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date: 26 May 2019

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

Abstract and Keywords

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are members of the voltage-gated K+ channels family, but with unique properties. In stark contrast to close relatives, HCN channels are permeable to both Na+ and K+, and they are activated by hyperpolarization. Activation by hyperpolarization is indeed a pretty funny feature, to the point that the physiologists who first characterized HCN current in heart muscle cells named it “funny current” or If. Since then, the funny current has also been recorded from several neuronal types in both the central and peripheral nervous systems, as well as from some non-excitable cells, becoming progressively less “funny” over the years. In fact, HCN current goes now by the more serious designation of “Ih,” for “hyperpolarization-activated.” Forty years after the first current recording, it is now established that HCN channels, by virtue of their special properties and a host of modulatory mechanisms, are profoundly involved in many critical aspects of neuronal function in physiological and pathological conditions.

Keywords: hyperpolarization-activated cyclic nucleotide-gated channels, HCN channels, Ih, nervous system, spontaneous activity, synaptic transmission, neurological disorders

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels belong to the voltage-gated pore-loop channels family. The HCN subfamily comprises four subunits, encoded by four distinct genes, named HCN1, 2, 3, and 4. All subunits are made up of six transmembrane domains (S1–S6), a voltage-sensing segment in S4, and the pore-loop element between S4 and S5. Functional channels are typically composed of homo- or hetero-tetramers. Altogether, these traits match the identikit of six-transmembrane domain, voltage-gated K+ channels. What sets HCN channels apart is their peculiar ionic selectivity and their unusual gating properties. In fact, unlike their close relatives, HCN channels are activated by hyperpolarization and permeable to both Na+ and K+. These properties, first described in the late 1970s by Brown, DiFrancesco, and Noble in heart muscle cells, were seen as pretty unusual features, even funny, thus the current was nicknamed If, where “f” stands for “funny.” Since then, the physiological relevance of HCN current has been well established in the heart, where it is now the target of a drug with therapeutic use. In the meantime, HCN channels have been discovered in several neuronal types in both central and peripheral districts of the nervous system and studied extensively with multiple approaches. The interest of neurophysiologists in HCN current was prompted by the unique biophysical features, which appeared suited to explain some remarkable electrical properties of single neurons and complex neural systems, such as intrinsically generated rhythmic firing and network oscillations. Two decades of extraordinary experimental effort have now established that HCN channels are key elements of neuronal physiology. In the attempt to review the role of HCN channels in the normal operation of neurons, as well as in pathological states, this chapter will start by discussing general molecular and biophysical concepts, will then focus on the most prominent and well-documented functions at cellular and network levels in physiological conditions, and will finally overview some remarkable examples of HCN channel–related pathologies affecting nerve cells.

Basic Facts About HCN Channels in the Nervous System

To understand the multiple roles played by HCN channels in physiological and pathological states of the nervous system, it is necessary to start from the expression pattern of the four pore-forming subunits and to overview the defining molecular and biophysical properties of HCN channels in nerve cells.

Expression Pattern at Regional, Cellular, and Subcellular Level

Available data on HCN channel expression at the supracellular level comes from in situ hybridization and immunohistochemistry studies performed in brain sections from both adult and developing rodent brains (Bender et al., 2001; Monteggia, Eisch, Tang, Kaczmarek, & Nestler, 2000; Moosmang, Biel, Hofmann, & Ludwig, 1999; Notomi & Shigemoto, 2004; Santoro et al., 2000). Single-cell reverse transcription-polymerase chain reaction (RT-PCR ) and electron microscopy have provided further details on the expression pattern of HCN transcripts and proteins with cellular and subcellular resolution (Bender et al., 2007; Brewster et al., 2007; Dufour, Woodhouse, & Goaillard, 2014; Franz, Liss, Neu, & Roeper, 2000; Huang et al., 2011; Luján, Albasanz, Shigemoto, & Juiz, 2005; Paspalas, Wang, & Arnsten, 2013; Ramakrishnan, Drescher, Khan, Hatfield, & Drescher, 2012). All four HCN isoforms are expressed in the brain, with the relative abundance of each subunit relating to the area, neuronal type, and cellular compartment. Despite the different methodologies and animal models employed, all reports addressing HCN distribution show fairly consistent results. In the central nervous system (CNS), HCN1 is highly expressed in the neocortex, hippocampus, cerebellar cortex, brainstem, and spinal cord. HCN2 has a diffuse expression pattern across the CNS, with highest density in thalamic and brainstem nuclei. Conversely, HCN4 expression is highly localized, with few areas exhibiting strong expression levels, such as the olfactory bulb and the thalamus, with a distribution topography that appears complementary to that of HCN1. The expression of HCN3 is sparse throughout the brain. All isoforms are present in the retina (Fyk-Kolodziej & Pourcho, 2007; Muller et al., 2003). In the peripheral nervous system (PNS), all HCN subunits are expressed. HCN1 is the isoform with highest expression in dorsal root ganglia (Chaplan et al., 2003), although a prominent function for HCN2 in the transmission of painful stimuli has also been reported (Emery, Young, Berrocoso, Chen, & McNaughton, 2011). Last but not least, HCN current has been recorded in many ganglion neurons of the autonomic nervous system (Doan et al., 2004; Galligan, Tatsumi, Shen, Surprenant, & North, 1990; Kullmann et al., 2016; Lamas, 1998; Zhang & Cuevas, 2002).

