Physiology and Pharmacology of The Cardiac Pacemaker (Bfunnyq) Current

Pharmacology & Therapeutics 107 (2005) 59 79
www.elsevier.com/locate/pharmthera
Associate editor: G.E. Billman
Physiology and pharmacology of the cardiac pacemaker (bfunnyQ) current

Mirko Baruscotti, Annalisa Bucchi, Dario DiFrancescoT
Laboratory of Molecular Physiology and Neurobiology, Department of Biomolecular Sciences and Biotechnology, University of Milano,
via Celoria 26, 20133 Milan, Italy
Abstract
First described over a quarter of a century ago, the cardiac pacemaker bfunnyQ (I f) current has been extensively characterized since, and its
role in cardiac pacemaking has been thoroughly demonstrated. A similar current, termed I h, was later described in different types of neurons,
where it has a variety of functions and contributes to the control of cell excitability and plasticity. I f is an inward current activated by both
voltage hyperpolarization and intracellular cAMP. In the heart, as well as generating spontaneous activity, f-channels mediate autonomicdependent modulation of heart rate: h-adrenergic stimulation accelerates, and vagal stimulation slows, cardiac rate by increasing and
decreasing, respectively, the intracellular cAMP concentration and, consequently, the f-channel degree of activation. Four isoforms of
hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have been cloned more recently and shown to be the molecular
correlates of native f-channels in the heart and h-channels in the brain. Individual HCN isoforms have kinetic and modulatory properties
which differ quantitatively. A comparison of their biophysical properties with those of native pacemaker channels provides insight into the
molecular basis of the pacemaker current properties and, together with immunolabelling and other detection techniques, gives information on
the pattern of HCN isoform distribution in different tissues. Because of their relevance to cardiac pacemaker activity, f-channels are a natural
target of drugs aimed at the pharmacological control of heart rate. Several agents developed for their ability to selectively reduce heart rate act
by a specific inhibition of f-channel function; these substances have a potential for the treatment of diseases such as angina and heart failure.
In the near future, devices based on the delivery of f-channels in situ, or of a cellular source of f-channels (biological pacemakers), will likely
be developed for use in therapies for diseases of heart rhythm with the aim of replacing electronic pacemakers.
D 2005 Elsevier Inc. All rights reserved.
Keywords: I f current; Funny current; f-Channels; HCN; Bradycardic agents
Abbreviations: CNBD, cyclic nucleotide-binding domain; CNG, cyclic nucleotide-gated; HCN, hyperpolarization-activated, cyclic nucleotide-gated; SAN,
sinoatrial node.
Contents
1.
2.
3.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Sinoatrial I f current and the mechanism of cardiac pacemaking . .
2.1. Early experiments . . . . . . . . . . . . . . . . . . . . . .
2.2. Biophysical properties . . . . . . . . . . . . . . . . . . . .
2.2.1. Voltage dependence of I f kinetics . . . . . . . . .
2.2.2. Kinetics. . . . . . . . . . . . . . . . . . . . . . .
2.2.3. Ionic nature. . . . . . . . . . . . . . . . . . . . .
2.3. Contribution to automaticity. . . . . . . . . . . . . . . . .
2.4. Autonomic modulation . . . . . . . . . . . . . . . . . . .
2.5. Single f-channel recording . . . . . . . . . . . . . . . . .
Molecular determinants of the I f current . . . . . . . . . . . . . .
3.1. Hyperpolarization-activated, cyclic nucleotide-gated clones .
T Corresponding author. Tel.: +39 02 50314931; fax: +39 02 50314932.

E-mail address: dario.difrancesco@unimi.it (D. DiFrancesco).
0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2005.01.005
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
60
61
61
61
61
63
64
64
64
65
65
65
60
M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979
3.2.
Structurefunction relationships for hyperpolarization-activated,

cyclic nucleotide-gated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. Structures involved in voltage-dependent gating . . . . . . . . . . . . . . . .
3.2.2. Structures involved in cAMP-dependent gating . . . . . . . . . . . . . . . .
3.3. Hyperpolarization-activated, cyclic nucleotide-gated isoforms in sinoatrial node tissue
4. f-Channel blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Alinidine (ST567). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Zatebradine (UL-FS 49) and cilobradine (DK-AH269) . . . . . . . . . . . . . . . . .
4.3. ZD-7288 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Ivabradine (S16257) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Future directions: the biological pacemaker . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
In mammals, cardiac pacemaking originates in a highly
specialized structure located in the wall of the right atrium,
the sinoatrial node (SAN). SAN myocytes generate spontaneous action potentials; these propagate, through specialized
conduction systems, first to the atria and then to the
ventricles, and thus drive cardiac rhythmic contractions.
The control of cardiac rate is a process of major
physiological relevance, and it is not surprising that the
identification of the electrical events underlying sinoatrial
pacemaker activity has attracted the constant interest of
cardiac physiologists.
During diastole, corresponding to mechanical relaxation,
myocytes of the working myocardium (atrial and ventricular
cells) lack electrical activity and normally rest at a hyperpolarized voltage level. Spontaneously beating SAN myocytes, on the contrary, are characterized by the presence of a
bslow diastolicQ depolarization phase of the action potential.
At the termination of one SAN action potential, the
membrane voltage does not stabilize to a negative level
but slowly creeps up with an approximately constant slope,
until it reaches the threshold for a new SAN action potential
(DiFrancesco, 1993).
The slow diastolic bpacemakerQ depolarization is responsible for the generation of repetitive activity and has
therefore been the focus of intense studies aimed to the
understanding of the cellular mechanisms that generate it.
Although the cellular processes ultimately contributing,
directly or indirectly, to the pacemaker depolarization are
many, and their involvement is still a subject of investigation and debate (DiFrancesco & Robinson, 2002;
Vinogradova et al., 2002), there is now general agreement
that a major role in the generation and control of this phase
is played by the so-called bpacemakerQ (funny, I f) current.
Since its discovery in the SAN (Brown et al., 1979), the
funny current has been thoroughly investigated in cardiac
pacemaker, and detailed knowledge of several of its
properties has been obtained. Importantly, a hyperpolarization-activated current similar to I f (I h current) has also been
described in a large number of different types of neurons,
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
66
68
69
69
70
71
71
72
72
74
75
75
75
where it contributes to a wide range of physiological

functions such as rhythmic firing, regulation of neuronal
excitability, sensory transduction, synaptic plasticity, and
more (Pape, 1996; Robinson & Siegelbaum, 2003). Despite
the early description of the funny current in the SAN, the
molecular components of f-channels were particularly
elusive, and their cloning was only achieved by chance in
the late 1990s. The first clone to be obtained (BCNG1), was
originally thought to be a novel K+ channel related to Eag
and cyclic nucleotide-gated (CNG) channels (Santoro et al.,
1997) and only later found to have properties expected from
f/h-channel subunits (Santoro et al., 1998). Further cloning
revealed the existence of four isoforms forming a new
family of hyperpolarization-activated, cyclic nucleotidegated channels (HCN1 to 4) and belonging to the superfamily of voltage-dependent K+ (Kv) and cyclic nucleotidegated (CNG) channels (see reviews by Ludwig et al., 1999a;
Santoro & Tibbs, 1999; Kaupp & Seifert, 2001; Accili et al.,
2002; Robinson & Siegelbaum, 2003). A thorough investigation has shown that the 4 HCN isoforms have different
properties and variable patterns of expression, and that
different isoforms can colocalize to form heteromultimers
with specific kinetic and modulatory properties.
Given its specific functional relevance in cardiac pacemaking and rate regulation, the funny current has long been
considered a primary target for the development of drugbased therapeutic strategies aiming to a selective control of
heart rate. Drugs targeting this current would selectively
alter the diastole, avoiding undesirable and potentially
proarrhythmic effects on other phases of the action
potential. The selection of drugs specifically interacting
with funny channels has advanced rapidly in the last few
years and is now moving from basic research towards
clinical application. A successful search for these drugs may
have a significant impact on specific cardiac therapies. The
use as pharmacological tools of drugs interacting with ion
channels or with their function is indeed widespread, since
several diseases such as epilepsy, long QT syndrome, cystic
fibrosis, myopathies, and others have been found to depend
on abnormalities of genes coding for channel proteins or
auxiliary subunit proteins.
In this review we will focus on the properties of cardiac

f- channels, most of which are analyzed in SAN pacemaker
cells, and address their physiological role, molecular
composition, localization, and interaction with drugs.
Potential applications to the therapeutic use of the functional
role of funny channels in the generation of spontaneous
activity and heart-rate regulation will also be addressed.
2. Sinoatrial I f current and the

mechanism of cardiac pacemaking
2.1. Early experiments
Early experiments investigating the nature of the
diastolic depolarization were conducted in Purkinje fibres
and led to the hypothesis that this process was due to the
decay of the delayed K+ conductance activated during the
preceding action potential (Weidmann, 1951). Results
apparently compatible with this assumption were obtained
by measurements of conductance changes in voltage clamp
conditions (Vassalle, 1966). The idea received further
confirmation with the finding in Purkinje fibres of the socalled bpacemakerQ (I K2) current, described as a pure K+
current activated upon depolarization in the diastolic range
of voltages (Noble & Tsien, 1968; Peper & Trautwein,
1969); the assumption of a pure K+ ionic nature for I K2
depended essentially on evidence for a current reversal near
the expected K+ equilibrium potential. Finally, the relevance
of I K2 to pacemaking was strengthened by evidence that this
component was modified by catecholamines and was
responsible for the acceleration of rate caused by sympathetic stimulation (Hauswirth et al., 1968).
The experimental evidence ruling in favour of the bK+
current-decayQ hypothesis was therefore apparently incontrovertible, and for over a decade, the I K2 current was
considered as one of the most typical K+ cardiac components (Hille & Schwarz, 1978). Yet, as we now know, the
I K2 interpretation and, consequently, the K+-current decay
hypothesis were deeply incorrect. In the late 1970s and early
1980s, a new set of experimental data appeared which led to
the demonstration that the Purkinje fibre pacemaker current
was not at all an outward current activated on depolarization, it was no less than just the opposite, an inward current
activated on hyperpolarization (DiFrancesco, 1981a,b). The
main reason for the incorrect interpretation was the presence
of a bfakeQ reversal potential close to the expected K+
equilibrium potential during voltage-clamp hyperpolarization, due to the superimposition of an inward activating
current (I f) and a large inward decaying component caused
by the depletion of K+ ions from the extracellular clefts
(DiFrancesco & Ojeda, 1980).
A crucial finding that contributed essentially to this
reintrepretation was the discovery of the bfunnyQ current in
the mammalian SAN. In 1979, the first detailed report of
this current appeared, describing its elementary properties
61
and involvement in the generation and catecholamineinduced acceleration of SAN spontaneous activity (Brown
et al., 1979). Among the unusual features which justified the
name bfunnyQ were a mixed Na+ and K+ permeability,
activation on hyperpolarization, and very slow kinetics
(Brown & DiFrancesco, 1980; Yanagihara & Irisawa, 1980).
Although uncommon, these features were quite appropriate
for I f to function as an efficient bpacemakerQ current
involved in the generation of diastolic depolarization and
in rate control by h-adrenergic stimulation. The Purkinje
fibres pacemaker current I K2 was then re-interpreted and
shown to be identical to I f in the SAN based on several bits
of evidence, including the abolishment of I K2 reversal near
the K+ equilibrium potential by the simple perfusion with
Ba2+, a K+ current blocker (DiFrancesco, 1981a). This latter
result was particularly impressive in that it unmasked the
real inward nature of the Purkinje fibres pacemaker current
and allowed, for the first time, to visualize the conversion of
I K2 into an inward, hyperpolarization-activated current. The
inward ionic nature of I K2 was confirmed by ion-substitution experiments (DiFrancesco, 1981b), revealing a mixed
Na+ and K+ permeability similar to that of the nodal I f.
The novel description of I f and the reinterpretation of the
Purkinje fibres I K2 confirmed the identity of the pacemaker
mechanisms in the 2 cardiac tissues and paved the way to an
integrated view of cardiac pacemaking in the different
pacing regions of mammalian heart. According to this view,
the diastolic depolarization simply reflects the slow activation of f-channels taking place when, at the termination of
an action potential, the membrane voltage enters the range
of I f activation (DiFrancesco, 1985, 1993).
2.2. Biophysical properties
The typical features of the I f current include hyperpolarization-induced activation, slow kinetics of activation
and deactivation, Na+ and K+ ionic nature, and modulation
by cAMP. Following its original description in the SAN,
several studies identified I f also in atrial and ventricular
myocytes (Carmeliet, 1984; Zhou & Lipsius, 1992, 1993;
Yu et al., 1993, 1995; Cerbai et al., 1994; Porciatti et al.,
1997; Hoppe & Beuckelmann, 1998; Zorn-Pauly et al.,
2004). We will now discuss these properties in the cardiac
SAN and extend the analysis to other cardiac tissues where
appropriate.
2.2.1. Voltage dependence of I f kinetics
The voltage range where diastolic depolarization occurs
in pacing myocytes is determined primarily by the voltage
range of I f activation and by its kinetics. This explains, for
example, why diastolic depolarization in SAN myocytes
occurs at more depolarized voltages than in Purkinje fibres,
where the I f activation range is normally tens of millivolts
more negative than in the SAN (see Table 1). Even within
the SAN area, the I f activation range varies from cell to cell,
shifting, for example, to more negative levels when moving
62
from the central to the peripheral node, reflecting the

phenotypic and functional tissue heterogeneicity of this
cardiac region (Boyett et al., 2000). It is therefore not
surprising that a large variability of I f activation threshold
and mid-activation voltages has been reported. Table 1 lists
the I f kinetic and modulatory parameters reported for
different types of cardiac myocytes in various experimental
conditions.
Typical values of mid-activation voltages of I f currents
recorded in the SAN cells are in the range of 60 to 70
mV, in agreement with the function of I f in the pacemaker

