The Hyperpolarization-Activated Cyclic Nucleotide–Gated Channels: from Biophysics to Pharmacology of a Unique Family of Ion Channels

i. HCN in SAN Cells and Its Contribution to Cardiac Pacemaking: Membrane and Calcium Clocks

HCN channels have long been recognized for their primary role in pacemaker impulse generation and regulation. However, the specific contribution of HCN has considerably evolved in recent years. Two main mechanisms, the voltage clock and the Ca²⁺ clock, mainly sustained by HCN current and Ca²⁺ release from sarcoplasmic reticulum, respectively, are supposed to contribute to a coordinated system that jointly drives spontaneous electrical activity in SAN. The relative contribution of the two mechanisms to basal rhythm and its adaption to autonomic balance have been the object of an intense debate; to have a taste of different—sometimes conflicting—views, the reader is referred to a lively point–counterpoint exercise by the leading scientists in the field (DiFrancesco and Noble, 2012; Maltsev and Lakatta, 2012) and the enlightening accompanying comment (Rosen et al., 2012).

In brief, in the SAN during the diastolic phase, the fraction of open HCN channels provides a steady-state inward current driving membrane potential (−70/−40 mV, depending on cells) to depolarize toward the threshold required to generate a spontaneous AP (DiFrancesco et al., 1986). A key role of HCN channels, particularly the HCN4 isoform, is suggested—besides other arguments—by bradycardia (or severe bradycardia) consequent to the following: 1) loss of HCN4 function in KO mouse models or patients carrying HCN4 mutations (see later in this section), and 2) the effect of selective HCN blockers (see section Pharmacology of HCN Channels). It is well recognized that the steepness of the diastolic depolarization in pacemaker cells also results from the concerted (simultaneous or sequential) work of other membrane ionic conductances: NCX, L- and T-type Ca²⁺ channels, Na⁺/K⁺ ATPase, voltage-dependent K⁺ and background Na⁺ currents—just to mention some relevant ones. The point by DiFrancesco and Noble (2012) is that these conductances concert to set the pacing rate—the membrane clock—with I_h being the conductor, also in force of its exquisite sensitivity to autonomic balance (via intracellular cAMP). Lakatta’s group (Vinogradova et al., 2010) named calcium clock the periodic, spontaneous submembrane calcium release from sarcoplasmic reticulum, triggering Ca²⁺ extrusion via NCX current, which in turns depolarizes the membrane, activates T- and L-type calcium current, and finally triggers APs. The regular sequence of APs and its adaption to autonomic input might be the result not of a main conductor such as I_h, rather of the interplay between the rate of spontaneous Ca²⁺ release—roughly periodic—and the fine adjustment operated by the balance of inward/outward membrane conductances, including I_h. At variance with ivabradine, there are not blockers of calcium-clock players specific for SAN cells; in fact, their loss of function impairs atrial and ventricular contractility in vivo. The scientific debate has been also fueled by discrepant results obtained in mouse models undergoing genetic deletion of sinoatrial HCN4, ranging from lethal bradycardia (Baruscotti et al., 2011) to minor rhythm alterations (Herrmann et al., 2007). Although experimental strategies of genetic manipulation as well as the contribution of different HCN isoforms may help explain different results in HCN4-KO mice, it is worth mentioning the following: 1) KO of STIM1, a key regulator of calcium dynamics, also causes severe bradycardia in mice (Zhang et al., 2015), and 2) complete ablation of I_f by a dominant-negative HCN4 mutant channel expressed in SAN cells alters membrane excitability and calcium cycling (i.e., both clocks), pacemaking and conduction impairment being partially rescued by additional KO of the muscarinic G protein–activated (GIRK4) channels (Mesirca et al., 2014). Interestingly, the latter model confirmed the prominent role of I_h in SAN as sensor of the autonomic nervous system input.

ii. HCN Expression and Function in Subsidiary Pacemakers

In the AVN the function of HCN channels does not diverge substantially; in fact, although the specific role played by HCN channels in this region is less investigated, robust experimental evidence indicates that HCN channels are implicated in AVN pacemaking and conduction (Liu et al., 2008; Marger et al., 2011; Verrier et al., 2014, 2015). Of note, abolition of HCN4 sensitivity through conditional expression of dominant-negative HCN4 channels lacking cAMP sensitivity reduces the spontaneous activity of AVN cells under basal conditions, but does not impair the maximal response to β-adrenergic stimulation, suggesting that HCN4 channels influence AVN basal activity, but are not obligatory for β-adrenergic regulation (Marger et al., 2011).

Transgenic mouse models have further consolidated the functions of HCN channels in SAN and AVN cells. In fact, inducible cardiac ablation of HCN4 in mice leads to progressive severe bradycardia, followed by AVN block, eventually resulting in cardiac arrest and death (Baruscotti et al., 2011).

The physiologic role of HCN channels in the healthy working myocardium remains an issue for which a conclusive function is still unclear. Since the early evidence obtained in human atrial appendage fibers, I_h has been hypothesized to support the spontaneous electrical activity observed in the atrial tissue. Subsequently, a series of studies investigated the specific properties of HCN current in atrial myocytes, showing that voltage dependence, activation kinetics, and ionic selectivity are similar to those retrieved in SAN cells (Carmeliet, 1984). The role of HCN channel in the atria differs from that in SAN because most healthy atrial cardiomyocytes have a stable resting membrane potential and infrequently display spontaneous electrogenesis, in line with a low contribution of HCN current to resting membrane potentials (−80/−70 mV) and absence of spontaneous automaticity. However, when the integrity of the intracellular milieu is preserved, some human atrial myocytes display a clear diastolic depolarization phase, suggesting that HCN current in the atria may overtly influence electrogenesis, in particular when favoring conditions are present, such as reduced repolarizing currents or increased adrenergic tone (Cerbai and Mugelli, 2006).