Biophysical Features of Neuronal HCN Channels

As stated before, this chapter will attempt to explain how HCN channels impart specific electrical properties to the single neuron, and how these impinge on neuronal input–output properties, synaptic transmission, and the operation of neuronal ensembles. Therefore, only some essential concepts on basic HCN channels’ biophysics will be provided here as tools for the reader to fully grasp the neurophysiological significance of HCN current. The brave reader who is willing to penetrate the multitude of studies that have addressed this matter over the past 40 years will find full satisfaction browsing the reference section of some excellent reviews (Biel, Wahl-Schott, Michalakis, & Zong, 2009; He, Chen, Li, & Hu, 2014; Sartiani, Mannaioni, Masi, Novella Romanelli, & Cerbai, 2017).

As mentioned, HCN subunits display all the standard structural features of voltage-gated K+ channels (KV), with which they share remarkable aminoacidic sequence homology, especially in functional domains. Nonetheless, HCN channels have a few remarkable peculiarities. These lie essentially in (i) the function of the voltage sensor, (ii) the selectivity filter, (iii) the absence of an inactivation gate, and (iv) the presence of a highly specialized cyclic nucleotide binding domain (CNBD) at the C-terminus. Universally present in voltage-gate ion channels, the voltage sensor is a typical α helix motif enriched with positively charged aminoacidic residues (Lys, Arg). These residues enable the segment to respond with outward or inward displacement following changes in magnitude of the membrane potential (VM). In “classic” depolarization-activated channels, depolarization causes outward motion of the voltage sensor and opening of the pore (Bahring, Barghaan, Westermeier, & Wollberg, 2012; Yellen, 1998). In contrast, HCN channels are stabilized in the closed state by depolarization (VM ≥ –50 mV), whereas hyperpolarization (VM ≤ –60 mV) causes opening of the pore (Prole & Yellen, 2006). Such a striking exception from the rule seems to depend on the uniquely large size of the S4 helix. In depolarization, the S4 helix compresses the S5–S6 helices, thus causing pore obstruction, while hyperpolarization causes an inward shift of the S4 sensor and unblocking of the pore. Once the channel is open, both Na+ and K+ ions are allowed through the pore. Such loose selectivity is due to the presence of only two binding sites for K+, as opposed to the four present in KV channels (Lee & MacKinnon, 2017). Because of this structural feature, K+:Na+ selectivity drops from 1000:1 to 4:1. Although a certain preference for K+ over Na+ is maintained, since HCN channels open at potentials that are closer to the equilibrium potential of K+ (~ –90 mV) than to that of Na+ (~ +60 mV), the electrochemical gradient driving Na+ inside is much greater than that driving K+ outside when channels are open, with the consequence that HCN current is, under physiological conditions, an inward depolarizing current.

The half-activation potential (V1/2, an index of voltage dependence) of HCN channels lies between –75 and –90 mV and varies based on the intrinsic properties of HCN isoforms, host cell type, and experimental conditions. Gating kinetics—i.e., the speed at which the channel opens when the membrane is hyperpolarized—is also voltage dependent, with greater hyperpolarization leading to faster activation. In this respect, compared to the majority of voltage-gated ion channels, HCN channels are exceptionally slow. In fact, with the exception of a minor initial component termed “instantaneous current,” which reaches steady-state amplitude within few milliseconds and whose molecular aspects are still unclear, the main component takes from hundreds of milliseconds to several seconds to reach the steady state. Gating kinetics are isoform and voltage dependent, with stronger hyperpolarization pulses leading to faster gating. In this regard, HCN1 is the isoform showing the fastest kinetics, reaching full activation in fewer than 100 milliseconds. Finally, HCN current does not inactivate—i.e., the current maintains steady-state amplitude until the membrane potential returns to depolarized values. This feature is also very unusual among voltage-gated channels, and it entails a number of important consequences, which will be discussed later in the chapter.

