency of the ventricular
muscle to develop ectopic, “pacemaker-like” action potentials and, thereby, contribute to arrhythmia and to
sudden death (72, 73, 139, 182, 374, 452).
In rodents and humans, upregulation of Ih is mirrored
by an increased expression of HCN channels. Examination of mRNA levels in hypertrophied atrial and ventricular myocytes revealed an upregulation of HCN2 and
HCN4 in different animal models (72, 73, 139, 177, 182,
374, 375, 452). In addition, upregulation of HCN4 was
found in human patients with end-stage heart failure (45).
The degree of hypertrophy positively correlates with increased Ih density (73) and the expression levels of HCN
channels (139, 336). While these findings strongly support
a direct link between upregulation of HCN channels and
ventricular dysfunction, the signaling pathways underlying upregulation of Ih are only poorly understood so far.
Both nerval and humoral factors are probably involved in
the process. Notably, high angiotensin II levels, which are
well-known to promote ventricular remodeling (382), as
well as aldosterone (284) seem to play a pivotal role in
HCN channel upregulation. This is supported by the finding that antagonists of the type I angiotensin II receptor
(AT1-receptor) not only reduce cardiac hypertrophy but
also hamper Ih and HCN2/HCN4 mRNA overexpression
(74, 75, 177). Interestingly, Ih is abundantly expressed in
spontaneously active fetal and neonatal ventricular myocytes (77, 325, 434). During maturation, these cells lose
their capacity to generate spontaneous activity. Both in
rat and mouse, this is accompanied by a progressive
decrease of Ih expression (77, 434). Thus it is tempting to
assume that cardiac hypertrophy provokes a reentry of
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Peripheral neuropathic pain is a complex pain condition that is characterized by spontaneous pain, hyperalgesia, and allodynia (68). The disease originates from
the injury of peripheral nerves. Nerve injury can have
many etiologies, among which are trauma, viral infections
(e.g., Herpes zoster), metabolic diseases (e.g., diabetes
mellitus), vascular diseases (e.g., stroke), autoimmune
diseases (e.g., multiple sclerosis), cancer, and exposure to
radiation or chemotherapy. The pathomechanism of neuropathic pain is complex and involves both peripheral and
central sensitization. A hallmark of the disease is the
generation of abnormal spontaneous (ectopic) discharges.
These discharges have strong rhythmic components and are
generated from injured dorsal root ganglion (DRG) somata
or axons, as well as from adjacent uninjured nerves. Previous work has shown that altered expression levels of several
types of voltage-gated Na⫹ channels contribute to the generation and persistence of spontaneous discharges (252, 417,
418).
Several findings suggest that HCN channels also play
a prominent role in neuropathic pain (for recent review,
see Ref. 197). First, Ih has been identified in DRG neurons,
particularly in large- and medium-sized neurons where
most ectopic discharges are produced (253, 339). Second,
in situ hybridization and immunohistochemistry revealed
the presence of HCN1-3 channels in different types of
mouse and rat DRGs (80, 276). Third, increased densities
of Ih were found in large- and/or medium-sized DRG
neurons after experimentally induced injuries (80, 206,
432). Fourth, low concentrations of the Ih blocker ZD7288
reverse both pain behavior and the spontaneous discharges in injured nerve fibers (80, 220, 380). Taken together, these findings strongly suggest that upregulation
of Ih that leads to an increased excitability of the cell is
causative to the generation of ectopic firing. The mechanism underlying upregulation of Ih remains to be investigated in more detail. Interestingly, Chaplan et al. (80)
found a downregulation of HCN1 and HCN2 in DRGs after
injury, although Ih densities were increased. It is unclear
how the discrepancy between current densities and protein expression can be explained. Potential mechanisms
include a positive shift of V0.5 of the Ih (leading to an
increased open probability), altered HCN channel subunit
assembly, and increased cell surface expression of HCN
channel proteins after injury.
3. Cardiac remodeling and arrhythmia
FUNCTION OF HCN CHANNELS
cells into a fetal program and the reinitiation of the corresponding gene expression patterns which includes upregulation of HCN2 and HCN4. Recently, it was found in
rat models of ventricular hypertrophy that upregulation of
HCN2 and HCN4 is accompanied by pronounced reduction of two microRNAs, miR-1 and miR-133. Conversely,
forced expression of these miRNAs prevented overexpression of HCN2/4 in hypertrophic cardiomyocytes
(229). These findings suggest that downregulation of
these two miRNAs may play an important role in the
reexpression of Ih during normal heart development but
also during remodeling of the diseased heart.