Similar observations are drawn for HCN channels in the healthy ventricle, where HCN current is readily detectable in most cells displaying a stable resting membrane potential (Cerbai and Mugelli, 2006; Sartiani et al., 2015). Interestingly, recent evidence has uncovered a different function for HCN in the mouse ventricle, where the channel appears involved in the prolongation of AP repolarization. This led to propose that HCN channels, and particularly HCN3, might mediate a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization (Fenske et al., 2011). These findings partially agree with a different study in the mouse, where HCN2 and HCN4 isoforms appear to predominate in controlling the late phase of repolarization (Hofmann et al., 2012).

i. Altered HCN Properties and Dysfunction of Sinus and Atrioventricular Nodes

The understanding of pacemaker alterations leading to cardiac arrhythmias is rapidly enlarging the genetic basis; currently, the combined efforts of clinical practice and appropriate transgenic models have helped to identify some modifications of HCN channel functions and regulatory proteins associated with dysfunctional cardiac pacemaking and/or conduction (Verkerk and Wilders, 2014).

Screening analysis performed in patients with idiopathic bradycardia has uncovered different loss-of-function mutations of HCN4 gene leading to impaired impulse generation capacity of SAN cells associated or not with conduction dysfunctions (AVN block and altered chronotropic response) (Fig. 4). Deep bradycardia and AVN block are also found in adult transgenic mice with inducible cardiac ablation of HCN4 (Baruscotti et al., 2011), further corroborating the pathophysiological implications of HCN4 reduction in cardiac pacemaker and conduction. Interestingly, HCN4 mutations have been identified in families with bradycardia and left ventricular noncompaction cardiomyopathy, a complex clinical phenotype that associates HCN alterations to cardiac structural abnormalities (Milano et al., 2014). Recently, a novel loss-of-function mutation of HCN4 channel has been identified during a screening in patients with sick sinus and Brugada syndromes (Biel et al., 2016), a finding that further complicates the understanding of proarrhythmic role of HCN channel dysfunctions.

In the healthy heart, a proper sensitivity of HCN4 to cAMP is also important to set basal HCN current magnitude and the contribution of HCN to resting pacemaker automaticity. In fact, transgenic mice expressing human mutated HCN4 gene lacking the cAMP binding site (CNBD) results in basal bradycardia and reduced heart rate sensitivity to adrenergic stimulation (Alig et al., 2009). However, the same model proved that rate adaption during physical activity is preserved, thus indicating that different mechanisms, alone or in combination with HCN channels, are enrolled to increase heart rate following adrenergic stimulation (Alig et al., 2009; Rosen et al., 2012). Of note, adrenergic regulation of heart rate is also preserved, at least partially, in condition of HCN channel blockade, because the state amplifies the effects of any autonomic stimuli on cycle length (Zaza and Lombardi, 2001).

As opposed to the above reports, a recent study in patients with inappropriate sinus tachycardia has identified a gain-of-function mutation (R524Q) in cardiac HCN4 channel. Mutant channels display a higher sensitivity to cAMP and mediate a larger than normal current during the diastolic depolarization, a finding in line with the enhanced cardiac rate detected in these patients (Baruscotti et al., 2016). As a confirmation of the role of I_h in human SAN pacemaking, a recent computational approach shows that all HCN4 mutations associated with a loss of function slow the pacemaker rate down in simulated APs, with negligible effects on other AP parameters, whereas the only gain-of-function mutation described to date has opposite consequences (Fabbri et al., 2017).

Regulatory proteins also have a role in HCN channel dysfunction in pacemaker centers. Currently, a single mutation in the MiRP1/KCNE2 gene has been associated with symptomatic severe sinus bradycardia and suppression of HCN current in vitro, an effect conceivable with an altered interaction between the regulatory subunit and HCN channels (Nawathe et al., 2013).

Sinus bradycardia may also be attributed to nongenetic causes, such as those occurring physiologically with endurance training (Dobrzynski et al., 2013; D’Souza et al., 2014) or ageing (Monfredi and Boyett, 2015). Both conditions are associated with complex and still incompletely defined modifications of SAN and atria that may predispose to develop atrial tachyarrhythmias. On the other side, pathologic conditions such as AF (Jackson et al., 2017) and heart failure (Wang and Hill, 2010) may also lead to sinus node dysfunctions associated with anatomic and electrical changes.

ii. Atrial Remodelling and Fibrillation

HCN current constitutively present in the human atria has since long been proposed to sustain atrial arrhythmias associated with different cardiac pathologies or triggered by various modulatory signals (Opthof, 1998) (Fig. 4).

In diseased conditions, such as cardiac hypertrophy and failure, atrial dysfunction and arrhythmias may occur because of adverse remodeling triggered by chronic impairment of ventricular function. In the context of chronic heart failure, experimental data have documented an altered expression of HCN channels in right atrial tissue, where HCN4 transcript was found significantly increased (Zicha et al., 2005). In line with this study, in atrial tissue from failing human hearts, HCN2 and HCN4 transcripts and proteins were also increased and most likely related to the enhanced HCN current and the positive voltage shift of channel activation (Stillitano et al., 2008). Interestingly, in a different study (Lai et al., 1999), the overexpression of HCN2 in the left atria is positively related with left atrial filling pressure, an indicator of congestive heart failure, further corroborating the link between left ventricular dysfunction and atrial electrical remodeling.