Functional Modulation by Multiple Heterogeneous Mechanisms

HCN channels are true voltage-gated channels. In other words, voltage is always sufficient to gate the channel. Nevertheless, gating properties may be modulated by several physiological mechanisms. These include covalent modifications, physical interaction with scaffold proteins and lipidic membrane constituents, and sensitivity to protons and other ions (He et al., 2014; Sartiani et al., 2017). This chapter will mainly focus on the examples for which strong evidence of physiological relevance in a native neuronal environment has been provided.

The modulation by intracellular cyclic nucleotides is a defining feature of HCN channels. The CNBD is highly conserved across isoforms and exerts a steric blocking action on the channel pore in the absence of the ligand. Cyclic adenosine monophosphate (cAMP) binding relieves self-inhibition, resulting in a positive shift of the activation curve and acceleration of gating kinetics. The overall effect exerted by cAMP-dependent modulation is an increase in current availability. Of note, cAMP promotes channel opening by increasing its voltage sensitivity; thus the same effect can be produced experimentally by increasing the amplitude of the hyperpolarizing test stimulus (Biel et al., 2009). Sensitivity to cAMP concentration, as well as the magnitude of the V1/2 shift (“intrinsic activity”), varies to a large extent across isoforms: HCN2 and 4 are the most responsive (10–20 mV shift), while HCN1 is much less responsive (2–6 mV shift). As a striking exception, HCN3, although equipped with a normal CNBD, is not activated by cAMP. Rather, there is evidence for a negative shift of V1/2 in this isoform. Cyclic guanosine monophosphate (cGMP) and cyclic cytosine monophosphate (cCMP) have also been reported to activate HCN channels, yet with lower potency and intrinsic activity. It is important to point out that a large majority of the studies addressing basic HCN channels’ properties were carried out in heterologous cellular systems. Hence, the actual significance of HCN current modulation by cyclic nucleotides in native conditions is still incompletely understood.

HCN channels are sensitive to lipidic acids, normal constituents of the plasma membrane. Phosphatidylinositol 4,5 bisphosphate (PIP2) depolarizes the V1/2 by ~20 mV, thus greatly contributing to the physiological relevance of HCN current. The molecular mechanism underlying PIP2 action on HCN channels is unknown, although it is certainly independent from the presence of cyclic nucleotides or the CNBD itself, as shown by experiments performed in CNBD-lacking mutants. In dopaminergic neurons of the substantia nigra pars compacta, expressing mainly HCN2 and 4, pharmacological inhibition of PIP2 synthesis shifts the V1/2 from –65 to –77 mV and reduces the frequency of autonomous firing (Zolles et al., 2006).

The presence of a protonable histidine residue near the S4 helix makes murine HCN2 sensitive to intracellular pH. Acidic (pH~6) and alkaline (pH~9) environments affect gating by shifting V1/2 to more negative or less negative potentials, respectively. This mechanism is thought to apply to other HCN isoforms and to contribute to the physiological role of these in the regulation of respiratory rhythm (Hawkins et al., 2015).

Several transmembrane and cytosolic proteins have been shown to interact with HCN channels and to modulate their expression and function. In nerve cells, the most important among these ancillary proteins is the tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). TRIP8b is a brain-specific cytosolic scaffold protein capable of interacting with the C-terminus domain of HCN channels. TRIP8b has several N-terminal splice variants that determine the heterogeneous subcellular localization of HCN channels. TRIP8b global knock-out (KO) mice display alterations in thalamic excitability, partly resembling the epileptic phenotype of HCN2 KO, but without cardiac phenotype. In the hippocampus, TRIP8b deletion causes overall reduction of HCN1 expression levels with disruption of the distinctive somatodendritic expression gradient (Lewis, Estep, & Chetkovich, 2010). HCN channel topology and basic current properties are illustrated in Figure 1.

Hyperpolarization-Activated Cyclic Nucleotide-Gated ChannelsClick to view larger

Figure 1. A. HCN channels are present at cell surface as homo- or hetero-tetrameric structures. Individual subunits are formed by 6 transmembrane domains (1–6, from left to right). Voltage-sensing domain is located in S4, the pore-loop between S5 and S6, while most regulatory domains are found in the intracellular tail at C-terminus. B. Basic electrophysiological properties of HCN current. Negative voltage steps elicit an instantaneous current (Iinst), followed by a main, voltage-dependent, slow component. Gating kinetics (τ) are related to magnitude of voltage pulse. C. Normalized current-voltage curve highlighting V1/2 and VREV of HCN current in relation to critical membrane potential values such as resting (VM) and AP threshold (VTHR). D. Intracellular cAMP concentration modulates the availability of HCN current by shifting the activation curve towards a more negative (-cAMP) or more positive (+cAMP) range of potentials. In addition, cAMP accelerates gating kinetics (right hand).