depolarization, although more positive and more negative
values are reported. Reasons for this variability have been
discussed elsewhere (DiFrancesco, 1991). I f, when detected,
activates at more negative voltages in atrial and ventricular
myocytes, reflecting the lack of a functional role in
physiological conditions. Interestingly, the expression of I f
in the working myocardium changes with the developmental
stage and in pathological conditions. In newborn rats, for
example, I f is expressed at significant levels and is activated
Table 1
Data refer to microelectrode or whole-cell recordings (33 8C V T V 37 8C), except in shaded rows, where data are from cell-attached or inside-out patch
recordings (room T); values extracted by eye from publication are in italic
Threshold
(mV)
Rabbit SAN
-50
-40
V1/2
(mV)
-58
-66.3
-61.9
t1/2
(s)
0.707 @ -70
0.319 @ -100
-50
-32
-60
-55
-70
Cat
SAN
Porc SAN
Mouse
SAN
Calf Purk.
Canine
Purkinje
Human
atrium
Canine
ventricle
Rat
ventricle
-80.5
-50
-78.7
-88.3
-87
-70
-80
-100
-50
-60/-70
-137
-120
-120
-70
Cholinergic
-91
-106.1
-86.7
-89.3
-140.6
-150
-92.9
-93.9
-87.9
-108
-60
-138
-89.5
-120
-80
-60
-145
3
30
12
11
1
10
0.35 @ -84
0.25 @ -94
13.2
11
10.2
11.1
0.74 @ -100
0.28 @ -120
8
22
32
15
15
14
4
31
32
26
21
1.07 @ -90
0.70 @ -120
0.91 @ -100
2.55 @ -100
9
29
19
23
19
6.1
6.8
21
26
24
5
2.32 @ -120
0.306 @ -100
0.152 @ -120
16.0 (1)
4.2 (2)
7.8
10.4
-80.6
13
2
18
-9.9
-6
3.2 @ -70
0.808 @ -100
-8.4
Notes
cAMP
-12.6
-80
-96.5
-93.1
-91.2
-121.9
-50
-113 adult
-72 newborn
-80
-60
Mouse
embr. vent
G. pig vent
Human
ventricle
-68
-65.6
-52
5.3
14.8
5.8
9.62
-67.1
Ref.
Shift activation curve (mV)

Adrenergic
1, 2
1
1
2, 3
2
4
4
4
1
5
1
1
25
25
24
6
5
6
16
27
5
7
24
20
7
5
1, 5, 8
at physiological voltages, but its function is lost with

adulthood (Robinson et al., 1997). On the other hand, in an
animal model of cardiac hypertrophy (spontaneous hypertensive rats), adult I f expression increases substantially
relative to control animals (Cerbai et al., 1996).
As well as by cAMP, which mediates autonomicdependent modulation (see below), I f kinetics and activation
range are modified by other mechanisms, such as auxiliary
subunits (Qu et al., 2004), phosphorylation-dependent
processes (Chang et al., 1991; Accili et al., 1997), and
interacting structural proteins that can affect the channel
sub-membrane localization (Barbuti et al., 2004; Gravante et
al., 2004). These mechanisms may act synergistically to
fine-tune the current activation range and kinetics and thus
set the amount of current that can be recruited at appropriate
times during cell activity. It is likely that additional, still
unidentified processes play a role in the control of I f
activation range. Evidence for such processes comes, for
example, from the presence of current run-down during
whole-cell recording in SAN myocytes, involving a leftward
shift of the current activation curve (DiFrancesco et al.,
1986), and the abrupt negative shift of the activation curve
when membrane macro-patches are excised from the
63
membrane during transition from cell-attached to inside

out configuration (DiFrancesco & Mangoni, 1994). These
phenomena suggest that cytoplasmic and/or cytoskeleton
integrity is a necessary requirement for a correct channel
functioning and that dilution by patch pipette or bath
solution of the channel intracellular micro-environment
affects the channel properties. This view is strengthened
by more recent evidence indicating the existence of a
bcontext dependenceQ of pacemaker channel properties,
based on the observation that identical pacemaker channel
isoforms (see Section 3) have quantitatively different
properties when expressed in different expression systems
(Qu et al., 2002).
2.2.2. Kinetics
Several types of mechanisms have been used in the
literature to describe I f activation and deactivation processes, including single- (DiFrancesco & Noble, 1985;
McCormick & Pape, 1990) and double-exponential HodgkinHuxley kinetics (Noble et al., 1989; van Ginneken &
Giles, 1991; Demir et al., 1994), and more complex, nonHodgkinHuxley kinetics (DiFrancesco, 1984), often
reflecting the different kind of accuracy required for specific
Notes to Table 1:
V 1/2 is the half-activation voltage and t 1/2 is the half-activation time upon stepping to a given potential.
Notes: (1) Isoprenaline 110 AM; (2) acetilcholine 110 AM; (3) saturating effect from Hill plot; (4) cAMP 10100 AM; (5) room T; (6) h1 stimulation
(noradrenaline 1 AM), h2 stimulation (isoprenaline 1 AM); (7) forskolin 10 AM; (8) carbachol 100 AM.
References for table:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Accili et al. (1996). Pflugers Arch 431, 757

Accili et al. (1997). Am J Physiol 272, H1549
Accili et al. (1997). J Physiol 500, 643
Bois et al. (1997). J Physiol 501, 565
Cerbai et al. (1996). Circulation 94, 1674
Cerbai et al. (1999). Cardiovasc Res 42, 121
Cerbai et al. (2001). J Mol Cell Cardiol 33, 441
Denyer et al. (1990). J Physiol 428, 405
DiFrancesco (1981). J Physiol 314, 377
DiFrancesco et al. (1986). J Physiol 377, 61
DiFrancesco et al. (1989). Science 243, 669
DiFrancesco et al. (1991). Pflugers Arch 417, 611
DiFrancesco et al. (1991). Nature 351, 145
Fares et al. (1998). J Physiol 506, 73
Frace et al. (1992). J Physiol 453, 307
Hagiwara et al. (1989). J Physiol 409, 121
Hoppe et al. (1998). Cardiovasc Res 38, 788
Hoppe et al. (1998). Circulation 97, 55
Mangoni et al. (2001). Cardiovasc Res 52, 51
Maruoka et al. (1994). J Physiol 477, 423
Porciatti et al. (1997). Br J Pharmacol 122, 963
Ranjan et al. (1998). Biophys J 74, 1850
Robinson et al. (1997). Pflugers Arch 433, 533
Shibata et al. (1999). Br J Pharmacol 128, 1284
Yasui et al. (2001). Circ Res 88, 536
Yu et al. (1993). Circ Res 72, 232
Yu et al. (1995). J Physiol 485, 469
Zaza et al. J Physiol 491, 347
Zhou et al. (1992). J Physiol 453, 503
Zhou et al. (1993). Pflugers Arch 423, 442
64
analysis. Detailed investigation reveals that several kinetic

features of I f cannot be accommodated by simple Hodgkin
Huxley type of gating and require complex multistate
kinetic modelling based on the existence of distinct
bdelayingQ and proper bgatingQ processes (DiFrancesco,
1984). A similar approach has been used to describe the
kinetics of HCN channels. As is further discussed below
(see Section 3.2), an ballostericQ dual voltage and cAMPdependent kinetic model, which views HCN channels as
tetramers carrying voltage sensors that can be gated
individually by voltage and undergoes concerted open/
closed allosteric transitions, can reproduce most of their
kinetic properties (DiFrancesco, 1999; Altomare et al.,
2001).
2.2.3. Ionic nature
Early evidence on the ion permeation properties of
pacemaker channels derives from experiments in Purkinje
fibres and in isolated rabbit SAN myocytes (DiFrancesco,
1981b; DiFrancesco et al., 1986). Values of the reversal
potential measured in these experiments were in the range of
10 to 20 mV, pointing to a mixed ionic permeability.
Ionic substitution experiments indeed identified Na+ and K+
ions as the physiological carriers of the current, with a Na+/
K+ permeability ratio of about 0.27 (DiFrancesco, 1981b;
Frace et al., 1992). The conductance of f-channels was also
shown to increase with external K+ concentration (DiFrancesco, 1981b) in a way similar to that of other K+-permeable
channels (Hille, 2001). This effect is physiologically
relevant since a higher external K+ concentration tends to
decrease, by depolarization, the fraction of f-channels
activated at the termination of an action potential and thus
decrease substantially the slope of diastolic depolarization
and heart rate; an increase in the I f conductance may thus
compensate, at least partially, for the bradycardic effect of
hyperkalemia.
stages and disappear together at later stages of development,

and in zebrafish heart, where a bradycardic mutant was
found to express, among all current systems investigated,
only a reduced I f in cardiac myocytes (Baker et al., 1997;
Warren et al., 2001).
A more recent investigation based on the overexpression
of HCN2 channels, one of the isoforms composing native fchannels (see Section 3), in embryonic rat ventricular
myocytes has shown a large increase of the rate of
spontaneous activity correlated to the expression of pacemaker channels (Qu et al., 2001). Further, the overexpression of a nonfunctional HCN2 isoform induced dominantnegative suppression of native pacemaker channel activity
and strongly depressed pacing in newborn ventricular
myocytes (Er et al., 2003).
The use of molecules specifically inhibiting I f also
provides evidence for a strict correlation between I f current
availability and the presence of spontaneous activity in
pacemaker cells. For example, drugs which block f-channels
with a high degree of selectivity, such as UL-FS49
(zatebradine), ZD7288, and S16257 (ivabradine), act as
pure bheart rate-reducingQ agents by decreasing the slope of
diastolic depolarization and, as such, have a potential for
therapeutic use in cardiac diseases whose prognosis can be
alleviated by moderate bradycardia (DiFrancesco & Camm,
2004). Heart rate-reducing drugs slow heart rate in a
concentration-dependent way, reflecting the proportionality
between I f inhibition and slowing effect. This aspect is
further developed below (see Section 4).
Finally, a direct demonstration of the I f role in pacemaker
generation and control is illustrated by the action of
autonomic neurotransmitters. As is outlined in the Section
2.4, opposite chronotropic effects exerted by sympathetic
and parasympathetic stimuli, particularly at low levels of
autonomic activity, are mediated by cAMP-dependent
modulation of I f, which underlies rate changes by the
modification of the slope of diastolic depolarization.
2.3. Contribution to automaticity

2.4. Autonomic modulation
Several direct and indirect experimental observations
substantiate the key role of I f in cardiac pacemaking. These
include the actual ionic and kinetic properties of I f
mentioned above, which are particularly appropriate for
the generation of the slow diastolic depolarization in the
pacemaker range of voltages, and the correlation between I f
expression and the presence of spontaneous activity in
cardiac cells. This correlation is evident by simply comparing different cell types in an adult mammalian heart, where
pacemaking cells such as SAN or atrio-ventricular cells do,
while atrial and ventricular myocytes, quiescent if not
stimulated, do not normally express I f at physiological
voltages. Further, the expression of I f correlates with
spontaneous activity in the developing newborn chick
(Satoh & Sperelakis, 1993) or mammalian (Robinson et
al., 1997; Yasui et al., 2001) ventricular cells, where I f and
spontaneous activity are simultaneously present at early
The mammalian SAN region is richly innervated by the

autonomic nervous system, which exerts a direct control of
pacemaker activity. Sympathetic stimulation accelerates,
and parasympathetic stimulation slows, heart rate acting
through h-adrenergic and muscarinic receptors, respectively.
The original description of I f already indicated the involvement of this current system not only in the generation, but
also in the positive chronotropic effect of adrenaline (Brown
et al., 1979). Subsequent work showed that this action is due
to a shift of the I f activation curve to more positive voltages
(DiFrancesco et al., 1986; DiFrancesco & Mangoni, 1994).
These results were in agreement with previous indications
on I K2 in Purkinje fibres: despite the incorrect interpretation
of I K2 as a pure K+ current, investigation of the action of
adrenaline had provided evidence for a rightward shifting
action of adrenaline on the I K2 activation curve (Hauswirth
et al., 1968), an effect mediated by increased intracellular

cAMP (Tsien et al., 1972).
How does therefore adrenaline accelerate the heart? The
adrenaline-induced shift of the I f activation curve to more
positive voltages, caused by increased cAMP levels
subsequent to hAR-stimulation of adenylate cyclase,
increases the degree of steady-state current activated at
any potential (within the activation range), thus increasing
the current availability during diastolic depolarization and,
as a consequence, the rate of development of diastolic
depolarization itself (DiFrancesco, 1993).
The realization of the three basic steps of the mechanism
by which sympathetic activity increases I f and accelerates
cardiac rate, i.e. (1) hAR stimulation and coupling to the
stimulatory G-protein Gs, (2) activation of adenylate cyclase
and cAMP increase, and (3) activation of f-channels and rate
acceleration, naturally posed the question whether this
mechanism could operate in the opposite direction, i.e. to
decrease I f, thereby slowing the rate. Indeed, later work
showed that, as well as being activated by adrenaline, I f is
also inhibited by acetylcholine (DiFrancesco & Tromba,
1987). The stimulation of muscarinic receptors was shown
to shift the I f activation curve to more negative voltages,
according to a process exactly opposite to that of hAR
stimulation, and involving Gi-protein-dependent inhibition
of cAMP synthesis (DiFrancesco & Tromba, 1988a,b). ACh
induced a negative shift of the I f activation curve without
changing the fully activated I/V relation, indicating a
modification of gating, but not of the channel conductance
(DiFrancesco & Tromba, 1988a).
The results above suggested a major role for I f in the
negative chronotropic action of vagal activity. However,
early work had identified an ACh-activated K+ current
(I K,ACh) as the main process underlying slowing (Sakmann
et al., 1983). What was the relative importance of the 2
processes? This issue was resolved with the demonstration
that ACh acts to inhibit I f at much (20-fold) lower
concentrations than those required to activate I K,ACh, and
that concentrations of ACh inhibiting I f were effective to
slow rate (DiFrancesco et al., 1989). The comparison
between ACh action on I f and I K,ACh was the first
demonstration that cardiac rate slowing by low ACh doses
(i.e. moderate vagal activity) is due to muscarinic-induced I f
inhibition, caused by decreased cAMP levels and a negative
shift of the I f activation curve, and the consequent reduction
of the rate of diastolic depolarization.
Data collected by experimentation in the SAN had
therefore provided evidence for a key role of I f in both
the adrenergic and cholinergic modulation of cardiac rate
mediated by the increase and decrease, respectively, of
intracellular cAMP (DiFrancesco et al., 1986; DiFrancesco
& Tromba, 1987, 1988a,b). This evidence was in accordance with the established notion that, in cardiac cells, the
stimulation of h-adrenergic receptors activates, and of
muscarinic receptors inhibits, adenylate cyclase and cAMP
production; it also identified a role for cAMP, which, by
65
promoting a depolarizing shift of the I f activation curve, acts

