1-s2.0-S0163725805000252-main

8/20/2019 1-s2.0-S0163725805000252-main

1/21

Associate editor: G.E. Billman

Physiology and pharmacology of the cardiac pacemaker (bfunny Q ) 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 bfunny Q ( 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 autonomic-

dependent 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. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

2. Sinoatrial I f current and the mechanism of cardiac pacemaking . . . . . . . . . . . . . . . . . . 61

2.1. Early experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.2. Biophysical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612.2.1. Voltage dependence of I f kinetics . . . . . . . . . . . . . . . . . . . . . . . . . 61

2.2.2. Kinetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

2.2.3. Ionic nature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

2.3. Contribution to automaticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

2.4. Autonomic modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64

2.5. Single f-channel recording . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

3. Molecular determinants of the I f current . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

3.1. Hyperpolarization-activated, cyclic nucleotide-gated clones. . . . . . . . . . . . . . . . . 65

0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.pharmthera.2005.01.005

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

E-mail address: [email protected] (D. DiFrancesco).

Pharmacology & Therapeutics 107 (2005) 59–79

www.elsevier.com/locate/pharmthera

8/20/2019 1-s2.0-S0163725805000252-main

2/21

3.2. Structure–function relationships for hyperpolarization-activated,

cyclic nucleotide-gated channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

3.2.1. Structures involved in voltage-dependent gating. . . . . . . . . . . . . . . . . . . 68

3.2.2. Structures involved in cAMP-dependent gating . . . . . . . . . . . . . . . . . . . 69

3.3. Hyperpolarization-activated, cyclic nucleotide-gated isoforms in sinoatrial node tissue . . . 69

4. f-Channel blockers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.1. Alinidine (ST567) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714.2. Zatebradine (UL-FS 49) and cilobradine (DK-AH269) . . . . . . . . . . . . . . . . . . . . 71

4.3. ZD-7288 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

4.4. Ivabradine (S16257) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

5. Future directions: the biological pacemaker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

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 sponta-

neous 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 hyper-

polarized voltage level. Spontaneously beating SAN myo-

cytes, on the contrary, are characterized by the presence of a

bslow diastolic Q 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 b pacemaker Q depolarization is respon-

sible 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 inves-

tigation 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 b pacemaker Q (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 hyperpolariza-

tion-activated current similar to I f ( I h current) has also been

described in a large number of different types of neurons,

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). Despitethe 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 nucleotide-

gated channels (HCN1 to 4) and belonging to the super-

family of voltage-dependent K + (Kv) and cyclic nucleotide-

gated (CNG) channels (see reviews by Ludwig et al., 1999a;

Santoro & Tibbs, 1999; Kaupp & Seifert, 2001; Accili et al.,

2002; Robinson & Siegelbaum, 2003). A thorough inves-

tigation 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 pace-

making and rate regulation, the funny current has long been

considered a primary target for the development of drug-

based 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.

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 59–7960

8/20/2019 1-s2.0-S0163725805000252-main

3/21

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). Resultsapparently 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 so-

called b pacemaker Q ( 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 K2depended 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 sympa-

thetic stimulation (Hauswirth et al., 1968).

The experimental evidence ruling in favour of the bK +

current-decay Q hypothesis was therefore apparently incon-

trovertible, and for over a decade, the I K2 current was

considered as one of the most typical K + cardiac compo-

nents (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 depolariza-

tion, 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 bfake Q reversal potential close to the expected K +

equilibrium potential during voltage-clamp hyperpolariza-

tion, 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 bfunny Q current in

the mammalian SAN. In 1979, the first detailed report of

this current appeared, describing its elementary properties

and involvement in the generation and catecholamine-

induced acceleration of SAN spontaneous activity (Brown

et al., 1979). Among the unusual features which justified the

name bfunny Q 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 appropriatefor I f to function as an efficient b pacemaker Q current

involved in the generation of diastolic depolarization and

in rate control by h-adrenergic stimulation. The Purkinje

fibre’s 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 fibre’s pacemaker current

and allowed, for the first time, to visualize the conversion of

I K2 into an inward, hyperpolarization-activated current. Theinward ionic nature of I K2 was confirmed by ion-substitu-

tion 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 fibre’s 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 activa-

tion 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 hyper-

polarization-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 whereappropriate.