Properties and Function of HCN Channels in the Nervous System

This section will overview the contribution of HCN channels to critical neuronal functions such as the regulation of resting membrane potential and intrinsic excitability, generation of rhythm, synaptic excitability. The influence of HCN current on the normal operation of neurons is determined by subunit stoichiometry, interplay with other membrane conductances and action of modulatory mechanisms.

Neurophysiological Implications of the Unique Biophysical Properties of HCN Channels

A large body of literature is available on the role of HCN channels in nerve cells. The basic aspects of HCN channels’ physiology in the CNS have been the topic of some recent, exhaustive reviews and will not be treated here in detail (He et al., 2014; Shah, 2016). However, in order to fully comprehend the relevance of HCN current in the physiology and pathology of the nervous system, it is necessary to review its basic biophysical properties proper, and in the context of neuronal and network excitability. In essence, these can be narrowed down to the following hallmarks: permeability to both Na+ and K+ (a); reversal potential (VREV) lying close to action potential (AP) threshold (b) and at the bottom of the activation curve (c); slow gating kinetics (d). Because of properties a and b, HCN channels mediate a tonic inward current, which, at potentials below ~50 mV, depolarizes the membrane, promoting intrinsic AP firing. In the same conditions, however, HCN current diminishes membrane resistance, thus opposing synaptically generated depolarizations. This dual action accounts for the complex consequences of HCN channels’ mutations for neuronal excitability reported in cellular and animal models. Property c implies that HCN current drives the membrane potential towards values where HCN channels are closed. In other words, HCN current is self-limiting, thus exerting a stabilizing force at subthreshold potentials that actively opposes both downward and upward shifts. Finally, property d confers a preference for low-frequency inputs to the membrane potential–stabilizing effect of HCN current. In other words, HCN current will effectively oppose, and thus filter out, slow (or low-frequency) voltage oscillations, while fast (or high-frequency) oscillations will escape filtering. In electronics, this process is called “high-pass filtering,” and in neurons, HCN current serves as a high-pass filter.

The concepts outlined here concern general and constitutive aspects of neuronal HCN channels. The resulting impact on the electrical activity of the neuron varies greatly, based on membrane expression levels and relative HCN subunit prevalence. Furthermore, the influence of many heterogeneous factors, including ionic species, second messengers, membrane lipids, and auxiliary/scaffolding proteins, shapes the activity of HCN channels and their influence on intrinsic excitability and synaptic transmission. Because of these regulatory mechanisms, HCN channels may serve as final effectors of important signaling pathways initiated by circulating or synaptically released chemical messengers. The main contributions of HCN current to resting membrane potential, AP firing frequency, and synaptic excitability are summarized in Figure 2.

Hyperpolarization-Activated Cyclic Nucleotide-Gated ChannelsClick to view larger

Figure 2. A. Stabilizing action exerted by HCN current on membrane potential following voltage perturbations. Top, injection of positive current +I causes fast depolarization of the membrane potential. However, deactivation of HCN current causes a rebound effect towards more negative potential V1. Symmetrically, activation of HCN current promotes a positive rebound of membrane potential V2 from initial hyperpolarization following injection of negative current –I (bottom). B. HCN current at somatic level (a) promotes firing in autonomous spiking neurons. Removal of HCN current hyperpolarizes the neuron and reduces firing rate. At somatodendritic level (b), HCN current accelerates the decay kinetics of EPSPs, thus promoting the ability of the neuron to resolve individual synaptic events (left). After removal of HCN current (right), temporal precision is impaired and summation of EPSPs occurs.

Regulation of Resting Membrane Potential and Intrinsic Excitability

The influence exerted by HCN current on the intrinsic excitability of neurons is complex. At –65 mV, a fraction of HCN channels are constitutively open (Kase & Imoto, 2012), sustaining a persistent Na+ inflow that keeps the membrane relatively depolarized and opposes hyperpolarizing inputs. Vice-versa, depolarization causes channel deactivation, and the resulting cessation of the inward current has a hyperpolarizing effect. As a result, HCN current acts as a membrane potential stabilizer in the subthreshold range. Although blockage of HCN current hyperpolarizes the neuron and thereby reduces its intrinsic excitability, for the same reason, the response to depolarizing synaptic inputs becomes potentiated. This dual action exerted by HCN current on overall neuronal excitability has been clearly established in many neuronal types and brain areas, including the thalamus, the hippocampus, and the cerebellum, and it has important repercussions on higher-order brain functions such as learning and memory, control of circadian rhythm, and sleep–wakefulness state (Gasparini & DiFrancesco, 1997; Maccaferri, Mangoni, Lazzari, & DiFrancesco, 1993; McCormick & Pape, 1990; Nolan, Dudman, Dodson, & Santoro, 2007).