as a second messenger in f-channel modulation. The mode
of action of cAMP was investigated by the use of insideout
macro-patch analysis in SAN cells and led to the surprising
finding that f-channels are activated by cAMP through the
direct binding of cAMP molecules to channels, and not by
phosphorylation, as it occurs with other channels such as,
for example, the L-type Ca2+ channels (DiFrancesco &
Tortora, 1991). This was the first evidence that funny
channels, as well as being voltage-gated, shared with
another class of channels, the cyclic nucleotide-gated
(CNG) channels of sensory neurons, the property of being
directly gated by cyclic nucleotides.
2.5. Single f-channel recording
There are few reports of single-channel recording for the
pacemaker current, performed in isolated SAN myocytes
(DiFrancesco et al., 1986; DiFrancesco & Mangoni, 1994).
This limited amount of data likely reflects the intrinsic
difficulty of measuring tiny, single-channel events (order of
0.050.1 pA) given the small single-channel conductance of
f-channels (about 1 pS, DiFrancesco, 1986). Despite the
paucity of data, single f-channel recording has allowed us to
understand features of the molecular mechanisms underlying f-channel dual gating by voltage hyperpolarization
and cAMP, and the neurotransmitter-induced f-channel
modulation.
For example, single-channel data have shown that the
hAR-induced current activation is due to an increased open
channel probability rather than to an increased singlechannel conductance, in agreement with the action of hAR
stimulation in whole-cell conditions, which leads to a shift
of the current activation curve to more positive voltages
without modification of the fully activated I/V relation
(DiFrancesco et al., 1986). cAMP was shown to activate fchannels by binding directly to the intracellular aspect of the
channel (DiFrancesco & Tortora, 1991), which, in accordance to the whole-cell data, resulted in an increase of singlechannel open probability, with no changes of channel
conductance (DiFrancesco & Mangoni, 1994).
3. Molecular determinants of the I f current

3.1. Hyperpolarizationactivated, cyclic nucleotide-gated clones
A major progress in the understanding of the molecular
basis of the properties of pacemaker channels was achieved
with the cloning of the Hyperpolarization-activated, Cyclic
Nucleotide-gated (HCN) family of channels in the late
1990s (Zagotta et al., 2003). The search for the pacemaker
channel clone had been a major task in several laboratories
for decades, but no direct approach yielded useful results
until, as it sometimes happens, the crucial step towards
66
cloning was made by chance while looking for proteins

interacting with the SH3 domain of the neural form of Src
(Santoro et al., 1997). The sequence thus identified was
homologous to eag K+ and cyclic nucleotide-gated (CNG)
channels and, although not immediately, was later used to
identify a complete ORF and recognized as a pacemaker
channel subunit based on its biophysical properties (Santoro
et al., 1998). Other members of the same family were soon
cloned in mammalian and non-mammalian tissues (Gaug et
al., 1998; Ludwig et al., 1998, 1999b; Ishii et al., 1999;
Seifert et al., 1999; Vaccari et al., 1999). In mammals, four
isoforms have been cloned (HCN1-4); human genes coding
for the 4 isoforms are all located in different chromosomes
(HCN1: 5p12; HCN2: 19p13.3, Vaccari et al., 1999; HCN3:
1q22; HCN4: 15q24-25, Seifert et al., 1999). In Fig. 1, the
alignment of the four human HCN isoforms is shown.
Based on their sequence, HCN channels are classified as
members of the superfamily of voltage-gated K+ (Kv) and
CNG channels; as such, they appear to have a tetrameric
composition (Ulens & Siegelbaum, 2003; Zagotta et al.,
2003), and are characterized by the presence of 6 transmembrane domains (S16) with a voltage sensor located in
the positively charged S4 domain, the GYG pore sequence
typical of K+-permeable channels, and a cyclic nucleotidebinding domain (CNBD) homologous to that of CNG
channels located in the C-terminus.
Sequence alignment revealed that the HCN bcoreQ
region, comprising the transmembrane and CNB domains,
is highly conserved among the different isoforms (z80%
identity), while sequences diverge at the N- and C-termini,
suggesting that the terminal regions are responsible for some
of the differences in the biophysical properties among
isoforms (Viscomi et al., 2001). Indeed, the properties of
different isoforms differ quantitatively. For example, the
activation/deactivation kinetics of HCN2 are faster than
those of HCN4 and slower than those of HCN1; typical
values of activation time constant at 95 mV are 0.11, 1.13,
and 2.52 s for HCN1, HCN2, and HCN4, respectively
(Altomare et al., 2001). HCN2 has a more negative
activation threshold than either HCN1 or HCN4 does;
typical values of the half-maximal activation voltage are
73, 81, and 92 mV for HCN1, HCN4, and HCN2,
respectively, when channels are expressed in HEK293 cells
(Accili et al., 2002), but several conditions may alter
significantly these values (see below). Finally, as with
native f-channels, cAMP activates HCN channels by
shifting the activation curve to more positive voltages, but
maximal shifts vary among isoforms, with HCN1 being
much less responsive (range 4.35.8 mV) than either HCN2
(range 16.919.2 mV) or HCN4 (range 11.123 mV; Ishii et
al., 1999; Ludwig et al., 1999b; Seifert et al., 1999; Moroni
et al., 2000; Viscomi et al., 2001; Wainger et al., 2001;
Wang et al., 2001; Altomare et al., 2003; Zagotta et al.,
2003). These differences appear to be determined by
differential inhibitory interactions of the C-termini with
the core transmembrane domains in the various isoforms,
more than by a variable cAMP binding affinity to the CNBD

(Wang et al., 2001). The properties of the HCN3 isoform
have been only partially investigated so far; HCN3 kinetics
are intermediate between those of HCN2 and HCN4
(Moosmang et al., 2001).
The different kinetic and modulatory properties of native
currents in various regions of the heart and brain (DiFrancesco, 1993; Pape, 1996) may thus simply reflect a different
tissue distribution of HCN isoforms. However, simple
electrophysiological analysis is not sufficient to reveal the
isoform composition of native channels, since several
conditions can contribute to modify the channel properties.
For example, native channels can be formed by heteromultimers of different isoforms, with properties intermediate
between those of individual components (Chen et al.,
2001b; Ishii et al., 2001; Ulens & Tytgat, 2001; Xue et
al., 2002; Altomare et al., 2003); also, HCN activity can be
modified by interaction with auxiliary subunits such as
MiRP1 (Qu et al., 2004) or with scaffold proteins such as
filamin-A (for HCN1, Gravante et al., 2004), or by specific
subcellular compartmentation such as the caveolar compartmentation of HCN4 in SAN cells (Barbuti et al., 2004);
finally, the expression of a given isoform may yield
quantitatively different biophysical properties according to
whether the isoform is expressed in heterologous or in
homologous expression systems, suggesting that a bcontextdependentQ modulation occurs (Qu et al., 2002). A more
detailed assessment of tissue distribution of specific HCN
isoforms therefore requires message and protein detection
assays such as Northern blot, RNase protection assay, and
immunolabelling. In heart HCN1, HCN2, and HCN4 are all
expressed, with HCN4 being the major component in the
pacemaker region, although low expressions of HCN1 and
HCN2 have also been reported (Santoro et al., 1998; Shi et
al., 1999; Moroni et al., 2001).
3.2. Structurefunction relationships for
hyperpolarization-activated, cyclic nucleotide-gated channels
Among the peculiar properties of bfunnyQ channels are
the dual dependence on voltage and cAMP, and the
hyperpolarization-induced activation (DiFrancesco, 1999).
These properties are typical of HCN channels and are not
shared by the other members of the superfamily of Kv and
CNG channels, which are gated by voltage only and cAMP
only, respectively; also, Kv channels open on depolarization, which raised the intriguing question of how similar
voltage sensor (S4) sequences could give rise to opposite
voltage dependences of gating.
The effect of cAMP is to shift the I f activation curve (i.e.
the single-channel open-probability curve) to more positive
voltages and to accelerate activation and slow deactivation
kinetics (DiFrancesco et al., 1986; DiFrancesco & Tortora,
1991; DiFrancesco & Mangoni, 1994). How does cAMP
cause these changes? The presence of a hyperpolarizing
shift of voltage dependence suggests that the gating
67
Fig. 1. Amino acid sequence alignment of the 4 human HCN isoforms. Transmembrane domains S1 to S6, pore helix P and the CNBD are indicated.
mechanism operated by voltage hyperpolarization and the

one operated by cAMP are the same, and that HCN channels
behave in the presence of a given cAMP concentration
simply as if they were experiencing a stronger voltage drop.
Indeed, the shifting action of cAMP can be explained by

assuming that channels behave according to an allosteric
model of channel kinetics (DiFrancesco, 1999; Altomare et
al., 2001). In this model, HCN channels are described as
68
tetramers whose four subunits undergo simultaneous,

concerted open/closed transitions according to an allosteric
reaction, and where each of the four subunits carries a
voltage sensor which is independently gated by voltage. The
channel open probability p o can then be described by a
modified Boltzmann equation:

1
po 1= 1 exp V V1=2 s =v
where V is voltage, V 1/2 is the half-activation voltage, v is
the inverse slope-factor, and s is a cAMP concentrationdependent term which represents the effects of cAMP on the
channel open/closed equilibrium (DiFrancesco, 1999). s is
clearly a shift of the voltage dependence of p o, showing that
the allosteric model assumptions are able to correctly
interpret the action of cAMP experimentally observed.
The shift depends on the cAMP concentration according
to the equation:
scAMP v ln1 cAMP=Ko =1 cAMP=Kc
2
where K o and K c are the dissociation constants of cAMP
binding to the channel in the open and closed states,
respectively. As illustrated in Fig. 2, this relation accounts
quantitatively for the observed sigmoidal dependence of the
shift on cAMP concentration (DiFrancesco & Tortora, 1991;
DiFrancesco, 1999). It is interesting to note that according
to this interpretation, the action of cAMP derives simply by
Fig. 2. Activation of f-channels by cAMP as explained according to an

allosteric model of channel gating. (A) The f-channel open probability
curve ( P o) measured by the voltage-ramp protocol in an inside-out macropatch in control conditions and during perfusion with 10 AM cAMP. The
action of cAMP is to shift the P o curve to more positive voltages (by about
14 mV in this case), thereby increasing the current availability for the
generation of diastolic depolarization, hence steepening its rate. (B) Dose
response relationship of the cAMP-induced shifts (squares, mean F SEM)
from 47 macro-patches. The full line is a best fit to data performed with Eq.
(2), yielding the values K o = 0.0578 AM and K c = 0.5416 AM. (C) Cartoon
illustrating the basic scheme of channel modulation by cAMP. Panels A and
B are modified from DiFrancesco (1999, with permission).
the assumption that cAMP molecules have a higher binding

affinity to open than to closed channel states (K obK c).
Several structurefunction studies have addressed these
aspects by investigating the HCN channel domains
involved in hyperpolarization-dependent and cAMPdependent gating.
3.2.1. Structures involved in voltage-dependent gating
HCN channels share several structural similarities with
voltage-gated K+ (Kv) channels, among which a positively
charged S4 domain, the putative voltage sensor which, in
HCN channels, includes 10 basic residues whose mutation
strongly affects the channel voltage dependence (Chen et
al., 2000; Vaca et al., 2000); however, while Kv channels
open on depolarization, HCN channels open on hyperpolarization. This difference could be due to an binvertedQ
movement of the voltage sensor of HCN vs. Kv channels in
response to the same voltage change, or to an binvertedQ
coupling between S4 movement and channel gating. The
latter possibility appears more likely since cysteine accessibility experiments have shown that, like in Kv channels,
hyperpolarization induces an inward movement of the S4
segment in HCN channels (Mannikko et al., 2002).
Although details of the mechanism coupling S4 to gating
are still not fully understood, there are indications for the
involvement of the S4S5 linker. These studies are based on
the observation that a point mutation in the HERG K+
channel S4S5 linker (D540K) is able to induce hyperpolarization-dependent activation (Sanguinetti & Xu, 1999).
A thorough investigation of the amino acid residues of the
S4S5 linker in HCN2 channels by an alanine-scanning
mutagenesis supports a critical role of this linker in
hyperpolarization-induced opening (Chen et al., 2001a).
The mutation of most of these residues was shown to
modify the channel voltage dependence and kinetics;
specifically, the mutation of E324, Y331, and R339 induced
a disruption of channel closure. The location in the S4S5
linker makes it unlikely that these residues are a structural
part of the gating machinery and suggests that they act as
coupling elements between the S4 voltage sensor and the
channel gate. The region at the boundary between S4 and
the S4S5 linker contains a histidine residue and is
important also in the pHi sensitivity of HCN channels
(Zong et al., 2001).
Differences in the kinetics of HCN isoforms can also
be exploited to gain insight into the gating mechanisms.
This approach takes advantage of the fact that, as
mentioned above, different isoforms can form heteromultimeric channels with properties intermediate between
those of homomeric channels and that chimeric channels
can be constructed by swapping various domains between
components.
The replacement of the HCN4 C-terminus, but not of the
N-terminus, by corresponding C- or N-termini from HCN1
led to a substantial acceleration of channel kinetics and loss
of cAMP sensitivity, demonstrating a role of the C-terminus
69
in voltage- and cAMP-dependent gating (Viscomi et al.,