A limited number of studies have investigated the issue in a defined arrhythmic state, such as chronic AF. In this setting, a significant increase of HCN current has been reported in atrial cardiomyocytes from diseased patients compared with control (Stillitano et al., 2013). In particular, at voltage values around myocyte resting membrane potential (≅−70 mV), HCN fractional activation is 10% larger (from 20% to 30%) because of a positive voltage shift in channel activation, suggesting that chronic AF modifies atrial electrogenesis also by enhancing the contribution of HCN current. The molecular counterpart of this modification is not obvious, consisting of unchanged transcript levels for HCN1/HCN2 and reduction of HCN4 and MiR-1; however, protein amounts are preserved, most likely because of post-transcriptional processing. The modifications of HCN transcripts are in agreement with those reported in a previous study performed on patients in chronic AF (Lezoualc’h et al., 2007). The mechanism underlying the increase of HCN current in chronic AF is not clear; it is possible to hypothesize the occurrence of modifications of HCN regulatory subunits that affect channel function. In this regard, the lack of quantitative modifications of caveolin-3 possibly escludes changes of the interaction between caveolin-3 and HCN channels in the diseased atria.

At variance with the above reports, recent studies in a canine model and in humans (Li et al., 2014b, 2015b) have identified an age-related increased expression of HCN2 and HCN4 transcripts and proteins in the atria. These modifications are associated with proarrhythmic alterations in the canine atria and propensity to AF. In humans, similar modifications were found to be associated with reduction of MiR-1 and MiR-133A that were greater in patients with AF (Li et al., 2015b).

The arrhythmogenic potential of adverse cardiac remodeling can be amplified or reduced by different triggers present at local or systemic levels. Some of those having important implications for AF, such as catecholamines, serotonin, atrial natriuretic peptide (ANP), and adenosine, also modulate the function of HCN channels via activation of different types of G protein–coupled receptors (Table 2). In the human atrium, serotonin and catecholamines interact with and activate G_s-coupled receptors, namely subtype-4 serotonin receptor and β₁- and β₂-adrenoceptors (Pino et al., 1998; Lonardo et al., 2005). Following increase of intracellular cAMP, both serotonin and catecholamines exert a stimulatory effect on HCN amplitude, shifting the activation voltage to positive values. Differently, ANP is synthesized and stored as a prohormone in the atria, and, upon distension, it is released, allowing activation of two receptors (peptide receptors A and B). Both of them stimulate a guanylyl-cyclase activity, thereby increasing intracellular concentration of cGMP; in this case, intracellular cAMP level rises because of cGMP-mediated inhibition of cAMP phosphodiesterases (Lonardo et al., 2004). It has been hypothesized that, in conditions of HCN channel gain of function, as those determined by adverse remodeling, these mediators may promote the function of HCN channels at physiologic potentials, enhancing the propensity to arrhythmias. Among the observations corroborating the hypothesis are the persistence of all mediator activity and associated signaling during AF and the values of affinity constants of serotonin and ANP for the corresponding receptors, which are closer to the physiologic concentrations of serotonin or ANP derived by local release during platelet aggregation or cell stretching. Further investigations are needed to better clarify the pathophysiological implications of these pathways in atrial arrhythmias and to evaluate the interplay of concomitant excitatory effects exerted on HCN channels expressed in atrial myocytes and in SAN cells.

iii. Ventricular Hypertrophy and Failure

Over the last decades, much attention has focused on HCN current in nonpacemaker cells and its potential role in triggering ventricular arrhythmias. The first evidences were obtained in the spontaneous hypertensive rat, which develops age-related cardiac hypertrophy associated with several electrophysiological alterations at ventricular level, including an unusual diastolic depolarization phase (Barbieri et al., 1994). Subsequently, this peculiar trait was associated with the occurrence of an atypical I_h that resulted to linearly relate to the severity of cardiac hypertrophy (Cerbai et al., 1994). Ventricular I_h exhibits electrophysiological properties and sensitivity to pharmacological blockade similar to those described in atrial and SAN cells. However, as described for the atrial current, voltage dependence is shifted to more negative potentials compared with SAN cells, being activation threshold at approximately −70 mV. The value is conceivable with a definite contribution of HCN current to ventricular resting membrane potential, ranging from −80 to −90 mV. The atypical voltage dependence of HCN current in the ventricle most likely arises from specific isoform assembling into functional channels, as well as the multiplicity of secondary subunits that influence HCN current properties, analogously to what was reported for nodal and immature cardiomyocytes (Cerbai et al., 1999b; Barbuti et al., 2004, 2007, 2012; Bosman et al., 2013).

HCN current in the ventricle exhibits a typical sensitivity to autonomic transmitters, mimicking that described in the SAN cells (Cerbai et al., 1999a). Upon adrenergic stimulation, the marked positive shift of HCN voltage dependence most likely reflects a major contribution of HCN2 and HCN4 to tetramer assembly, being these isoforms most sensitive to cAMP-mediated effects (Stillitano et al., 2008; Wahl-Schott et al., 2014). The occurrence and density of HCN current in human cardiac ventricular myocytes isolated from explanted hearts are larger compared with control ventricle and related to disease etiology, being more prominent in ischemic than in dilated cardiomyopathy (Cerbai et al., 1997, 2001; Hoppe and Beuckelmann, 1998). In this setting, the gain of function of HCN current relates to increased levels of HCN4 and HCN2 proteins and transcripts (Fernández-Velasco et al., 2003; Stillitano et al., 2008; Suffredini et al., 2012) (Figs. 3 and 4).

