martin biel et al- hyperpolarization-activated cation channels: from genes to function

Hyperpolarization-Activated Cation Channels: From Genesto Function

MARTIN BIEL, CHRISTIAN WAHL-SCHOTT, STYLIANOS MICHALAKIS, AND XIANGANG ZONG

Center for Integrated Protein Science CIPS-M and Zentrum fur Pharmaforschung, Department Pharmazie,

Pharmakologie fur Naturwissenschaften, Ludwig-Maximilians-Universitat Munchen, Munich, Germany

I. Introduction 848II. A Short Overview on Basic Biophysical Properties of Ih 848

A. Channel gating by membrane hyperpolarization 849B. Modulation by cyclic nucleotides 850C. Ion selectivity 850D. Pharmacological profile 851

III. The HCN Channel Family 851A. Transmembrane segments and voltage sensor 852B. Pore loop and selectivity filter 852C. Cyclic nucleotide-binding domain and C-linker 852D. Dual channel gating 853E. Functional differences between HCN channel types 854

IV. Regulation of HCN Channels 856A. Regulation by acidic lipids 856B. Regulation by protons 857C. Regulation by chloride 857D. Regulation by Src kinase-mediated tyrosine phosphorylation 857E. Regulation by p38-mitogen-activated protein kinase 858F. Transmembrane and cytosolic proteins interacting with HCN channels 858

V. Tissue Expression of HCN Channels 859VI. Physiological Roles of HCN Channels in Neurons 860

A. Principles 860B. Role of Ih in dendritic integration 861C. Role of Ih in working memory 863D. Role of Ih in constraining hippocampal LTP 863E. Role of Ih in motor learning 864F. Role of Ih in synaptic transmission 864G. Role of Ih in resonance and oscillations 864H. Role of Ih in the generation of thalamic rhythms 865

VII. Role of Ih Channels in Cardiac Rhythmicity 867A. HCN4 868B. HCN2 869C. Conclusions and open questions 869

VIII. Role of Ih in Disease 870A. Inherited channelopathies 870B. Transcriptional channelopathies 870

IX. HCN Channels as Novel Drug Targets 873A. Heart rate-reducing agents 873B. Blockers of neuronal Ih 874

X. Conclusions and Future Directions 874

Biel M, Wahl-Schott C, Michalakis S, Zong X. Hyperpolarization-Activated Cation Channels: From Genes toFunction. Physiol Rev 89: 847–885, 2009; doi:10.1152/physrev.00029.2008.—Hyperpolarization-activated cyclic nu-cleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cationchannels. In mammals, the HCN channel family comprises four members (HCN1-4) that are expressed in heart andnervous system. The current produced by HCN channels has been known as Ih (or If or Iq). Ih has also been

Physiol Rev 89: 847–885, 2009;doi:10.1152/physrev.00029.2008.

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designated as pacemaker current, because it plays a key role in controlling rhythmic activity of cardiac pacemakercells and spontaneously firing neurons. Extensive studies over the last decade have provided convincing evidencethat Ih is also involved in a number of basic physiological processes that are not directly associated with rhythmicity.Examples for these non-pacemaking functions of Ih are the determination of the resting membrane potential,dendritic integration, synaptic transmission, and learning. In this review we summarize recent insights into thestructure, function, and cellular regulation of HCN channels. We also discuss in detail the different aspects of HCNchannel physiology in the heart and nervous system. To this end, evidence on the role of individual HCN channeltypes arising from the analysis of HCN knockout mouse models is discussed. Finally, we provide an overview of theimpact of HCN channels on the pathogenesis of several diseases and discuss recent attempts to establish HCNchannels as drug targets.

I. INTRODUCTION

Since their first discovery in sinoatrial node cells (49,51, 113, 116, 430) and neurons (169, 234) in the late 1970sand early 1980s, hyperpolarization-activated currents (Ih)have sparked continuing interest of physiologists and re-searchers working in biomedical disciplines. The excep-tional position of Ih among ionic currents mainly arisesfrom its unique ion selectivity and gating properties. Ih isa mixed cationic current carried by Na� and K�. Since itsreversal potential is around �20 mV at physiological ionicconditions (114), Ih is inwardly directed at rest and,hence, depolarizes the membrane potential. However, un-like the vast majority of cellular conductances that areactivated upon membrane depolarization, Ih is activatedby hyperpolarizing voltage steps to potentials negative to�55 mV, near the resting potential of cells. In addition,activation of Ih is facilitated by cAMP in a direct, proteinkinase A (i.e., phosphorylation)-independent fashion. Be-cause of its quite unusual biophysical profile, the hyper-polarization-activated current was designated “funny”current (If) in heart (49), while other researchers used theterm Iq (for “queer”) instead of Ih for the neuronal current(169). In this review, we will generally use the term Ih.

Many physiological roles have been attributed to Ih.First and foremost, the properties of Ih suggested that thecurrent plays an important role in the initiation and reg-ulation (49, 117) of the heart beat (“pacemaker cur-rent”)(51, 113, 430). However, even now, 30 years afterthis hypothesis has been raised, strong controversy overthe exact role of Ih in cardiac pacemaking exists. Geneticstudies in men and mice (see sect. VII) will be very instru-mental in solving this physiological conundrum (171, 174,226, 370). Ih is also involved in the control of rhythmicactivity in neuronal circuits (128, 221, 222, 258, 261, 355)(e.g., in thalamus) and contributes to several other basicneuronal processes, including determination of restingmembrane potential (98, 125, 226, 230, 266, 291–293, 300),dendritic integration (239–241, 423), synaptic transmis-sion (32, 33), and the temporal processing of visual signalsin the retina (105, 106, 209, 247).

The ion channels underlying Ih have been discoveredabout a decade ago (153, 192, 211, 227, 228, 251, 332, 333,341, 344, 400). With reference to their complex dual gating

mode (7, 93, 112), these proteins were termed hyperpo-larization-activated cyclic nucleotide-gated (HCN) chan-nels.

In mammals, the HCN channel family comprises fourdistinct members (HCN1-4). Currents obtained after het-erologous expression of the cDNAs of HCN1-4 channelsreveal the principal features of native Ih, confirming thatHCN channels indeed represent the molecular correlateof Ih.

In the first three sections of this review, we give abrief overview on the biophysical properties of Ih/HCNcurrents and correlate these properties with structuraldomains of HCN channels. Readers who are interested ingetting a more detailed and complete overview on thementioned topics are referred to a number of excellentreviews that extensively deal with these issues and alsodescribe the discovery of Ih and the advance in the Ih fieldfrom a more historical point of view (29, 40, 82, 93, 152,226, 300, 324, 334). Section IV is a first important focus ofthis review, presenting recent data on the cellular regula-tion of HCN channels. Section V briefly summarizes thedata available on the tissue expression of HCN channels.The major focus of this review is on the roles of HCNchannels in neuronal function and cardiac rhythmicity(sects. VI and VII). We discuss these issues in the context ofrecent results from the analysis of HCN channel knockoutmice. Finally, we give an overview on the role of HCNchannels in the pathologies of human diseases (sect. VIII)and discuss pharmacological agents interacting withthese proteins (sect. IX).

II. A SHORT OVERVIEW ON BASIC

BIOPHYSICAL PROPERTIES OF Ih

Native Ih as well as the currents induced by hetero-logously expressed HCN channels are characterized byfour major hallmark properties: 1) channel activation bymembrane hyperpolarization, 2) facilitation of channelactivation by direct interaction with cAMP, 3) permeationof Na� and K�, and 4) a specific pharmacological profilethat includes sensitivity to external Cs� concurred withrelative insensitivity to Ba2� (Fig. 1).

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A. Channel Gating by Membrane Hyperpolarization

In general, Ih currents activate with hyperpolarizingsteps to potentials negative to �50 to �60 mV (Fig. 1A).Unlike most other voltage-gated currents, Ih does notdisplay voltage-dependent inactivation. Typically, two ki-netic components can be distinguished upon activation ofIh: a minor instantaneous current (IINS) (236, 237, 312),which is fully activated within a few milliseconds, and themajor slowly developing component (ISS) that reaches itssteady-state level within a range of tens of milliseconds toseveral seconds under fully activating conditions. Whilethere is no doubt that ISS is generated by cations passingthe well-characterized pore of HCN channels, the ionicnature of IINS is a matter of current dispute. IINS is notconsistently observed in all measurements of Ih and, if so,its amplitude is usually small.

However, in some neurons, a large IINS lacking time-dependent ISS has been reported (12, 100, 326). Specula-tions on the nature of IINS reach from models where thiscurrent represents a leak conductance or an experimentalartifact to models in which IINS is caused by a second porethat is found within the same HCN channel that producesISS or a second channel population associated with HCNchannels (236, 237, 312). Depending on the cell type, theactivation of ISS can be empirically described by either asingle (120, 136, 200, 261, 264, 355, 393, 425) or doubleexponential function (17, 104, 131, 175, 248, 402). Asmentioned above, kinetics of ISS are quite variable (111,118). While most Ih channels activate quite slowly withtime constants (�act) ranging between hundreds of milli-seconds and seconds (131) (Fig. 1B, inset), there are alsocurrents with profoundly faster activation (17, 169, 175,

234, 248, 264), for example, in hippocampal CA1 neurons,where �act values in the range of 30–50 ms have beenfound (146, 300, 324). The diversity of �act probably resultsfrom an interplay of several factors. First of all, the dif-ferences reflect the diverse intrinsic activation propertiesof distinct HCN channel isoforms underlying the Ih of agiven cell type. Second, there is growing evidence that thecellular microenvironment that fine-tunes HCN channelactivity (e.g., auxiliary subunits, concentration of cellularfactors, etc.) can profoundly vary from cell type to celltype. Third, Ih measurements are highly sensitive to ex-perimental conditions (e.g., pH, temperature, patch con-figuration, expression system, ionic composition of solu-tions, etc.), a circumstance that may explain that even forthe same channel different kinetics have been found bydifferent laboratories. As mentioned above, Ih activates ataround resting membrane potential. The voltage depen-dence of activation shows a typical S-shaped dependencethat can be fit using Boltzmann functions (20, 25, 131, 175,200, 216, 234, 253, 261, 354, 357, 393) (Fig. 1B). Such fitsreveal half-maximal activation (“midpoint”) potentials(V0.5) of around �70 to �100 mV in most cell types.However, like for �act, V0.5 values can vary profoundlyin vivo (28, 111, 118). For example, V0.5 of Ih recorded insinoatrial node cells is in the range of �60 to �70 mV(3–5, 121, 124, 167) while currents found in ventricularcardiomyocytes activate at much more hyperpolarizedvoltages with V0.5 being negative to �90 to �140 mV (138,319, 346). Like for �act, the strikingly large range of V0.5

values probably results from the combination of intrinsicparameters such as the HCN channel isoform and modu-latory factors present in a given cell type, and extrinsic

FIG. 1. A: voltage dependence ofHCN2 activation. Family of currenttraces (bottom panel) and pulse protocol(top panel). B: activation curve of HCN2currents in the absence of cyclic nucleo-tides (open circles) or after intracellularperfusion with 1 mM cAMP (solid circles)or 1 mM cGMP (triangles), respectively.Inset: time course of HCN2 activation inthe absence (black line) and the presenceof cAMP (red line). C: dose-response re-lationship for the shift in the HCN2 acti-vation curve as a function of cAMP con-centration. D: current-voltage relation-ship for the fully activated HCN2 channeldetermined at 5.4 mM (solid circles) and30 mM (open circles) extracellular K�,respectively. E: extracellular block ofwhole cell current by 2 mM Cs�, 2 mMBa2�, 20 mM tetraethylammonium (TEA),and 1 mM 4-aminopyridine (4-AP), respec-tively. F: percentage inhibition of steady-statecurrent at �140 mV by 20 mM TEA, 1 mM4-AP, 2 mM Ba2�, and 2 mM Cs�.

FUNCTION OF HCN CHANNELS 849


factors such as the experimental settings during Ih mea-surements.

B. Modulation by Cyclic Nucleotides

The second key feature of Ih is its regulation by cyclicnucleotides (122; Fig. 1B). Hormones and neurotransmit-ters that elevate cAMP levels facilitate activation of Ih byshifting V0.5 values to more positive values and by accel-erating the opening kinetics (25, 41, 49, 50, 122, 136, 151,203, 216–218, 260, 262, 303, 391). It has been shown thatthe acceleration of the opening kinetics with cAMP can beattributed to the shift in voltage dependence of activation(412). Thus, in the presence of high cAMP concentrations,Ih channel opening is faster and more complete than atlow cAMP levels (Fig. 1B, inset). Conversely, neurotrans-mitters that downregulate cAMP inhibit Ih activation byshifting its activation curve to more hyperpolarized volt-ages (117, 123, 124, 147, 190, 299, 315, 318). The range ofV0.5 shift induced by saturating cAMP concentrations isquite large (0–20 mV), depending on the cell type (25, 41,49, 50, 122, 136, 151, 203, 216–218, 260, 262, 303, 384, 391)and the expressed Ih channel isoform (192, 227, 228, 333,341). The regulation by cellular cAMP level is of keyimportance for the specific role that Ih channels fulfill inphysiological settings, since it enables these proteins tooperate as transmembrane integrators of electrical (mem-brane potential) and chemical (hormones and neurotrans-mitters) inputs. Most notably, the cAMP-mediated modu-lation of Ih channel activity is considered to play a majorrole in the up- or downregulation of the heart rate duringsympathetic stimulation and muscarinic regulation ofheart rate at low vagal tone (49, 50, 117, 122–124).

As mentioned above, it was this specific physiologi-cal function that earned Ih the name “pacemaker” current.There is also good evidence that cAMP-dependent modu-lation of Ih is of crucial significance in some neuronalcircuits, e.g., in sleep-related thalamocortical circuits(231). There are a few studies showing that Ih can also beregulated by nitric oxide (NO)-mediated increase ofcGMP levels in brain (302) and heart (283). However, sofar the physiological role of this kind of modulation re-mains unclear. Like cAMP, cGMP shifts the voltage de-pendence of channel activation to more positive values(Fig. 1B) (153, 227). While the extent of the shift is similarfor both cyclic nucleotides (i.e., both cyclic nucleotideshave the same efficacy at least in mammalian Ih), theapparent affinities of Ih are �10- to 100-fold higher forcAMP (range of Ka � 60–500 nM; Fig. 1C; Ref. 227) thanfor cGMP (Ka � 6 �M for HCN2; Ref. 227). The principalmechanism by which cyclic nucleotides regulate Ih chan-nel gating was uncovered by DiFrancesco and Tortora(122) in the early 1990s using current recordings in ex-cised patches of sinoatrial node cells. It came as a big

surprise that in contrast to many other ion channels thatare regulated by cAMP via protein kinase A (PKA)-medi-ated serine or threonine phosphorylation (304, 337, 433),Ih channels are activated by cAMP independent of phos-phorylation (122). Like in the structurally related cyclicnucleotide-gated (CNG) channels (39, 93, 201), Ih chan-nels are activated by cAMP binding to a cyclic nucleotide-binding domain (CNBD) on the COOH terminus of thechannel (443). The molecular details underlying the mod-ulation of Ih channels by cAMP have been extensivelystudied when cloned Ih(HCN) channels were availableand will be discussed later. There also are some reportson PKA-mediated phosphorylation of Ih channels (79,403); however, phosphorylation could not yet be con-firmed on the molecular level in HCN channels. Moreover,the physiological relevance of PKA-induced Ih phosphor-ylation remains elusive.