2001). These results were in agreement with previous data
on native f-channels of SAN myocytes demonstrating that
the perfusion of pronase onto the intracellular side of
insideout patches, acting by the cleavage of internal
portions of channels, causes a dramatic shift of the channel
open probability curve to more positive voltages (+56 mV)
and eliminates the cAMP dependence (Barbuti et al., 1999).
The above experiments introduced the concept of a basal
inhibitory action exerted by a proteolysis-sensitive internal
domain (possibly in the C-terminus) on channel gating,
which could be removed by either hyperpolarization or
cAMP binding, a concept that was later confirmed by a
study using HCN1 and HCN2 CNBD-deletion mutants
(Wainger et al., 2001) or chimeras (Wang et al., 2001; see
below).
HCN channel kinetics can be modified by mutations in
several channel regions, including S1, the S1S2 linker, S6,
and the S3S4 linker, in the latter probably by a surfacecharge action (Ishii et al., 2001; Henrikson et al., 2003).
Residues in the S4S5 linker appear to be specifically
relevant to the coupling of S4-mediated voltage sensing and
channel activation in HCN2 (Chen et al., 2001a), while the
C-linker (i.e. the region linking S6 to the CNBD) is
important in mediating an inhibitory action of the CNBD
on channel activation through an interaction with core
transmembrane regions (Wang et al., 2001).
Although the outermost pore rim affects the gating of
HCN channels (Xue & Li, 2002), the voltage-dependent
gate appears to be located at the intracellular side of the
channel (Shin et al., 2001). This conclusion is based on
evidence that the HCN channel blocker ZD7288, applied
from the intracellular side, can enter and leave the channel
pore only when it is open. A similar reasoning applies to the
blocking action of other HCN channel blockers such as
ivabradine (Bucchi et al., 2002).
The present data therefore suggest that gating is a
complex mechanism involving interactions among several
regions, which include the CNBD, the C-linker, the S4S5
linker, and the core transmembrane regions.
Homology with the CNBD structure of both the bacterial

catabolite-activating protein and the regulatory subunit of
cAMP-dependent protein kinase, and the more recent data
from X-ray crystallography of the C-terminus of HCN2,
have contributed to a more detailed identification of
functionally relevant sub-domains of the C-terminus and
specifically of the CNBD (Weber & Steitz, 1987; Su et al.,
1995; Wainger et al., 2001; Zagotta et al., 2003).
The CNBD structure comprises a h-roll sub-domain,
acting as a constitutive inhibitor of the channel, and an ahelix located at the C-terminal end (termed C-helix), which
contributes to the interaction with the purine ring of cAMP.
cAMP binds with a greater affinity to the open, rather than
the closed state of the channel, and the mutation of residues
in the C-helix of HCN2 and CNG channels uncouple ligand
binding from gating modulation (Varnum et al., 1995;
Matulef et al., 1999; Wainger et al., 2001; Zagotta et al.,
2003).
The deletion of the C-helix of HCN1 and HCN2
abolished the cAMP response, but did not alter normal
gating (Wainger et al., 2001). The deletion of the whole
CNBD shifted the activation curves of HCN1 and HCN2 by
different amounts, such that the half-activation voltages of
the truncated channels were nearly coincident. These data
provide a quantitative confirmation of the idea that cAMP
removes a basal inhibitory mechanism operated by the Cterminus on HCN channel activation, originally proposed
for native f-channels of SAN myocytes (Barbuti et al.,
1999), and shows that the reduced cAMP sensitivity of
HCN1 can be simply explained by a reduced basal
inhibition of the CNBD on gating for HCN1 channels
(Wainger et al., 2001).
Additional information revealed by crystallographic
analysis provided further ground to the evidence for a
tetrameric nature of HCN channels and revealed that the Clinkers are responsible for subunitsubunit interactions
(Zagotta et al., 2003).
3.2.2. Structures involved in cAMP-dependent gating

cAMP exerts its modulatory effects by directly binding to
HCN channels (DiFrancesco & Tortora, 1991), and its
binding is more likely to occur when channels are in the
open state. The binding of cAMP to the channel thus
stabilizes the open configuration by partial removal of a
tonic inhibition, thereby shifting the channel distribution
equilibrium towards the open state (DiFrancesco, 1999).
The region involved in ligand binding and functional
transfer of cAMP-mediated channel gating is located in the
C-terminus (Viscomi et al., 2001; Wang et al., 2001). The Cterminus can be subdivided into different regions, including
a central cyclic nucleotide-binding domain (CNBD), and a
C-linker (about 80 aa long) that connects the C-terminal part
of S6 to the CNBD.
The identification of the molecular components of native

pacemaker channels in different tissues, and specifically in
the cardiac pacemaker region, is important not only for a
more detailed understanding of channel gating and interaction with other components, but also for pharmacological
and genetic therapy approaches.
As a first step in the identification of the molecular
composition of native f- channels, their properties can be
compared with those of individual HCN isoforms. However,
this comparison does not always enable the identification of
the channel composition. For example, the kinetics and
cAMP modulation of HCN2 are not too dissimilar from
those of native f-channels of SAN myocytes, but the
position of the activation curve is far too negative relative
to that of the I f current (see Section 3.1); similarly, f-
3.3. Hyperpolarization-activated, cyclic

nucleotide-gated isoforms in sinoatrial node tissue
70
channels are unlikely to be homotetramers of either HCN1

or HCN4 subunits only, since HCN1 has too fast kinetics
and weak cAMP sensitivity, while the HCN4 kinetics are
too slow (Altomare et al., 2003). Complementary approaches, based on RNase protection assay, Northern
blotting, and in situ hybridization techniques, indicate that,
in the SAN region, HCN4 is the prevalent isoform (about
80% of the total HCN signal), while HCN1 and HCN2 are
expressed weakly; also, the expression of HCN1 and HCN2
appears to be species dependent (Shi et al., 1999; Ishii et al.,
2001; Moosmang et al., 2001; Moroni et al., 2001; Stieber et
al., 2003).
As mentioned above, several mechanisms may contribute
to modify the functional properties of HCN channels,
including the heteromerization of different isoforms, as
well as interaction with auxiliary proteins and with the
intracellular environment. Studies using heterologous coexpression of different isoforms (HCN1 and HCN2 or HCN1
and HCN4), individually or in concatenated form, or
dominant-negative channel constructs, together with studies
using the yeast 2-hybrid system, clearly revealed the ability
of HCN isoforms to form heterotetrameric complexes, with
properties distinct from those of individual isoforms (Chen
et al., 2001b; Ulens & Tytgat, 2001; Proenza et al., 2002;
Xue et al., 2002; Altomare et al., 2003; Er et al., 2003;
Much et al., 2003). Heteromeric channel constructs exist in
vivo (Much et al., 2003), demonstrating the physiological
relevance of heteromerization. It is interesting to note that
HCN2HCN3 heteromers cannot be formed, suggesting that
specific channel domains may be required for the stabilization of subunit interactions (Much et al., 2003). The
tetramerization domains must be different from the Nterminal domains shown to underlie channel assembly and
trafficking to the plasma membrane, since the latter are
conserved among HCN isoforms (Proenza et al., 2002; Tran
et al., 2002).
The hypothesis that f-channels of rabbit SAN myocytes
are composed of HCN4HCN1 heteromers was investigated
by constructing tandem HCN4HCN1 constructs, with the
HCN4 C-terminus covalently linked to the HCN1 Nterminus (Altomare et al., 2003). The expression of this
construct generated currents with kinetics similar to those of
I f, but did not reproduce the same voltage dependence of
activation and cAMP sensitivity. A normal cAMP sensitivity could be obtained, on the other hand, with the mirror
tandem construct HCN1HCN4, with the HCN4 N-terminus now linked to the HCN1 C-terminus, suggesting that a
restricted mobility of the C-terminus reduces the response to
cAMP. The position of the activation curves of HCN
constructs was, however, always more negative than that of
I f. These data, together with the observation of a 4:1 ratio of
HCN4 vs. HCN1 mRNA (Shi et al., 1999), indicate that fchannels may indeed be heteromultimers of HCN4 and
HCN1, with a prevalence of HCN4 (Altomare et al., 2003),
but that a bcontextQ-dependent mechanism also likely
modulates the channel properties (Qu et al., 2002).
Most voltage-dependent ionic channels are composed by

a main pore-forming a-subunit, able to generate functional
channels, and by additional accessory h-subunits. hsubunits do not have channel-like properties per se but
interact functionally with a-subunits and modulate the
properties of the channel. The minK-related protein 1
(MiRP1), a single transmembrane-spanning protein highly
expressed in the SAN, acts as a modulatory h-subunit of the
K+ channels responsible for the currents I Kr, I Ks, and I to
(Abbott et al., 1999; Tinel et al., 2000; Zhang et al., 2001).
The effects of MiRP1 on HCN currents heterologously
expressed have been investigated, but the results in the
literature are not uniform. Yu et al. (2001) coexpressed
MiRP1 with HCN1 or HCN2 in Xenopus oocytes and
observed an enhanced protein expression and acceleration
of the activation kinetics. Altomare et al. (2003), on the
other hand, did not have evidence for an effect of MiRP1 on
either HCN1, HCN4, or a tandem HCN4HCN1 construct
when coexpressed in HEK293 cells. When MiRP1 modulation of HCN4 currents was analyzed by the expression in
CHO cells and in Xenopus oocytes, the results included an
increase of maximal current, a slowing of activation
kinetics, and a negative shift of the voltage dependence of
activation (Decher et al., 2003). The reason for the observed
differences is still unclear and awaits further investigation.
More recently, Qu et al. (2004) investigated the effect
of MiRP1 on HCN2 channels homologously expressed in
neonatal rat ventricle myocytes, thus using a physiological
cell substrate. In these experiments MiRP1 strongly
enhanced (4-fold) the maximal conductance and accelerated the rates of activation and deactivation; also, structural
interaction was demonstrated by co-immunoprecipitation.
A possible interpretation of the results with homologous
vs. heterologous channel expression is therefore that the
interaction between HCN and h-subunits depends on the
intracellular environment according to still unknown
mechanisms.
4. f-Channel blockers
Molecules interfering specifically with ion channels are
important tools in the characterization of their structural and
functional properties. Ion channel blockers are molecules
which inhibit ionic flow through channel pores; when their
action is specific for a given channel, their use allows the
pharmacological dissection of the contribution of this
channel to cellular electrical activity. Ion channels dysfunctions are the basis of several types of diseases known as
channelopathies, and the development of drugs specifically
targeting ion channels has naturally become a rapidly
growing concern of pharmacological research. It is not a
case that a large number of drugs on the market today are
targeted to modify ion channel functions.
The relevance of I f to cardiac pacemaking makes it an
obvious target for the search of drugs able to interfere
selectively with f-channels and thus control the pacemaker

function. If the concept of I f activation as the primary
mechanism responsible for the generation and autonomic
modulation of spontaneous activity is true, then a specific
alteration of I f is expected to affect, via a modification of
the slope of diastolic depolarization, the cardiac chronotropic state, without modifying other cardiovascular
parameters.
In the last few years, substances able to act as specific
blockers of the pacemaker current have been developed.
These molecules, originally known as bpure bradycardicQ
agents and termed today as bheart rate-loweringQ agents, are
potentially important therapeutic agents able to induce rate
slowing without the inotropic side effects typical of drugs
currently used to slow heart rate, such as Ca2+ antagonists or
h-blockers (Yusuf & Camm, 2003; DiFrancesco & Camm,
2004). Because of the early incorrect interpretation of the
origin of the pacemaker depolarization and of the pacemaker
current (see Section 2.1 above), heart rate-lowering agents
were sometimes thought to act as Ca2+-channel blockers
(Doerr & Trautwein, 1990) and were shown to be specific I f
blockers only based on more careful experimentation (Van
Bogaert & Goethals, 1987; DiFrancesco, 1994).
Moderate reduction of heart rate is therapeutically
beneficial in a variety of cardiovascular conditions, including chronic angina, ischaemic heart disease, and heart
failure. A lower heart rate decreases oxygen demand and
improves myocardial perfusion during a prolonged diastole,
thus improving myocardial oxygen balance. Also, the
concept of heart-rate reduction as a valid therapeutic
intervention in a range of cardiovascular diseases is
validated by several studies showing a link between
elevated heart rate and mortality in patients with coronary
heart disease and in the general population (DiFrancesco &
Camm, 2004).
Specific heart rate-lowering agents include ST567
(alinidine), UL-FS49 (zatebradine), ZD-7288, and S16257
(ivabradine; Van Bogaert & Goethals, 1987; BoSmith et al.,
1993; DiFrancesco, 1994; Gasparini & DiFrancesco, 1997;
Bucchi et al., 2002; Yusuf & Camm, 2003) and their
properties are discussed below.
4.1. Alinidine (ST567)
The term bspecific bradycardic agentQ was originally
proposed as a way to define drugs acting directly on
pacemaker function and able to decrease heart rate at doses
having minimal or no side effects (Kobinger, 1985).
Alinidine (N-allyl-clonidine), the first member of the family
of specific bradycardic agents, is an imidazoline compound
derived from the antihypertensive drug clonidine. Although
structurally similar to clonidine, it has different pharmacological effects, including analgesic (Stockhaus, 1977) and
bradycardic properties (Kobinger et al., 1979; Traunecker &
Walland, 1980; Millar & Vaughan-Williams, 1981; Lillie &
Kobinger, 1983).
71
In a study in rabbit SAN, 0.3 Ag/ml alinidine induced the