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

M. Baruscotti et al. / Pharmacology & Therapeutics 107 (2005) 59–79 61

8/20/2019 1-s2.0-S0163725805000252-main

4/21

8/20/2019 1-s2.0-S0163725805000252-main

5/21

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 hyper-

tensive rats), adult I f expression increases substantially

relative to control animals (Cerbai et al., 1996).

As well as by cAMP, which mediates autonomic-

dependent modulation (see below), I f kinetics and activationrange 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 duringwhole-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

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 dependence Q 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 pro-

cesses, including single- (DiFrancesco & Noble, 1985;

McCormick & Pape, 1990) and double-exponential Hodg-kin–Huxley kinetics ( Noble et al., 1989; van Ginneken &

Giles, 1991; Demir et al., 1994), and more complex, non-

Hodgkin–Huxley 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 1–10 AM; (2) acetilcholine 1–10 AM; (3) saturating effect from Hill plot; (4) cAMP 10–100 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. Accili et al. (1996). Pflugers Arch 431, 757

2. Accili et al. (1997). Am J Physiol 272, H1549

3. Accili et al. (1997). J Physiol 500, 6434. Bois et al. (1997). J Physiol 501, 565

5. Cerbai et al. (1996). Circulation 94, 1674

6. Cerbai et al. (1999). Cardiovasc Res 42, 121

7. Cerbai et al. (2001). J Mol Cell Cardiol 33, 441

8. Denyer et al. (1990). J Physiol 428, 405

9. DiFrancesco (1981). J Physiol 314, 377

10. DiFrancesco et al. (1986). J Physiol 377, 61


12. DiFrancesco et al. (1989). Science 243, 669

13. DiFrancesco et al. (1991). Pflugers Arch 417, 611

14. DiFrancesco et al. (1991). Nature 351, 145


16. Fares et al. (1998). J Physiol 506, 73

17. Frace et al. (1992). J Physiol 453, 307

18. Hagiwara et al. (1989). J Physiol 409, 121

19. Hoppe et al. (1998). Cardiovasc Res 38, 788

20. Hoppe et al. (1998). Circulation 97, 55

21. Mangoni et al. (2001). Cardiovasc Res 52, 51

22. Maruoka et al. (1994). J Physiol 477, 423

23. Porciatti et al. (1997). Br J Pharmacol 122, 963

24. Ranjan et al. (1998). Biophys J 74, 1850

25. Robinson et al. (1997). Pflugers Arch 433, 533

26. Shibata et al. (1999). Br J Pharmacol 128, 1284

27. Yasui et al. (2001). Circ Res 88, 536

28. Yu et al. (1993). Circ Res 72, 232

29. Yu et al. (1995). J Physiol 485, 469

30. Zaza et al. J Physiol 491, 347

31. Zhou et al. (1992). J Physiol 453, 503

32. Zhou et al. (1993). Pflugers Arch 423, 442


8/20/2019 1-s2.0-S0163725805000252-main

6/21

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

bdelaying Q and proper bgating Q 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 ballosteric Q dual voltage and cAMP-

dependent 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, 1981 b;

Frace et al., 1992). The conductance of f-channels was also

shown to increase with external K + concentration (DiFran-

cesco, 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.

2.3. Contribution to automaticity

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 compar-

ing 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

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 f-channels (see Section 3), in embryonic rat ventricular

myocytes has shown a large increase of the rate of

spontaneous activity correlated to the expression of pace-

maker channels (Qu et al., 2001). Further, the overexpres-

sion of a nonfunctional HCN2 isoform induced dominant-

negative 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-channelswith a high degree of selectivity, such as UL-FS49

(zatebradine), ZD7288, and S16257 (ivabradine), act as

pure bheart rate-reducing Q 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.

2.4. Autonomic modulation

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 actingthrough h-adrenergic and muscarinic receptors, respectively.