Following on these pieces of evidence, a number of studies have extensively documented that increase in HCN channel expression is a common trait of different models of cardiac hypertrophy (Stilli et al., 2001; Fernández-Velasco et al., 2003) and myocardial infarction (Sartiani et al., 2006; Suffredini et al., 2012). Hence, HCN channels most likely have their place in the fetal/embryonic gene pattern re-expressed during functional remodeling of the diseased ventricle (Swynghedauw, 1999). Interestingly, the expression of the HCN channels appears more pronounced in ventricular regions with the greatest overload, suggesting the possibility of mechano‐sensitive mechanism of channel expression (Fernández-Velasco et al., 2003).

Despite the emerging appraisal of HCN channel as representative marker of cardiac remodeling, its arrhythmogenic role in the ventricle has long remained unproven both in experimental and clinical studies. A recent study in a bradycardic model of heart failure has provided insight into the role of HCN channels in the initiation of triggered activity. The model, expressing a dominant-negative form of the transcriptional repressor neuron‐restrictive silencing factor, a regulator of the fetal cardiac gene program, develops heart failure and dilated cardiomyopathy. Along this process, HCN2 and HCN4 channels are upregulated, leading to enhanced HCN current density associated with ventricular tachycardia, premature ventricular contractions, and sudden cardiac death (Kuwabara et al., 2013; Yamada et al., 2014). Accordingly, β‐adrenergic stimulation increased the susceptibility of myocytes to early after‐depolarizations and spontaneous APs. These findings are in agreement with another study (Hofmann et al., 2012) showing that HCN activity increases the arrhythmogenic potential in the failing myocytes through prolongation of the repolarization phase in ventricular APs.

The consolidated knowledge on the pathways involved in cardiac remodeling has led to investigate whether abnormal neurohumoral signals, known to trigger the onset and progression of adverse cardiac modifications, are also involved in HCN channel overexpression. Among the most important signals, adrenergic and renin-angiotensin-aldosterone systems are responsible for increased levels of hormones, which are released systemically and locally (Swynghedauw, 1999; De Mello, 2004) and have a well-established role in the proarrhythmic alterations of the ventricle, such as the reduction of transient outward potassium channel and the prolongation of AP duration. Indeed, in similar experimental context, chronic administration of angiotensin II receptor antagonists, losartan and irbesartan, is effective in restoring normal electrogenesis in the ventricle, including the expression and function of HCN channels (Cerbai et al., 2000, 2003).

More recently, a different pharmacological strategy has emerged from clinical trials demonstrating the efficacy of pacing to modify positively the progression and worsening of cardiac failure (Rao et al., 2007; Fox et al., 2008). That high heart rate was an independent risk factor for cardiovascular mortality and morbidity was demonstrated since the early Framingham studies (Kannel et al., 1987). Accordingly, growing clinical evidence also prompted studies aimed at testing heart-rate–reducing drugs to reverse and/or ameliorate the functional remodeling of the hypertrophic ventricle in animal models (Dedkov et al., 2007). In line with these observations, experimental findings obtained in the postinfarcted rat demonstrate that chronic treatment of ivabradine, the only bradycardic agent in clinical use, is able to ameliorate cardiac function and to counteract the global electrophysiological remodeling of the ventricle, including the overexpression of HCN channels (Ceconi et al., 2011; Suffredini et al., 2012). The mechanism(s) responsible for the benefit of heart-rate–lowering drugs in heart hypertrophy and failure will be further discussed later (section Ivabradine in Angina and Heart Failure).

i. Regulation of Resting Membrane Potential and Intrinsic Excitability

The influence exerted by HCN current on the intrinsic excitability of the neuron has a complex nature. At −65 mV, a fraction of HCN channels is tonically open (Kase and Imoto, 2012). This results in persistent sodium inflow, which depolarizes the membrane and counteracts hyperpolarization. Depolarization causes channel deactivation, and the consequent pause in current flow has a hyperpolarizing effect. Hence, by opposing negative and positive perturbations, tonic HCN current stabilizes membrane potential in the subthreshold range. Therefore, HCN loss of function causes membrane hyperpolarization and reduction of intrinsic excitability. At the same time, however, input resistance at subthreshold potentials increases and the neuron becomes more responsive toward synaptic stimulation. This dual action exerted by HCN current on neuronal function has been clearly established in many diverse brain areas, including the thalamus, the hippocampus, and the cerebellum, and has important repercussions on higher-order brain functions such as learning and memory, control of circadian rhythm, and sleep/wakefulness state (McCormick and Pape, 1990b; Maccaferri et al., 1993; Gasparini and DiFrancesco, 1997; Nolan et al., 2003). Very recently, a novel role for HCN1 in the control of the neuron’s intrinsic excitability has been proposed. In the axon initial segment of medial superior olive principal neurons, HCN1-mediated current reduces spike probability by elevating the firing threshold. The 5-HT_1A receptor signaling shifts HCN channel activation curve in the hyperpolarizing direction, thereby relieving the brake on spike probability (Ko et al., 2016). Serotonin-dependent modulation of HCN current is also involved in the control of respiratory rhythm in the retrotrapezoid nucleus. Activation of the 5-HT₇ receptor causes a cAMP-dependent depolarizing shift in the activation curve of HCN current, increasing the firing rate of retrotrapezoid nucleus neurons in vitro and respiratory rhythm in vivo (Hawkins et al., 2015).