slowing of spontaneous activity accompanied by a prolongation of the action potential duration by about 10% (Satoh
& Hashimoto, 1986). No effects were observed on the
maximum diastolic potential (MDP), maximal dV/dt, and
action potential amplitude. Further experiments in sheep
Purkinje fibres showed that this drug is able to slow heart
rate by reducing the steepness of diastolic depolarization,
with limited side effects on action potential duration and
inotropic state (Snyders & Van Bogaert, 1987). In these
experiments, alinidine (28 AM) was reported to inhibit I f by
shifting its activation curve to more negative voltages (by
7.8 mV) and reducing its fully activated conductance (by
27.0%). No use or frequency dependence was observed,
suggesting that the drug binds equally well to open and
closed channel states.
Although alinidine was the first substance shown to act
as a bradycardic agent with little inotropic side effects, it
was not pursued as a pharmacological tool because its
selectivity for phase 4 changes was limited and partial
prolongation of action potential duration was observed, as
mentioned above, at moderate drug concentrations (0.3 Ag/
ml). This effect implied that alinidine, as well as inhibiting
f-channels, was acting as an inhibitor of other types of
channels, namely K+ channels involved in repolarization, at
only slightly higher doses.
4.2. Zatebradine (UL-FS 49) and cilobradine (DK-AH269)
The search for more specific bradycardic agents led to a
second group of molecules derived from the Ca2+ channel
inhibitor verapamil, among which falipamil (AQ-A39),
zatebradine (UL-FS49), and cilobradine (DK-AH26) have
been investigated with some detail. Like alinidine, these
drugs slow heart rate by the inhibition of the I f current. A
slowing of spontaneous activity was observed in Purkinje
fibres, intact SAN tissue, and single pacemaker cells (Van
Bogaert & Goethals, 1987, 1992; Van Bogaert et al., 1990;
Goethals et al., 1993; DiFrancesco, 1994; Thollon et al.,
1994; Bois et al., 1996; Van Bogaert & Pittoors, 2003).
In contrast to alinidine and derivatives, zatebradine and
cilobradine do not modify the position of the I f activation
curve but reduce the maximal conductance in a use- and
frequency-dependent fashion (Goethals et al., 1993; DiFrancesco, 1994). Preclinical studies were promising, and for
this reason, the specific action of zatebradine was analyzed
extensively. Zatebradine acts from the cytoplasmic side of
the membrane (Van Bogaert et al., 1990; DiFrancesco,
1994).
In a study in SAN myocytes (DiFrancesco, 1994), the
development of UL-FS49-induced I f block was slow, with
time constants of the order of several tens of seconds; also,
block only developed when the channel was open.
Apparently in contrast with the requirement of negative
voltages for channel opening, hyperpolarization relieved the
block. These data indicated that UL-FS49 behaves as an
72
bopen channel blockerQ, and that block occurs when the

protonated form of the drug binds at a site located ~39% of
the way across the membrane electrical field from the
intracellular side of the membrane. Thus, use and frequency
dependence of block arises from the need of drug molecules
to move through part of the voltage drop across the channel
pore in order to reach its binding site (DiFrancesco, 1994).
Zatebradine is a more specific f-channel blocker than
alinidine is. According to one report in SAN cells, 1 AM
zatebradine caused a strong (6595%) use-dependent block
of I f and only small reductions of sinoatrial Ca2+ and delayed
K+ currents (Goethals et al., 1993). Other reports confirmed
these findings (BoSmith et al., 1993; Thollon et al., 1994;
Bois et al., 1996; Valenzuela et al., 1996). Zatebradine
reduced heart rate both at rest and during exercise, but these
results were not accompanied by a significant prevention of
angina (Frishman et al., 1995). In addition, some undesired
effects on vision, including the persistence of images in the
visual field and flashes (Frishman et al., 1995; Usui et al.,
1995; Glasser et al., 1997), further hindered the use of
zatebradine as a cardiac selective drug. Experimentation in
photoreceptors showed that this side effect of zatebradine
was still due to the block of h-channels, the neuronal type of
f-channels, which are expressed in several different types of
neurons (Pape, 1996; Robinson & Siegelbaum, 2003),
including photoreceptors and retinal bipolar and ganglion
cells. Incidentally, these data allowed an interpretation of the
function of hyperpolarization-activated channels in the
retina, since their inhibition by zatebradine led to a reduced
ability to respond to high-frequency sinusoidal stimuli
(explaining the persistence of images), thus indicating the
involvement of I h in the response to rapid changes of light
intensity (Gargini et al., 1999).
A newer congener of zatebradine, cilobradine (DKAH269), was later found to induce a more effective and
faster block of I f in SAN myocytes; for example, the block
due to 1 AM cilobradine was over 2-fold higher (82% vs.
36%) and developed faster than that due the same concentration of zatebradine (Van Bogaert & Pittoors, 2003).
4.3. ZD-7288
Early data on the action of ZD-7288, a compound
originally named ICI D7288, were obtained in isolated
beating atria and intact SAN tissue of the guinea pig
(Marshall et al., 1993) and in anaesthetized dog (Rouse &
Johnson, 1994). The results showed that in intact right atria,
ZD-7288 (0.1100 AM) reduced spontaneous rate (maximal
slowing was 50%) but did not modify the contractile force
of electrically stimulated left atria (Marshall et al., 1993);
similar effects were observed in intact SAN tissue (slowing
of 61% at 0.3 AM concentration, BoSmith et al., 1993),
although a small prolongation of the action potential
duration was also present (10%).
The slowing of heart rate suggested the possibility that
ZD-7288 has an inhibitory action on f-channels, and
experiments on single cells isolated from the guinea pig

SAN confirmed this hypothesis (BoSmith et al., 1993). The
I f inhibitory effect of ZD-7288 (0.3 AM) consisted of a
negative shift of the current activation curve (16.2 mV) and
a decrease of the maximal conductance (by 52%; BoSmith
et al., 1993). Furthermore, ZD-7288, like alinidine but
unlike zatebradine, blocked I f in a use- and frequencyindependent way; the simplest interpretation of this property
is that ZD-7288 interacts with its channel binding site
independently from the channel gating configuration (Gasparini & DiFrancesco, 1997). The development of ZD7288-induced I f block in SAN myocytes and in other cell
types was slow (BoSmith et al., 1993; Harris & Constanti,
1995; Gasparini & DiFrancesco, 1997), suggesting an
intracellular block like that of zatebradine.
The selectivity of ZD-7288 for f-channels was investigated by testing its action on Ca2+ and K+ currents.
Experiments in isolated SAN cells from the guinea pig
(BoSmith et al., 1993) showed that both Ca2+ and K+
currents were only slightly affected by the drug. Concentrations equal or less than 1 AM elicited a Ca2+ current
reduction of only 18%, while I f was strongly reduced (82%
reduction); 0.1 AM ZD-7288 decreased the delayed K+
current by about 1%.
When the effects of ZD-7288 were evaluated in the
central nervous system, substantial block of the hyperpolarization-activated current (I h) was observed in guinea
pig substantia nigra neurons, rat hippocampal CA1 cells, cat
ventrobasal thalamocortical neurons, and newt photoreceptors (Harris & Constanti, 1995; Gasparini & DiFrancesco,
1997; Williams et al., 1997; Satoh & Yamada, 2002). The
high drug sensitivity of the neuronal pacemaker current is a
serious limitation to its use as a specific heart rate-limiting
agent. It is, however, worth noting that the block of
hippocampal pacemaker current appears to require higher
drug doses than those blocking I f in the SAN, which may be
due to the different HCN isoform composition of native
channels in these 2 tissues (Gasparini & DiFrancesco,
1997). The different heteromeric composition of f-channels
in different tissues is the basis for a molecular approach
aimed to develop drugs with tissue-specific properties,
which explains the interest of knowing the precise isoform
distribution in target tissues.
4.4. Ivabradine (S16257)
Ivabradine is, at present, the only member of the family
of specific heart rate-reducing agents to have completed
clinical development for stable angina. Its viability for
clinical use in ischaemic heart disease and cardiac failure
has been carefully investigated by both in vitro and in vivo
studies; its rate-reducing action is entirely attributable to a
specific and selective inhibition of I f (DiFrancesco &
Camm, 2004).
Early studies comparing the action of ivabradine and
zatebradine in rabbit Purkinje fibres and in guinea pig
papillary muscles showed that the 2 drugs are nearly

equipotent in slowing rate, and that their effect is mediated
by a reduced steepness of the slow diastolic depolarization
(Thollon et al., 1994). The same analysis revealed that the
action potential duration increases by about 29% with 3 AM
zatebradine, and by only about 9% with the same dose of
ivabradine.
Investigation in isolated SAN myocytes showed that the
reduction of the slope of diastolic depolarization is due to
the use-dependent intracellular blockade of f-channels (Bois
et al., 1996). In this study, 3 AM ivabradine did not affect
either T-type or L-type Ca2+ currents, nor did it modify the
73
delayed K+ current, whereas I f was strongly reduced (about

60%). The overall effect of ivabradine is similar to that of
zatebradine and cilobradine since all drugs affect I f by
inhibiting the maximal conductance without modifications
of the voltage dependence of current activation (Bois et al.,
1996).
Additional studies of ivabradine on f-channels in SAN
cells have shed light on some of the molecular aspects of its
blocking mechanism (Bucchi et al., 2002). These studies
have shown that bindingunbinding reactions are restricted
to the open state of the f-channel, implying that ivabradine is
an open channel blocker like zatebradine. Also, similar to
Fig. 3. Properties of the I f block by ivabradine. (A) I f block induced by ivabradine during repetitive stimulation (100 mV/+5 mV) is partially removed by a
long hyperpolarizing step to 100 mV (compare traces a and c in inset). (B) When the same protocol is applied in the presence of Cs+, no block removal
occurs, indicating that current flow is required for block removal. (C) The voltage dependence of block shifts to more negative voltages when the external Na+
concentration is reduced, and the shift is similar to that of the I f reversal potential, as measured by plotting fully activated I/V relations in the 2 solutions
(bottom panel); also, there is a steep change of block efficiency across the reversal potentials in both conditions. This confirms that the block depends on
current flow rather than on voltage per se. (D) The current dependence could be due to the interaction of ivabradine with permeating ions within the pore
(ivabradine structure courtesy Dr. Peglion Servier). Panels A through C, modified from Bucchi et al. (2002, with permission).
74
other heart rate-reducing agents, ivabradine blocks fchannels more efficiently at depolarized voltages and blocks
them from the intracellular side of the membrane. This
property reflects the positively charged nature of the
blocking molecule, which includes a quaternary ammonium
ion, and its tendency to enter the channel from the internal
side more easily at depolarized than at hyperpolarized
voltages. Ivabradine, however, has a distinctive property in
that its blocking action changes with voltage not because of
an intrinsic voltage dependence, but because of a dependence on the ion flow across the channel pore; ivabradine
block of f-channels is therefore bcurrentQ dependent (Bucchi
et al., 2002).
Evidence for the current dependence of block is
illustrated in Fig. 3. In panels A and B, full block was
induced by a repetitive activation/deactivation protocol in
the presence of 3 AM ivabradine before the application of a
long hyperpolarizing step in the presence (A) or in the
absence of 5 mM Cs+ (B), a known extracellular blocker of
the I f current (DiFrancesco, 1982). While in the absence of
Cs+ the long hyperpolarization clearly removes part of the
block (compare in the inset the current records just before
(A) and just after (C) the long step), in the presence of Cs+
no block removal occurs. A simple interpretation of these
data suggests that voltage hyperpolarization alone is not
enough to induce block removal, and that an inward ionic
flow is required (Bucchi et al., 2002). Additional evidence
for an bion flow-dependentQ block is presented in panel C,
where the voltage dependence of fractional block induced
by 3 AM ivabradine is plotted for 2 different external Na+
solutions. The 2 curves do not coincide, and in both cases, a
steep change of block occurs across the reversal potential of
the current for the 2 Na+ concentrations, indicating that the
electrochemical gradient, more than absolute voltage,
determines the extent of the block. The bcurrentQ dependence of a block is an atypical property of ivabradine that is
not shared by other heart rate-limiting agents. A biophysical
analysis of the current dependence suggests that f-channels
are multi-ion, single-file pores and that ivabradine blocks
ion flow by entering channels from the intracellular side and
competing with permeating ions in their binding to specific
ion-binding sites in the permeation pathway (Fig. 3D;
Bucchi et al., 2002).
Preclinical tests have confirmed that the in vitro
specificity of ivabradine as an f-channel blocker correlates
with a selective heart rate-reducing action with little or no
cardiac side effects. In experimental animals, the heart rate
was reduced without modification of the QT interval or
myocardial contractility; in addition, ivabradine decreased
oxygen consumption during exercise and preserved coronary vasodilatation in conscious dogs (Thollon et al., 1994;
Simon et al., 1995; Monnet et al., 2001; Colin et al., 2002,
2004; Camm & Lau, 2003; Vilaine et al., 2003). Trials
conducted with ivabradine on experimental animal models
of cardiac ischemia and on patients with stable angina
confirmed a cardioprotective therapeutic efficacy and
showed an improved recovery of the regional contractility

of the stunned myocardium; most importantly, drug
concentrations affecting rate did not elicit undesired
symptoms typical of other specific bradycardic agents,
except for limited visual disturbances, which were anyway
compatible with clinical tolerability and voluntary continuation of the therapy (Monnet et al., 2001; Borer et al., 2003;
Vilaine et al., 2003).
5. Future directions: the biological pacemaker