C. Ion Selectivity

Ih is a mixed cation current that is carried by both K�

and Na� under normal physiological conditions (110, 116,145, 227, 333, 424). The ratio of the Na� to K� permeabil-ity of the channel (PNa:PK) ranges from 1:3 to 1:5, yieldingvalues for the reversal potential between �25 and �40mV (Fig. 1D) (20, 25, 31, 60, 95, 103, 113, 131, 175, 180, 200,203, 227, 253, 261, 264, 301, 333, 341, 354, 357, 383, 391,393, 402, 424). As a consequence, activation of Ih at rest-ing membrane potentials results in an inward currentcarried mainly by Na�, which depolarizes the membranetoward threshold for firing of action potentials. Ih is al-most impermeable to Li� (180, 424) and is blocked by Cs�

(109, 137, 227). K� is not only a permeating cation, it alsoaffects the permeation of Na� (20, 95, 103, 109, 132, 145,175, 200, 227, 234, 248, 250, 261, 354, 357, 383, 385, 424).Both the current amplitude and the PNa:PK ratio of Ih

channels depend on the extracellular K� concentration(Fig. 1D) (227). An increase in extracellular K� concen-tration results in a strongly increased current amplitudeand in a slightly reduced selectivity for K� over Na� (145,424). The interdependence of Na� and K� permeation ofIh channels is illustrated by the finding that the channelsconduct little, if any, Na� in the absence of K�. In con-trast, current modifications upon reduction of extracellu-lar Na� levels are merely the result of the altered drivingforce (116, 300). Although Ih channels do not conductanions, their conductance is sensitive to external Cl�

levels (144, 411) (see sect. IVC for further discussion). Fora long time, only monovalent cations were expected topermeate through the Ih channels. However, there is re-cent evidence for a small but significant Ca2� permeabil-ity of these channels (440, 441). At 2.5 mM external Ca2�,the fractional Ca2� current of native Ih as well as that ofheterologously expressed HCN2 and HCN4 channels is

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�0.5% (440, 441). The functional relevance of Ca2� entrythrough Ih channels is not clear at the moment. It wasassumed that in dorsal root ganglion neurons, Ca2� influxthrough Ih channels at negative potentials contributes toactivity-evoked secretion (441).

There is an ongoing controversy on the size of thesingle-channel conductance of Ih. Originally, single-chan-nel conductance was found to be very low, in the range of�1 pS (110). This estimate is in good agreement with veryrecent data (210). However, single-channel conductancesthat are 10–30 times higher have been reported for clonedHCN channels (267) as well as for native cardiac (267)and neuronal Ih (350). At this point, it is unclear how thismajor discrepancy can be explained.

Furthermore, single-channel conductance as well asother biophysical parameters may be dynamically regu-lated by modulatory factors and proteins assembled withHCN channels in vivo. However, extreme care must betaken before a final conclusion should be drawn on thisissue. It will be necessary to rigorously prove that therecorded currents are indeed HCN channel currents. Forexample, one problem with single-channel recording re-ported by Michels and co-workers (267, 268) is that theensemble records of single-channel activity poorly matchknown kinetic properties of HCN channel current basedon whole cell recordings. This issue needs to be clarifiedin the future.

D. Pharmacological Profile

Ih channels are almost completely blocked by lowmillimolar concentrations of Cs� (Fig. 1, E and F) (109,137, 227). Inwardly rectifying K� currents that activateover a similar voltage range as Ih are also blocked by thiscation (99, 288, 289). However, in contrast to these cur-rents, Ih is insensitive to millimolar concentrations ofexternal Ba2� and tetraethylammonium (TEA) (227). Ih isalso insensitive to 4-aminopyridine, a blocker of voltage-gated K� channels (227). A number of organic blockershave been described to block Ih channels. Among these,the most specific ones are bradycardic agents such asivabradine that block the Ih channels in the low micro-molar range (see sect. IX).

III. THE HCN CHANNEL FAMILY

HCN channels represent the molecular correlate ofIh. Together with CNG channels and the Eag-like K�

channels (39, 93, 201), HCN channels form the subgroupof cyclic nucleotide-regulated cation channels within thelarge superfamily of the pore-loop cation channels (437).HCN channels have been cloned from vertebrates andseveral invertebrates (153, 192, 211, 227, 228, 251, 332,

333, 341, 344, 400) but are missing in Caenorhabditis

elegans, yeast, and prokaryotes.In all mammals investigated so far, four homologous

HCN channel subunits (HCN1-4) exist. HCN1-4 are alsopresent in the genome of fishes (e.g., Tetraodon nigro-

viridis and Takifugu rubripes). Analysis of genomic se-quences suggests that HCN2–4 genes underwent duplica-tions in the fish lineage increasing the number of potentialHCN species (194). HCN homologs have also been clonedfrom several invertebrates including arthropods [e.g.,spiny lobster (157), insects (158, 211, 251)] and sea ur-chins (149, 153). The overall characteristics of the Ih

currents from these species are strikingly similar to Ih

currents from mammals. Notably, HCN channels are notpresent in C. elegans and yeast and also have not beenfound in a prokaryotic genome.

Like other pore-loop channels, HCN channels arecomplexes consisting of four subunits that are arrangedaround the centrally located pore (Fig. 2). These subunitsform four different homotetramers with distinct biophys-ical properties (192, 227, 228, 333, 341). There is evidencethat the number of potential HCN channel types is in-creased in vivo by the formation of heterotetramers (8, 85,135, 278, 397, 420, 428). So far, splicing variants of verte-brate HCN channels were not identified. In contrast, al-ternative splicing of HCN channel transcripts was re-ported for some invertebrates, including spiny lobster,

C-linker

FIG. 2. Structure of HCN channels. HCN channels are tetramers.One monomer is composed of six transmembrane segments includingthe voltage sensor (S4) and the pore region between S5 and S6. The poreregion contains the selectivity filter carrying the GYG motif. The COOH-terminal channel domain is composed of the C-linker and the cyclicnucleotide-binding domain (CNBD). The C-linker consists of six �-heli-ces, designated A� to F�. The CNBD follows the C-linker domain andconsists of �-helices A-C with a �-roll between the A- and B-helices(thick gray line). Human mutations involved in Ih channelopathies areindicated as gray circles. The N-glycosylation site between S5 and thepore loop is indicated as a Y. For details, see text.



Drosophila melanogaster, and Apis mellifera (156, 159,298). Each HCN channel subunit consists of three princi-pal structural modules: the transmembrane core and thecytosolic NH2-terminal and COOH-terminal domains (Fig.2). The transmembrane core harbors the gating machin-ery and the ion-conducting pore while the proximal partof the cytosolic COOH-terminal domain consisting of theCNBD and the peptide that connects the CNBD with thetransmembrane core (the “C-linker”) confers modulationby cyclic nucleotides (93) (Fig. 2). The transmembranecore and the proximal COOH terminus allosterically in-teract with each other during channel gating and reveal ahigh degree of sequence conservation within the HCNchannel family (sequence identity of �80–90% betweenHCN1-4) (28, 202). In contrast, cytosolic NH2 termini andthe sequence downstream of the CNBD vary considerablyin their length and share only modest to low homologybetween various HCN channels (28).

A. Transmembrane Segments and Voltage Sensor

The transmembrane channel core of HCN channelsconsists of six �-helical segments (S1-S6) and an ion-conducting pore loop between S5 and S6 (Fig. 2). A highlyconserved asparagine residue in the extracellular loopbetween S5 and the pore loop is glycosylated (N380 inmurine HCN2; Fig. 2). This posttranslational channelmodification was shown to be crucial for normal cellsurface expression (278). The voltage sensor of HCNchannels is formed by a charged S4-helix carrying ninearginine or lysine residues regularly spaced at every thirdposition (81, 399). Positively charged S4 segments arefound in all voltage-dependent members of the pore-loopcation channel superfamily (436). However, inward move-ment of S4 charges through the plane of the cell mem-brane leads to opening of HCN channels while it triggersthe closure of depolarization-activated channels such asthe Kv channels (245). The molecular determinants un-derlying the different polarity of the gating mechanism ofHCN and depolarization-gated channels remain to be clar-ified. However, there is initial evidence that the loopconnecting the S4 with the S5 segment plays a crucial rolein conferring the differential response to voltage (102,224, 314) (Fig. 2).

Recently, it was shown that the voltage-dependentactivation of the HCN channel cloned from sea urchinsperm (spHCN) can shift between two modes dependingon the previous activity (53, 133, 246). In mode I, gatingcharge movement and channel opening occur at verynegative potentials, while in mode II, both processes areshifted to �50 mV more positive potentials (133). Thetransition from mode I to mode II is favored in the openstate, while the transition from mode II to mode I prefer-entially occurs in the closed state. The shift between

these two modes also affects the kinetics of the channelactivation and deactivation. The fact that these channelsare differentially sensitive to the direction of the voltagechange and that the activation curves in mode I and modeII are separated by up to 50 mV is defined as “voltagehysteresis.” This interesting gating behavior of spHCN isprobably the result of a slow conformational change at-tributable to lateral movement of S4 that stabilizes theinward position of S4 upon hyperpolarization (52, 53, 133,245, 246, 405). HCN1 channels behave similarly as spHCNchannels with respect to hysteresis behavior and modeshift (246). In contrast, for HCN2, the voltage hysteresis isless pronounced, but the effects of the mode shift on thedeactivation kinetics are present. For HCN4, only thechanges in deactivation kinetics are observed under cer-tain conditions using high K� concentrations in the extra-cellular recording solution. It has been suggested that themode shift is important for short-term, activity-dependentmemory in HCN channels (53).

B. Pore Loop and Selectivity Filter

As pointed out before, Ih channels slightly select K�

over Na� and carry an inward Na� current under physi-ological conditions (Fig. 2). Given this particular ion se-lectivity profile, it came as a big surprise that the poreloop sequence of HCN channels is closely related to thatof highly selective K� channels (436). Notably, the porecontains the glycine-tyrosine-glycine (GYG) motif that inK� channels forms the selectivity filter for K� (129, 448)(Fig. 2). Thus, based on sequence analysis, HCN channelsare expected to be selective for K� and to exclude Na�

from permeation. Also, conduction of divalent cations isnot consistent with current models, since a ring of acidicresidues (glutamate or aspartate) that is present in thepore of Ca2�-permeable channels is missing in HCN chan-nels (343, 431, 442). Several attempts have been made,mainly using site-directed mutagenesis approaches, toidentify residues in the pore of HCN channels that conferthe unique permeation properties of these channels (19,135, 237, 278, 428). However, these efforts were quiteunsuccessful. A major problem is that the pore regionturned out to be extremely sensitive to mutagenesis andthat even subtle mutations can lead to nonconductingchannels. Clearly, a high-solution crystal structure will berequired to establish a conclusive model of ion perme-ation in HCN channels.

C. Cyclic Nucleotide-Binding Domain and C-Linker

Regulation of HCN channels by cAMP is mediated bythe proximal portion of the cytosolic COOH terminus(443) (Fig. 2). This part of the channel contains a cyclic

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nucleotide-binding domain composed of �120 amino ac-ids (CNBD) and an 80-amino acid-long C-linker regionthat connects the CNBD with the S6 segment of thechannel core (Figs. 2 and 3). Evolutionarily, the CNBD isan ancient protein domain that has been conserved acrossa wide range of proteins, including bacterial transcriptionfactors (e.g., CAP; Ref. 419), cAMP- and cGMP-dependentprotein kinases (307, 387), cAMP-dependent guanine nu-cleotide exchange factors (Epac; Ref. 321), and CNGchannels (39, 201). The determination of the crystal struc-ture of the C-linker-CNBD of HCN2 in its cAMP- andcGMP-bound form (443) and of spHCN in its cAMP-boundform (142) has provided important insights into themolecular determinants of cyclic nucleotide-dependentchannel modulation. So far, high-resolution structures ofunliganded CNBDs from HCN channels are not available.The structures of the two liganded C-linker-CNBD do-

mains of HCN2 were virtually identical, with the onlydifference that cAMP binds in its anti conformation whilecGMP binds in the syn conformation (443). The CNBDshows a highly conserved fold consisting of an initial�-helix (A-helix), followed by an eight-stranded antipar-allel �-roll (�1-�8), a short B-helix, and a long C-helix(Figs. 2 and 3). The C-linker consists of six �-helices(A�–F�) (Figs. 2 and 3). Four C-linker CNBDs are assem-bled to a complex with a fourfold axis of symmetry that isconsistent with the tetrameric architecture of pore-loopchannels. Most of the subunit-subunit interactions in thetetramer are mediated by amino acid residues in theC-linker. The binding pocket for cAMP is formed by anumber of residues at the interface between the �-roll andthe C-helix (142, 443, 446). Within the binding pocket ofHCN2, a group of seven amino acids interacts with ligand(446) (Fig. 3).

Three of these residues are located in the �-roll(R591, T592, and E582 in HCN2) and four in the C-helix(R632, R635, I636, and K638 in HCN2). Among these, onlyone key residue of the C-helix (R632) was identified tocontrol the efficacy by which cyclic nucleotides enhancechannel opening (446). Four residues in the C-helix (e.g.,R632, R635, I636, and K638) contribute to the higherselectivity of HCN2 channels for cAMP than for cGMP(446). However, it is not clear how these residues gener-ate cAMP selectivity. It was suggested that cAMP selec-tivity at least partially is not due to its preferential con-tacts with the protein, but rather reflects the greaterhydration energy of cGMP relative to cAMP. This couldresult in a greater energetic cost for cGMP binding to thechannel (447).

The CNBD is not only an important modulatory do-main in HCN channels, like in the structurally relatedEag-like K� channels (18), it may also be important fornormal cell surface expression of these proteins (6, 313).Recently, a four-amino acid motif (EEYP) in the B-helix ofHCN2 was identified that strongly promotes channel ex-port from the endoplasmatic reticulum and targeting tothe cell membrane (287).

D. Dual Channel Gating

Unlike CNG channels, which obligatorily requirebinding of cAMP to open (39, 201), HCN channels areprincipally operated by voltage, i.e., membrane hyperpo-larization is necessary and sufficient to activate thesechannels. cAMP and cGMP can be considered as stimu-latory modulators (or coagonists) of HCN channels thatdo not open the channels in the absence of membranehyperpolarization but rather facilitate voltage-dependentactivation by shifting the voltage dependence of activa-tion to more positive voltages (122). Electrophysiologicalstudies in excised patches from sinoatrial node cells in-

FIG. 3. Sequence alignment of HCN2 and spHCN COOH terminus.Residues that have been reported by Zhou and Siegelbaum (446) tointeract with cAMP are indicated in red. Residues that have been iden-tified by Flynn et al. (142) to control cGMP efficacy in spHCN areindicated by arrows. Tyrosine residues that are phosphorylated by cSrckinase are shown in blue (Y453 in HCN2 corresponds to Y531 in HCN4).�-Helices are indicated as green boxes, and the eight �-sheets areindicated as green arrows and numbered from 1–8.



dicated that the modulatory effect of cAMP is due to itsdirect binding to the channel rather than being conferredby protein phosphorylation (122). The identification ofCNBDs in the primary sequence of HCN channels stronglysupported this model and suggested that mechanisticallythere were probably similarities between the action ofcyclic nucleotides in HCN and CNG channels. As dis-cussed in the previous section, crystal structure dataalong with site-directed mutagenesis and molecular dy-namics simulations allowed to identify structural deter-minants controlling cAMP and cGMP binding (142, 412,414, 443, 446, 447).