In the axon initial segment of medial superior olive principal neurons, HCN1 current reduces spike probability by elevating the firing threshold. Serotonin 5-HT1A receptor engagement shifts HCN channel V1/2 in the hyperpolarizing direction, thereby relieving the brake on spike probability (Ko, Rasband, Meseguer, Kramer, & Golding, 2016). Serotonin-dependent modulation of HCN current is also involved in the control of respiratory rhythm in the retrotrapezoid nucleus (RTN). Here, activation of the 5-HT7 serotonin receptor causes a cAMP-dependent depolarizing shift in V1/2, increasing the firing rate of RTN neurons in vitro and the frequency of respiratory rhythm in vivo (Hawkins et al., 2015).

The ability of HCN channels to set the membrane potential plays an important role in the physiology of the retina. Early in situ hybridization studies have shown high levels of HCN1 in retinal photoreceptors (Moosmang et al., 2001). Later, functional studies have uncovered an important role exerted by HCN current in the regulation of the electrical events following phototransduction. In the outer segment of rod photoreceptors, a light flash shuts the inward, depolarizing (“dark”) current mediated by cyclic nucleotide-gated (CNG) channels, leading to hyperpolarization of the membrane. This causes a strong reduction in synaptic release at the rod–bipolar cell synapse. Hyperpolarization also activates HCN1 current located in the membrane of the inner segment, in turn promoting the rebound of membrane potential back to the resting, depolarized state. Thus, synaptic activity recovers, reducing rod output and preventing saturation of the downstream retinal network (Seeliger et al., 2011). This regulatory action on photoreceptor function is at the basis of the side effects produced by ivabradine, an HCN channel blocker with medical applications in cardiovascular pathologies (Cangiano, Gargini, Della Santina, Demontis, & Cervetto, 2007).

Rhythmogenesis

In a functional interplay with other membrane ionic mechanisms, the activation-deactivation cycle of HCN channels helps setting the pace of subthreshold membrane oscillations, which determines AP discharge rate and, ultimately, neuronal output. HCN current cooperates with a persistent, subthreshold Na+ current in setting a 4–10 Hz (“theta-like”) rhythm in pyramidal and stellate cells of the entorhinal cortex (EC) layer II (Alonso & Llinas, 1989). Both cell types in this area are critically involved during spatial navigation tasks. These cells are named “grid cells,” because they show periodic, hexagon-shaped firing locations that scale up progressively along the dorsal–ventral axis of the medial entorhinal cortex (Moser, Rowland, & Moser, 2015). In this structure, relative HCN1/HCN2 expression ratio and oscillation frequency decrease following a dorsal–ventral gradient (Notomi & Shigemoto, 2004). Such a dorsal–ventral frequency gradient correlates with the different 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 theta modulation, are expanded (Giocomo & Hasselmo, 2009; Giocomo et al., 2011).

The firing pattern of thalamocortical neurons is governed by HCN current, here mediated by HCN2 channels. Adrenergic and serotonergic stimulation causes cAMP-mediated modulation of HCN current. During wakefulness and REM sleep, thalamocortical neurons fire in a single-spike, or “transmission,” mode. In this state, information is effectively transmitted to the cortex. During certain physiological and pathological states, such as non-REM sleep and absence seizures, thalamocortical neurons fire in “burst” mode, and the transfer of signals to the cortex is thought to be less effective (McCormick & Pape, 1990). In agreement, global HCN2 deletion causes absence epilepsy with typical “spike-and-wave” discharges in EEG recordings (Ludwig et al., 2003).

A similar role has been described for HCN3 in leaflet neurons of the intergeniculate nucleus, a retino-recipient thalamic structure implicated in orchestrating the circadian rhythm. Here, HCN3-mediated current drives low-threshold burst firing and spontaneous oscillations and is bi-directionally modulated by PIP2. Depletion of PIP2 or pharmacological block of HCN current results in profound inhibition of excitability (Ying et al., 2011).

Synaptic Excitability and Plasticity

Due to the properties described previously, HCN current shapes the amplitude and temporal dynamics of synaptic potentials. This deeply affects the integrative properties of the somatodendritic compartment and the ability of the neuron to express different forms of synaptic plasticity.