The development of f-channel-specific drugs acting
selectively on heart rate and having a potential for
therapeutic use represents an important clinical application
of the concept of bpacemakerQ current. The way these bheart
rate-reducingQ drugs act is to inhibit, by partial channel
block, the I f function and thus slow pacemaker activity.
Ideally, however, the concept of bpacemakerQ current should
be applicable not only to the aim of inhibiting pacemaking,
but also to the opposite purpose, i.e. induce pacemaking.
The question then becomes, is it possible to enhance
pacemaking by means of an increased contribution of funny
channels to activity?
This idea forms the basis for the development of another
potentially important clinical application of the concept of
bpacemakerQ current, the so-called bbiologicalQ pacemaker.
In essence, this new application relies on the possibility to
engineer a biological substrate that can be transferred in
vivo to identified cardiac locations and enhance, or induce,
automaticity. Ideally, such a device could replace the
electronic pacemakers used today.
The feasibility of this approach is supported by several
bproofs of conceptQ, which have been reviewed elsewhere,
along with the evaluation of advantages and drawbacks of
biological vs. electronic pacemakers (Rosen et al., 2004). A
first demonstration that HCN channels can be used to
enhance pacemaker activity was provided by evidence that
the overexpression of HCN2 in neonatal rat ventricular
myocytes by adenoviral infection substantially accelerated
their spontaneous rate (Qu et al., 2001). The same approach
used in canine hearts in vivo showed that the overexpression
of HCN2 was able to induce spontaneous left-atrium rhythm
after sinus arrest (Qu et al., 2003).
Cell-based methods have been adopted more recently.
One method relies on the in vitro differentiation of stem
cells towards a pacemaker-like phenotype; differentiated
cells can eventually be implanted in vivo onto the heart, by
grafting, to provide a new pacemaker source. Although in
origin the new pacemaker site is exogenous, the implanted
cells should, in principle, be able to integrate with the
surrounding cardiac tissue. An obvious advantage of this
approach is that the new pacemaker might be able to
respond to autonomic modulation if the new cells express
the whole chain of proteins mediating adrenergic and
cholinergic stimulation.
Some of the crucial evidence showing the feasibility of

this approach has been provided. Kehat et al. (2004) and
Xue et al. (2005) have shown that human Embryonic Stem
Cells (hESCs) can differentiate following specific treatments into spontaneously pacing myocytes, which, once
implanted into the heart of recipient animals (pigs or guinea
pigs) are able to pace the heart.
Another approach uses human Mesenchymal Stem
Cells (hMSCs) genetically engineered to express, by stable
transfection, high levels of functional HCN2 channels
(Potapova et al., 2004). HCN2-expressing cells lack the
intrinsic ability to generate action potentials but, when
implanted in the left ventricular wall of canine heart, are
able to form a functional syncytium with endogenous cells
and to provide depolarizing current during diastole. Thus,
the mutually interacting native and implanted cells can act
functionally as a single multicellular pacemaker.
Both cell-based strategies face major concerns, such as
immune responses induced by heterologous implanted cells,
neoplasia, and cell migration, and these problems need to be
safely solved before these approaches can be applied for
therapeutic use.
6. Conclusion
The properties of the bfunny Q current have been
described in detail in cardiac and neuronal cells since its
original finding over a quarter of a century ago, and the
molecular basis for these properties are now partially known
following the cloning of HCN channels. More molecular
details of channel gating, permeation, and block by specific
molecules will be available in the near future, along with a
more complete description of the channel structure based on
crystallization.
The functional role of I f in the heart is established. I f is
responsible for the generation of spontaneous activity and is
the main mechanism mediating the control of cardiac rate by
the autonomic neurotransmitters. Because of this role, fchannels are a natural target of drugs aiming to pharmacological control of heart rate, such as the heart rate-reducing
agents; these drugs are potentially beneficial in the treatment
of several cardiac diseases. It is to be expected that a
pharmacological use of f-channel-specific drugs will also be
possible in the future in other tissues and, specifically, in the
brain. Finally, new approaches using stable in situ transfection of HCN channels or pacemaking cells derived from
stem cells represent a novel opportunity for the development
of biological pacemakers.
Acknowledgments
Work from the laboratory is supported by grants
02.00652.ST97, 2003052919, and RBNE01LNX7 from
the Ministry of University and Scientific and Technological
75
Research to DD. We thank Richard B. Robinson for critical

reading.
References
Abbott, G. W., Sesti, F., Splawski, I., Buck, M. E., Lehmann, M. H.,
Timothy, K. W., et al. (1999). MiRP1 forms IKr potassium channels
with HERG and is associated with cardiac arrhythmia, Cell 97,
175 187.
Accili, E. A., Redaelli, G., & DiFrancesco, D. (1997). Differential control
of the hyperpolarization-activated (I f) current by cAMP and phosphatase inhibition in rabbit sino-atrial node myocytes, J Physiol (Lond)
500, 643 651.
Accili, E. A., Proenza, C., Baruscotti, M., & DiFrancesco, D. (2002). From
funny current to HCN channels: 20 years of excitation, News Physiol
Sci 17, 32 37.
Altomare, C., Bucchi, A., Camatini, E., Baruscotti, M., Viscomi, C.,
Moroni, A., et al. (2001). Integrated allosteric model of voltage gating
of HCN channels, J Gen Physiol 117, 519 532.
Altomare, C., Terragni, B., Brioschi, C., Milanesi, R., Pagliuca, C.,
Viscomi, C., et al. (2003). Heteromeric HCN1HCN4 channels: a
comparison with native pacemaker channels from the rabbit sinoatrial
node, J Physiol (Lond) 549, 347 359.
Baker, K., Warren, K. S., Yellen, G., & Fishman, M. C. (1997). Defective
bpacemakerQ current (I h) in a zebrafish mutant with a slow heart rate,
PNAS 94, 4554 4559.
Barbuti, A., Baruscotti, M., Altomare, C., Moroni, A., & DiFrancesco, D.
(1999). Action of internal pronase on the f-channel kinetics in the rabbit
SA node, J Physiol (Lond) 520, 737 744.
Barbuti, A., Gravante, B., Riolfo, M., Milanesi, R., Terragni, B., &
DiFrancesco, D. (2004). Localization of pacemaker channels in lipid
rafts regulates channel kinetics, Circ Res 94, 1325 1331.
Bois, P., Bescond, J., Renaudon, B., & Lenfant, J. (1996). Mode of action
of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial
node cells, Br J Pharmacol 118, 1051 1057.
Borer, J. S., Fox, K., Jaillon, P., Lerebours, G., & Ivabradine Investigators
Group (2003). Antianginal and antiischemic effects of ivabradine, an I f
inhibitor, in stable angina: a randomized, double-blind, multicentered,
placebo-controlled trial, Circulation 107, 817 823.
BoSmith, R. E., Briggs, I., & Sturgess, N. C. (1993). Inhibitory actions of
ZENECA ZD7288 on whole-cell hyperpolarization activated inward
current (I f) in guinea-pig dissociated sinoatrial node cells, Br J
Pharmacol 110, 343 349.
Boyett, M. R., Honjo, H., & Kodama, I. (2000). The sinoatrial node, a
heterogeneous pacemaker structure, Cardiovasc Res 47, 658 687.
Brown, H., & DiFrancesco, D. (1980). Voltage-clamp investigations of
membrane currents underlying pace-maker activity in rabbit sino-atrial
Brown, H. F., DiFrancesco, D., & Noble, S. J. (1979). How does adrenaline
accelerate the heart? Nature 280, 235 236.
Bucchi, A., Baruscotti, M., & DiFrancesco, D. (2002). Current-dependent
block of rabbit sino-atrial node I f channels by ivabradine, J Gen Physiol
120, 1 13.
Camm, A. J., & Lau, C. P. (2003). Electrophysiological effects of a single
intravenous administration of ivabradine (S 16257) in adult patients
with normal baseline electrophysiology, Drugs 4, 83 89.
Carmeliet, E. (1984). Existence of pacemaker current I f in human atrial
appendage fibres, J Physiol (Lond) 357, 125P.
Cerbai, E., Barbieri, M., & Mugelli, A. (1994). Characterization of the
hyperpolarization-activated current, I f, in ventricular myocytes isolated
from hypertensive rats, J Physiol (Lond) 481, 585 591.
Cerbai, E., Barbieri, M., & Mugelli, A. (1996). Occurrence and properties
of the hyperpolarization-activated current I f in ventricular myocytes
from normotensive and hypertensive rats during aging, Circulation 94,
1674 1681.
76
Chang, F., Cohen, I. S., DiFrancesco, D., Rosen, M. R., & Tromba, C.
(1991). Effects of protein kinase inhibitors on canine Purkinje fibre
pacemaker depolarization and the pacemaker current I f, J Physiol 440,
367 384.
Chen, J., Mitcheson, J. S., Lin, M., & Sanguinetti, M. C. (2000). Functional
roles of charged residues in the putative voltage sensor of the HCN2
pacemaker channel, J Biol Chem 275, 36465 36471.
Chen, J., Mitcheson, J. S., Tristani-Firouzi, M., Lin, M., & Sanguinetti, M.
C. (2001a). The S4S5 linker couples voltage sensing and activation of
pacemaker channels, PNAS 98, 11277 11282.
Chen, S., Wang, J., & Siegelbaum, S. A. (2001b). Properties of hyperpolarization-activated pacemaker current defined by coassembly of
HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide,
J Gen Physiol 117, 491 504.
Colin, P., Ghaleh, B., Hittinger, L., Monnet, X., Slama, M., Giudicelli, J. -F.,
et al. (2002). Differential effects of heart rate reduction and betablockade on left ventricular relaxation during exercise, Am J Physiol
282, H672 H679.
Colin, P., Ghaleh, B., Monnet, X., Hittinger, L., & Berdeaux, A. (2004).
Effect of graded heart rate reduction with ivabradine on myocardial
oxygen consumption and diastolic time in exercising dogs, J Pharmacol
Exp Ther 308, 236 240.
Decher, N., Bundis, F., Vajna, R., & Steinmeyer, K. (2003). KCNE2
modulates current amplitudes and activation kinetics of HCN4:
influence of KCNE family members on HCN4 currents, Pflugers Arch
446, 633 640.
Demir, S. S., Clark, J. W., Murphey, C. R., & Giles, W. R. (1994). A
mathematical model of a rabbit sinoatrial node cell, Am J Physiol 266,
C832 C852.
DiFrancesco, D. (1981a). A new interpretation of the pace-maker current in
calf Purkinje fibres, J Physiol (Lond) 314, 359 376.
DiFrancesco, D. (1981b). A study of the ionic nature of the pace-maker
current in calf Purkinje fibres, J Physiol (Lond) 314, 377 393.
DiFrancesco, D. (1982). Block and activation of the pace-maker channel in
calf Purkinje fibres: effects of potassium, caesium and rubidium,
J Physiol (Lond) 329, 485 507.
DiFrancesco, D. (1984). Characterization of the pace-maker current kinetics
in calf Purkinje fibres, J Physiol (Lond) 348, 341 367.
DiFrancesco, D. (1985). The cardiac hyperpolarizing-activated current, I f.
Origins and developments, Prog Biophys Mol Biol 46, 163 183.
DiFrancesco, D. (1986). Characterization of single pacemaker channels in
cardiac sino-atrial node cells, Nature 324, 470 473.
DiFrancesco, D. (1991). The contribution of the dpacemakerT current (I f) to
generation of spontaneous activity in rabbit sino-atrial node myocytes,
DiFrancesco, D. (1993). Pacemaker mechanisms in cardiac tissue, Annu
Rev Physiol 55, 451 467.
DiFrancesco, D. (1994). Some properties of the UL-FS 49 block of the
hyperpolarization-activated current (I f) in sino-atrial node myocytes,
Pflugers Arch 427, 64 70.
DiFrancesco, D. (1999). Dual allosteric modulation of pacemaker (f)
channels by cAMP and voltage in rabbit SA node, J Physiol (Lond)
515, 367 376.
DiFrancesco, D., & Camm, J. A. (2004). Heart rate lowering by
specific and selective I f current inhibition with ivabradine: a new
therapeutic perspective in cardiovascular disease, Drugs 64,
1757 1765.
DiFrancesco, D., & Mangoni, M. (1994). Modulation of single hyperpolarization-activated channels (I f) by cAMP in the rabbit sino-atrial
DiFrancesco, D., & Noble, D. (1985). A model of cardiac electrical activity
incorporating ionic pumps and concentration changes, Philos Trans R
Soc Lond B 307, 353 398.
DiFrancesco, D., & Ojeda, C. (1980). Properties of the current I f in the
sino-atrial node of the rabbit compared with those of the current I K2, in
Purkinje fibres, J Physiol (Lond) 308, 353 367.
DiFrancesco, D., & Robinson, R. B. (2002). Beta-modulation of pacemaker