ii. Rhythmogenesis

In a functional interplay with other conductances, the activation/deactivation cycle of HCN channels sets the pace of subthreshold membrane oscillations, which may eventually generate AP firing. HCN current cooperates with a subthreshold, persistent Na⁺ current to determine a 4–10 Hz (θ-like) rhythm in layer II grid cells of the entorhinal cortex (EC) (Alonso and Llinas, 1989). Entorhinal grid cells show periodic, hexagonally-patterned firing locations that scale up progressively along the dorsal–ventral axis of medial EC, which are critically involved in spatial navigation (Moser et al., 2015). In this structure, relative HCN1/HCN2 expression ratio goes down, along with oscillation frequency, following a dorsal–ventral gradient (Notomi and Shigemoto, 2004). This frequency gradient is consistent with the distinct time constants of HCN1 and HCN2 isoforms. In HCN1-KO mice, the dorsal–ventral gradient of the grid pattern is preserved, but the size and spacing of the grid fields, as well as the period of the accompanying θ modulation, are expanded (Giocomo and Hasselmo, 2009; Giocomo et al., 2011). In thalamocortical neurons, HCN current mainly results from HCN2 expression. Adrenergic and serotonergic stimulation causes a cAMP-dependent modulation of HCN2-mediated current, which in turn determines the firing mode of thalamocortical neurons. During wakefulness and rapid eye movement sleep, thalamocortical neurons fire in a single-spike, or transmission mode. In this state, information is effectively transmitted to the cortex. During non-rapid eye movement sleep and absence seizures, thalamocortical neurons fire in a burst mode, and transfer of signals to the cortex is believed to be not as effective (McCormick and Pape, 1990a, b). In agreement, global HCN2 deletion causes absence epilepsy with typical spike-and-wave discharges in electroencephalogram (EEG) recordings (Ludwig et al., 2003). In the intergeniculate leaflet neurons, a retino-recipient thalamic structure implicated in orchestrating circadian rhythm, a role for HCN3 subunit has been described. In this work, HCN3-mediated current drives low-threshold burst firing and spontaneous oscillations and is bidirectionally modulated by PI(4,5)P2. Depletion of PI(4,5)P2 or pharmacologic block of HCN current results in a profound inhibition of excitability (Ying et al., 2011).

iii. Synaptic Excitability and Plasticity

Functional HCN channels are strongly expressed along the dendritic arborizations of neocortical and hippocampal neurons with a soma-to-dendrites expression gradient (Bender et al., 2001; Lörincz et al., 2002; Harnett et al., 2015). In these areas, HCN current constitutes a shunt conductance accelerating the membrane’s time constant and the decay phase of excitatory postsynaptic potentials (EPSPs). As a corollary, temporal summation during an EPSP sequence is minimized. This function, discovered and characterized in pyramidal neurons of the hippocampal CA1 region and somatosensory cortex (Magee, 1998, 1999; Williams and Stuart, 2000; Berger et al., 2003), has then been described in subcortical structures (Ying et al., 2007; Engel and Seutin, 2015; Masi et al., 2015). In the dendritic compartment of CA1 pyramidal neurons, the shunting effect exerted by HCN current limits the activation of voltage-dependent calcium entry, with important consequences on synaptic excitability (Tsay et al., 2007). The nonuniform distribution of HCN current is also responsible for a phenomenon termed “site independence” of synaptic potentials, whereby the decay time constant of distally-generated EPSPs is similar to that of proximally-generated EPSPs (Williams and Stuart, 2000). As a rule, HCN current accelerates the kinetics of both forward- and backward-propagating depolarization waves, thus increasing the precision with which the dendrite detects the concurrence of salient events, including EPSP–AP coincidence, an event leading to plasticity (Pavlov et al., 2011). In several brain areas, long-term potentiation and associated cognitive functions may be constrained by normal HCN function. Indeed, HCN1-KO mice show improved hippocampal-dependent learning and memory performance. In these mice, proximal synapses (CA3–CA1 contacts) function normally, whereas they are potentiated at distal locations (EC layer III–CA1 contacts), as predicted by stronger expression of HCN channels in this compartment (Nolan et al., 2004). HCN current affects cortical functions such as cognition, movement planning, and execution by setting the connectivity strength within local cortical microcircuits. HCN current is abundantly expressed in dorsal–lateral prefrontal cortex (PFC) layer III neurons, a population crucially involved in spatial working memory. In these neurons, HCN channels are modulated by the opposite action of α₂-adrenergic receptor (α₂-AR) and dopaminergic receptor type 1 (D1) stimulation, via cAMP signaling. When α₂-AR stimulation inhibits cAMP–HCN channel signaling, neurons are more tightly connected to recurring microcircuit activity and performance is optimal. In contrast, exposure to stress causes a D1-mediated enhancement of cAMP–HCN activity, leading to a functional disconnection from the local network and impairment of spatial working memory (Wang et al., 2007; Arnsten and Jin, 2014). In the mouse primary motor cortex, HCN expression is specifically elevated in corticospinal neurons of layer V. HCN current confers these neurons a 4 Hz–resonance preference and gates synaptic inputs from layer II/III pyramidal neurons, determining the efficacy of signal transmission between the two layers. In this context as well, α₂-AR stimulation modulates I_h function (Sheets et al., 2011).

