Hyperpolarization-activated Cyclic Nucleotide-gated Isoforms Research Articles

Exploring characteristic features for effective HCN1 channel inhibition using integrated analytical approaches: 3D QSAR, molecular docking, homology modelling, ADME and molecular dynamics.

Neuropathic pain (NP) is characterized by hyperalgesia, allodynia, and spontaneous pain. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel involved in neuronal hyperexcitability, has emerged as an important target for the drug development of NP. HCN channels exist in four different isoforms, where HCN1 is majorly expressed in dorsal root ganglion having an imperative role in NP pathophysiology. A specific HCN1 channel inhibitor will hold the better potential to treat NP without disturbing the physiological roles of other HCN isoforms. The main objective is to identify and analyze the chemical properties of scaffolds with higher HCN1 channel specificity. The 3D-QSAR studies highlight the hydrophobic & hydrogen bond donor groups enhance specificity towards the HCN1 channel. Further, the molecular interaction of the scaffolds with the HCN1 pore was studied by generating an open-pore model of the HCN1 channel using homology modelling and then docking the molecules with it. In addition, the important residues involved in the interaction between HCN1 pore and scaffolds were also identified. Moreover, ADME predictions revealed that compounds had good oral bioavailability and solubility characteristics. Subsequently, molecular dynamics simulation studies revealed the better stability of the lead molecules A7 and A9 during interactions and ascertained them as potential drug candidates. Cumulative studies provided the important structural features for enhancing HCN1 channel-specific inhibition, paving the way to design and develop novel specific HCN1 channel inhibitors.

HCN1 hyperpolarization-activated cyclic nucleotide–gated channels enhance evoked GABA release from parvalbumin-positive interneurons

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the cationic Ih current in neurons and regulate the excitability of neuronal networks. The function of HCN channels depends, in part, on their subcellular localization. Of the four HCN isoforms (HCN1-4), HCN1 is strongly expressed in the dendrites of pyramidal neurons (PNs) in hippocampal area CA1 but also in presynaptic terminals of parvalbumin-positive interneurons (PV+ INs), which provide strong inhibitory control over hippocampal activity. Yet, little is known about how HCN1 channels in these cells regulate the evoked release of the inhibitory transmitter GABA from their axon terminals. Here, we used genetic, optogenetic, electrophysiological, and imaging techniques to investigate how the electrophysiological properties of PV+ INs are regulated by HCN1, including how HCN1 activity at presynaptic terminals regulates the release of GABA onto PNs in CA1. We found that application of HCN1 pharmacological blockers reduced the amplitude of the inhibitory postsynaptic potential recorded from CA1 PNs in response to selective optogenetic stimulation of PV+ INs. Homozygous HCN1 knockout mice also show reduced IPSCs in postsynaptic cells. Finally, two-photon imaging using genetically encoded fluorescent calcium indicators revealed that HCN1 blockers reduced the probability that an extracellular electrical stimulating pulse evoked a Ca2+ response in individual PV+ IN presynaptic boutons. Taken together, our results show that HCN1 channels in the axon terminals of PV+ interneurons facilitate GABAergic transmission in the hippocampal CA1 region.

Comparison of cyclic AMP binding activity within HCN channel subfamily using atomistic molecular dynamics simulations of isolated cyclic nucleotide binding domains.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a subfamily of homotetrameric, voltage gated potassium/sodium channels whose activity is modulated by cyclic AMP (cAMP). The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region which is induced by the binding of cAMP is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that affects conductance. Although various static structures of the four HCN isoforms with and without bound cyclic nucleotide have been solved and deposited on the Protein Data Bank, there is still a need to understand the precise binding interactions which lead to different sensitivities and selectivities for cyclic nucleotides within the subfamily. Using atomistic molecular dynamics, we have simulated the isolated CNBD of HCN1-4 bound to cAMP and have used both equilibrium simulations and alchemical free energy perturbation to characterize and compare the binding activities of the different isoforms. These simulations highlight the differential cyclic nucleotide binding behavior between HCN channels.