There is now good evidence that “disinhibition” is thefundamental principle underlying the cAMP-dependentmodulation of HCN channels. The C-linker-CNBD is anautoinhibitory domain that in the absence of cAMP lowersopen probability. Binding of cAMP increases channel ac-tivity by removing tonic channel inhibition that is con-ferred by this domain (412, 414). The exact sequence ofmolecular events leading from initial cyclic nucleotidebinding to facilitation of voltage-gated channel opening isstill an unsolved issue. Addressing this problem is com-plicated by the fact that in the available crystal structuresthe C-linker seems to be caught in the resting conforma-tion (i.e., the conformation that is occupied in the absenceof cAMP) while the CNBD is in its active (i.e., cAMP-bound) conformation (93, 94, 198). According to a recentmodel (93), in the absence of cAMP, the C-linker isthought to be in a “compact” conformation that producesan inhibitory effect on channel opening. Binding of cAMPin turn induces a conformational change in the CNBDinvolving the C-helix. The conformational change in theC-helix is then coupled to the C-linker that occupies amore “loose” conformation leading to an alteration of theintersubunit interface between helices of neighboringsubunits. The resulting change in the quaternary confor-mation removes the inhibition of the COOH terminus anddestabilizes the closed state, thus promoting the openingof the channel. One would expect that the describedactivation process comes along with large translocationsof subdomains in the C-linker-CNBD. However, recentdata suggest that this may not be necessarily the case.Fluorescent resonance energy transfer (FRET) measure-ments in a CNG channel suggested that movements withinthe C-linker are probably subtle, involving only limitedrearrangements (386).

The finding that cAMP facilitates activation of HCNchannels by affecting their voltage dependence suggeststhat the gating mechanism operated by hyperpolarizationand the one operated by cAMP are closely related to eachother, if not identical. In other words, HCN channelsbehave in the presence of a given cAMP concentrationsimply as if they were experiencing a stronger voltagedrop. Several kinetic models have been proposed to de-scribe the tight interconnection between voltage- and cy-

clic nucleotide-dependent gating (for a recent overview,see Refs. 28, 93). Among these, cyclic allosteric modelsderived from the classic Monod-Wyman-Changeux(MWC) model developed for hemoglobin (274), currentlygive the best approximation to HCN channel behavior (7,112). In its basic form, the MWC model assumes that eachof the four subunits of the tetrameric channel is indepen-dently gated by voltage. Every time a voltage sensorswitches to the activated state, the probability for channelopening increases. The opening/closing reactions occurallosterically and involve concerted transitions of all foursubunits. This transition occurs if the channel is unligan-ded, partially liganded, or fully liganded and is energeti-cally stabilized by a constant amount for each cAMPbound. The model further assumes that cAMP has ahigher binding affinity to open than to closed channels (7,112, 413). The model reproduces the characteristic fea-tures of HCN channel gating like the voltage dependenceand the activation by cyclic nucleotides. In addition, sev-eral kinetic features are well described by the model,including the delay in current activation and deactivation.However, in its original form, the model also has severallimitations. For example, there is evidence (86) that thefinal allosteric open/close transition in HCN2 channels isnot voltage dependent as suggested previously (7). Inaddition, the mode shift and the voltage hysteresis of HCNchannels cannot be explained by simple MWC models. Tosolve these issues, several extensions of the cyclic allo-steric model have been proposed among which the dimer-of-dimers model (396), the circular four-state model byMannikko et al. (246), and an extension of the MWCmodel made by Zhou and Siegelbaum (446) are the mostrecent ones. Despite the progress that clearly has beenmade in mathematically modeling HCN channel gating, itis obvious that these approaches have to be accompaniedby new high-resolution structures of HCN channels inclosed and open state(s) to achieve a full understandingof channel behavior.

E. Functional Differences Between HCN

Channel Types

All four mammalian HCN channel subtypes havebeen expressed in heterologous expression systems andshown to induce currents that display the principal bio-physical properties of native Ih (153, 192, 211, 227, 228,333, 341, 400). However, the individual HCN currentsquantitatively differ from each other with respect to theirrespective activation time constants (�), their steady-statevoltage dependence, and the extent of cAMP-dependentmodulation. Before we will discuss these issues in moredetail, it is important to recall that at least some of thereported differences may not reflect the intrinsic biophys-ical diversity of individual HCN channel types. Instead,

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these differences rather can be attributed to the fact thatmeasurements of Ih are very sensitive to experimentalparameters and that the same HCN channel may be em-bedded in quite diverse modulatory networks, dependingon the expression system or cell type (111, 118). Theintrinsic sensitivity to external and internal factors alsomakes it difficult to unambiguously match the Ih character-ized in a native type with Ih currents induced by a heterolo-gously expressed HCN channel isoform. Assembly of differ-ent HCN channel subtypes to heterotetrameric channelcomplexes may further increase heterogeneity of Ih chan-nels.

Within the HCN channel family, HCN1 is the subtypewith the fastest kinetics (191, 331, 372). Like in native Ih

activation kinetics of HCN1 is strongly voltage dependent,with �act values ranging from 30 to 300 ms at �140 to �95mV (191, 331, 372). HCN1 is also the HCN channel typewith the most positive V0.5 value (range �70 to �90 mV)(8, 28, 372). On the other hand, compared with HCN2 andHCN4, HCN1 reveals only a weak shift of the activationcurve in the presence of saturating cAMP concentrations(�2 to �7 mV) (8, 372, 409, 412, 414). Taken together,these findings suggest that HCN1 activation is energeti-cally favored compared with the other channels. Onereason for this finding is that the tonic channel inhibitionproduced by the C-linker-CNBD is weaker in HCN1 thanin HCN2 or HCN4. Indeed, deletion of the CNBD in HCN1or HCN2 which is equivalent with a total removal of tonicinhibition resulted in channels with almost identical V0.5

values (412). However, HCN1 lacking a CNBD still acti-vates five- to sixfold faster than HCN2 lacking this do-main. This shows that gating differences between bothchannels are not solely determined by the different degreeof tonic inhibition conferred by the CNBD but that theopening of the core HCN2 channel is also inherentlyslower than that of HCN1 (412).

While HCN1 is the fastest member within the HCNchannel family, HCN4 is by far the channel with theslowest opening kinetics (192, 228, 341). �act values forthis channel range between a few hundred milliseconds atstrongly hyperpolarized voltages (�140 mV) up to manyseconds at normal resting potential (�70 mV) (192, 228,341). HCN2 occupies an intermediate position with �act

ranging from 150 ms to 1 s (228, 372). V0.5 values between�70 and �100 mV have been reported for HCN2 andHCN4 (8, 28, 372). Both channels also have in commonthat their steady-state activation curves are very sensitiveto cAMP with shifts of V0.5 of 10–25 mV (8, 192, 228, 277,341, 409, 412, 414, 443).

So far, electrophysiological measurements of HCN3-mediated currents have been only reported by a fewgroups (80, 271, 372). A major obstacle to a full charac-terization of this particular channel type is that the HCN3protein tends to accumulate in intracellular compart-ments and reveals only weak cell surface expression (170;

unpublished observations). V0.5 of HCN3 ranges between�80 and �95 mV. Activation constants of 250–400 ms at�140 mV place the kinetics of HCN3 in between those ofHCN2 and HCN4 (271, 372). Remarkably and unlike in allother HCN channels, cyclic nucleotides do not induce apositive shift of V0.5 in HCN3. Human HCN3 was found tobe insensitive to cAMP or cGMP (372), while the murinechannel seems to be even slightly inhibited by cyclicnucleotides (shift of V0.5 by �5 mV) (271). The structuraldeterminants underlying this unexpected behavior areunclear. Interestingly, when the CNBD of HCN4 is re-placed by the corresponding domain of HCN3, cAMPsensitivity is fully maintained, suggesting that the CNBDof HCN3 is principally able to bind cAMP and to mediatecAMP-dependent gating (372). Thus, within the HCN3channel, the CNBD may be functionally silenced by astructural change in channel domains that communicatecAMP binding with channel gating. A similar mechanismmay explain the lack of cAMP sensitivity in members ofthe Erg K� channel family that also contain a CNBD (54,184, 443).

The properties of an HCN channel cloned from seaurchin sperm (spHCN) differ from mammalian HCN chan-nels (153). In the absence of cAMP, the spHCN currentdevelops with a sigmoidal time course, and then decays toa much lower degree. In contrast, in the presence ofsaturating cAMP, hyperpolarizing voltage steps producelarge currents of sigmoidal waveform lacking inactiva-tion, which are virtually identical to those of mammalianHCN currents. The large augmentation of spHCN chan-nels by cAMP is produced by removal of inactivation withlittle or no shift in steady-state voltage dependence (142,153, 347). Mutation of a conserved phenylalanine residuein the S6-helix of spHCN to a leucine (F459L; F431 inHCN2) is sufficient to convert spHCN to a channel thatbehaves very much like HCN2, i.e., it shows no inactiva-tion and its voltage dependence is positively shifted bycAMP (347). The ability to accomplish this conversionwith a single point mutation in S6 argues strongly that thetwo channel types use the same fundamental mechanismsfor gating and that the different phenotypes representdifferences in the precise relationship between the ener-getics and kinetics of the different gating processes.

There is one other property in which spHCN differsfrom its mammalian homologs. In HCN1-4, saturating con-centration of either cAMP or cGMP induces the samemaximal current, i.e., both cyclic nucleotides are full ago-nists of these channels (202). In contrast, cGMP behavesas a partial agonist on the spHCN channel, activating only�50% of the maximal current obtained at saturating cAMPconcentrations (202). Using a combination of X-ray crys-tallography and electrophysiology, Flynn et al. (142) re-vealed that the efficacy of cyclic nucleotides in channelactivation is controlled by complex interactions of theseligands with residues in the �-roll and the C-helix of the



CNBD. Replacement of a valine in the �-sheet to threo-nine (V621T) in conjunction with another replacement inthe C-helix (I665D) (Fig. 3) is sufficient to convert cGMPinto a full agonist of spHCN. The V621T mutation proba-bly leads to binding of cGMP in the syn conformation(like in HCN2), while the aspartate in the C-helix is nec-essary to stabilize the movement of the C-helix afterprimary cGMP binding, a process which is coupled to theopening conformational change in the channel pore. Allfour mammalian HCN channels carry a threonine residueat the position equvialent to V621 in spHCN, which mayexplain why cGMP is a full agonist of these channels.Interestingly, however, mammalian channels also harboran isoleucine at the position equivalent to I665 in spHCN.Flynn et al. (142) speculated that in mammalian HCNchannels the opening allosteric conformational changemay be less energetically costly than in spHCN. There-fore, unlike in spHCN, in mammalian channels an inter-action with an aspartic acid residue in the C-helix may notbe required to promote full activation.

IV. REGULATION OF HCN CHANNELS

HCN channels are tightly regulated by interactingproteins as well as by low molecular factors (e.g., protons,chloride ions) in the cytosol and the extracellular space(Fig. 4). These molecules control the functional proper-ties of the channels in the plasma membrane, regulatetheir cell surface expression (i.e., the number of func-tional channels in the membrane), and control their tar-geting to defined cellular compartments. In the followingsection, we give an overview on HCN channel regulatorsthat have been identified in the last couple of years.

A. Regulation by Acidic Lipids

It has been long known that native Ih as well asheterologously expressed HCN currents display a rapidrundown when measured in excised patches or duringprolonged whole cell recordings (43, 85, 118, 119, 122,227). This effect is caused by an �30 to 50 mV hyperpo-larizing shift of the activation curve. It was speculatedthat washout or depletion of cAMP may account for up to20 mV of this shift; however, the mechanistic basis of therest of the voltage shift was unclear (85, 119). Recently,two groups provided convincing evidence that phospha-tidylinositol 4,5-bisphosphate (PIP2) is probably the miss-ing factor that underlies the so far unexplained gap in thevoltage shift (308, 449).

PIP2 is not a novel entry in the list of ion channelregulators but has been found to control numerous iontransporters and ion channels including many K� chan-nels, TRP channels, and Ca2� channels (for recent re-views, see Refs. 150, 378). In HCN channels, PIP2 acts asan allosteric activator from the intracellular site that fa-cilitates channel activation by shifting V0.5 �20 mV to-ward more positive potentials. Importantly, this action isindependent of the presence of cyclic nucleotides. As aresult, PIP2 adjusts HCN channel opening to a voltagerange relevant for the physiological role of Ih channels.PIP2-mediated regulation of HCN channels may be ofphysiological significance for the function in neuronalcircuits, as enzymatic degradation of phospholipids re-duces channel activation and slows down firing frequencyof neurons. Different levels of PIP2 may also contribute tothe profound variations in the half-maximal activationvoltages of Ih in cardiac cells of different developmentalstage or distinct regional distribution (77, 316, 325). Themolecular determinants conferring the effect of PIP2 onHCN channel gating are not yet known. Most likely, HCNchannels are activated by an electrostatic interaction be-tween the negatively charged head groups of phosphoinosi-tides and the channel protein. A similar mechanism forPIP2 modulation of gating was identified in voltage-gatedand inwardly rectifying K� channels (30, 186, 297, 349). Itwas proposed that the interaction between PIP2 and Ih

channels relieves an inhibition of channel opening con-ferred by an inhibitory channel domain. This “PIP2 re-sponsive” domain is clearly different from the CNBD be-cause the PIP2 effect is still present in channels lackingthe CNBD.

There is recent evidence that in addition to PIP2

other acidic lipids may also serve as allosteric modulatorsof HCN channels. Fogle et al. (143) showed that phospha-tidic acid and arachidonic acid, which are products ofdiacylglycerol kinase/phospholipase A2 signaling path-ways, directly facilitate HCN channel gating by shiftingthe V0.5 to more positive values (shift of 5–10 mV inHCN2). The molecular mechanism of this facilitation re-

FIG. 4. Regulation of HCN channels by interacting proteins and lowmolecular factors. Only one subunit of the tetrameric channel complexis shown. Proteins interacting with HCN channels and low molecularfactors regulating HCN channels are shown. KCR1, MiRP1, Filamin A,TRIP8b, S-SCAM, Tamalin, Mint2, cSrc, Cl�, and H� interact with dif-ferent channel portions as indicated. The tyrosine phosphorylation sitein the C-linker is shown as a circle with a “P” in it.

856 BIEL ET AL.


mains to be elucidated, but there is initial evidence that itinvolves direct binding of both metabolites to the channeland is independent of cAMP pathways. It was speculatedthat the regulation of the voltage dependence of Ih byacidic lipids may be involved in the fine tuning of sub-threshold properties of excitable cells (143).

B. Regulation by Protons

The activity of HCN channels depends on both intra-cellular (281, 451) and extracellular (369) concentrationsof protons. Intracellular protons shift the voltage depen-dence of channel activation to more hyperpolarized po-tentials and slow down the speed of channel opening. Inthe murine HCN2, a protonable histidine residue (His321)localized at the boundary between the voltage-sensingS4-helix and the cytoplasmatic S4-S5-linker has been iden-tified to confer intracellular pH (pHi) sensitivity (451)(Fig. 4). At acidic (pHi 6.0) and alkaline pHi (pHi 9.0), themidpoint potential of HCN2 activation is shifted by �10mV to more hyperpolarized and depolarized potentials,respectively, compared with physiological pHi (pH 7.4)(451). The modulation of HCN channels by intracellularprotons may have an important physiological impact onthe modulation of HCN channel activity in the brain (281),for example, for the regulation of thalamic oscillationsand the respiratory frequency. The protective action ofcarbonic anhydrase inhibitors in generalized seizures hasbeen attributed to the high sensitivity of HCN channels topHi (282). Inhibition of the carbonic anhydrase causes anincrease in pHi and augments Ih in thalamocortical neu-rons. As a result, these neurons are depolarized, and theirengagement in synchronized paroxysmal discharges isreduced. Inhibition of HCN channels by intracellular aci-dosis could also be pathophysiologically relevant duringcardiac ischemia and heart failure (70, 451).

Acidic extracellular pH (pHe �5.0) was found toactivate Ih in a subset of rat taste cells. It was assumedthat this mechanism contributes to sour taste transduc-tion in these cells (369). Heterologous expression of thetwo HCN channel isoforms present in taste cells, HCN1and HCN4, verified the findings from native cells. Acidifi-cation to pH 3.9 profoundly sped up channel kinetics,shifted the threshold of activation by up to �50 mV, andinduced a depolarizing shift of V0.5 by up to �35 mV withrespect to neutral pH. The structural determinants in theHCN1 or HCN4 protein underlying regulation by externalpH have not been reported so far.