iv. Resonance Properties

HCN current in the dendritic compartment also confers the neuron-specific resonance properties, i.e., the ability to respond preferentially to inputs at a certain frequency, and thus determines to what extent the neuron responds to synchronous network activity. Due to its slow gating kinetics, HCN current has high-pass filtering properties that, combined with the low-pass filtering action exerted by membrane time constant (Bedard et al., 2006), result in a resonance preference of 1–10 Hz (Hutcheon et al., 1996). The increased power of θ oscillation due to HCN1 deletion and potentiation of perforant path–CA1 pyramidal neurons synapse has been proposed as the physiologic background of hippocampal-dependent learning and memory storage (Nolan et al., 2004). The ability to resonate in a HCN-dependent manner has been reported for other areas, but the functional significance at higher-order level has yet to be established (Ulrich, 2002; Wang et al., 2006; Xue et al., 2012b; Borel et al., 2013).

v. Neurotransmitter Release

HCN current at synaptic terminals has been reported to control efficacy of vesicle release. HCN1 and HCN2 isoforms are present at GABAergic terminals of pallidal axon collaterals (Boyes et al., 2007) as well as glutamatergic terminals onto EC layer III pyramidal neurons (Huang et al., 2011). In both settings, pharmacological or genetic inactivation leads to elevation of spontaneous synaptic release. Huang et al. (2011) have suggested that HCN current inhibitory action is exerted by setting the potential to values where Cav3.2 N-type channels are less active. A follow-up study from the same authors shows that HCN1 channels restrict the rate of exocytosis from a subset of cortical synaptic terminals within the EC and constrain non-AP–dependent and AP-dependent spontaneous release, as well as synchronous, evoked release (Huang et al., 2017).

Despite a large mass of information on the cellular effects of HCN channels, our knowledge of their role in more integrate CNS functions and behavior is rather indirect and derived from pathophysiological studies, as described below. An interesting insight comes from recent evidence in Aplysia californica, whose neuroanatomy and physiology are by far less complex than the mammalian brain (Yang et al., 2015). A. californica motor neurons possess only one HCN isoform, acHCN; I_h appears to be involved in classic conditioning upon stimulation by nitric oxide/cGMP signaling and subsequent enhancement of a N-methyl-D-aspartate (NMDA)-like current pathway, similarly to the mammalian hippocampal neurons (Neitz et al., 2014).

i. Epilepsy

The majority of studies have reported that increased neuronal excitability, eventually leading to epileptic state, is associated with loss-of-function mutations in HCN1 and HCN2 isoforms (Nava et al., 2014; DiFrancesco and DiFrancesco, 2015). HCN1-null mice are more susceptible to kainic acid–induced seizures, and, in spite of the hyperpolarization in resting membrane potential, synaptic excitability of cortical and hippocampal neurons is increased in these mice (Huang et al., 2009). Global HCN2 deletion results in the appearance of absence seizures (Ludwig et al., 2003). Mice carrying a C terminus–truncated HCN2 protein manifest generalized spike–wave absence seizures. Next-generation sequencing analysis on patients’ cohorts has revealed association with single-point mutations leading to loss-of-function phenotype in HCN2 isoform and idiopathic generalized epilepsy (Tang et al., 2008; DiFrancesco et al., 2011). Another study, however, reported a single-point mutation in HCN2 gene leading to a gain of function, showing a significant association with febrile seizures (Dibbens et al., 2010). In agreement with the hypothesis that epilepsy correlates with a negative functional modulation of HCN current is the evidence that the established anticonvulsants such as lamotrigine (Poolos et al., 2002), gabapentin (Surges et al., 2003), and acetazolamide (Munsch and Pape, 1999) activate HCN current in vitro. However, it is not clear whether HCN current upregulation contributes to the antiepileptic efficacy of these compounds. HCN current loss of function may also result from alterations in the expression of auxiliary subunits, such as TRIP8b. TRIP8b-KO mice feature spontaneous spike–wave discharges on EEG that are the electrographic hallmark of absence seizures, resembling the scenario of a global HCN2 deletion. Compared with global HCN2 KOs, TRIP8b KOs have no cardiac phenotype and less severe seizure phenotype. Mechanistically, TRIP8b-KO mice have significantly reduced HCN channel expression and function in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but preserved function in inhibitory neurons of the reticular thalamic nucleus (Heuermann et al., 2016). Finally, a number of studies report a remodelling of HCN1/2 expression following experimental seizure induction, suggesting a reciprocal cause–effect relation between HCN channels and epilepsy (Brewster et al., 2002; Bender et al., 2003). HCN channels are upregulated in rat CA1 pyramidal neurons following hyperthermia-induced seizures. Interestingly, an adaptive increase in GABAergic activity has been reported in this paradigm. GABAergic inhibitory postsynaptic potentials at high frequency (∼50 Hz) tend to summate and to activate HCN channels enough to cause rebound firing at the end of the inhibitory train. This mechanism has been proposed as an explanation for the paradoxical increase in GABAergic inhibition in experimental models of epilepsy (Chen et al., 2001; Dyhrfjeld-Johnsen et al., 2009). In summary, a relatively large number of preclinical and human genetic studies supports the evidence linking HCN channelopathy to epilepsy. These studies do not always hint at a univocal cause–effect relation between HCN alteration and disease. In fact, both up- and downregulation have been reported in association to epileptic states, and HCN expression remodelling is sometimes a consequence rather than the cause of seizures. For these reasons, the hypothesis that HCN channels may serve as new-generation targets for the development of antiepileptic drugs, yet promising, deserves further experimental testing.

ii. Autism Spectrum Disorder

It has been recently established that a reduction in I_h density is one of the alterations determined by Shank3 haploinsufficiency in reprogrammed human neurons (Yi et al., 2016). Shank3 is a ubiquitous scaffolding protein enriched at postsynaptic densities associated with autism spectrum disorder and other neuropsychiatric disorders. Similar reduction in I_h density was found in hippocampal neurons from transgenic mice carrying a similar Shank3 mutation. Finally, chronic treatment of developing mouse neurons with ZD7288 reproduces some of the morphologic abnormalities caused by mutant Shank3.