Positive chronotropic action of HCN channel antagonism in human collecting lymphatic vessels.

Spontaneous action potentials precede phasic contractile activity in human collecting lymphatic vessels. In this study, we investigated the expression of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels in human collecting lymphatics and by pharmacological inhibition ex vivo tested their potential role in controlling contractile function. Spontaneous and agonist‐evoked tension changes of isolated thoracic duct and mesenteric lymphatic vessels—obtained from surgical patients with informed consent—were investigated by isometric myography, and ivabradine, ZD7288 or cesium were used to inhibit HCN. Analysis of HCN isoforms by RT‐PCR and immunofluorescence revealed HCN2 to be the predominantly expressed mRNA isoform in human thoracic duct and mesenteric lymphatic vessels and HCN2‐immunoreactivity confirmed protein expression in both vessel types. However, in functional experiments ex vivo the HCN inhibitors ivabradine, ZD7288, and cesium failed to lower contraction frequency: conversely, all three antagonists induced a positive chronotropic effect with concurrent negative inotropic action, though these effects first occurred at concentrations regarded as supramaximal for HCN inhibition. Based on these results, we conclude that human collecting vessels express HCN channel proteins but under the ex vivo experimental conditions described here HCN channels have little involvement in regulating contraction frequency in human collecting lymphatic vessels. Furthermore, HCN antagonists can produce concentration‐dependent positive chronotropic and negative inotropic effects, which are apparently unrelated to HCN antagonism.

Long-term If current blockade increases right atrial mRNA expression of HCN4, encoding proteins for If

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS - UEFISCDI, within PNCDI III Background Numerous long-term pharmacologic manipulations of cardiac ion channels have shown potential to modify the expression of genes encoding proteins for those channels. Purpose We aimed to investigate whether long-term pharmacologic inhibition of the hyperpolarization-activated inward current (If) using ivabradine affects the right atrial expression of genes encoding for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, responsible for If. Methods The right atrial mRNA expression of HCN1, HCN2, and HCN4, and of the neuron-specific HCN3 isoform was quantified in 6 control (Control) and 12 ivabradine-treated (IVA; 10 mg/kg, 4 weeks) adult male Wistar rats. The expression levels of the target genes were normalized with GAPDH housekeeping gene levels and compared between the two groups. Results Right atrial HCN1 and HCN2 expression levels were both similar (both p >0.05) between the IVA and the Control rats. There was also no significant difference in the right atrial expression of the neuron-specific HCN3 isoform between the two groups (p = 0.22). However, the right atrial expression of HCN4, the most highly expressed HCN isoform in the sinus node, was significantly higher in the ivabradine-treated compared to the non-treated rats (p = 0.02). Conclusions Our study shows that chronic If blockade using ivabradine significantly up-regulates right atrial HCN4 expression. Although the examined samples contained both nodal and non-nodal tissue, since HCN4 is highly expressed in the nodal pacemaker cells and only sparsely in the remaining atrial myocardium, it is likely that the HCN4 changes observed in our study reflect sinus node alterations. HCN4 and a consequent If up-regulation could render the sinus node less sensitive to acute vagal inputs and protect against excessive vagal-induced bradycardia. Further studies will have to evaluate this hypothesis.

Decreased Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Activity in a Rat Model of Absence Epilepsy and the Effect of ZD7288, an Ih Inhibitor, on the Spike-and-Wave Discharges

Introduction: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents of Ih and absence epilepsy seizures are associated, but studies reveal differential results. Objective: In our study, we aimed to investigate the role of the HCN channels on the expression of spike-and-wave discharges (SWDs) using the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Methods: HCN isoform levels from isolated brains of both naïve nonepileptic Wistar and GAERS groups were evaluated by enzyme-linked immunosorbent assay. ZD7288, an Ih inhibitor as well as an HCN channel antagonist, was administered intracerebroventricularly to the adult GAERS groups, and to evaluate their SWD activities, electroencephalography was recorded. The effect of ZD7288 on the cumulative total duration and number of SWDs and the mean duration of each SWD complex was evaluated. Results: The HCN2 levels in the cortex and hippocampus of the GAERS group were lower compared to the naïve nonepileptic Wistar group (p < 0.05). ZD7288 increased the number of SWDs at the 20th and 120th min with the highest administered dose of 7 μg (p < 0.05). Conclusion: The Ih inhibitor ZD7288 increased the number of SWDs in a genetic absence epilepsy rat model, although this increase may not be significant due to the inconsistent time-dependent effects. In GAERS, the cortical and hippocampal HCN2 channel levels were significantly lower compared to the control group. Further studies are needed with higher doses of ZD7288 to determine if the effects will increase drastically.