A. Heart Rate-Reducing Agents
High heart rate positively correlates with an increased mortality in a variety of cardiac diseases including heart failure, arterial hypertension, and ischemic heart
disease (36, 130, 340). Lowering heart rate is therefore
one of the most important therapeutic approaches in the
treatment of these diseases. Currently used bradycardic
drugs including ␤-adrenoceptor antagonists, and some
Ca2⫹ channel antagonists efficiently reduce heart rate, but
their use is also limited by adverse reactions or contraindications (44, 127). Given the key role of Ih/HCN channels in cardiac pacemaking, these channels are very
promising pharmacological targets for the development
of novel and more specific heart-rate reducing agents
(44). Importantly, HCN channels are not expressed in
vascular and airway smooth muscle. Therefore, in contrast to ␤-adrenoceptor or Ca2⫹ channel antagonists,
drugs acting on HCN channels are not expected to have
major side effects on the vascular system or on pulmonary function. In the past, several agents inhibiting
cardiac Ih have been developed. Early drugs identified
as pure bradycardic agents include alinidine (ST567),
ZD7288, zatebradine (UL-FS49), and cilobradine (DKAH269). A more recent drug is ivabradine (S16257). The
principal action of all these substances is to reduce the
frequency of pacemaker potentials in the sinus node by
inducing a reduction of the diastolic depolarization
slope. In this section, we only briefly discuss these
drugs. We refer readers who are interested in more
detailed information to a number of recent reviews on
this issue (28, 42, 55).
Recently, ivabradine (S16257, Procoralan) was introduced into clinical use as the first therapeutic Ih blocker.
Ivabradine blocks cardiac Ih at low micromolar concentrations and has been approved as a treatment of chronic
stable angina pectoris (379). Electrophysiological studies
revealed that ivabradine acts by accessing Ih channels
Physiol Rev • VOL
from their intracellular side and by exerting a use- and
current-dependent block (56). The mechanism of channel
block by ivabradine was examined in heterologously expressed HCN channels (57, 388). Ivabradine blocks HCN4
and HCN1 channels with half-block concentrations (IC50)
of ⬃1–2 ␮M. Interestingly, ivabradine acts as open channel blocker in HCN4 (like in sinoatrial Ih), while block of
HCN1 requires channels either to be closed or in a transitional state between open and closed configuration (57).
The structural determinants underlying this difference are
still unknown.
Zatebradine and cilobradine are derived from the
L-type Ca2⫹ channel blocker verapamil. Low micromolar
concentrations of both blockers (IC50 between 0.5 and 2
␮M) inhibit sinoatrial Ih as well as heterologously expressed HCN channels in a use-dependent fashion (373,
401). Like with ivabradine, zatebradine block results from
drug molecules entering the channel pore from the intracellular site (115).
ZD7288 is probably the most widely used experimental blocker of Ih. Like the agents discussed so far, ZD7288
blocks channels from the intracellular site (348). However, the block is use independent and is associated with
an ⬃15 mV shift of V0.5 to more negative potentials and
with a decrease of the maximal channel conductance
(46). Recently, two amino acid residues were identified in
the S6-helix of HCN2 (A425 and I432) and that confer
high-affinity binding of ZD7288 (88).
The well-known ␣2-adrenoceptor agonist clonidine,
which is chemically related to the bradycardic agent alinidine (353), was recently shown to block sinoatrial Ih
(208). In mice lacking all three types of ␣2-receptors,
clonidine still exerts a profound bradycardic activity, indicating that inhibition of cardiac Ih contributes significantly to the netto-bradycardic effect of clonidine. Electrophysiological recordings revealed that clonidine
blocks HCN4 and HCN2 (IC50 of ⬃10 ␮M) and also,
though with less sensitivity, HCN1 (IC50 of ⬃40 ␮M). Like
ZD7288, clonidine also shifts the voltage dependence of
the channel by 10 –20 mV to more hyperpolarizing potentials (208).
With the exception of ivabradine, HCN channel blockers
were not introduced to therapy so far. A major obstacle of
most existing agents is that they are not specific enough
for sinoatrial (mainly HCN4-mediated) Ih, but also block
neuronal Ih in several regions of the nervous system. For
example, visual disturbances that result from block of
retinal Ih have been reported for several of these drugs
(78). Moreover, some of the blockers may also interact
with other ion channels. For example, recently it was
reported that the widely used “specific” Ih blocker ZD7288
inhibits T-type Ca2⫹ currents in rat hippocampal pyramidal cells (330).
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IX. HCN CHANNELS AS NOVEL
DRUG TARGETS
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BIEL ET AL.
slows the activation of native and expressed HCN channels (67, 233).
B. Blockers of Neuronal Ih
Physiol Rev • VOL
X. CONCLUSIONS AND FUTURE DIRECTIONS
Thirty years after the first discovery of Ih and 10
years after the cloning of HCN channels, the field of
hyperpolarization-activated cation channels has emerged
into one of the most exciting areas of ion channel research. Starting as an electrophysiological curio (the
“funny” current), Ih channels are now considered as important regulators of many fundamental processes in nervous system, as well as in the heart. Extensive studies
with native and heterologously expressed HCN channels,
combined with modeling approaches, biochemistry, and
X-ray crystallography, have dramatically increased our
knowledge on the structural determinants of HCN channel function. Moreover, genetic mouse models are now
available to study the physiological role of individual HCN
channel types in vivo. Despite the progress made, numerous key questions are still unsolved and will have to be
addressed by future approaches. In the following, a small
selection of important problems is listed.