iii. Schizophrenia

Schizophrenia is characterized by deficits in PFC function and alteration in cAMP signaling pathway. In layer II/III of the PFC, HCN1 and cAMP/phosphodiesterase signaling machinery colocalizes to the neck of thin spines to regulate gating of afferent inputs during the execution of working memory tasks. As discussed earlier, unbalance of cAMP–HCN signaling results in network disconnection and reduced performance in these tasks (Arnsten, 2011). Consistently, D1 stimulation in rat or monkey PFC impairs working memory performance, and HCN channel blockade in PFC prevents this impairment in rats exposed to either stress or D1 receptor stimulation. These findings suggest that D1 stimulation or stress weakens PFC function via opening of HCN channels at network synapses. This effect can be counterbalanced by elevation of cAMP levels with type-4 phosphodiesterase inhibitors (Paspalas et al., 2013; Gamo et al., 2015). Rescue of synaptic connectivity in the PFC seems to be the rationale at the basis of the clinical efficacy of the α_2A agonist guanfacine in PFC-related disorders (Arnsten and Jin, 2014).

iv. Substances of Abuse and Addiction

As a conductance involved in the rhythmic activity of the DA reward system, HCN current has been implicated in pathophysiology of addiction. Modulation of HCN current has been associated with exposure to ethanol (Okamoto et al., 2006), cocaine (Arencibia-Albite et al., 2012, 2017; Goertz et al., 2015), and methamphetamine (Gonzalez et al., 2016). It has recently been proposed that the reported impairment of long-term potentiation and spatial memory formation caused by cannabinoids requires activation of cannabinoid receptor type 1 and cGMP-dependent upregulation of HCN current in superficial CA1 pyramidal cells (Maroso et al., 2016).

v. Mood Disorders

Recent studies have suggested a role for HCN channels in animal models of depression. Chetkovich and coworkers (Lewis et al., 2011) have generated a KO mouse lacking the HCN channel auxiliary subunit TRIP8b that significantly regulates the voltage gating and kinetics of I_h. TRIP8b deletion dramatically reduces I_h expression, disrupting subcellular distribution, in hippocampal pyramidal neurons. Behaviorally, mice lacking TRIP8b demonstrate motor-learning deficits and enhanced resistance to multiple tasks of despair with high predictive validity for antidepressant efficacy. Of note, similar resistance to behavioral despair was observed in distinct mutant mice lacking HCN1 or HCN2 (Lewis et al., 2011). Similarly, another study has shown that HCN current–mediated depressant effects are localized in the dorsal hippocampus, as focal infusion of lentiviral particles carrying HCN1 short hairpin RNA has anxiolytic- and antidepressant-like effects. Indeed, knocking down HCN1 channels increases cellular excitability and results in physiologic changes consistent with a reduction of HCN current. Moreover, treated animals display antidepressant- and anxiolytic-like behaviors associated with widespread enhancement of hippocampal activity and upregulation of brain-derived neurotrophic factor–mammalian target ofrapamycin signaling pathways (Kim et al., 2012). HCN-dependent hyperactivity of ventral tegmental area (VTA) DA neurons has been causally associated with susceptibility in social defeat stress paradigms in rats and mice. In the rat study, chronic treatment with the selective serotonin reuptake inhibitor and antidepressant fluoxetine, besides exerting the expected effects, normalizes both HCN current amplitude and VTA firing rate, suggesting a link between the actions at behavioral and cellular levels. The effects of fluoxetine are in part mimicked by intracerebral infusion of ZD7288, thus suggesting a mechanistic involvement of HCN current in the development of depressive state associated with social defeat stress (Cao et al., 2010). In the mouse, upregulated HCN current causes hyperactivity of VTA DA neurons and susceptibility to depression. Paradoxically, further enhancement of HCN function with lamotrigine or optogenetic hyperactivation of VTA discharge rate is able to rescue susceptibility to social defeat stress (Friedman et al., 2014).

vi. Fear and Anxiety

HCN currents control the excitability of principal neurons in the basolateral amygdala by reducing synaptic excitability (Park et al., 2007). In a recent study, HCN1 was identified in one of three loci associated with conditioned fear acquisition and expression in a mouse genetic reference panel. This discovery was validated using behavioral pharmacology and revealed that HCN current in the basolateral amygdala is required for fear acquisition and expression (Knoll et al., 2016). Collectively, these data indicate that HCN channels support negative mood states in animal models in area- and subunit-specific fashion. Direct or indirect negative modulation of HCN current in the brain areas subtending anxiety level, fear, and depression may have a potential therapeutic efficacy.