The HCN channel as a pharmacological target: Why, where, and how to block it

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, expressed in a variety of cell types and in all tissues, control excitation and rhythm. Since their discovery in neurons and cardiac pacemaker cells, they attracted the attention of medicinal chemistry and pharmacology as novel targets to shape (patho)physiological mechanisms. To date, ivabradine represents the first-in-class drug as specific bradycardic agent in cardiac diseases; however, new applications are emerging in parallel with the demonstration of the involvement of different HCN isoforms in central and peripheral nervous system. Hence, the possibility to target specific isoforms represents an attractive development in this field; indeed, HCN1, HCN2 or HCN4 specific blockers have shown promising features in vitro and in vivo, with remarkable pharmacological differences likely depending on the diverse functional role and tissue distribution. Here, we show a recently developed compound with high potency as HCN2–HCN4 blocker; because of its unique profile, this compound may deserve further investigation.

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod, Gadus morhua (Linnaeus, 1758).

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels as Drug Targets for Neurological Disorders.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that critically modulate neuronal activity. Four HCN subunits (HCN1-4) have been cloned, each having a unique expression profile and distinctive effects on neuronal excitability within the brain. Consistent with this, the expression and function of these subunits are altered in diverse ways in neurological disorders. Here, we review current knowledge on the structure and distribution of the individual HCN channel isoforms, their effects on neuronal activity under physiological conditions, and how their expression and function are altered in neurological disorders, particularly epilepsy, neuropathic pain, and affective disorders. We discuss the suitability of HCN channels as therapeutic targets and how drugs might be strategically designed to specifically act on particular isoforms. We conclude that medicines that target individual HCN isoforms and/or their auxiliary subunit, TRIP8b, may provide valuable means of treating distinct neurological conditions.

The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

Ivabradine for the Treatment of Cardiovascular Diseases.

Higher heart rate (HR) is independently related to worse outcomes in various cardiac diseases, including hypertension, coronary artery disease, and heart failure (HF). HR is determined by the pacemaker activity of cells within the sinoatrial node. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 4 channel, one of 4 HCN isoforms, generates the If current and plays an important role in the regulation of pacemaker activity in the sinoatrial node. Ivabradine is a novel and only available HCN inhibitor, which can reduce HR and has been approved for stable angina and chronic HF in many countries other than Japan. In this review, we summarize the current knowledge of the HCN4 channel and ivabradine, including the function of HCN4 in cardiac pacemaking, the mechanism of action of If inhibition by ivabradine, and the pharmacological and clinical effects of ivabradine in cardiac diseases as HF, coronary artery disease, and atrial fibrillation.

Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain

A prominent role of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels has been suggested based on their expression and (dys)function in dorsal root ganglion (DRG) neurons, being likely involved in peripheral nociception. Using HCN blockers as antinociceptive drugs is prevented by the widespread distribution of these channels. However, tissue-specific expression of HCN isoforms varies significantly, HCN1 and HCN2 being considered as major players in DRG excitability. We characterized the pharmacological effect of a novel compound, MEL55A, able to block selectively HCN1/HCN2 isoforms, on DRG neuron excitability in-vitro and for its antiallodynic properties in-vivo. HEK293 cells expressing HCN1, HCN2, or HCN4 isoforms were used to verify drug selectivity. The pharmacological profile of MEL55A was tested on mouse DRG neurons by patch-clamp recordings, and in-vivo in oxaliplatin-induced neuropathy by means of thermal hypersensitivity. Results were compared to the non-isoform-selective drug, ivabradine. MEL55A showed a marked preference toward HCN1 and HCN2 isoforms expressed in HEK293, with respect to HCN4. In cultured DRG, MEL55A reduced Ih amplitude, both in basic conditions and after stimulation by forskolin, and cell excitability, its effect being quantitatively similar to that observed with ivabradine. MEL55A was able to relieve chemotherapy-induced neuropathic pain. In conclusion, selective blockade of HCN1/HCN2 channels, over HCN4 isoform, was able to modulate electrophysiological properties of DRG neurons similarly to that reported for classical Ih blockers, ivabradine, resulting in a pain-relieving activity. The availability of small molecules with selectivity toward HCN channel isoforms involved in nociception might represent a safe and effective strategy against chronic pain.