The original description of I f already indicated the involve-

ment 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 (Hauswirt h


8/20/2019 1-s2.0-S0163725805000252-main

7/21

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 negativeshift 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 accord-

ance 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

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 inside–out

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

0.05–0.1 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 under-

lying 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 single-

channel 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 f-

channels by binding directly to the intracellular aspect of the

channel (DiFrancesco & Tortora, 1991), which, in accord-

ance to the whole-cell data, resulted in an increase of single-

channel open probability, with no changes of channel

conductance (DiFrancesco & Mangoni, 1994).

3. Molecular determinants of the I f current

3.1. Hyperpolarization-

activated, 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


8/20/2019 1-s2.0-S0163725805000252-main

8/21

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 (Santor oet 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 tetramericcomposition (Ulens & Siegelbaum, 2003; Zagotta et al.,

2003), and are characterized by the presence of 6 trans-

membrane domains (S1–6) with a voltage sensor located in

the positively charged S4 domain, the GYG pore sequence

typical of K +-permeable channels, and a cyclic nucleotide-

binding domain (CNBD) homologous to that of CNG

channels located in the C-terminus.

Sequence alignment revealed that the HCN bcore Q

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 4.3–5.8 mV) than either HCN2

(range 16.9–19.2 mV) or HCN4 (range 11.1–23 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 (DiFran-cesco, 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 heteromul-

timers 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 asfilamin-A (for HCN1, Gravante et al., 2004), or by specific

subcellular compartmentation such as the caveolar compart-

mentation 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 bcontext-

dependent Q 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. Structure–function relationships for

hyperpolarization-activated, cyclic nucleotide-gated channels

Among the peculiar properties of bfunny Q 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 andCNG channels, which are gated by voltage only and cAMP

only, respectively; also, Kv channels open on depolariza-

tion, 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 & Tortor a,

1991; DiFrancesco & Mangoni, 1994). How does cAMP

cause these changes? The presence of a hyperpolarizing

shift of voltage dependence suggests that the gating


8/20/2019 1-s2.0-S0163725805000252-main

9/21

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

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.


8/20/2019 1-s2.0-S0163725805000252-main

10/21

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 po can then be described by a

modified Boltzmann equation:

po ¼ 1= 1 þ exp V V 1=2 s

=v

ð1Þ

where V is voltage, V 1/2 is the half-activation voltage, v is

the inverse slope-factor, and s is a cAMP concentration-

dependent 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 po, 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:

s cAMP½ ð Þ ¼ v ln 1 þ cAMP½ = K o

ð Þ= 1 þ cAMP½ = K c

ð Þð Þ

ð2Þ

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

the assumption that cAMP molecules have a higher binding

affinity to open than to closed channel states ( K ob K c).

Several structure–function studies have addressed these

aspects by investigating the HCN channel domains

involved in hyperpolarization-dependent and cAMP-

dependent 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 hyper-

polarization. This difference could be due to an binverted Q

movement of the voltage sensor of HCN vs. Kv channels in

response to the same voltage change, or to an binverted Q

coupling between S4 movement and channel gating. Thelatter possibility appears more likely since cysteine acces-

sibility 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 S4–S5 linker. These studies are based on

the observation that a point mutation in the HERG K +

channel S4–S5 linker (D540K) is able to induce hyper-

polarization-dependent activation (Sanguinetti & Xu, 1999).

A thorough investigation of the amino acid residues of the

S4–S5 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 S4–S5

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 S4–S5 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 heteromul-

timeric 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

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 macro-

patch 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).


8/20/2019 1-s2.0-S0163725805000252-main

11/21

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

inside–out 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 S1–S2 linker, S6,

and the S3–S4 linker, in the latter probably by a surface-charge action (Ishii et al., 2001; Henrikson et al., 2003).

Residues in the S4–S5 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 S4–S5

linker, and the core transmembrane regions.

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 C-

terminus 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.

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 a-

helix 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 HCN2abolished 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 C-

terminus 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 C-

linkers are responsible for subunit–subunit interactions

(Zagotta et al., 2003).