rate: novel mechanism or novel mechanics of an old one? Circ Res 90,
E69.
DiFrancesco, D., & Tortora, P. (1991). Direct activation of cardiac
pacemaker channels by intracellular cyclic AMP, Nature 351,
145 147.
DiFrancesco, D., & Tromba, C. (1987). Acetylcholine inhibits activation of
the cardiac hyperpolarizing-activated current, I f, Pflugers Arch 410,
139 142.
DiFrancesco, D., & Tromba, C. (1988a). Inhibition of the hyperpolarization-activated current (I f) induced by acetylcholine in rabbit sino-atrial
node myocytes, J Physiol (Lond) 405, 477 491.
DiFrancesco, D., & Tromba, C. (1988b). Muscarinic control of the
hyperpolarization-activated current (I f) in rabbit sino-atrial node
myocytes, J Physiol (Lond) 405, 493 510.
DiFrancesco, D., Ferroni, A., Mazzanti, M., & Tromba, C. (1986).
Properties of the hyperpolarizing-activated current (I f) in cells
isolated from the rabbit sino-atrial node, J Physiol (Lond) 377,
61 88.
DiFrancesco, D., Ducouret, P., & Robinson, R. B. (1989). Muscarinic
modulation of cardiac rate at low acetylcholine concentrations, Science
243, 669 671.
Doerr, T., & Trautwein, W. (1990). On the mechanism of the bspecific
bradycardic actionQ of the verapamil derivative UL-FS 49, NaunynSchmiedebergs Arch Pharmakol 341, 331 340.
Er, F., Larbig, R., Ludwig, A., Biel, M., Hofmann, F., Beuckelmann, D. J.,
et al. (2003). Dominant-negative suppression of HCN channels
markedly reduces the native pacemaker current I f and undermines
spontaneous beating of neonatal cardiomyocytes, Circulation 107,
485 489.
Frace, A. M., Maruoka, F., & Noma, A. (1992). External K+ increases Na+
conductance of the hyperpolarization-activated current in rabbit cardiac
pacemaker cells, Pflugers Arch 421, 97 99.
Frishman, W. H., Pepine, C. J., Weiss, R. J., & Baiker, W. M. (1995).
Addition of zatebradine, a direct sinus node inhibitor, provides no
greater exercise tolerance benefit in patients with angina taking
extended-release nifedipine: results of a multicenter, randomized,
double-blind, placebo-controlled, parallel-group study, J Am Coll
Cardiol 26, 305 312.
Gargini, C., Demontis, G. C., Bisti, S., & Cervetto, L. (1999). Effects of
blocking the hyperpolarization-activated current (I h) on the cat electroretinogram, Vision Res 39, 1767 1774.
Gasparini, S., & DiFrancesco, D. (1997). Action of the hyperpolarizationactivated current (I h) blocker ZD 7288 in hippocampal CA1 neurons,
Pflugers Arch 435, 99 106.
Gaug, R., Seifert, R., & Kaupp, B. U. (1998). Molecular identification of a
hyperpolarization-activated channel in sea urchin sperm, Nature 393,
583 587.
Glasser, S. P., Michie, D. D., Thadani, U., & Baiker, W. M. (1997). Effects
of zatebradine (ULFS 49 CL), a sinus node inhibitor, on heart rate and
exercise duration in chronic stable angina pectoris, Am J Cardiol 79,
1401 1405.
Goethals, M., Raes, A., & Van Bogaert, P. -P. (1993). Use-dependent block
of the pacemaker current I f in rabbit sinoatrial node cells by zatebradine
(UL-FS 49): on the mode of action of sinus node inhibitors, Circulation
88, 2389 2401.
Gravante, B., Barbuti, A., Milanesi, R., Zappi, I., Viscomi, C., &
DiFrancesco, D. (2004). Interaction of the pacemaker channel HCN1
with filamin A, J Biol Chem 279, 43847 43853.
Harris, N. C., & Constanti, A. (1995). Mechanism of block by ZD
7288 of the hyperpolarization-activated inward rectifying current in
guinea pig substantia nigra neurons in vitro, J Neurophysiol 74,
2366 2378.
Hauswirth, O., Noble, D., & Tsien, R. W. (1968). Adrenaline: mechanism
of action on the pacemaker potential in cardiac Purkinje fibers, Science
162, 916 917.

Henrikson, C. A., Xue, T., Dong, P., Sang, D., Marban, E., & Li, R. A.
(2003). Identification of a surface charged residue in the S3-S4 linker of
the pacemaker (HCN) channel that influences activation gating, J Biol
Chem 278, 13647 13654.
Hille, B. (2001). Ion Channels of Excitable Membranes, 3rd ed.
Sunderland7 Sinauer.
Hille, B., & Schwarz, W. (1978). Potassium-channels as multi-ion singlefile pores, J Gen Physiol 72, 409 442.
Hoppe, U. C., & Beuckelmann, D. J. (1998). Characterization of the
hyperpolarization-activated inward current in isolated human atrial
myocytes, Cardiovasc Res 38, 788 801.
Ishii, T. M., Takano, M., Xie, L. H., Noma, A., & Ohmori, H.
(1999). Molecular characterization of the hyperpolarization-activated
cation channel in rabbit heart sinoatrial node, J Biol Chem 274,
12835 12839.
Ishii, T. M., Takano, M., & Ohmori, H. (2001). Determinants of activation
kinetics in mammalian hyperpolarization-activated cation channels,
Kaupp, U. B., & Seifert, R. (2001). Molecular diversity of pacemaker ion
channels, Annu Rev Physiol 63, 235 257.
Kehat, I., Khimovich, L., Caspi, O., Gepstein, A., Shofti, R., Arbel, G.,
et al. (2004). Electromechanical integration of cardiomyocytes
derived from human embryonic stem cells, Nat Biotechnol 22,
1282 1289.
Kobinger, W. (1985). Specific bradycardic agent, a new approach to therapy
in angina pectoris? Progress in Pharmacology (pp. 89 100). Stuttgart7
Fischer.
Kobinger, W., Lillie, C., & Pichler, L. (1979). N-allyl-derivative of
clonidine, a substance with specific bradycardic action at a cardiac
site, Naunyn-Schmiedebergs Arch Pharmakol 306, 255 262.
Lillie, C., & Kobinger, W. (1983). Comparison of the bradycardic effects of
alinidine (St 567), AQ-A 39 and verapamil on guinea-pig sinoatrial
node superfused with different Ca2+ and NaCl solutions, Eur J
Pharmacol 87, 25 33.
Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F., & Biel, M. (1998). A
family of hyperpolarization-activated mammalian cation channels,
Nature 393, 587 591.
Ludwig, A., Zong, X., Hofmann, F., & Biel, M. (1999a). Structure and
function of cardiac pacemaker channels, Cell Physiol Biochem 9,
179 186.
Ludwig, A., Zong, X., Stieber, J., Hullin, R., Hofmann, F., & Biel, M.
(1999b). Two pacemaker channels from human heart with profoundly
different activation kinetics, EMBO J 18, 2323 2329.
Mannikko, R., Elinder, F., & Larsson, H. P. (2002). Voltage-sensing
mechanism is conserved among ion channels gated by opposite
voltages, Nature 419, 837 841.
Marshall, P. W., Rouse, W., Briggs, I., Hargreaves, R. B., Mills, S. D., &
McLoughlin, B. J. (1993). ICI D7288, a novel sinoatrial node
modulator, J Cardiovasc Pharmacol 21, 902 906.
Matulef, K., Flynn, G. E., & Zagotta, W. N. (1999). Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels,
Neuron 24, 443 452.
McCormick, D. A., & Pape, H. -C. (1990). Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation
in thalamic relay neurones, J Physiol 431, 291 318.
Millar, J. S., & Vaughan-Williams, E. M. (1981). Pacemaker selectivity:
influence on rabbit atria of ionic environment and of alinidine, a
possible anion antagonist, Cardiovasc Res 15, 335 350.
Monnet, X., Ghaleh, B., Colin, P., De Curzon, O. P., Giudicelli, J. -F., &
Berdeaux, A. (2001). Effects of heart rate reduction with ivabradine on
exercise-induced myocardial ischemia and stunning, J Pharmacol Exp
Ther 299, 1133 1139.
Moosmang, S., Stieber, J., Zong, X., Biel, M., Hofmann, F., & Ludwig, A.
(2001). Cellular expression and functional characterization of four
hyperpolarization-activated pacemaker channels in cardiac and neuronal
tissues, Eur J Biochem 268, 1646 1652.
77
Moroni, A., Barbuti, A., Altomare, C., Viscomi, C., Morgan, J., Baruscotti,
M., et al. (2000). Kinetic and ionic properties of the human HCN2
pacemaker channel, Pflugers Arch 439, 618 626.
Moroni, A., Gorza, L., Beltrame, M., Gravante, B., Vaccari, T., Bianchi, M.
E., et al. (2001). Hyperpolarization-activated cyclic nucleotide-gated
channel 1 is a molecular determinant of the cardiac pacemaker current
I f, J Biol Chem 276, 29233 29241.
Much, B., Wahl-Schott, C., Zong, X., Schneider, A., Baumann, L.,
Moosmang, S., et al. (2003). Role of subunit heteromerization and
N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels, J Biol Chem 278,
43781 43786.
Noble, D., & Tsien, R. W. (1968). The kinetics and rectifier properties of
the slow potassium current in cardiac Purkinje fibres, J Physiol (Lond)
195, 185 214.
Noble, D., DiFrancesco, D., & Denyer, J. (1989). Ionic mechanisms in
normal and abnormal cardiac pacemaker activity, In J. W. Jacklet
(Ed.), Neuronal and Cellular Oscillators (pp. 59 85). New York7
Dekker.
Pape, H. -C. (1996). Queer current and the pacemaker: hyperpolarization-activated cation current in neurones, Annu Rev Physiol 58,
299 327.
Peper, K., & Trautwein, W. (1969). A note on the pacemaker current in
Purkinje fibers, Pflugers Arch 309, 356 361.
Porciatti, F., Pelzmann, B., Cerbai, E., Schaffer, P., Pino, R., Bernhart, E., et
al. (1997). The pacemaker current I f in single human atrial myocytes
and the effect of h-adrenoceptor and A1-adenosine receptor stimulation,
Br J Pharmacol 122, 963 969.
Potapova, I., Plotnikov, A., Lu, Z., Danilo, J. P., Valiunas, V., Qu, J., et al.
(2004). Human mesenchymal stem cells as a gene delivery system to
create cardiac pacemakers, Circ Res 94, 952 959.
Proenza, C., Tran, N., Angoli, D., Zahynacz, K., Balcar, P., & Accili, E.
A. (2002). Different roles for the cyclic nucleotide binding domain
and amino terminus in assembly and expression of hyperpolarizationactivated, cyclic nucleotide-gated channels, J Biol Chem 277,
29634 29642.
Qu, J., Barbuti, A., Protas, L., Santoro, B., Cohen, I. S., & Robinson, R. B.
(2001). HCN2 overexpression in newborn and adult ventricular
myocytes: distinct effects on gating and excitability, Circ Res 89,
E8 E14.
Qu, J., Altomare, C., Bucchi, A., DiFrancesco, D., & Robinson, R. B.
(2002). Functional comparison of HCN isoforms expressed in
ventricular and HEK 293 cells, Pflugers Arch 444, 597 601.
Qu, J., Plotnikov, A. N., Danilo Jr., P., Shlapakova, I., Cohen, I. S.,
Robinson, R. B., et al. (2003). Expression and function of a biological
pacemaker in canine heart, Circulation 107, 1106 1109.
Qu, J., Kryukova, Y., Potapova, I. A., Doronin, S. V., Larsen, M.,
Krishnamurthy, G., et al. (2004). MiRP1 modulates HCN2 channel
expression and gating in cardiac myocytes, J Biol Chem 279,
43497 43502.
Robinson, R. B., & Siegelbaum, S. A. (2003). Hyperpolarization-activated
cation currents: from molecules to physiological function, Annu Rev
Physiol 65, 453 480.
Robinson, R. B., Yu, H., Chang, F., & Cohen, I. S. (1997). Developmental
change in the voltage-dependence of the pacemaker current, I f, in rat
ventricle cells, Pflugers Arch 433, 533 535.
Rosen, M. R., Brink, P. R., Cohen, I. S., & Robinson, R. B. (2004).
Genes, stem cells and biological pacemakers, Cardiovasc Res 64,
12 23.
Rouse, W., & Johnson, I. R. (1994). Haemodynamic actions of a novel
sino-atrial node function modulator, ZENECA ZD7288, in the
anaesthetized dog: a comparison with zatebradine, atenolol and
nitrendipine, Br J Pharmacol 113, 1064 1070.
Sakmann, B., Noma, A., & Trautwein, W. (1983). Acetylcholine activation
of single muscarinic K-channels in isolated pacemaker cells of the
mammalian heart, Nature 303, 250 253.
78
Sanguinetti, M. C., & Xu, Q. P. (1999). Mutations of the S4S5 linker alter
activation properties of HERG potassium channels expressed in
Xenopus oocytes, J Physiol (Lond) 514, 667 675.
Santoro, B., & Tibbs, G. R. (1999). The HCN gene family: molecular basis
of the hyperpolarization-activated pacemaker channels, Ann N Y Acad
Sci 868, 741 764.
Santoro, B., Grant, S. G. N., Bartsch, D., & Kandel, E. R. (1997).
Interactive cloning with the SH3 domain of N-src identifies a new brain
specific ion channel protein, with homology to Eag and cyclic
nucleotide-gated channels, PNAS 94, 14815 14820.
Santoro, B., Liu, D. T., Yao, H., Bartsch, D., Kandel, E. R., Slegelbaum, S.
A., et al. (1998). Identification of a gene encoding a hyperpolarizationactivated pacemaker channel of brain, Cell 93, 717 729.
Satoh, H., & Hashimoto, K. (1986). Electrophysiological study of alinidine
in voltage clamped rabbit sino-atrial node cells, Eur J Pharmacol 121,
211 219.
Satoh, H., & Sperelakis, N. (1993). Developmental changes and modulation
through G-proteins of the hyperpolarization-activated inward current in
embryonic chick heart, Ann N Y Acad Sci 707, 413 416.
Satoh, T. O., & Yamada, M. (2002). Multiple inhibitory effects of
zatebradine (UL-FS 49) on the electrophysiological properties of retinal
rod photoreceptors, Pflugers Arch 443, 532 540.
Seifert, R., Scholten, A., Gauss, R., Mincheva, A., Lichter, P., & Kaupp, U.
B. (1999). Molecular characterization of a slowly gating human
hyperpolarization- activated channel predominantly expressed in
thalamus, heart, and testis, PNAS 96, 9391 9396.
Shi, W., Wymore, R., Yu, H., Wu, J., Wymore, R. T., Pan, Z., et al. (1999).
Distribution and prevalence of hyperpolarization-activated cation
channel (HCN) mRNA expression in cardiac tissues, Circ Res 85,
e1 e6.
Shin, K. S., Rothberg, B. S., & Yellen, G. (2001). Blocker state dependence
and trapping in hyperpolarization-activated cation channels: evidence
for an intracellular activation gate, J Gen Physiol 117, 91 101.
Simon, L., Ghaleh, B., Puybasset, L., Giudicelli, J. -F., & Berdeaux, A.
(1995). Coronary and hemodynamic effects of S 16257, a new
bradycardic agent, in resting and exercising conscious dogs,
J Pharmacol Exp Ther 275, 659 666.
Snyders, D. J., & Van Bogaert, P. -P. (1987). Alinidine modifies the
pacemaker current in sheep Purkinje fibers, Pflugers Arch 410,
83 91.
Stieber, J., Herrmann, S., Feil, S., Loster, J., Feil, R., Biel, M., et al. (2003).
The hyperpolarization-activated channel HCN4 is required for the
generation of pacemaker action potentials in the embryonic heart, PNAS
100, 15235 15240.
Stockhaus, K. (1977). Investigations concerning the profile of action of 2[N-allyl-N(2,6-dichlorophenyl)-amino]-2-imidazoline-hydrobromide
(St567-Br), a compound with analgesic activity, Problems of Drug
Dependence (pp. 355 366). Cambridge7 National Academy of
Sciences.
Su, Y., Dostmann, W. R., Herberg, F. W., Durick, K., Xuong, N. H., Ten
Eyck, L., et al. (1995). Regulatory subunit of protein kinase A:
structure of deletion mutant with cAMP binding domains, Science
269, 807 813.
Thollon, C., Cambarrat, C., Vian, J., Prost, J. -F., Peglion, J. L., & Vilaine,
J. P. (1994). Electrophysiological effects of S 16257 a novel sino-atrial
node modulator, on rabbit and guinea-pig cardiac preparations:
comparison with UL-FS 49, Br J Pharmacol 112, 37 42.
Tinel, N., Diochot, S., Borsotto, M., Lazdunski, M., & Barhanin, J. (2000).
KCNE2 confers background current characteristics to the cardiac
KCNQ1 potassium channel, EMBO J 19, 6326 6330.
Tran, N., Proenza, C., Macri, V., Petigara, F., Sloan, E., Samler, S., et al.
(2002). A conserved domain in the NH2 terminus important for
assembly and functional expression of pacemaker channels, J Biol
Chem 277, 43588 43592.
Traunecker, W., & Walland, A. (1980). Haemodynamic and electrophysiologic actions of alinidine in the dog, Arch Internat Pharmacodyn
Ther 244, 58 72.
Tsien, R. W., Giles, W., & Greengard, P. (1972). Cyclic AMP mediates the
effects of adrenaline on cardiac Purkinje fibres, Nature 240, 181 183.
Ulens, C., & Siegelbaum, S. A. (2003). Regulation of hyperpolarizationactivated HCN channels by cAMP through a gating switch in binding
domain symmetry, Neuron 40, 959 970.
Ulens, C., & Tytgat, J. (2001). Functional heteromerization of HCN1 and
HCN2 pacemaker channels, J Biol Chem 276, 6069 6072.
Usui, S., Kamiyama, Y., Ogura, T., Kodama, I., & Toyama, J. (1995).
Effects of zatebradine (UL-FS49) on the vertebrate retina, Recent
Progress in Electropharmacology of the Heart (pp. 37 46). Boca
Raton, FL7 CRC Press.
Vaca, L., Stieber, J., Zong, X., Ludwig, A., Hofmann, F., & Biel, M. (2000).
Mutations in the S4 domain of a pacemaker channel alter its voltage
dependence, FEBS Lett 479, 35 40.
Vaccari, T., Moroni, A., Rocchi, M., Gorza, L., Bianchi, M. E., Beltrame,
M., et al. (1999). The human gene coding for HCN2, a pacemaker
channel of the heart, Biochim Biophys Acta 1446, 419 425.
Valenzuela, C., Delpon, E., Franqueza, L., Gay, P., Perez, O., Tamargo, J.,
et al. (1996). Class III antiarrhythmic effects of zatebradine: time-,
state-, use-, and voltage-dependent block of hKv1.5 channels,
Circulation 94, 562 570.
Van Bogaert, P. -P., & Goethals, M. (1987). Pharmacological influence of
specific bradycardic agents on the pacemaker current of sheep cardiac
Purkinje fibres. A comparison between three different molecules, Eur
Heart J 8, 35 42.
Van Bogaert, P. P., & Goethals, M. (1992). Blockade of the pacemaker
current by intracellular application of UL-FS 49 and UL-AH 99 in
sheep cardiac Purkinje fibers, Eur J Pharmacol 229, 55 62.
Van Bogaert, P. P., & Pittoors, F. (2003). Use-dependent blockade of cardiac
pacemaker current (I f) by cilobradine and zatebradine, Eur J Pharmacol
478, 161 171.
Van Bogaert, P. P., Goethals, M., & Simoens, C. (1990). Use- and
frequency-dependent blockade by UL-FS 49 of the I f pacemaker current
in sheep cardiac Purkinje fibres, Eur J Pharmacol 187, 241 256.
van Ginneken, A. C. G., & Giles, W. (1991). Voltage clamp measurements
of the hyperpolarization-activated inward current I f in single cells from
rabbit sino-atrial node, J Physiol (Lond) 434, 57 83.
Varnum, M. D., Black, K. D., & Zagotta, W. N. (1995). Molecular
mechanism for ligand discrimination of cyclic nucleotide-gated
channels, Neuron 15, 619 625.
Vassalle, M. (1966). Analysis of cardiac pacemaker potential using a
voltage-clamp technique, Am J Physiol 210, 1335 1341.
Vilaine, J. P., Bidouard, J. P., Lesage, L., Reure, H., & Peglion, J. L. (2003).
Anti-ischemic effects of ivabradine, a selective heart rate-reducing
agent, in exercise-induced myocardial ischemia in pigs, J Cardiovasc
Pharmacol 42, 688 696.
Vinogradova, T. M., Bogdanov, K. Y., & Lakatta, E. G. (2002). Betaadrenergic stimulation modulates ryanodine receptor Ca2+ release
during diastolic depolarization to accelerate pacemaker activity in
rabbit sinoatrial nodal cells, Circ Res 90, 73 79.
Viscomi, C., Altomare, C., Bucchi, A., Camatini, E., Baruscotti, M.,
Moroni, A., et al. (2001). C-terminus-mediated control of voltage and
cAMP gating of hyperpolarization-activated cyclic nucleotide-gated
channels, J Biol Chem 276, 29930 29934.
Wainger, B. J., DeGennaro, M., Santoro, B., Siegelbaum, S. A., & Tibbs, G.
R. (2001). Molecular mechanism of cAMP modulation of HCN
pacemaker channels, Nature 411, 805 810.
Wang, J., Chen, S., & Siegelbaum, S. A. (2001). Regulation of hyperpolarization-activated HCN channel gating and cAMP modulation due
to interactions of COOH terminus and core transmembrane regions,
J Gen Physiol 118, 237 250.
Warren, K. S., Baker, K., & Fishman, M. C. (2001). The slow mo mutation
reduces pacemaker current and heart rate in adult zebrafish, Am J
Physiol 281, H1711 H1719.
Weber, I. T., & Steitz, T. A. (1987). Structure of a complex of catabolite
gene activator protein and cyclic AMP refined at 2.5 2 resolution,
J Mol Biol 198, 311 326.