C. Regulation by Chloride

Although Ih is a pure cationic current, its conduc-tance is affected by the concentration of small anions,notably by Cl� (144, 411). Frace et al. (144) showed that

the amplitude of rabbit sinoatrial Ih decreased when ex-tracellular Cl� was replaced by larger anions such asaspartate. The molecular basis of the regulation by exter-nal Cl� was studied in heterologously expressed HCNchannels (411). The effect of Cl� was found to be pro-nounced for HCN2 and HCN4, while it is rather weak forHCN1 (411). A single amino acid residue in the poreregion was identified as molecular determinant of extra-cellular Cl� sensitivity (411) (Fig. 4). Channels with highCl� sensitivity (HCN2 and HCN4) carry an arginine resi-due at this position (R405 in HCN2; R483 in HCN4), whileHCN1 carries an alanine (A352). The regulation of HCNchannels by Cl� is probably relevant for heart (patho)physiology. A reduction of the amplitude of sinoatrial Ih

could be involved in the generation of arrhythmias ob-served in hypochloremia.

Cl� regulates HCN channel function also from theintracellular site. In a recent study, it was shown thatintracellular Cl� acts as a physiological suppressor of theinstantaneous component of Ih (IINS) (272). An increaseof intracellular Cl� from physiological concentrations (10mM) to high concentrations (140 mM) almost completelyabolished IINS while it had no significant effect on thesteady-state component of Ih. The physiological relevanceof this regulation remains to be determined.

D. Regulation by Src Kinase-Mediated

Tyrosine Phosphorylation

On the basis of experiments with pharmacologicalblockers, it was assumed that cardiac Ih is regulated bytyrosine kinases of the Src family (426). This hypothesiswas corroborated by yeast two-hybrid screens that pro-vided evidence for direct interaction between Src andHCN1 (332), as well as between Src and HCN2 (450).Direct binding of Src to HCN2 (450) and to HCN4 (13) wasalso shown by coimmunoprecipitation in heterologousexpression systems and native tissue.

Mapping experiments in HCN2 revealed that Srcbinds via its SH3 domain to the C-linker-CNBD and phos-phorylates the channel at this domain (450). As a conse-quence, the activation of the channel is accelerated. Con-versely, inhibition of Src by pharmacological agents orcotransfection of a dominant-negative Src mutant slowsdown channel kinetics probably via dephosphorylation bynot yet defined cellular phosphatases. The residue confer-ring modulation by Src (Y476) has been identified inHCN2 by mass spectrometry. The residue is localized inthe B�-helix of the C-linker (Figs. 3 and 4) and is con-served in all HCN channel isoforms, suggesting that reg-ulation by Src may be a commonality of the HCN channelclass. In agreement with this notion, replacement of Y476by phenylalanine in either HCN2 or HCN4 yields to chan-nels that are no longer regulated by Src. A recent study



identified an additional tyrosine residue in HCN4 (Y531 inA�-helix of the C-linker) that may also be involved inSrc-mediated channel regulation (223) (Fig. 3). In agree-ment with findings of Arinburg et al. (13), this study alsofound that Src not only speeds up channel kinetics ofHCN4 but also induces a �10 to 15 mV shift of the V0.5. Atthis point, one can only speculate on the mechanism bywhich Src-mediated phosphorylation facilitates channelopening. It is tempting to assume that the presence of thebulky negatively charged phosphate group in the B�-helixof the C-linker destabilizes the interaction between neigh-boring C-linkers within the HCN channel tetramer. As aconsequence, the C-linker tetramer would occupy a lesscompact conformation and would impose a weaker inhib-itory impact on the channel gate as if present in the morecompact dephosphorylated conformation.

Regulation of Ih by tyrosine phosphorylation throughSrc kinase has been demonstrated under physiologicalconditions in sinoatrial pacemaker cells in the murine(450) and rat heart (13, 439) as well as in neurons (450).These results support the notion that the control of thephosphorylation status is indeed an important regulatorymechanism to adjust the properties of Ih to the specificrequirements of different types of neurons and heart cells.

E. Regulation by p38-Mitogen-Activated

Protein Kinase

In addition to tyrosine kinases, HCN channels arealso regulated by the serine/threonine kinase, p38-mito-gen-activated protein (MAP) kinase (310). In hippocampalpyramidal neurons, activation of p38-MAP kinase signifi-cantly shifts the voltage-dependent activation towardsmore positive potentials. This regulation may functionallyaffect temporal summation and neuronal excitability. It isnot clear whether p38 induces the observed effects bydirect phosphorylation of the HCN channel protein or byphosphorylation of another protein interacting with thesechannels.

F. Transmembrane and Cytosolic Proteins

Interacting With HCN Channels

There is growing evidence that ion channels usuallyare macromolecular protein complexes that in addition tothe principal pore-forming subunit contain auxiliary pro-teins that are required for the fine-tuning of electrophys-iological properties, the functional coupling to signalingpathways, and trafficking to specific cellular compart-ments. HCN channels are no exception from this rule. Inthe last couple of years, several proteins interacting withHCN channels have been identified (Fig. 4).

1. Regulation by MiRP1

The MinK-related protein MiRP1 (encoded by thegene KCNE2) was reported to interact with several HCNchannel types (101, 317, 438). MiRP1 is a member of afamily of single transmembrane-spanning proteins and isan established auxiliary subunit of the HERG delayedrectifier K� channel (2, 390, 445). MiRP1 was found tointeract with HCN2 in rat neonatal cardiomyocytes andcanine sinoatrial node tissue (317). Overexpression stud-ies in Xenopus oocytes (438) and neonatal rat cardio-myoctes (317) showed that MiRP1 increases current den-sities and accelerates activation kinetics of HCN2. Incontrast, MiRP1 did not affect voltage dependence of acti-vation. In overexpression systems, MiRP1 was also foundto interact with HCN1 (438) and HCN4 (101). MiRP1increased current densities of HCN4, but unlike in coex-pression experiments with HCN1 or HCN2, it sloweddown kinetics and induced a negative shift of the activa-tion curve of this channel (101). Collectively, these resultsmay suggest that MiRP1 is an auxiliary subunit of HCNchannels. However, extreme care should be taken beforea final decision on this issue is made. It will be necessaryto verify the interaction between MiRP1 and HCN chan-nels including its functional implications in native tissues.Furthermore, the specificity of antibodies used for immu-noprecipitations must be confirmed in adequate knockoutmodels.

2. Regulation by KCR1

Very recently, another transmembrane protein(KCR1) was reported to interact with HCN2 and nativecardiac Ih channels (268). KCR1 is a plasma membrane-associated protein with 12 putative transmembrane re-gions that like MiRP1 can associate with the HERG K�

channel (183, 213). Upon overexpression in CHO cells,KCR1 reduces HCN2 current densities and affects single-channel current parameters of this channel. Overexpres-sion in rat cardiomyocytes also reduced current densitiesof native Ih and suppressed spontaneous action potentialactivity of these cells. From these experiments, it wasconcluded that KCR1 is an inhibitory auxiliary subunit ofHCN channels and serves as a regulator of cardiac auto-maticity. As discussed for MiRP1, further experimentswill be required to verify this hypothesis.

3. Regulation by neuronal scaffold proteins

Several scaffold proteins interact with the COOHterminus of HCN channels. These proteins were mainlyidentified in neurons and may regulate channel targetingto distinct subcellular compartments (e.g., dendrites orsynapses). A brain-specific protein termed TRIP8b (TPR-containing Rab8b interacting protein) interacts through aconserved tripeptide sequence in the COOH terminus of

858 BIEL ET AL.


HCN channels (335). TRIP8b is thought to play an impor-tant role in the trafficking of vesicles to their final targets(444). TRIP8b colocalizes with HCN1 in dendrites of cor-tical and hippocampal pyramidal cells. Functional coex-pression of TRIP8b with HCN protein, either in nativecells or heterologous systems, results in a strong down-regulation of HCN channels in the plasma membrane. Onthe basis of these experiments and the analysis of HCN1knockout mice, it was speculated that TRIP8 is involvedin the generation of somatodendritic HCN1 channel gra-dients in cortical layer V pyramidal neurons (335). HCN2,but not other HCN channel types, interacts with the neu-ronal scaffold protein tamalin, mostly through a PDZ-likebinding domain (205). In addition, HCN2 interacts withthe scaffold proteins Mint2 and S-SCAM via distinct pro-tein-binding domains at the COOH-terminal tail. In COS-7cells, HCN2 levels were increased upon coexpressionwith Mint2, suggesting that this protein is a positive reg-ulator of cell surface expression of HCN channels (205).

4. Regulation by filamin A

HCN1, but not HCN2 or HCN4, was found to bindfilamin A via a 22-amino acid sequence downstream of theCNBD (161). Filamin A is a putative cytoplasmic scaffoldprotein that binds actin and thereby links transmembraneproteins, among those are the K� channels Kv4.2 and Kir2.1, to the actin cytoskeleton (305, 329). On the basis ofheterologous overexpression in filamin A-expressing andfilamin A-deficient cell lines, it was proposed that filaminA causes clustering and slows down activation and deac-tivation kinetics of HCN1 (161).

5. Regulation by caveolin-3

Ih channels localize to membrane lipid rafts in sino-atrial myocytes and in HEK293 cells expressing HCN4, themajor HCN channel isoform contributing to native sino-atrial Ih (26). Coimmunoprecipitation experiments indi-cate an interaction between HCN4 and caveolin-3, whichis a marker protein for so-called caveolae (27). Caveolaerepresent a morphologically distinct type of lipid rafts. Incardiomyocytes and pacemaker cells, several elements ofthe �-adrenergic signaling pathway including �2-adreno-ceptors that regulate HCN channel activity are localized incaveolae. It was proposed that clustering of HCN4 incaveolae is essential for normal function and regulation ofthis channel. Indeed, disruption of lipid rafts by choles-terol depletion caused a redistribution of HCN channelswithin the membrane and modified their kinetic proper-ties (26).

V. TISSUE EXPRESSION OF HCN CHANNELS

Throughout the nervous system and the heart, thehyperpolarization-activated current Ih as well as HCN

channel expression has been identified. In addition, Ih

was also found in some other tissues such as B cells ofpancreatic Langerhans islets (134), enteric (37, 179, 429),lymphatic smooth muscle (254), uterine smooth muscle(296), smooth muscle cells of the bladder (163) or portalvein (164), testis (341), and the enteric nervous system(427). However, HCN channel transcripts have only beenreported in some of these tissues or cell types (134, 164,341, 427). Moreover, in most of these tissues, HCN chan-nels are expressed at low levels, making it very difficult todefine the physiological role of Ih, if there is any. Anotherproblem is that in several cell types, HCN channels havebeen detected only on the mRNA level, e.g., using RT-PCRor in situ hybridization, while protein expression has notbeen shown so far. Given these drawbacks, in this reviewwe will concentrate on HCN channels in heart and ner-vous system. We will also give only a rough outline oftissue distribution of HCN1-4. Readers who are interestedin a more complete description of the expression patternare referred to a number of excellent studies that havereported on this issue in great detail (126, 226, 275, 276,294, 331, 332, 344, 345).

Briefly, in the brain, HCN1 is expressed in the neo-cortex, hippocampus, cerebellar cortex, and brain stem(270, 275, 294, 331, 332). In addition, HCN1 expressionwas reported in the spinal cord (270). HCN2 is distributednearly ubiquitously throughout most brain regions, withthe highest expression in the thalamus and brain stemnuclei (275, 294, 331). In contrast, HCN3 is expressed atvery low levels in the central nervous system. Moderate tohigh expression has only been detected in the olfactorybulb and in some hypothalamic nuclei (275, 294). HCN4 isstrongly expressed in some parts of the brain, e.g., invarious thalamic nuclei and in the mitral cell layer of theolfactory bulb (275, 294, 331). In other brain regions, theexpression of HCN4 is much lower. All four HCN channelisoforms are expressed differentially in the retina (69, 193,209, 276, 280). In the peripheral nervous system, all fourHCN subtypes have been reported in the dorsal rootganglion, with HCN1 being the most abundant one (80).

All four HCN channel isoforms have been detectedin the heart. The expression levels of these isoforms stronglydepend on the cardiac region and, in addition, seem tovary between species. In the sinoatrial node, in all speciesanalyzed so far (e.g., human, rabbit, guinea pig, mouse,and dog), HCN4 is the major isoform accounting for �80%of Ih (276, 344, 388). The remaining fraction of this currentis species dependent. In rabbits, this fraction of Ih isdominated by HCN1 (344), whereas in mice and humans,HCN2 accounts for this fraction (226, 388). In addition, inthe mouse sinoatrial node, a significant amount of HCN1mRNA was identified (249).

In other parts of the cardiac conduction system,HCN4 is also the major isoform, with expression in theatrioventricular node (126) and the Purkinje fibers (345).



HCN1 expression has been reported in the atrioventricu-lar node (249). For HCN3, either no expression (276) oronly very low expression levels have been reported in theconduction system (249). HCN channels are also presentin atrial and ventricular myocytes. In these cells, HCN2 isthe dominant isoform displaying a rather ubiquitous dis-tribution. Transcripts of HCN1, HCN3, and HCN4 havealso been detected in heart muscle (344, 375). In general,expression levels of HCN channels are low in normalheart muscle compared with cells of the conduction sys-tem. However, upregulation of HCN channels may occurduring heart diseases (72, 73, 139, 374) (see also sect. VIII).

VI. PHYSIOLOGICAL ROLES OF HCN

CHANNELS IN NEURONS

A. Principles

Ih displays a set of unique biophysical features that isessential to control excitability and electrical responsive-ness of cells. With respect to these physiological roles,two properties of Ih are of particular relevance. First, Ih

channels are partially open at rest. As a consequence, thechannels contribute to the setting of the membrane po-tential in many cells (98, 125, 226, 230, 266, 291–293, 300).Second, Ih channels possess an inherent negative-feed-back property because they can counteract both mem-brane hyperpolarization and depolarization by producinga depolarizing inward current (due to Ih channel activa-tion) or by facilitating hyperpolarization (resulting from Ih

channel deactivation), respectively. Thus Ih can activelydampen both inhibitory and excitatory stimuli arriving atthe cell membrane and thus helps to stabilize the mem-brane potential (31, 253, 291, 354). We will now discuss insome more detail these two key properties of Ih whichtogether provide the Leitmotiv for the specific functionsof this current in various physiological settings.

1. Activation of Ih at the resting membrane potential

In sinoatrial pacemaker cells of the heart as well as inmany neurons of the central nervous system, Ih channelsare constitutively open at voltages near the resting mem-brane potential (12, 226, 291–293, 326), pass a depolariz-ing noninactivating inward current, and hence set themembrane potential to more depolarized voltages. In ad-dition, constitutively open Ih channels per se stabilize theresting membrane potential by lowering the membraneresistance (Rm), which is defined as the ratio of a voltagechange and the required current (226, 291–293). There-fore, in the presence of Ih, any given input current evokesa smaller change in membrane potential than in the ab-sence of Ih. Constitutively open Ih channels seem to func-tion as a slow “voltage clamp” (291), tending to stabilize

the membrane potential by opposing depolarizing or hy-perpolarizing inputs (185). This effect suppresses low-frequency fluctuations in membrane potential (291). Indendrites, constitutively open Ih channels influence thepassive propagation of excitatory postsynaptic potentials(EPSPs) (239–241). In the absence of active voltage-de-pendent ion channels, the passive properties of a dendrite(Fig. 5A) attenuate the amplitude and slow down thekinetics of an EPSP (Fig. 5B) as it spreads from its site oforigin in the dendrites to the soma (Fig. 5, A–C). Thisprocess is comparable to a wave traveling across water.Just as a wave moving across water widens and dimin-ishes in height over distance, EPSP signals can degradewith distance along the neuronal membrane (Fig. 5B). Thepresence of Ih in dendrites lowers Rm and thereby furtherincreases the amplitude attenuation of EPSPs. In addi-tion, Ih counteracts kinetic filtering of propagating EPSPsalso by lowering Rm. Therefore, the EPSP upstroke anddecline are faster (Fig. 5C).