Functional HCN channels are strongly expressed along the dendritic arborisation of neocortical and hippocampal neurons with an increasing soma-to-dendrites expression gradient (Bender et al., 2001; Harnett, Magee, & Williams, 2015; Lorincz, Notomi, Tamas, Shigemoto, & Nusser, 2002). Here, HCN current constitutes a shunt conductance accelerating the decay of excitatory post-synaptic potentials (EPSPs). As a result, temporal summation during EPSP sequences is reduced, and the ability of the neuron to resolve individual inputs at the somatic level is enhanced. This function, first discovered and characterized in pyramidal neurons of the hippocampal CA1 region and somatosensory cortex (Berger & Luscher, 2003; Magee, 1998, 1999; Williams & Stuart, 2000), has since been described in subcortical structures (Carbone, Costa, Provensi, Mannaioni, & Masi, 2017; Engel & Seutin, 2015; Masi et al., 2015; Ying et al., 2007). In the dendritic compartment of CA1 pyramidal neurons, the shunting effect exerted by HCN current limits voltage-dependent Ca2+ entry, with important consequences for synaptic excitability (Tsay, Dudman, & Siegelbaum, 2007). The non-uniform distribution of HCN current is also responsible for a phenomenon termed “site independence” of synaptic potentials, whereby the decay time of distally generated EPSPs is similar to that of proximally generated EPSPs (Williams & Stuart, 2000).

HCN current accelerates the decay time of both forward- and back-propagating depolarization waves, thus increasing the precision with which the dendrite detects the coincidence of salient electrical events. These include the coincidence of EPSPs and APs, an event leading to synaptic potentiation and, presumably, memory formation (Pavlov, Scimemi, Savtchenko, Kullmann, & Walker, 2011). In several brain areas, HCN function constrains long-term potentiation, thus affecting the associated cognitive functions. In keeping with brain-slice physiology experiments, transgenic animals have provided evidence of a role for HCN current in cognition. Indeed, HCN1 KO mice show improved hippocampal-dependent learning and memory performance. In these mice, proximal CA3–CA1 synapses function normally, whereas distal EC Layer III-CA1 contacts are potentiated, as predicted by stronger expression of HCN channels in this compartment in normal conditions (Nolan et al., 2004).

It has been suggested that HCN current affects higher cortical functions such as cognition, movement planning, and execution by setting the strength of connectivity within local microcircuits. HCN current is abundantly expressed in dorsal–lateral prefrontal cortex (dlPFC) layer III neurons, a population crucially involved in spatial working memory. Working memory is a short-term memory form engaged during the execution of complex cognitive tasks that requires the transient activation of intracortical microcircuits. In dlPFC layer III neurons, HCN channels are modulated by the opposite action of α2-adrenergic (α2-AR) and type-1 dopaminergic receptor (D1R) stimulation of cAMP signaling. When α2-AR stimulation inhibits the cAMP-HCN channel pathway, neurons are more excitable and more tightly connected to recurring microcircuit activity, thus performance is optimal. In contrast, negative affective states induced by stress cause a D1R-mediated enhancement of the cAMP-HCN pathway, leading to a functional disconnection from the local network and impairment in working memory performance (Arnsten & Jin, 2014; M. Wang et al., 2007).

In the primary motor cortex of the mouse, HCN expression is specifically elevated in corticospinal neurons of layer V. HCN current confers to 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 and thus the transmission efficacy of motor inputs. Also in this context, α2-AR stimulation modulates HCN function (Sheets et al., 2011).

Resonance Properties

Dendritic HCN current confers to the neuron specific “resonance” properties; i.e., the ability to respond preferentially to inputs at a certain frequency, and determines the extent to which the neuron takes part to synchronous network activity. Due to its slow gating kinetics, HCN current has high-pass filtering properties, which, combined to the low-pass filtering action exerted by the membrane time constant, contribute to impart a resonance frequency in the 1–10 Hz range to sensorimotor neurons (Hutcheon, Miura, & Puil, 1996). In the hippocampus, the power of theta oscillation and the strength of the perforant path-CA1 pyramidal neuron synapses, both influenced by HCN1 function, are required for hippocampal-dependent learning and memory storage (Nolan et al., 2004). The ability to resonate in an HCN-dependent manner has been reported for other areas (Borel, Guadagna, Jang, Kwag, & Paulsen, 2013; Ulrich, 2002; Wang et al., 2006; Xue et al., 2012). Although the role of HCN current in oscillating activity and resonance properties has been clearly established at single cell or local network level, its actual significance for higher-order functions remains undetermined.