1) What is the structural basis for the “inverse” gating
and the mixed K⫹/Na⫹ permeability of HCN channels?
How is binding of cAMP to the CNBD coupled to the
activation gate of the channel?
2) Which cellular mechanisms control the subunit
stoichiometry of HCN channels? Can HCN channel subunits freely assemble to (hetero)tetramers or are there
restraints limiting free assembly?
3) Like other transmembrane proteins, HCN channels are probably associated with cellular proteins in
large macromolecular complexes. It is important to identify the constituents of these complexes in different types
of neurons and heart cells to understand how HCN channels are regulated in vivo under physiological conditions
and in diseased states.
4) Which factors control the expression, the cellular
processing, and the targeting of HCN channels to specific
cellular compartments?
5) What is the detailed role of HCN channels in
various diseases? The analysis of advanced genetic mouse
models in conjunction with studies in human patients will
be required to reach this goal.
6) Finally, given that HCN channels are relevant as
disease factors, the full potential of these proteins as drug
targets has to be further explored. To this end, highaffinity, subtype-specific HCN channel blockers must be
developed.
Last but not least, HCN channels themselves may be
useful as therapeutic agents. Currently, many laboratories
develop strategies to generate so-called “biological” pacemakers (for recent reviews, see Refs. 90, 244, 351). Bio-
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Modulation of Ih may also be a promising approach
for treatment of disease processes in the central and
peripheral nervous systems. In the previous section we
discussed that upregulation of Ih in DRGs and peripheral
nerves is an important process in the pathogenesis of
neuropathic pain. Therefore, blockers of Ih may be beneficial to analgesic therapy. In principle, the existing Ih
blockers could be used to treat neuropathic pain. However, if applied systemically, all known blockers would
also exert bradycardic effects. To circumvent this problem, agents with high sensitivity to the HCN channel types
relevant for neuropathic pain (mainly HCN1 and HCN2)
would be wishful. Conversely, these blockers should not
interfere with the function of sinoatrial HCN4. Drugs
displaying this pharmacological profile do not exist so far,
but given the wealth of chemical structures relevant to Ih
channel block it should be a feasible task to develop
subtype-specific blockers. The number of potential chemical entities related to Ih block is increased by agents that
have been shown to block Ih in addition to their wellknown primary receptor. For example, it was recently
found that loperamide, a potent ␮-opioid-receptor agonist, blocks Ih of DRGs with quite high affinity (IC50
between 5 and 10 ␮M depending on the DRG type) (404).
In addition, capsazepine, a well-known inhibitor of the
vanilloid-receptor (TRPV1), blocks HCN1 in a concentration-dependent manner (IC50 ⫽ 8 ␮M) (154). The development of structural analogs of the mentioned agents may
yield compounds that selectively inhibit specific HCN
channel types.
The use of Ih blockers has also been implicated in
therapy of epilepsies. However, given the complexity and
diversity of the cellular mechanisms leading to these diseases, a clear concept for a rational design of antiepileptic
Ih channel modulators has not yet emerged. A major
conceptual problem is that both inhibition and activation
of Ih may be beneficial depending on the type of epilepsy.
For example, stereotactic injection of the Ih blocker
ZD7288 reduced the generation of hippocampal epileptiform discharges in rabbit (207). On the other site, there is
evidence that part of the antiepileptic activity of lamotrigine (an established blocker of voltage-gated Na⫹
channels) is caused by an upregulation of Ih in dendrites
of pyramidal neurons (309, 311). Similarly, the antiepileptic drug gabapentin, which was shown to inhibit Ca2⫹
currents by binding to the ␣2␦-subunit, may also act in
part via upregulation of Ih (381).
Finally, HCN channels may contribute to the clinical
actions of general anesthetics. Native neuronal Ih as well
as heterologously expressed HCN channels are inhibited
by clinically relevant concentrations (ⱕ0.5 mM) of the
inhalational anesthetics halothane and isoflurane (59, 87).
Similarly, the intravenous anesthetic propofol inhibits and
FUNCTION OF HCN CHANNELS
ACKNOWLEDGMENTS
We apologize to all our colleagues whose studies have not
been cited because of space constraints.
Addresses for reprint requests and other correspondence:
M. Biel, Center for Integrated Protein Science CIPS-M and Zentrum für Pharmaforschung, Department Pharmazie, LudwigMaximilians-Universität München, Butenandtstr. 5-13, D-81377
Munich, Germany (e-mail: [email protected]); C.
Wahl-Schott, Molekulare Pharmakologie, Zentrum für Pharmaforschung, Department Pharmazie, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377 Munich, Germany
(e-mail: [email protected]).
GRANTS
This work was supported by the Deutsche Forschungsgemeinschaft, the Center for Integrated Protein Science CIPS-M,
and the EU research project NORMACOR (LSHM-CT-2006018676) within the Sixth Framework Programme of the European Union.
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