In the absence of Ih (e.g., after blockade of Ih by thespecific Ih blocker ZD7288 or after genetic deletion), so-matic EPSPs rise and decay more slowly if they aregenerated in distal dendrites compared with proximaldendrites (239–241, 423) (Fig. 5C).

2. Negative-feedback properties of Ih

As mentioned above, voltage-dependent activationand deactivation of Ih actively oppose deviations of themembrane potential away from the resting membranepotential (31, 253, 291, 354). This property is a conse-quence of the unusual relation between the activationcurve and the reversal potential of Ih. In contrast tovoltage-gated Ca2� and Na� channels (Fig. 5E), the rever-sal potential of Ih falls close to the base of its activationcurve (189) (Fig. 5E). If membrane hyperpolarization in-creases the fraction of open Ih channels, the depolarizinginward current drives the membrane potential back to-wards the reversal potential of Ih and thereby to the initialvalue close to the resting membrane potential (Fig. 5D).This characteristic effect is called “depolarizing voltagesag” (300, 324). Conversely, a depolarizing input causesdeactivation of the Ih that was active at rest. The loss ofa tonic depolarizing current causes a hyperpolarization(“hyperpolarizing voltage sag”; Refs. 300, 324) again re-turning membrane potential toward rest (Fig. 5D, right).

Ih is involved in the control of a variety of basic andcomplex neuronal functions, including dendritic integra-tion, long-term potentiation, learning, and neuronal pace-making, just to mention a few of them. In the followingsections we will review the role of Ih in these diversefunctions in more detail. Wherever possible, we will dis-cuss the role of Ih in the light of recent findings obtainedfrom the analysis from HCN channel-deficient mouselines. These studies have allowed defining the specific

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contribution of distinct HCN channel subtypes to differ-ent physiological functions.

B. Role of Ih in Dendritic Integration

Dendritic integration is a process that is crucial forsignal processing in most neurons. Typical isolated EPSPsare too small to bridge the gap between the resting mem-brane potential and the action potential threshold. There-fore, generation of action potentials at the soma usuallyrequires the integration of multiple synaptic inputs. Toensure high fidelity of information processing, integrationof EPSP must be tightly controlled both in space and time.Dendritic integration has been extensively studied in CA1hippocampal and neocortical pyramidal neurons (241).There is good evidence that in these neurons Ih and mostnotably the Ih component encoded by HCN1 has an im-portant role in regulating dendritic integration (239–241,394, 423). The way incoming EPSPs are summed up intime largely depends on kinetic filtering by passive cableproperties of the dendrites. As mentioned above, den-

dritic filtering slows down the time course of EPSPsresulting in somatic EPSPs that rise and decay moreslowly if they are generated in distal dendrites comparedwith proximal dendrites (Fig. 5, B and C). As a conse-quence of filtering, one would expect that repetitiveEPSPs arising from more distal synapses should summateat the soma to a greater extent and over a longer timecourse than EPSPs generated in more proximal dendrites.However, in many neurons, including CA1 (239, 240) andneocortical layer 5 pyramidal cells (38, 377, 423), thislocalization dependence of temporal summation is notobserved. This discrepancy is probably explained by agradient of Ih whose density rises progressively by morethan sixfold with distance from the soma (225, 239–241,423) (Fig. 6). This gradient efficiently counteracts den-dritic filtering. During the rising phase of an EPSP, Ih

channels rapidly deactivate. Turning off the inward cur-rent carried by Ih leaves an effective net outward currentthat hyperpolarizes the plasma membrane and acceler-ates the decay of each EPSP (Fig. 5, B and C). Because thedensity of Ih is higher in distal dendrites, distal EPSPs

FIG. 5. Basic properties of Ih. A: equivalent electrical circuit of an idealized cylindrical segment of a dendrite. Three electrical componentsare responsible for the passive features of a dendrite: the specific membrane resistance (Rm), the specific membrane capacitance (Cm), and theintracellular resistance (Ri). For simplicity, the resistance of the extracellular space has been neglected. B: the passive properties of a dendriteattenuate the amplitude (arrow) of an excitatory postsynaptic potential (EPSP) as it spreads from dendrites to the soma (EPSP at the distal synapse,black line; the same EPSP after propagation to the soma, red line). C: EPSP recorded at the soma after propagation from a distal dendrite beforeand after blockade of Ih with ZD 7288. In the presence of Ih (black line), the EPSP rise and decay faster than after blockade of Ih with ZD 7288 (redline). D: Ih actively opposes changes in membrane voltage. Cartoon of a current-clamp experiment in a neuron. In the presence of Ih, ahyperpolarizing current step (�200 pA) induces a depolarizing voltage sag (arrow, left panel) and a depolarizing current step (200 pA) induces ahyperpolarizing voltage sag (arrow, right panel). Blockade of Ih by Cs� eliminates the sag (red traces). E: the reversal potential (Vrev) of Ih falls nearthe base of its activation curve. Therefore, Ih actively opposes changes in membrane voltage. Partial activation of the current drives the membranepotential toward the reversal potential on the base of the activation curve (left, arrow in the activation curve) and thus is self limiting and stabilizingfor the membrane potential. In contrast, voltage-gated currents, whose reversal potential falls near the top of their activation curve (e.g., INa; right),are destabilizing and self-regenerative. Partial activation drives the membrane potential towards the reversal potential at the top of the activationcurve (right, arrow in the activation curve).



decay faster and are therefore shorter (Fig. 5C). As aconsequence, after propagation to the soma, the temporalsummation is more dampened for distal than for proximalinputs. Therefore, the temporal summation of all inputsreaching the soma is about equal (Fig. 6A). However, thenormalization of somatic EPSP time course by Ih comesat a cost. As mentioned in section VIA, the activation of Ih

increases the dendrosomatic attenuation of EPSP ampli-tude and thus the dependence of somatic EPSP amplitudeon synapse location (Fig. 5B).

Collectively, these results suggest that the proposedrole of Ih in dendritic integration critically depends on anincreasing somatodendritic Ih gradient. However, thismay not apply to all types of neurons. For example, thereis a shallower Ih gradient or even a reversed Ih distribu-

tion in a subset of CA1 pyramidal cells that show a similardendritic integration and temporal summation as pyrami-dal cells with a distal enrichment of Ih (61, 160). More-over, location independence of EPSP summation is ob-served in cerebellar Purkinje cells, although Ih exhibits auniform dendritic density in these neurons (11). Thusnormalization of temporal summation may occur in avariety of Ih distribution patterns. Using a modeling ap-proach, Angelo et al. (11) concluded that it is the totalnumber of Ih channels, not their distribution, that governsthe degree of temporal summation of EPSPs. Finally,other voltage-gated ion channels also have importantroles in dendritic integration. For example, a gradient ofA-type potassium channels rising from proximal to distaldendrites (34, 181, 199, 377) may be involved in dendritic

�

��

FIG. 6. Proposed roles of Ih in various neuronal functions. A: the somato-dendritic gradient of Ih effectively counteracts kinetic filtering bydendrites and normalizes the localization dependence of temporal summation. Summation of EPSPs from proximal (black trace) and distal (graytrace) dendrites recorded at the soma after propagation. The voltage recordings are shown before (top panel) and after (bottom panel) inhibitionof Ih by the selective Ih blocker ZD 7288. B: hypothetical model of �2-adrenoceptor-cAMP-HCN regulation in prefrontal cortex neuron networksinvolved in the control of spatial working memory. For details, see text. �2-R, �2-adrenoceptor; Gi, Gi protein. C: a model for the role of HCN1channels in motor learning through history-independent integration of inputs by cerebellar Purkinje cells. Input-output function of wild-type (black;top panel) and HCN1-KO Purkinje cells (gray; bottom panel). Presynaptic spike trains evoke synaptic currents in Purkinje cells that are integratedto produce output spikes. The input-output function of wild-type Purkinje cells (black) is independent of the history of activity. In contrast, theinput-output function of Purkinje cells from HCN1-KO mice (gray) depends on whether the neuron is initially in an active (down arrow) or silent(up arrow) state. D: HCN1 constrains LTP in perforant path synapses on distal dendrites of CA1 neurons (gray line, HCN1-deficient mice; black line,WT mice), but not in Schaffer collateral synapses on proximal dendrites (not shown).

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integration. In addition, it has been shown that the densityof AMPA glutamate receptors is increased in distal den-drites of several neurons, increasing the strength of distalsynapses (10, 242, 352). Thus the relative importance ofindividual ion channels and receptors to the complexprocess of dendritic integration may vary among differentneurons.

C. Role of Ih in Working Memory

A recently discovered role of Ih is the control of thespatial working memory (415) (Fig. 6B). This form ofmemory depends on prefrontal cortical networks thathave the unique ability to represent information that is nolonger present in the environment and to use this “repre-sentational knowledge” to guide behavior. In monkeysperforming a spatial working memory task, prefrontalcortical neurons increase firing in response to visual stim-uli presented in the preferred direction, while they de-crease firing when stimuli are presented in the nonpre-ferred direction. This property is called spatial tuning. Itwas proposed that spatial tuning is regulated by theopposing effects of adrenoceptors and the D1-receptoron intracellular cAMP signaling (15, 407). It has beensuggested that HCN channels are important downstreamtargets for cAMP in this signaling pathway (415). HCNchannels colocalize and functionally interact with �2A-adrenoceptors on dendritic spines of prefrontal neurons. Thefunctional interaction was explained according to thefollowing model (Fig. 6B) (415): �2A-adrenoceptors areactivated by relatively low levels of norepinephrine re-leased during alert and wakefulness (16). The activationof �2A-adrenoceptors inhibits the production of cAMP viaGi signaling and thus reduces the open probability of HCNchannels. As a result, Rm, the efficiency of synaptic input,and the network activity of these neurons increase. Onthe basis of this model, it was speculated that the reducedHCN channel activity on spines receiving inputs fromneurons with similar spatial properties increases the fir-ing to preferred spatial directions and thereby augmentsrelevant information (15). In contrast, activation of D1-receptors increases the production of cAMP via Gs signal-ing. As a result, HCN channels open, reduce the mem-brane resistance, and, thereby, decrease inputs to thespines (Fig. 6B). This could selectively disconnect spi-nous inputs to the dendrites. Thus activation of D1-receptors on spines receiving inputs from neurons withdissimilar spatial properties could suppress irrelevant in-formation (“noise”) from the nonpreferred direction (15,407, 422). So far, the regulation of HCN channels byD1-receptors has not directly been demonstrated andtherefore remains hypothetical (15). During stress, higherconcentrations of norepinephrine may be present andactivate low-affinity �1- and very-low-affinity �1-adreno-

ceptors giving rise to strongly increased cAMP levels (16).In this situation, cAMP levels could significantly increasethe open probability of HCN channels and functionallydisconnect spinous inputs to the dendrites (15). Thusconnections of the prefrontal cortex would be function-ally cut off.

An alternative modulation of HCN channels by �2-adrenoceptors in prefrontal neurons has been proposedby a different group (71). Carr et al. (71) suggested that�2-adrenoceptor activation suppresses HCN channels inprefrontal neurons through a cAMP-independent signal-ing pathway that appears to be mediated by PLC-PKCsignaling.

D. Role of Ih in Constraining Hippocampal LTP

Mice in which the HCN1 gene has been selectivelydeleted from forebrain show an improvement of hip-pocampal-dependent learning and memory formation,compared with wild-type mice (292, 394) (Fig. 6D). Elec-trophysiological recordings indicated that this surprisingeffect is concurred and probably also caused by a specificenhancement of long-term synaptic plasticity (LTP) anddendritic excitability in hippocampal CA1 neurons. CA1neurons provide the major output of the hippocampusand receive two major sources of excitatory synapticinputs (Fig. 7A). One set of inputs, the Schaffer collater-als, comes from hippocampal CA3 pyramidal neurons andterminates on regions of CA1 neuron dendrites that arerelatively close to the cell body, an area with only mod-erate HCN1 expression. The second set of inputs, theperforant path, represents a direct connection from layerIII neurons of the entorhinal cortex and terminates on thedistal dendritic regions of the CA1 neurons, an area whereHCN1 expression is very high. At the more proximalSchaffer collateral pathway, both synaptic transmissionand long-term plasticity are relatively unaffected by thedeletion of HCN1, consistent with the relatively low ex-pression of HCN1 in this dendritic region (292)(Fig. 6D).In contrast, at distal perforant path synapses, a significantenhancement in the integration of synaptic potentials andan increase in LTP was observed, consistent with therelatively high expression of HCN1 in this region (292)(Fig. 6D). From these results, it was concluded that HCN1channels impair spatial learning because they exert aninhibitory constraint on dendritic integration and synapticlong-term plasticity at the perforant path inputs to CA1pyramidal neurons. It was proposed that high levels ofHCN1 may interfere with generation of Ca2� spikes, aprocess that is critical for the induction of LTP at den-dritic synapses (394). These Ca2� spikes are triggered bysynaptic activation of AMPA- and NMDA-receptors (394).The activation of these glutamate receptors generates aninitial depolarization that activates most likely T-type



(Cav3.3) and N-type (Cav2.2) voltage-dependent Ca2�

channels. Because HCN1 passes a depolarizing inwardcurrent at rest, it will make the resting membrane poten-tial more positive and, hence, shift a substantial fractionof voltage-gated T- and N-type channel to their respectiveinactivated states. Thus the availability of these channelsto participate in an action potential is decreased and Ca2�

influx is reduced. In other words, HCN1 can be consid-ered as a partial brake that constrains dendritic Ca2�

spikes. Quantitatively, the inhibitory effect of HCN1 is afunction of the protein amount present in the membrane,which may explain why it is only observed in distal syn-apses displaying high HCN1 expression levels.

E. Role of Ih in Motor Learning

The role of Ih in motor learning has been recentlyuncovered in mice with a global deletion of HCN1 (293)(Fig. 6C). Deletion of HCN1 causes profound learning andmemory deficits in behavioral tests that require complexand repeated coordination of motor output by the cere-bellum (e.g., in visible platform, water maze, and rotarodtasks). In contrast, the deletion of HCN1 does not modify

acquisition or extinction of eyelid conditioning, a discretemotor behavior that also involves cerebellar synapticplasticity. The actions of HCN1 were tested in cerebellarPurkinje cells, the key component of the cerebellar circuitrequired for learning of correctly timed movements (293).In these spontaneously spiking neurons, HCN1 mediates adepolarizing inward current that counteracts inputs thathyperpolarize the membrane below the threshold forspontaneous spiking. By stabilizing the integrative prop-erties of Purkinje cells, HCN1 enables the Purkinje cellsto maintain an input-output relation that is independent ofthe neuron’s previous history of activity (Fig. 6C). Thisfunction of HCN1 is required for reliable encoding ofinformation and ensures accurate decoding of input pat-terns.

F. Role of Ih in Synaptic Transmission

Electrophysiological recordings have demonstratedthe presence of Ih in several presynaptic terminals, includ-ing the crustacean neuromuscular junction (33), avianciliary ganglion (141), cerebellar basket cells, and thecalyx of Held in the auditory brain stem (356). It wassuggested that an important role of these presynaptic Ih

channels consists in controlling synaptic transmission. Insupport of this hypothesis, long-term facilitation of syn-aptic transmission in crustacean motor terminals wasshown to be conferred by cAMP-dependent upregulationof Ih (32, 33). The downstream pathway coupling Ih acti-vation to synaptic release is not yet known. However,there is initial evidence that Ih channels may directlyinteract with the release machinery, perhaps mediated bythe cytoskeleton (32, 33). In vertebrates, the functionalrelevance of Ih in regulating synaptic transmission is cur-rently disputed. Mellor et al. (263) reported that presyn-aptic Ih is involved in the generation of LTP in hippocam-pal mossy fiber synapses that terminate on CA3 pyramidalcells. However, this finding was challenged by anotherstudy that showed in the same system that LTP is inde-pendent of Ih (89).