Weidmann, S. (1951). Effect of current flow on membrane potential of
cardiac muscle, J Physiol 115, 227 236.
Williams, S. R., Turner, J. P., Hughes, S. W., & Crunelli, V. (1997). On the
nature of anomalous rectification in thalamocortical neurones of the cat
ventrobasal thalamus in vitro, J Physiol 505, 727 747.
Xue, T., & Li, R. A. (2002). An external determinant in the S5-P linker of
the pacemaker (HCN) channel identified by sulfhydryl modification,
J Biol Chem 277, 46233 46242.
Xue, T., Marban, E., & Li, R. A. (2002). Dominant-negative suppression of
HCN1- and HCN2-encoded pacemaker currents by an engineered
HCN1 construct: insights into structurefunction relationships and
multimerization, Circ Res 90, 1267 1273.
Xue, T., Cheol Cho, H., Akar, F. G., Tsang, S. -Y., Jones, S. P., Marban, E.,
et al. (2005). Functional integration of electrically active cardiac
derivatives from genetically engineered human embryonic stem cells
with quiescent recipient ventricular cardiomyocytes, Circulation 111,
11 20.
Yanagihara, K., & Irisawa, H. (1980). Inward current activated during
hyperpolarization in the rabbit sinoatrial node cell, Pflugers Arch 385,
11 19.
Yasui, K., Liu, W., Opthof, T., Kada, K., Lee, J. K., Kamiya, K., et al.
(2001). If current and spontaneous activity in mouse embryonic
ventricular myocytes, Circ Res 88, 536 542.
Yu, H., Chang, F., & Cohen, I. S. (1993). Pacemaker current exists in
ventricular myocytes, Circ Res 72, 232 236.
Yu, H., Chang, F., & Cohen, I. S. (1995). Pacemaker current I f in adult
canine cardiac ventricular myocytes, J Physiol 485, 469 483.
79
Yu, H., Wu, J., Potapova, I., Wymore, R. T., Holmes, B., Zuckerman, J., et
al. (2001). MinK-related peptide 1: a beta subunit for the HCN ion
channel subunit family enhances expression and speeds activation, Circ
Res 88, E84 E87.
Yusuf, S., & Camm, A. J. (2003). Sinus tachyarrhythmias and the specific
bradycardic agents: a marriage made in heaven? J Cardiovasc
Pharmacol Ther 8, 89 105.
Zagotta, W. N., Olivier, N. B., Black, K. D., Young, E. C., Olson, R., &
Gouaux, E. (2003). Structural basis for modulation and agonist
specificity of HCN pacemaker channels, Nature 425, 200 205.
Zhang, M., Jiang, M., & Tseng, G. N. (2001). minK-related peptide 1
associates with Kv4.2 and modulates its gating function: potential role
as beta subunit of cardiac transient outward channel? Circ Res 88,
1012 1019.
Zhou, Z., & Lipsius, S. L. (1992). Properties of the pacemaker current (I f)
in latent pacemaker cells isolated from cat right atrium, J Physiol (Lond)
453, 503 523.
Zhou, Z., & Lipsius, S. L. (1993). Effect of isoprenaline on I f current in
latent pacemaker cells isolated from cat right atrium: ruptured vs.
perforated patch whole-cell recording methods, Pflugers Arch 423,
442 447.
Zong, X., Stieber, J., Ludwig, A., Hofmann, F., & Biel, M. (2001). A single
histidine residue determines the pH sensitivity of the pacemaker
channel HCN2, J Biol Chem 276, 6313 6319.
Zorn-Pauly, K., Schaffer, P., Pelzmann, B., Lang, P., Machler, H., Rigler,
B., et al. (2004). I f in left human atrium: a potential contributor to atrial
ectopy, Cardiovasc Res 64, 250 259.

Physiology and Pharmacology of The Cardiac Pacemaker (Bfunnyq) Current

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Physiology and Pharmacology of The Cardiac Pacemaker (Bfunnyq) Current

Uploaded by

Copyright:

Available Formats

Pharmacology & Therapeutics 107 (2005) 59 79

Associate editor: G.E. Billman

Physiology and pharmacology of the cardiac pacemaker (bfunnyQ) current

T Corresponding author. Tel.: +39 02 50314931; fax: +39 02 50314932.

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

Structurefunction relationships for hyperpolarization-activated,

where it contributes to a wide range of physiological

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

In this review we will focus on the properties of cardiac

2. Sinoatrial I f current and the

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

from the central to the peripheral node, reflecting the

mV, in agreement with the function of I f in the pacemaker

Shift activation curve (mV)

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

at physiological voltages, but its function is lost with

membrane during transition from cell-attached to inside

Accili et al. (1996). Pflugers Arch 431, 757

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

analysis. Detailed investigation reveals that several kinetic

stages and disappear together at later stages of development,

2.3. Contribution to automaticity

The mammalian SAN region is richly innervated by the

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

et al., 1968), an effect mediated by increased intracellular

promoting a depolarizing shift of the I f activation curve, acts

3. Molecular determinants of the I f current

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

cloning was made by chance while looking for proteins

more than by a variable cAMP binding affinity to the CNBD

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

mechanism operated by voltage hyperpolarization and the

Indeed, the shifting action of cAMP can be explained by

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

tetramers whose four subunits undergo simultaneous,

Fig. 2. Activation of f-channels by cAMP as explained according to an

the assumption that cAMP molecules have a higher binding

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

in voltage- and cAMP-dependent gating (Viscomi et al.,

Homology with the CNBD structure of both the bacterial

3.2.2. Structures involved in cAMP-dependent gating

The identification of the molecular components of native

3.3. Hyperpolarization-activated, cyclic

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

channels are unlikely to be homotetramers of either HCN1

Most voltage-dependent ionic channels are composed by

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

selectively with f-channels and thus control the pacemaker

In a study in rabbit SAN, 0.3 Ag/ml alinidine induced the

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

bopen channel blockerQ, and that block occurs when the

experiments on single cells isolated from the guinea pig

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

papillary muscles showed that the 2 drugs are nearly

delayed K+ current, whereas I f was strongly reduced (about

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

showed an improved recovery of the regional contractility

5. Future directions: the biological pacemaker

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979

Some of the crucial evidence showing the feasibility of

Research to DD. We thank Richard B. Robinson for critical

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 5979