Neurotransmitter Release

HCN current at synaptic terminals has been reported to control the efficacy of vesicle release. HCN1 and HCN2 isoforms are present at GABAergic terminals of pallidal axon collaterals (Boyes, Bolam, Shigemoto, & Stanford, 2007) and at glutamatergic terminals making contacts onto EC layer III pyramidal neurons (Huang et al., 2011). In both cases, pharmacological or genetic KO of HCN function leads to elevation of spontaneous synaptic release. Huang et al. (2011) have suggested that HCN current exerts this inhibitory action by setting the potential to a value where N-type CaV3.2 Ca2+ channels are in a partially inactivated state. HCN deletion hyperpolarizes the terminal and removes Ca2+ channel inactivation. Thus, when the AP invades the terminal, a larger amount of Ca2+ flows in through CaV3.2 Ca2+ channels. A follow-up study by the same authors shows that HCN1 channels restrict the rate of exocytosis from a subset of cortical synaptic terminals within the EC and constrain spontaneous as well as evoked release (Huang, Li, Aguado, Lujan, & Shah, 2017).

HCN Channels in Pathological States of the Central Nervous System

To date, mutations in HCN genes have been clearly reported as primary causes of rare epilepsy forms. Furthermore, multiple lines of evidence suggest that acquired HCN current defects are associated to other pathological states as diverse as Parkinson's disease, neuropathic pain, substance use disorder. This last section will present some remarkable examples of HCN-associated pathologies and the mechanisms linking HCN abnormalities to disease.

Epilepsy

Epilepsy is the neurological disorder with strongest correlation with genetic or epigenetic alterations in HCN channels. In most HCN-linked epilepsies, increased neuronal excitability is associated with loss-of-function mutations in HCN1 and HCN2 isoforms (DiFrancesco & DiFrancesco, 2015; Nava et al., 2014). HCN1 null mice show increased susceptibility to kaynic acid-induced seizures. In addition, these mice show increased synaptic excitability of cortical neurons, in spite of the more negative membrane potential (Huang, Walker, & Shah, 2009). In contrast, HCN2 KO mice show absence seizures (Ludwig et al., 2003). Evidence of the role of HCN mutations in the pathophysiology of epilepsy are corroborated by genetic studies on human subjects. Next generation sequencing analysis on epileptic individuals point to a strong association between single-nucleotide mutations in HCN2 gene, leading to a loss-of-function phenotype, and idiopathic generalized epilepsy (DiFrancesco et al., 2011; Tang, Sander, Craven, Hempelmann, & Escayg, 2008). Another study, however, reported a significant association between febrile seizures and a single-point, gain-of-function mutation in HCN2 (Dibbens et al., 2010).

HCN loss of function phenotype may also result as a consequence of changes in the expression of auxiliary proteins, such as TRIP8b, that normally control channel surface expression or correct subcellular targeting. TRIP8b KO mice show spontaneous spike-wave discharges on EEG, the electrographic hallmark of absence seizures, which resemble the phenotype of global HCN2 deletion. At cellular and molecular levels, HCN2 channels are strongly reduced in thalamic-projecting cortical layer 5b neurons and thalamic relay neurons, but unaltered in inhibitory neurons of the reticular thalamic nucleus (Heuermann et al., 2016).

Interestingly, there is also evidence of the opposite relationship between HCN channels’ expression and epilepsy, as a number of studies have reported remodeling of HCN1 and 2 expression following experimental seizure induction. In pyramidal neurons of the hippocampus, HCN1 expression goes down, while HCN2 expression goes up, after febrile seizures (Brewster et al., 2002). Abnormal HCN channels’ expression has been detected in autopsy specimens from individuals suffering from temporal lobe epilepsy (Bender et al., 2003). The significance of such neuroadaptative change triggered by epileptic states and involving HCN expression remains to be clarified.

In summary, a relatively large number of preclinical and clinical studies support the evidence linking HCN channelopathy to epilepsy. However, they do not unambiguously point to a straightforward mechanistic link between HCN alteration and clinical manifestations. For these reasons, the exploitation of HCN channels as potential targets for antiepileptic medications requires further preclinical advancements.

Parkinson’s Disease

Parkinson’s disease (PD) is caused by massive degeneration of nigrostriatal dopaminergic (DA) neurons. Substantia nigra pars compacta (SNc) DA neurons express high levels of HCN2 and 4 (Dufour et al., 2014; Neu et al., 2002). Preclinical evidence has highlighted a correlative link between HCN loss of function and selective vulnerability of SNc DA neurons in PD models linked to mitochondrial failure. The Mitopark mouse, a transgenic PD model based on a DA neuron-targeted mitochondrial defect leading to selective nigrostriatal degeneration and PD-like phenotype, shows reduced HCN current density in SNc DA neurons (Branch et al., 2016; Good et al., 2011). Of note, HCN downregulation is an early event preceding nigrostriatal degeneration (Good et al., 2011). With brain slice patch clamp recordings, it has been reported that 1-Methyl-4-phenylpyridinium (MPP+), a mitochondrial toxin able to produce selective nigrostriatal degeneration, causes HCN current inhibition in SNc DA neurons. MPP+-induced HCN block leads to increased synaptic excitability (Masi, Narducci, Landucci, Moroni, & Mannaioni, 2013). In addition, in agreement with respective HCN expression levels, the effect of HCN block on synaptic excitability and Ca2+ entry is significantly greater in SNc compared to other DA subsets (Masi et al., 2015) and pharmacological blockade of HCN channels in vivo causes selective nigrostriatal DA degeneration (Carbone et al., 2017). These findings point to a significant correlation between altered HCN current and nigrostriatal vulnerability, with potential implications for the pathogenesis of PD and the development of disease-modifying medications.