G. Role of Ih in Resonance and Oscillations

1. Resonance

The term resonance describes the property of a neu-ron to selectively respond to inputs at a preferred fre-quency (189). In electrophysiological experiments, reso-nance can be induced by applying a current stimulus withlinearly increasing frequency (so-called ZAP current). Inmany neurons, the voltage answer shows a prominentresonant peak at the natural frequency or Eigenfrequencyof the neuron (Fig. 7B, arrow). To generate resonance, aneuron needs to have properties of a low-pass filter com-

FIG. 7. Proposed roles of Ih in resonance and oscillation. A: anat-omy of the hippocampal formation. For details, see text. EC, entorhinalcortex; CA1, CA1 pyramidal neurons; PP, perforant path input; DG,dentate gyrus; CA3, CA3 pyramidal neurons; SC, Schaffer collaterals.B: resonance is formed by the interaction of active and passive proper-ties in a neuron. The response to a ZAP current (current with linearilyincreasing frequency) shows a prominent resonant peak (arrows). C: Ih

and passive membrane properties of the membrane act in concert toproduce resonance. The broken line shows the contribution of Ih, andthe black line shows the contribution of the passive membrane proper-ties (�m) to the total impedance. The bell-shaped curve is the resultingimpedance curve for the combined system (gray line).

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bined with properties of a high-pass filter (65, 69, 187, 189)(Fig. 7C). The low-pass filtering is caused by the mem-brane time constant �m. The high-pass filtering is causedby the action of several voltage-gated currents includingIh. To act as a high-pass filter, these currents need to beable to actively oppose changes in membrane voltage(Fig. 5, D and E). In addition, these currents have toactivate slowly relative to the membrane time constant.Both requirements are met for Ih. At low frequencies, theIh channels have time to activate and actively opposechanges in membrane potential. At high frequencies,there is not enough time for the Ih channels to open. As aresult, high-frequency changes are not suppressed, ren-dering the neuron responsive for fast trains of spikes.Resonance arises at intermediate frequencies where in-put-induced voltage changes are too high to be opposedby Ih and too low to be filtered by �m. The importance ofIh for the generation of membrane resonance has beendemonstrated for several neurons, for example, in neo-cortical pyramidal cells of the rat (398), in subicular py-ramidal neurons of the rat (416), and in neurons from thesensorimotor cortex of juvenile rats (188).

2. Oscillations

Sustained rhythmic oscillations are a hallmark fea-ture of neuronal circuits in various brain regions (62, 65).Oscillations arise from synchronized activity of neuronsand are thought to play an essential role in informationprocessing in neuronal networks. There are several typesof oscillations in different frequency bands, called delta,theta, gamma, and fast “ripple” oscillations (62, 65). Oneoscillation, the theta frequency oscillation (4–12 Hz), isprominent in all areas of the hippocampal formation.These oscillations may be important for various cognitiveprocesses including processing, encoding, and storing ofspatial information (62) as well as for memory formationand retrieval (63, 219). Hippocampal theta oscillationshave been proposed to originate from reciprocal interac-tions between rhythmic inputs from the medial septum-diagonal band of Broca, the entorhinal cortex, and othersubcortical structures (Fig. 7A; Refs. 66, 306, 406). Theinputs from septal neurons to the hippocampus seem tobe mainly responsible for the generation of the thetarhythm (62, 306). In this process, the perforant path andvarious other brain regions are also involved (155, 327).

In mice with a forebrain-restricted deletion of HCN1,changes in hippocampal-dependent network oscillationshave been described (292). While low- and high-frequencynetwork oscillations appeared to be unchanged in knock-out mice, there was a significant enhancement in the thetafrequency band (4–9 Hz), during rapid-eye-movement(REM) sleep and during wheel running (292). Consistentwith these results, in CA1 neurons responses to low-frequency inputs at the theta range are preferentially in-

creased in HCN1-deficient mice. HCN1 channels cantherefore be thought of as a partial brake that contributesto resonance by preferentially dampening low-frequencycomponents of input waveforms at frequencies below thetheta range. Releasing the brake in knockout animalscauses a general enhancement in the voltage response tolow-frequency oscillatory currents.

In addition to the important role of Ih in the genera-tion of theta oscillation, Ih is also involved in the genera-tion of other types of oscillations. There are several stud-ies that demonstrated the importance of Ih for �-oscilla-tions in the hippocampus (97, 140), synchronized oscillationsin the inferior olive (21), and subthreshold oscillations in theentorhinal cortex (108, 166). Moreover, Ih has been sug-gested to influence network oscillations that play an im-portant role in learning and memory (140, 185, 230, 235).

H. Role of Ih in the Generation

of Thalamic Rhythms

Synchronized neuronal oscillations are produced inthalamocortical networks during sleep (64, 255, 363, 364),sensory processing (162), and seizures (256). The gener-ation and synchronization of complex oscillations (e.g.,spindle waves and slow waves) requires extensive synap-tic interactions within the thalamocortical network (361)(Fig. 8A). In contrast, there are other neuronal rhythms,for example, the delta frequency rhythm that can be gen-erated in single cells (255, 361, 367). We will first discussthe role of Ih in the generation of characteristic firingpatterns that arise in single thalamocortical cells and thendiscuss the role of Ih in oscillations that require moreextensive synaptic interactions. In our discussion, wefocus on data from in vitro slice preparations due to thecurrent paucity of in vivo data.

1. Single cell oscillations in thalamic relay neurons

In vitro (22–24, 96, 231, 232, 259) and in vivo (9, 23,107, 238, 328, 363) studies have shown that thalamocorti-cal neurons fire in two distinct firing modes called trans-mission mode and burst mode (22–24, 96, 231, 232, 259)(Fig. 8B). During wakefulness and REM sleep, thalamo-cortical neurons are depolarized by afferent inputs andswitch to the transmission or “single spike” mode (195,196, 257) (Fig. 8, B and D). During this mode informationis gated through the thalamus and forwarded to the cortex(255, 367). The transmission mode is characterized by thegeneration of repetitive single Na� spikes at depolarizedpotentials where T-type Ca2� channels are inactivated(Fig. 8D). During this mode, sufficient excitation repeti-tively discharges thalamocortical neurons. The output fre-quency of these neurons increases with increasing depo-larization induced by afferent excitatory inputs (165).



During the burst mode, thalamic networks display amonotonous repetitive firing pattern at hyperpolarizedmembrane potentials (178, 195, 196, 368, 389). This firingmode is characteristically seen during non-REM sleep orepileptic discharges and has been considered to decreasetransfer of sensory information to the cortex (368).In vitro studies have demonstrated that thalamic neuronsfire in the “burst mode” through the interaction of alow-threshold Ca2� current (IT) and Ih (Fig. 8, B and C)(255, 300). The following model has been proposed: theactivation of Ih by hyperpolarization beyond �65 mVslowly depolarizes the membrane potential until a re-bound low-threshold Ca2� spike is generated by the acti-vation of IT at more depolarized potentials. Because these“slow spikes” are depolarizing and last for tens of milli-seconds, typically a series of Na� spikes rides on them.The inactivation of IT terminates the low-threshold Ca2�

spike. During the spike, Ih is deactivated. As a result, thetermination of the spike is followed by a hyperpolarizing“overshoot.” In turn, Ih is activated, the cycle repeats, andcontinuous rhythmic burst firing is sustained. The intrin-sic interplay of these currents promotes delta rhythmicityin single thalamocortical neurons (128, 221, 222, 258, 261,355). These single cell oscillations are synchronized inlarge neuronal circuits and can be recorded in the EEG asdelta waves during non-REM sleep (128, 295, 366, 367).

Electrophysiological recordings in thalamic slicepreparations of HCN2-deficient mice revealed that thecurrent produced by HCN2 constitutes the major compo-nent underlying thalamocortical Ih (226). Deletion ofHCN2 reduced the current amplitude of Ih by �80% andresulted in a 12 mV hyperpolarizing shift of the restingmembrane potential. Thalamocortical neurons of HCN2-deficient mice displayed a higher susceptibility to fire inthe burst mode when experiencing excitatory inputs thanwild-type neurons (Fig. 9). Macroscopically, the alteredthalamic firing behavior of HCN2-deficient mice con-curred with the presence of spike-and-wave discharges,the clinical hallmark of absence epilepsy (Fig. 9A; see alsosect. VIII). The higher incidence of burst firing in HCN2-deficient mice (Fig. 9B) may simply result from the factthat the ratio of T-type channels present in the closed (butactivatable) versus inactivated state depends on the rest-ing membrane potential. In HCN2-deficient neurons thatdisplay a more hyperpolarized resting membrane poten-tial, the fraction of T-type channels present in the closedstate will be higher than in wild-type cells where moreT-type channels will be in the inactivated state. Thus thesusceptibility of HCN2-deficient neurons to produce aCa2� spike is higher than that of wild-type neurons be-cause the T-type channels present in these cells are easierto activate by excitatory inputs.

FIG. 8. The role of Ih in the generation of thalamic oscillations. A, top: coronal section through the mouse brain. The cortex, the reticularthalamic nuclei, and the thalamus are indicated. Bottom: schematic diagram of the thalamocortical loop: �, excitatory glutamatergic synapticcontacts; �, inhibitory GABAergic contacts. For detailed information, see text. B: firing modes of thalamocortical neurons. C and D: higher temporalresolution of sections shown in B. [Modified from McCormick and Bal (255).]

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2. Network oscillations

Ih is involved in the generation of a number of rhyth-mic oscillations in thalamocortical networks. During theearly state of non-REM sleep, synchronized oscillationsare generated in the thalamocortical network that giverise to spindle waves in the surface EEG (22, 363, 367).Spindle waves are characterized by a crescendo-decre-scendo type of oscillation at 6–14 Hz that lasts for 1–4 sfollowed by a refractory period of 5–20 s that terminatesthe oscillations (91, 204, 363, 367). These waves are as-sumed to have an essential functional role in synapticplasticity of cortical and thalamic neurons (368). Spindlewaves have been investigated in vivo (9, 92, 279, 362, 364,365) and also in slice preparations (410). These studiesindicate that spindle waves are generated by a cyclicinteraction between excitatory thalamocortical cellsand inhibitory thalamic reticular neurons (23, 24, 363,367, 410) (Fig. 8A). Thalamic reticular cells are the pace-

makers for the generation of spindle wave oscillations(148, 359). In these neurons, rhythmic bursts are gener-ated by low-threshold Ca2� spikes. Burst firing in tha-lamic reticular neurons induces rhythmic inhibitorypostsynaptic potentials (IPSPs) in thalamocortical cells.These IPSPs hyperpolarize thalamocortical cells, removeinactivation of the low-threshold Ca2� current, and at thesame time activate Ih. The resulting depolarization trig-gers a rebound Ca2� spike and a burst of action potentials(Fig. 8C). These bursts reexcite the thalamic reticularneurons and also stimulate cortical pyramidal cells. Thenearly simultaneous occurrence of spindle waves overwidespread cortical territories is produced by networksynchronization involving the cortex and the thalamus(358). This synchronized activity underlies the presenceof spindle waves in the EEG.

The silent period between spindle waves is largelydetermined by persistent Ih activation in thalamocorticalcells (22, 363, 367). This persistent activation of Ih resultsfrom an increase in intracellular Ca2� primarily triggeredby the rebound low-threshold Ca2� bursts that occurredduring the spindle wave generation. The Ca2� most likelyactivate a Ca2�-sensitive isoform of adenylyl cyclase, pro-ducing an increase in cAMP synthesis that enhances Ih

(231). The persistent activation of Ih in turn suppressesthe next spindle wave until Ih is slowly decayed. Spindlewaves disappear during wakening or REM sleep. Thetransition to these states is regulated by several neuro-transmitters such as norepinephrine and serotonin thatboth increase intracellular cAMP and lead to a consecu-tive upregulation of Ih (255).

VII. ROLE OF Ih CHANNELS

IN CARDIAC RHYTHMICITY

The heart beat originates from specialized pace-maker cells in the sinoatrial (SA) node region of the rightatrium. These cells generate a special kind of action po-tential (“pacemaker potential”) that is characterized bythe presence of a progressive diastolic depolarization(DD) in the voltage range between �65 to �45 mV (Fig. 10;see also Ref. 244 for an excellent recent overview on heartautomaticity). After the repolarization phase, it is the DDthat drives the membrane potential of a SA node cell backtoward threshold of Ca2� channel activation, therebymaintaining firing. The DD is generated by the concertedaction of several currents, among which Ih is consideredto play a prominent role because it is activated at negativepotentials and, therefore, potentially could serve as pri-mary initiator of the DD. In addition to Ih, other inwardcurrents including Ca2� currents (ICaT and ICaL) (168, 243,244, 371), as well as the sustained inward current (Ist)(273) whose molecular correlate is not yet known maycontribute to the DD. Furthermore, it was proposed that

FIG. 9. Absence epilepsy and spike-and-wave discharges inHCN2�/� mice. A: comparison of spontaneous EEG patterns betweenwild-type and HCN2�/� mice. One-minute EEG recordings (L, left chan-nel; R, right channel) are shown. Spike-and-wave discharges are indi-cated by asterisks. The horizontal bars demarcate the traces shown onan expanded time scale below the 1-min recordings. B: different intrinsicfiring properties of wild-type and HCN2�/� thalamocortical neurons.The firing patterns elicited by 1-s depolarizing current pulses at restingmembrane potential of wild-type and HCN2�/�neurons are shown. Theamount of current injected is indicated above. Inset: traces elicited byinjection of 100 pA at an expanded time scale.



the DD may be initiated by the decay of outward rectifierK� currents (IKr and IKs) or by mechanisms involving intra-cellular Ca2� release through ryanodine (214, 215, 408) orinositol 1,4,5-trisphosphate (IP3) receptors (265). Ih is notonly involved in principal rhythm generation, it also playsa key role in heart rate regulation by the autonomicnervous system. Sympathetic stimulation activates Ih and,hence, accelerates heart rate via �-adrenoceptor-triggeredcAMP production (49, 50, 122) (Fig. 10, C and D) whilelow vagal stimulation lowers heart rate via inhibition ofcAMP synthesis and an ensuing inhibition of Ih activity(117, 123, 124). High vagal tone most likely lowers theheart rate mainly via the activation of IKACh (117, 421).

A. HCN4

Recent genetic mouse models have made it possibleto evaluate the proposed roles of Ih in cardiac rhythmicityunder in vivo conditions. As mentioned above, HCN4makes up �80% of SA node Ih and has been considered tobe crucial for the generation of the heart beat (171, 172,370). In support of this notion, mice with global- or heart-specific disruption of the HCN4 gene die in utero betweenembryonic days 9.5 and 11.5 (370). Embryonic hearts ofHCN4-deficient mice analyzed before embryonic day 10show a reduction of the beating frequency of �40%. Im-portantly, these hearts do not respond to �-adrenergic

stimulation. Thus one may conclude that in the embryonicheart HCN4 is not required for principal heart beat gen-eration, at least not before embryonic day 11.5, but thatthis channel is absolutely crucial for autonomous heartrate regulation (370). In support of this conclusion, heartsof mice carrying a mutation in the CNBD that abolisheshigh-affinity cAMP binding (HCN4R669Q) also fail to re-spond to �-adrenergic stimulation (171). Further analysisrevealed that HCN4-deficient mice probably die from adevelopmental defect of the SA node (370). These micedevelop normal embryonic pacemaker cells that produceprimitive pacemaker potentials but fail to generate adult-type pacemaker cells. Obviously, the latter cells are cru-cially required to drive the heart beat after embryonic day11. Interestingly, HCN4R669Q mice are able to generate thiskind of pacemaker cells, but like HCN4-deficient micealso die around embryonic day 11.5 (171). Together, themouse studies indicate that the HCN4 protein is required1) for the formation of adult pacemaker cells duringembryonic heart development and 2) for conferringcAMP-dependent upregulation of embryonic heart rate.Both HCN4-mediated functions are dispensable in earlyheart function and early embryonic heart developmentbut are needed for the transition to late embryonic stages.