3.3. Hyperpolarization-activated, cyclic

nucleotide-gated isoforms in sinoatrial node tissue

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 amore detailed understanding of channel gating and inter-

action 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-


8/20/2019 1-s2.0-S0163725805000252-main

12/21

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 ap-

proaches, 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 coex-

pression 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

HCN2–HCN3 heteromers cannot be formed, suggesting that

specific channel domains may be required for the stabiliza-

tion of subunit interactions (Much et al., 2003). The

tetramerization domains must be different from the N-

terminal 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 HCN4–HCN1 heteromers was investigated

by constructing tandem HCN4–HCN1 constructs, with the

HCN4 C-terminus covalently linked to the HCN1 N-

terminus (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 sensi-tivity could be obtained, on the other hand, with the mirror

tandem construct HCN1–HCN4, with the HCN4 N-termi-

nus 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 f-

channels may indeed be heteromultimers of HCN4 and

HCN1, with a prevalence of HCN4 (Altomare et al., 2003),

but that a bcontext Q -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. h-

subunits 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 highlyexpressed 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 HCN4–HCN1 construct

when coexpressed in HEK293 cells. When MiRP1 modu-lation 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 acceler-

ated 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 moleculeswhich 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 dysfunc-

tions 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


8/20/2019 1-s2.0-S0163725805000252-main

13/21

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 chrono-

tropic 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 b pure bradycardic Q

agents and termed today as bheart rate-lowering Q 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 agentswere 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, includ-

ing 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 agent Q 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 pharmaco-

logical effects, including analgesic (Stockhaus, 1977) and

bradycardic properties (Kobinger et al., 1979; Traunecker &

Walland, 1980; Millar & Vaughan-Williams, 1981; Lillie &

Kobinger, 1983).

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

slowing of spontaneous activity accompanied by a prolon-

gation 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 itsselectivity 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; DiFran-cesco, 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


8/20/2019 1-s2.0-S0163725805000252-main

14/21

bopen channel blocker Q , 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 (65–95%) 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 (DK-

AH269), 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 concen-

tration 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 (0.1–100 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%; BoSmit h

et al., 1993). Furthermore, ZD-7288, like alinidine but

unlike zatebradine, blocked I f in a use- and frequency-independent way; the simplest interpretation of this property

is that ZD-7288 interacts with its channel binding site

independently from the channel gating configuration (Gas-

parini & DiFrancesco, 1997). The development of ZD-

7288-induced I f block in SAN myocytes and in other cell

types was slow (BoSmith et al., 1993; Harris & Constant i,

1995; Gasparini & DiFrancesco, 1997), suggesting an

intracellular block like that of zatebradine.

The selectivity of ZD-7288 for f-channels was inves-

tigated 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. Concen-

trations 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 hyper-

polarization-activated current ( I h) was observed in guinea

pig substantia nigra neurons, rat hippocampal CA1 cells, cat

ventrobasal thalamocortical neurons, and newt photorecep-

tors (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


8/20/2019 1-s2.0-S0163725805000252-main

15/21

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

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 SANcells have shed light on some of the molecular aspects of its

blocking mechanism (Bucchi et al., 2002). These studies

have shown that binding–unbinding 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 along 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 removaloccurs, 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).


8/20/2019 1-s2.0-S0163725805000252-main

16/21

other heart rate-reducing agents, ivabradine blocks f-

channels 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 hyperpolarizedvoltages. 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 depend-

ence on the ion flow across the channel pore; ivabradine

block of f-channels is therefore bcurrent Q 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-dependent Q 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 bcurrent Q depend-

ence 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 vitrospecificity 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 coro-

nary 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 continu-

ation 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 b pacemaker Q current. The way these bheart

rate-reducing Q drugs act is to inhibit, by partial channel

block, the I f function and thus slow pacemaker activity.

Ideally, however, the concept of b pacemaker Q

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

b pacemaker Q current, the so-called b biological Q 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

b proofs of concept Q , 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.


8/20/2019 1-s2.0-S0163725805000252-main

17/21

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 treat-

ments into spontaneously

Documents

1-s2.0-S0163725805000252-main