vii. Neurodegenerative Disorders

Parkinson disease (PD) is caused by the progressive degeneration of nigrostriatal DA neurons and resulting fall of dopamine levels. In midbrain DA neurons, HCN current results from HCN2 and HCN4 expression, and substantia nigra pars compacta (SNc) DA neurons show the highest expression levels (Neuhoff et al., 2002; Dufour et al., 2014). Interestingly, HCN current density is reduced in SNc DA neurons of MitoPark mice, a genetic mouse model in which a DA-targeted mitochondrial defect leads to selective nigrostriatal degeneration and PD-like phenotype (Good et al., 2011; Branch et al., 2016). Of note, this defect marks early, asymptomatic disease stages and is not due to HCN transcript downregulation. In addition, it has been reported that 1-methyl-4-phenylpyridinium, a toxin capable of inducing PD-like selective nigrostriatal degeneration in primates and rodents, is a voltage-dependent inhibitor of HCN channels in SNc DA neurons. The 1-methyl-4-phenylpyridinium–induced HCN block results in increased dendritic excitability and overall responsiveness to excitatory synaptic inputs (Masi et al., 2013). In agreement with relative HCN expression levels, the effect of HCN block on synaptic excitability is significantly greater in SNc compared with VTA DA neurons (Masi et al., 2015). Although preliminary, these findings suggest that altered HCN channel expression or function in experimental PD models deserves consideration in the context of differential vulnerability. A single study has investigated the electrophysiological actions of riluzole, a medication used to retard the onset of respiratory impairment in the treatment of amyotrophic lateral sclerosis, on the firing activity of hypoglossal motor neurons in rat brain slices, and reported that HCN current undergoes a negative shift in the activation curve following drug administration. HCN inactivation contributes to the riluzole-induced reduction in firing rate of hypoglossal motor neurons, the cellular mechanism regarded as the presumed basis of its therapeutic action (Bellingham, 2013). One study has described that HCN1 levels diminished in the temporal lobe of macaques during aging and Alzheimer’s disease patients. The study also suggests a causal role of HCN1 downregulation in the increased susceptibility of Alzheimer’s disease patients to epileptic seizures (Saito et al., 2012).

i. Retinitis Pigmentosa

Although HCN are expressed in the retina, HCN dysfunction was not associated with any retinal disorder until recently. It has been known that patients during prolonged treatment with I_h inhibitor ivabradine experienced visual symptoms such as phosphenes (described as sensations of enhanced brightness in a fully maintained visual field) (Cervetto et al., 2007). However, these symptoms were dose dependent and reverted to normal 1 week after discontinuation of ivabradine without any alteration of retinal morphology and modification of HCN distribution in healthy animals (Della Santina et al., 2010). Moreover, HCN inhibition in dystrophic mice had no effect on either extent or progression of retinal degeneration. However, more recent observations pointed out the importance of HCN1 in a mouse model of CNGB1-linked retinitis pigmentosa in the progression of the disease. Indeed, the absence of HCN1 in CNGB1-KO mice exacerbated photoreceptor degeneration, thus identifying HCN1 as a major modifier of photoreceptor degeneration. This observation suggested that pharmacological inhibition of HCN channels may enhance disease progression in retinitis pigmentosa and achromatopsia (Schön et al., 2016).

i. Synaptic Transmission in Dorsal Root Ganglion Neurons

Shortly after the first report of a hyperpolarization-activated queer current in hippocampal neurons (Halliwell and Adams, 1982), I_h was described also in neuronal cells of the embryonic mouse DRG (Mayer and Westbrook, 1983). In these neurons, it was noticed the presence of a time- and voltage-dependent conductance activated by membrane hyperpolarization owning similar characteristics of the “pacemaker current,” I_f, identified in SAN cells a few years before (Brown et al., 1979; Yanagihara and Irisawa, 1980). Its expression was shown to be most prominent in large- and medium-sized neurons, whereas only half of small sized DRG neurons displayed a functional I_h (Scroggs et al., 1994). Of all the four HCN isoforms, HCN1 and HCN2 are predominantly expressed in DRGs, whereas expression of HCN3 and HCN4 is low or undetectable (Acosta et al., 2012). In particular, HCN2 is mainly abundant in small-sized neurons, whereas HCN1 is the predominant subunit expressed in large neurons (Schnorr et al., 2014). Consistently, I_h in large- and medium-sized DRG neurons has a faster activation kinetic and is less cAMP-sensitive as compared with the characteristics of the current in small-diameter neurons (Gao et al., 2012).

HCN1- and HCN2-KO studies largely confirm HCN distribution in different DRG neuronal subtypes. Specifically, HCN1 deletion erased I_h in large DRG neurons, whereas slowly activating cAMP-sensitive I_h was still present in smaller DRG neurons (Momin et al., 2008). In contrast, genetic deletion of HCN2 removed the cAMP-sensitive component of I_h and abolished AP firing caused by an elevation of cAMP in nociceptors (Emery et al., 2011).

ii. HCN as Pacemakers of Pain

The precise role of I_h in DRG both in physiology and in pathology is not perfectly understood. Indeed, healthy sensory neurons seem largely unaffected by HCN blockade. Moreover, clinical studies with I_h blocker ivabradine did not show any adverse drug reaction such as dysesthesias and paresthesias (Herrmann et al., 2015). Therefore, I_h function seems to be mainly affected following pathologic states, such as in neuronal damage or in experimental model of inflammation (Papp et al., 2010; Acosta et al., 2012; Weng et al., 2012; Schnorr et al., 2014). Indeed, in these scenarios, HCN channels have been reported to increase their expression and/or function (Chaplan et al., 2003; Yao et al., 2003; Jiang et al., 2008). These observations suggested an association between HCN overexpression/overfunction and pathologic conditions, such as allodynia and hyperalgesia (Herrmann et al., 2015). Multiple mechanisms most likely underlie the phenomenon, including the following: 1) increased and modified gene expression (Papp et al., 2010; Descoeur et al., 2011; Schnorr et al., 2014); 2) PI(4,5)P2-mediated pathway interaction (Pian et al., 2006, 2007); 3) PKA overactivation (Cheng and Zhou, 2013); and 4) elevated intracellular levels of cAMP triggered by inflammatory substances such as prostaglandin E2, serotonin, and substance P (Jafri and Weinreich, 1998; Momin et al., 2008; Resta et al., 2016).