Given its vital role in embryonic heart, it came as abig surprise that mice in which HCN4 was deleted in adultSA node using a temporally controlled knock-out ap-

FIG. 10. Role of Ih in cardiac automaticity. A: anatomy and localization of the sinoatrial node. SAN, sinoatrial node (gray); CT, crista terminalis.B: structure of the sinoatrioal node (left; arrow) and morphology of an isolated pacemaker cell (right). C, top: idealized pacemaker potentials in theabsence (black) and presence (red) of adrenergic stimulation. DD, diastolic depolarization. Bottom: proposed Ih time course during the pacemakerpotential. D, left: sinoatrial Ih activates faster in the presence of cAMP (red) than in the absence (black) of cAMP at maximal activation voltage (�140mV). Right: activation curve of Ih in the absence (black) and the presence (red) of cAMP. In the presence of cAMP, the activation curve of Ih isshifted to the right.

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proach were viable and displayed a rather mild cardiacphenotype (172) (Fig. 11A). Like in embryonic pacemakercells of global HCN4-deficient mice, Ih in SA node cells ofthe adult HCN4-deficient mice was reduced by �80%(172). However, the lack of this current component didneither lead to a major impairment of pacemaker poten-tial generation nor did it interfere with �-adrenergic reg-ulation of heart rate (Fig. 11A). Unlike global knockoutmice, adult HCN4 knockout mice revealed a normal basalheart rate and normal sympathetic and vagal heart ratemodulation. However, adult HCN4-deficient mice dis-played a cardiac arrhythmia characterized by recurrentsinus pauses (Fig. 11B). Pacemaker cells of adult HCN4knockout mice were hyperpolarized by �8 mV and inmost cases did not fire spontaneously under basal condi-tions. However, this functional impairment could be com-pensated by �-adrenergic stimulation. In conclusion,these findings suggest that in the adult SA node, HCN4may serve as a kind of a stabilizer of the pacemakerpotentials. In most situations, and particularly duringsympathetic stimulation, HCN4 does not seem to be re-quired to promote stable pacemaking. However, after anincrease in repolarizing currents (e.g., vagal stimulationor transition from activated to basal cardiac state), HCN4is activated and provides a depolarizing current, keepingthe system well-balanced. The loss of this “depolarizationreserve” may explain the induction of recurrent sinuspauses in HCN4 knockout mice.

B. HCN2

With respect to the stabilizing function, HCN2 thatmakes up �20% of SA node Ih in mice may serve as acomplementary channel to HCN4 (226) (Fig. 11C). HCN2-deficient mice also reveal a sinus dysrhythmia that ischaracterized by varying peak-peak intervals in the elec-trocardiogram (226). Other parameters of the sinus rhythmincluding autonomous heart rate regulation are normal inthese mice. Like for HCN4, the maximal diastolic poten-tial (MDP) of HCN2-deficient SA node cells is slightlyhyperpolarized. It is important to note that HCN2 obvi-ously is not crucial for the development of cardiac con-duction system since HCN2-deficient mice do not displayincreased embryonic lethality.

C. Conclusions and Open Questions

In conclusion, genetic mouse studies indicate thatthe two components of SA node Ih carried by HCN4 andHCN2 are required for maintaining a stable cardiacrhythm. However, neither HCN2 nor HCN4 is needed forprincipal pacemaking and for autonomous rate regulationin the adult heart. Further experiments (e.g., analysis ofHCN2/4 double knockout mice) will be required to solid-ify these conclusions. Importantly, it remains to be eluci-dated why heart rate regulation in early embryos requiresHCN4 but is independent of this channel in adult animals.

FIG. 11. Effect of deletion of HCN4 and HCN2 on cardiac pacemaking. A: HCN4 is not required for upregulation of the heart rate. Isoproterenol(0.5 mg/kg) injected at time 0 increased the heart rate of control (n � 8; black curve) and knockout (n � 8; red curve) animals to similar maximumlevels. B: example ECG traces from control (top) and knockout mice (bottom) at 0.5 h (I) and 1.5 h (II) after isoproterenol injection. All traces aredisplayed at the same scale. C: sinus dysrhythmia in HCN2-deficient mice. Telemetric ECG recordings (lead II) were obtained simultaneously fromwild-type (con) and HCN2-deficient mice (HCN-KO) at rest.



In this context, it remains to be determined which cur-rent(s), if not Ih, are the downstream target(s) of cAMPsignaling in adult SA node cells. Finally, it should beconsidered that the importance of HCN channels for car-diac rhythm initiation and frequency modulation may bedifferent between species.

The analysis of human HCN4 channelopathies sup-ports this notion (269, 290, 338, 395). So far, four differentheterozygous HCN4 mutations have been identified inhumans (269, 290, 338, 395) (Fig. 2). The mutations lead toloss of cAMP-dependent modulation (HCN4-573X) (338),hyperpolarizing shift of the activation curve (S672R andG480R) (269, 290), or a severe reduction of cell surfaceexpression (D553R) (395). Interestingly, all patients suf-fering from these mutations have in common that theydisplay a more or less severe bradycardia, a clinical phe-notype that is not observed in HCN channel-deficientmouse models. Another issue that is difficult to explain atthis moment is the effect of bradycardic agents, such ascilobradine, ivabradine, and zatebradine (373), as well asof clonidine (208). It was proposed that these agentslower heart rate by blocking Ih channels and reducing thefiring frequency of SA node cells. In agreement with thishypothesis, cilobradine lowers heart rate in wild-typemice but does not do so in adult HCN4 knockouts (174).On the other hand, given that HCN4 confers the heart-ratelowering effect of cilobradine and related substances,why then do HCN4-deficient mice (which corresponds toa 100% pharmacological block) display no basal bradycar-dia? At present, there is no satisfying answer to thisconundrum; however, solving this issue will undoubtedlyprofoundly advance our understanding of the molecularbasis of rhythmicity in normal and diseased heart.

VIII. ROLE OF Ih IN DISEASE

Given the widespread expression and physiologicalimportance of HCN channels in nervous system and heart,it is obvious to assume that impaired expression or mal-function of these channels is associated with the genesisof human diseases. Indeed, evidence has accumulatedover the last couple of years that Ih is implicated in thepathologies of at least three classes of diseases: epilep-sies, neuropathic pain disorders, and cardiac arrhythmias.

A. Inherited Channelopathies

In a classical sense, the term channelopathy refers todiseases that are caused by inherited genomic mutationsin an ion channel gene that lead to loss, impaired expres-sion levels, or functional defects of the channel protein.Interestingly, despite numerous efforts 10 years after thediscovery of the HCN channel genes, only four inheritedHCN channel mutations have been described (Fig. 2). All

four are localized in the HCN4 gene and are associatedwith the induction of sinus bradycardia (see sect. VIIC andRef. 174). All patients identified so far are heterozygousfor the respective HCN4 mutation. A likely explanationfor this finding may be that like in mice disruption (370) orfunctional impairment (171) of both HCN4 alleles is asso-ciated with embryonic lethality due to the failure to de-velop a mature cardiac conduction system. Neuronal phe-notypes have not been reported in the four groups ofpatients. This is surprising given the expression of HCN4in CNS. So far, disease-causing mutations in humanHCN1-3 genes have not been reported. On the basis of theanalysis of murine HCN knockout models, it is expectedthat mutations in these genes would be implicated incomplex cardiac (HCN2) and/or neuronal (HCN1, HCN2)phenotypes. However, one would also predict that eventhe total loss of these individual channels does not lead tolethality in mice. Thus one could assume that mutations inHCN1-3 proteins have much more impact in humans thanin mice and are not detected because they are lethal.Alternatively, the mutations exist but have escaped detec-tion so far because they occur very rarely. More system-atical screening and sequencing approaches in patientswill be required to clarify this issue.

B. Transcriptional Channelopathies

Transcriptional (or acquired) channelopathies resultfrom pathological alterations of the expression or local-ization of a normal (nonmutated) ion channel (417). Suchdisorders occur more frequently than inherited monoge-netic channelopathies, which are usually very rare. How-ever, the analysis of transcriptional channelopathies isalso much more difficult and prone to misinterpretationsthan the analysis of monogenetic diseases. The majorobstacle in the analysis is that expression and cellularhandling of ion channels usually is an extremely dynam-ical process that is regulated by numerous intracellularand external factors. In general, this complexity makes itdifficult to decide whether an altered expression profile ofan ion channel 1) is causative to a disease, 2) is a com-pensatory process in response to a disease, or 3) repre-sents a normal physiological variation that is neither caus-ative nor compensatory to a disease. Having these limita-tions in mind, we will now summarize recent evidencesupporting a role of HCN channels in transcriptional dis-eases.

1. Epilepsy

First evidence connecting Ih and epileptic seizurescame from studies in a rat model of childhood febrileseizures (hyperthermia model of febrile seizure) (83, 84).At the age of 10 days, these rats are exposed to a singleperiod of hyperthermia lasting for �30 min. Such animals

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reliably develop epileptic convulsions originating in thehippocampus and reveal an increased tendency to de-velop seizures at adult stage. Electrophysiological record-ings revealed an enhanced Ih (increase of Imax by 40% at�70 mV) in hippocampal CA1 neurons of epileptic rats. Inaddition, the Ih current showed a slight positive shift ofthe V0.5 (�3 to �5 mV) and slowed activation kinetics,compared with wild-type rats. Functionally, the enhancedIh was coupled to an increased probability of rebounddepolarizations and action potential firing (83). Closerinspection of the molecular basis of Ih observed afterfebrile seizures revealed a downregulation of HCN1 inhippocampal pyramidal neurons while HCN2 was upregu-lated, resulting in a decrease of the ratio of HCN1 vs.HCN2 protein from 8:1 to �4:1 (47, 48). The mechanismunderlying changes in HCN channel expression levels inresponse to seizures is unclear. Reduction of HCN1 chan-nel expression is probably a transcriptionally regulatedprocess that involves activation of calmodulin (CaM) ki-nase II and Ca2� entry via AMPA receptors (322). Incontrast, upregulation of HCN2 was found to be indepen-dent of CaM kinase II (322). The consequences of up- anddownregulation of HCN channel subunits may be morecomplex than originally expected. A recent coimmuno-precipitation study indicated that altered hippocampalHCN1 and HCN2 expression levels are accompanied by along-lasting increase in the levels of heteromeric HCN1/2channels (48). Thus the Ih of hippocampal neurons isprobably conferred by a complex mixture of HCN1 andHCN2 homomers as well as by HCN1/2 heteromers.Pathological deviations from the normal ratio of the indi-vidual HCN channel types may play a critical role in thegeneration and the long-term maintenance of hippocam-pal hyperexcitability.

Altered HCN channel expression has also been foundin another class of seizures, the absence epilepsies. Thetypical absence is clinically defined by a sudden, briefimpairment of consciousness and behavioral arrest. Im-mediately following the absence, there appears to be littleif any disruption of cognitive abilities. Absences mainlyoriginate in the cortex; however, the thalamus is alsoinvolved in the pathology of the disease (256, 360). Inparticular, the generation of spike-and-wave discharges(SWDs), a diagnostic hallmark of absences, is correlatedwith an increased prevalence of burst firing and synchro-nized oscillatory activity in thalamocortical circuits (256).Given the key role of HCN channels in controlling thefiring behavior of thalamocortical neurons, abnormalHCN channel expression may contribute to the genera-tion of absences. In agreement with this hypothesis, intwo rat models for absence epilepsy [WAG/Rij (58, 376)and GAERS (212)], increased levels of HCN1 were foundin thalamocortical neurons. In contrast, expression ofHCN2-4 was not altered. In both rat models, the Ih ofthalamocortical neurons displayed a reduced sensitivity

to cAMP that is consistent with an increase of HCN1levels relative to HCN2. In the WAG/Rij model, but not inGAERS, V0.5 of Ih was also shifted to more hyperpolarizedvoltages. It was speculated that the hyperpolarizing shiftin combination with the reduced cAMP sensitivity locksthalamocortical neurons in the burst firing mode (58).

Perhaps the strongest evidence linking Ih channelswith absences relates to HCN2-deficient mice (226) (Fig.9). Like other absence models, HCN2-deficient mice dis-play frequent bilaterally synchronous SWDs in the EEGthat are accompanied with brief episodes of immobility(so-called behavioral arrest). In thalamocortical and tha-lamic reticular neurons of these mice, Ih was found to bealmost completely abolished (1, 226, 320, 435). The resid-ual Ih (max. 20% of wild-type Ih) had a very slow kineticsand a strongly hyperpolarized V0.5. As a consequence, theresting membrane potential of thalamocortical neuronsfrom HCN2-deficient mice was shifted to hyperpolarizedpotentials, which could well explain the increased burstactivity and network oscillations seen in these animals(see also sect. VI).

Downregulation of Ih was also found in a pharmaco-logical (kainic acid) rat model of temporal lobe epilepsy(TLE) (342). Layer III pyramidal neurons of the entorhinalcortex (EC) of these epileptic rats are hyperexcitable,giving rise to profound synchronous network activity.Electrophysiological recordings in EC neurons revealed asignificant reduction of dendritic Ih which was caused byreduced HCN1 and HCN2 protein levels. Mechanistically,hyperexcitability induced by downregulation of Ih couldbe put down to the key role Ih plays in dendritic integra-tion (see also sect. V). As predicted, reduction of Ih den-sities in dendrites leads to an increase of the dendriticinput resistance. This, in turn, enhances dendritic EPSPsummation and EPSP-spike coupling.

Rat and mouse models clearly indicated that im-paired HCN channel function or expression is associatedwith epileptiform activity. Such a clear correlation, al-though likely, is so far missing in humans. As mentioned,HCN channelopathies involving epilepsy have not beenreported so far. However, changes in HCN channel ex-pression have been found in the dentate gyrus from pa-tients with temporal lobe epilepsy and severe hippocam-pal sclerosis (35). Since upregulation of HCN1 mRNA wasfound only in cases of end-stage disease, long after theonset of epilepsy, it was suggested that it is not causativefor the epilepsy but rather represents a compensatorychange of the brain.

In conclusion, the role of Ih in epilepsies seems to beextremely complex and diverse. There is evidence that1) both up- or downregulation can be associated with thedisease, 2) different HCN types contribute to differentextents to the disease, 3) the role of an HCN channelstrongly depends on its cellular localization, and 4) find-ings from rodents may not necessarily be applicable to



human epilepsies. In most cases it remains unclearwhether an observed change in Ih produces hyperexcit-ability or represents a compensatory reaction to the stateof hyperexcitability. Thus the relevance of Ih in epilepto-genesis cannot be addressed by a simplified generalmodel but has to be defined for various pathophysiologi-cal settings in a very specific and distinct manner.

2. Peripheral neuropathic pain

Peripheral neuropathic pain is a complex pain con-dition that is characterized by spontaneous pain, hyper-algesia, and allodynia (68). The disease originates fromthe injury of peripheral nerves. Nerve injury can havemany etiologies, among which are trauma, viral infections(e.g., Herpes zoster), metabolic diseases (e.g., diabetesmellitus), vascular diseases (e.g., stroke), autoimmunediseases (e.g., multiple sclerosis), cancer, and exposure toradiation or chemotherapy. The pathomechanism of neu-ropathic pain is complex and involves both peripheral andcentral sensitization. A hallmark of the disease is thegeneration of abnormal spontaneous (ectopic) discharges.These discharges have strong rhythmic components and aregenerated from injured dorsal root ganglion (DRG) somataor axons, as well as from adjacent uninjured nerves. Previ-ous work has shown that altered expression levels of severaltypes of voltage-gated Na� channels contribute to the gen-eration and persistence of spontaneous discharges (252, 417,418).