Neuropathy

The precise role of HCN current in physiological somatosensory transmission is imperfectly understood. Indeed, healthy sensory neurons seem largely unaffected by pharmacological HCN blockade. Consistently, safety studies with HCN blocker ivabradine did not report any adverse reactions such as dysesthesias and paresthesias (Herrmann, Schnorr, & Ludwig, 2015). Therefore, it appears that HCN current acquires major functional relevance in pathological states. Multiple studies have suggested that HCN channels undergo changes in expression or function following neuronal damage or in experimental models of inflammation (Acosta et al., 2012; Chaplan et al., 2003; Jiang, Sun, Tu, & Wan, 2008; Papp, Holló, & Antal, 2010; Schnorr et al., 2014; Weng, Smith, Sathish, & Djouhri, 2012). These reports point to an association between HCN current upregulation and pathological pain conditions such as allodynia and hyperalgesia (Herrmann et al., 2015). HCN current upregulation seems to depend on multiple factors such as increased expression or functional modification (Descoeur et al., 2011; Papp et al., 2010; Resta et al., 2018; Schnorr et al., 2014), PIP2-mediated modulation (Pian, Bucchi, Decostanzo, Robinson, & Siegelbaum, 2007), protein kinase A (PKA) over-activation (Cheng & Zhou, 2013), and elevated intracellular levels of cAMP triggered by inflammatory mediators (Jafri & Weinreich, 1998; Momin, Cadiou, Mason, & McNaughton, 2008; Resta et al., 2016). Some of the works cited here suggest that targeting pathological HCN current alterations in sensory neurons with selective blockers devoid of activity on cardiac isoforms can be seen as a therapeutic avenue in the treatment of pathological pain.

Retinal Pathology

The first evidence of an HCN-related pathology in the retina was the discovery that prolonged treatment with ivabradine, an HCN inhibitor used in the treatment of angina pectoris and chronic heart failure, causes visual disturbances described as sensations of enhanced brightness, called “phosphenes” (Cervetto, Demontis, & Gargini, 2007). These symptoms revert following drug discontinuation and do not lead to modification of overall retinal morphology or HCN expression in animals (Della Santina et al., 2010).

Other reports have addressed the role of HCN in the pathophysiology of degenerative diseases of the retina. In one study, the absence of HCN1 in cyclic nucleotide-gated channel β1 (CNGB1)-KO mice exacerbated photoreceptor degeneration, thus suggesting that intact HCN1 functions may have a pro-survival role in this cell type (Schon et al., 2016).

Addiction

Natural and artificial rewarding stimuli cause activation of the ventral tegmental area (VTA) DA system. These neurons show slow, autonomous, regular firing, which is transiently interrupted by bursts or pauses during reward or punishment (Schultz, 2016). The antagonism of the excitatory action exerted by psychostimulants on the reward DA system is considered the most promising strategy to develop pharmacological therapies to treat addiction. In this respect, HCN current contributes to shape the firing pattern of VTA DA both in physiological conditions and in the presence of substances of abuse such as ethanol (Appel, Liu, McElvain, & Brodie, 2003; Okamoto, Harnett, & Morikawa, 2006), cocaine (Arencibia-Albite, Vazquez-Torres, & Jimenez-Rivera, 2017; Goertz et al., 2015), and methamphetamine (Gonzalez et al., 2016). The development of subunit- or cell type–specific HCN blockers or modulators may control the rewarding actions of these substances (Novella Romanelli et al., 2016). However, there is also convincing evidence that one of the long-term effects of drug abuse is the induction of a negative hypodopaminergic state that substance intake can transiently compensate for in drug addicts (Diana, 2011; Koob & Volkow, 2010). In this respect, HCN current density and spontaneous firing frequency of VTA DA neurons from mice previously exposed to a voluntary drinking paradigm is inversely correlated to ethanol intake (Juarez et al., 2017). Prospectively, restoring the normal tone of the DA system in drug abusers through functional upregulation of HCN current may be effective in relieving craving or preventing relapse. To achieve this aim, however, it will be necessary to develop compounds able to enhance HCN current with subunit and neuronal type specificity (Novella Romanelli et al., 2016).

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