Several findings suggest that HCN channels also playa prominent role in neuropathic pain (for recent review,see Ref. 197). First, Ih has been identified in DRG neurons,particularly in large- and medium-sized neurons wheremost ectopic discharges are produced (253, 339). Second,in situ hybridization and immunohistochemistry revealedthe presence of HCN1-3 channels in different types ofmouse and rat DRGs (80, 276). Third, increased densitiesof Ih were found in large- and/or medium-sized DRGneurons after experimentally induced injuries (80, 206,432). Fourth, low concentrations of the Ih blocker ZD7288reverse both pain behavior and the spontaneous dis-charges in injured nerve fibers (80, 220, 380). Taken to-gether, these findings strongly suggest that upregulationof Ih that leads to an increased excitability of the cell iscausative to the generation of ectopic firing. The mecha-nism underlying upregulation of Ih remains to be investi-gated in more detail. Interestingly, Chaplan et al. (80)found a downregulation of HCN1 and HCN2 in DRGs afterinjury, although Ih densities were increased. It is unclearhow the discrepancy between current densities and pro-tein expression can be explained. Potential mechanismsinclude a positive shift of V0.5 of the Ih (leading to anincreased open probability), altered HCN channel subunitassembly, and increased cell surface expression of HCNchannel proteins after injury.

3. Cardiac remodeling and arrhythmia

Structural and functional remodeling of the heartmuscle is a clinical hallmark of a variety of cardiovasculardiseases including cardiomyopathies, infarction, chronichypertension, and inflammation (e.g., myocarditis) (14).Initially, these adaptations are beneficial to maintain car-diac function (e.g., at pressure overload) but in laterstages of the diseases they contribute to contractile ab-normalities and sudden death. On the cellular level, irreg-ular expression of ion channels involved in the control ofcardiac contractility and automaticity plays an importantrole in remodeling (176, 285, 286, 323, 392).

Upregulation of Ih has been described in a variety ofanimal models of cardiac hypertrophy and heart failure aswell as in human patients suffering from these diseases(for reviews, see Refs. 76, 173, 244). A common feature ofthese diseases is the profound upregulation of Ih channelsin ventricular cardiomyocytes which normally expressonly very low levels of these channels. In contrast, theactivation threshold of the Ih of diseased cardiomyocytesis usually not shifted to more positive values comparedwith normal ventricular Ih (76, 139, 177, 182). Increased Ih

densities strongly raise the tendency of the ventricularmuscle to develop ectopic, “pacemaker-like” action po-tentials and, thereby, contribute to arrhythmia and tosudden death (72, 73, 139, 182, 374, 452).

In rodents and humans, upregulation of Ih is mirroredby an increased expression of HCN channels. Examina-tion of mRNA levels in hypertrophied atrial and ventric-ular myocytes revealed an upregulation of HCN2 andHCN4 in different animal models (72, 73, 139, 177, 182,374, 375, 452). In addition, upregulation of HCN4 wasfound in human patients with end-stage heart failure (45).The degree of hypertrophy positively correlates with in-creased Ih density (73) and the expression levels of HCNchannels (139, 336). While these findings strongly supporta direct link between upregulation of HCN channels andventricular dysfunction, the signaling pathways underly-ing upregulation of Ih are only poorly understood so far.Both nerval and humoral factors are probably involved inthe process. Notably, high angiotensin II levels, which arewell-known to promote ventricular remodeling (382), aswell as aldosterone (284) seem to play a pivotal role inHCN channel upregulation. This is supported by the find-ing that antagonists of the type I angiotensin II receptor(AT1-receptor) not only reduce cardiac hypertrophy butalso hamper Ih and HCN2/HCN4 mRNA overexpression(74, 75, 177). Interestingly, Ih is abundantly expressed inspontaneously active fetal and neonatal ventricular myo-cytes (77, 325, 434). During maturation, these cells losetheir capacity to generate spontaneous activity. Both inrat and mouse, this is accompanied by a progressivedecrease of Ih expression (77, 434). Thus it is tempting toassume that cardiac hypertrophy provokes a reentry of

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cells into a fetal program and the reinitiation of the cor-responding gene expression patterns which includes up-regulation of HCN2 and HCN4. Recently, it was found inrat models of ventricular hypertrophy that upregulation ofHCN2 and HCN4 is accompanied by pronounced reduc-tion of two microRNAs, miR-1 and miR-133. Conversely,forced expression of these miRNAs prevented overex-pression of HCN2/4 in hypertrophic cardiomyocytes(229). These findings suggest that downregulation ofthese two miRNAs may play an important role in thereexpression of Ih during normal heart development butalso during remodeling of the diseased heart.

IX. HCN CHANNELS AS NOVEL

DRUG TARGETS

A. Heart Rate-Reducing Agents

High heart rate positively correlates with an in-creased mortality in a variety of cardiac diseases includ-ing heart failure, arterial hypertension, and ischemic heartdisease (36, 130, 340). Lowering heart rate is thereforeone of the most important therapeutic approaches in thetreatment of these diseases. Currently used bradycardicdrugs including �-adrenoceptor antagonists, and someCa2� channel antagonists efficiently reduce heart rate, buttheir use is also limited by adverse reactions or contrain-dications (44, 127). Given the key role of Ih/HCN chan-nels in cardiac pacemaking, these channels are verypromising pharmacological targets for the developmentof novel and more specific heart-rate reducing agents(44). Importantly, HCN channels are not expressed invascular and airway smooth muscle. Therefore, in con-trast to �-adrenoceptor or Ca2� channel antagonists,drugs acting on HCN channels are not expected to havemajor side effects on the vascular system or on pulmo-nary function. In the past, several agents inhibitingcardiac Ih have been developed. Early drugs identifiedas pure bradycardic agents include alinidine (ST567),ZD7288, zatebradine (UL-FS49), and cilobradine (DK-AH269). A more recent drug is ivabradine (S16257). Theprincipal action of all these substances is to reduce thefrequency of pacemaker potentials in the sinus node byinducing a reduction of the diastolic depolarizationslope. In this section, we only briefly discuss thesedrugs. We refer readers who are interested in moredetailed information to a number of recent reviews onthis issue (28, 42, 55).

Recently, ivabradine (S16257, Procoralan) was intro-duced into clinical use as the first therapeutic Ih blocker.Ivabradine blocks cardiac Ih at low micromolar concen-trations and has been approved as a treatment of chronicstable angina pectoris (379). Electrophysiological studiesrevealed that ivabradine acts by accessing Ih channels

from their intracellular side and by exerting a use- andcurrent-dependent block (56). The mechanism of channelblock by ivabradine was examined in heterologously ex-pressed HCN channels (57, 388). Ivabradine blocks HCN4and HCN1 channels with half-block concentrations (IC50)of �1–2 �M. Interestingly, ivabradine acts as open chan-nel blocker in HCN4 (like in sinoatrial Ih), while block ofHCN1 requires channels either to be closed or in a tran-sitional state between open and closed configuration (57).The structural determinants underlying this difference arestill unknown.

Zatebradine and cilobradine are derived from theL-type Ca2� channel blocker verapamil. Low micromolarconcentrations of both blockers (IC50 between 0.5 and 2�M) inhibit sinoatrial Ih as well as heterologously ex-pressed HCN channels in a use-dependent fashion (373,401). Like with ivabradine, zatebradine block results fromdrug molecules entering the channel pore from the intra-cellular site (115).

ZD7288 is probably the most widely used experimen-tal blocker of Ih. Like the agents discussed so far, ZD7288blocks channels from the intracellular site (348). How-ever, the block is use independent and is associated withan �15 mV shift of V0.5 to more negative potentials andwith a decrease of the maximal channel conductance(46). Recently, two amino acid residues were identified inthe S6-helix of HCN2 (A425 and I432) and that conferhigh-affinity binding of ZD7288 (88).

The well-known �2-adrenoceptor agonist clonidine,which is chemically related to the bradycardic agent alini-dine (353), was recently shown to block sinoatrial Ih

(208). In mice lacking all three types of �2-receptors,clonidine still exerts a profound bradycardic activity, in-dicating that inhibition of cardiac Ih contributes signifi-cantly to the netto-bradycardic effect of clonidine. Elec-trophysiological recordings revealed that clonidineblocks HCN4 and HCN2 (IC50 of �10 �M) and also,though with less sensitivity, HCN1 (IC50 of �40 �M). LikeZD7288, clonidine also shifts the voltage dependence ofthe channel by 10–20 mV to more hyperpolarizing poten-tials (208).

With the exception of ivabradine, HCN channel blockerswere not introduced to therapy so far. A major obstacle ofmost existing agents is that they are not specific enoughfor sinoatrial (mainly HCN4-mediated) Ih, but also blockneuronal Ih in several regions of the nervous system. Forexample, visual disturbances that result from block ofretinal Ih have been reported for several of these drugs(78). Moreover, some of the blockers may also interactwith other ion channels. For example, recently it wasreported that the widely used “specific” Ih blocker ZD7288inhibits T-type Ca2� currents in rat hippocampal pyrami-dal cells (330).



B. Blockers of Neuronal Ih

Modulation of Ih may also be a promising approachfor treatment of disease processes in the central andperipheral nervous systems. In the previous section wediscussed that upregulation of Ih in DRGs and peripheralnerves is an important process in the pathogenesis ofneuropathic pain. Therefore, blockers of Ih may be bene-ficial to analgesic therapy. In principle, the existing Ih

blockers could be used to treat neuropathic pain. How-ever, if applied systemically, all known blockers wouldalso exert bradycardic effects. To circumvent this prob-lem, agents with high sensitivity to the HCN channel typesrelevant for neuropathic pain (mainly HCN1 and HCN2)would be wishful. Conversely, these blockers should notinterfere with the function of sinoatrial HCN4. Drugsdisplaying this pharmacological profile do not exist so far,but given the wealth of chemical structures relevant to Ih

channel block it should be a feasible task to developsubtype-specific blockers. The number of potential chem-ical entities related to Ih block is increased by agents thathave been shown to block Ih in addition to their well-known primary receptor. For example, it was recentlyfound that loperamide, a potent �-opioid-receptor ago-nist, blocks Ih of DRGs with quite high affinity (IC50

between 5 and 10 �M depending on the DRG type) (404).In addition, capsazepine, a well-known inhibitor of thevanilloid-receptor (TRPV1), blocks HCN1 in a concentra-tion-dependent manner (IC50 � 8 �M) (154). The devel-opment of structural analogs of the mentioned agents mayyield compounds that selectively inhibit specific HCNchannel types.

The use of Ih blockers has also been implicated intherapy of epilepsies. However, given the complexity anddiversity of the cellular mechanisms leading to these dis-eases, a clear concept for a rational design of antiepilepticIh channel modulators has not yet emerged. A majorconceptual problem is that both inhibition and activationof Ih may be beneficial depending on the type of epilepsy.For example, stereotactic injection of the Ih blockerZD7288 reduced the generation of hippocampal epilepti-form discharges in rabbit (207). On the other site, there isevidence that part of the antiepileptic activity of lam-otrigine (an established blocker of voltage-gated Na�

channels) is caused by an upregulation of Ih in dendritesof pyramidal neurons (309, 311). Similarly, the antiepilep-tic drug gabapentin, which was shown to inhibit Ca2�

currents by binding to the �2�-subunit, may also act inpart via upregulation of Ih (381).

Finally, HCN channels may contribute to the clinicalactions of general anesthetics. Native neuronal Ih as wellas heterologously expressed HCN channels are inhibitedby clinically relevant concentrations (�0.5 mM) of theinhalational anesthetics halothane and isoflurane (59, 87).Similarly, the intravenous anesthetic propofol inhibits and

slows the activation of native and expressed HCN chan-nels (67, 233).

X. CONCLUSIONS AND FUTURE DIRECTIONS

Thirty years after the first discovery of Ih and 10years after the cloning of HCN channels, the field ofhyperpolarization-activated cation channels has emergedinto one of the most exciting areas of ion channel re-search. Starting as an electrophysiological curio (the“funny” current), Ih channels are now considered as im-portant regulators of many fundamental processes in ner-vous system, as well as in the heart. Extensive studieswith native and heterologously expressed HCN channels,combined with modeling approaches, biochemistry, andX-ray crystallography, have dramatically increased ourknowledge on the structural determinants of HCN chan-nel function. Moreover, genetic mouse models are nowavailable to study the physiological role of individual HCNchannel types in vivo. Despite the progress made, numer-ous key questions are still unsolved and will have to beaddressed by future approaches. In the following, a smallselection of important problems is listed.

1) What is the structural basis for the “inverse” gatingand the mixed K�/Na� permeability of HCN channels?How is binding of cAMP to the CNBD coupled to theactivation gate of the channel?

2) Which cellular mechanisms control the subunitstoichiometry of HCN channels? Can HCN channel sub-units freely assemble to (hetero)tetramers or are thererestraints limiting free assembly?

3) Like other transmembrane proteins, HCN chan-nels are probably associated with cellular proteins inlarge macromolecular complexes. It is important to iden-tify the constituents of these complexes in different typesof neurons and heart cells to understand how HCN chan-nels are regulated in vivo under physiological conditionsand in diseased states.

4) Which factors control the expression, the cellularprocessing, and the targeting of HCN channels to specificcellular compartments?

5) What is the detailed role of HCN channels invarious diseases? The analysis of advanced genetic mousemodels in conjunction with studies in human patients willbe required to reach this goal.

6) Finally, given that HCN channels are relevant asdisease factors, the full potential of these proteins as drugtargets has to be further explored. To this end, high-affinity, subtype-specific HCN channel blockers must bedeveloped.

Last but not least, HCN channels themselves may beuseful as therapeutic agents. Currently, many laboratoriesdevelop strategies to generate so-called “biological” pace-makers (for recent reviews, see Refs. 90, 244, 351). Bio-

874 BIEL ET AL.


logical pacemakers are genetically engineered cells thatproduce spontaneous pacemaker potentials. Such cellscan be generated by direct transfer of HCN channel genesinto quiescent heart tissue or by transplantation of engi-neered cells expressing HCN channels. In any case, bio-logical pacemakers are an attractive alternative to elec-trical devices to stimulate automaticity in defined regionsof the heart. At this point, it is too early to pass a finaljudgment on the relevance, the long-term reliability, andthe safety of HCN channel-based therapies. Whatever theanswer to this question finally will be, the study of HCNchannels has profoundly increased our knowledge onimportant physiological and pathophysiological pro-cesses and, undoubtedly, will do so in the future.

ACKNOWLEDGMENTS

We apologize to all our colleagues whose studies have notbeen cited because of space constraints.

Addresses for reprint requests and other correspondence:M. Biel, Center for Integrated Protein Science CIPS-M and Zen-trum fur Pharmaforschung, Department Pharmazie, Ludwig-Maximilians-Universitat Munchen, Butenandtstr. 5-13, D-81377Munich, Germany (e-mail: [email protected]); C.Wahl-Schott, Molekulare Pharmakologie, Zentrum fur Phar-maforschung, Department Pharmazie, Ludwig-Maximilians-Uni-versitat Munchen, Butenandtstr. 5-13, D-81377 Munich, Germany(e-mail: [email protected]).

GRANTS

This work was supported by the Deutsche Forschungsge-meinschaft, the Center for Integrated Protein Science CIPS-M,and the EU research project NORMACOR (LSHM-CT-2006-018676) within the Sixth Framework Programme of the Euro-pean Union.

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