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.

Increased HCN Channel Activity in the Gasserian Ganglion Contributes to Trigeminal Neuropathic Pain

Orofacial neuropathic pain caused by trigeminal nerve injury is a debilitating condition with limited therapeutic options. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are involved in the development and maintenance of chronic pain. However, the effect of HCN channel activity in the Gasserian ganglion on trigeminal neuropathic pain has not been examined. We evaluated nociceptive behaviors after microinjection of the HCN channel blockers ZD7288 or ivabradine into the Gasserian ganglion in rats with trigeminal nerve injury. Both blockers dose-dependently ameliorated evoked and spontaneous nociceptive behavior in rats with trigeminal neuropathic pain. Moreover, the clinically available HCN channel blocker ivabradine showed a prolonged antinociceptive effect. In the Gasserian ganglion, HCN1 and HCN2 are major HCN isoforms. After trigeminal nerve injury, the counts of HCN1 as well as HCN2 immuno-positive punctae were increased in the ipsilateral Gasserian ganglions. These results indicate that the increased HCN channel activity in the Gasserian ganglion directly contributes to neuropathic pain resulting from trigeminal nerve injury. PerspectiveTrigeminal nerve damage-induced orofacial pain is severe and more resistant to standard pharmacological treatment than other types of neuropathic pain. Our study suggests that targeting HCN channel activities in the Gasserian ganglion may provide an alternative treatment of trigeminal neuropathy including trigeminal neuralgia.

Kir2 inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity.

Spontaneous rhythmic action potential or pacemaking activity of pacemaker cells controls rhythmic signaling such as heartbeat. The mechanism underlying the origin of pacemaking activity is not well understood. In this study, we created human embryonic kidney (HEK) 293 cells that show pacemaking activity through heterologous expression of strong inward rectifier K+ subfamily 2 isoform 1 (Kir2.1) channels, hyperpolarization-activated cyclic nucleotide-gated isoform 2 (HCN2) nonselective cation channels, and voltage-gated Na+ subfamily 1 isoform 5 or Ca2+ subfamily 3 isoform 1 (Nav1.5 or Cav3.1) channels. A range of relative levels of Kir2.1 and HCN2 currents dynamically counterbalance, generating spontaneous rhythmic oscillation of resting membrane potential between -64 and -34 mV and determining oscillation rates. Each oscillation cycle begins with an autodepolarization phase, which slowly proceeds to the threshold potential that activates Nav1.5 or Cav3.1 channels and triggers action potential, causing engineered HEK293 cells to exhibit pacemaking activity at a rate of ≤67 beats/min. Engineered HEK293 cells with Kir2.1 and either HCN3 or HCN4 also show the oscillation. Engineered HEK293 cells expressing HCN2 and other Kir2 channels, which lack Kir2.1-like complete inward rectification, do not show the oscillation. Therefore, Kir2.1-like inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate spontaneous rhythmic action potential and form an origin of pacemaking activity; Kir2 and HCN channels play essential roles in pacemaking activity.-Chen, K., Zuo, D., Wang, S.-Y. Chen, H. Kir2 inward rectification-controlled precise and dynamic balances between Kir2 and HCN currents initiate pacemaking activity.

Selective HCN1 block as a strategy to control oxaliplatin-induced neuropathy

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is the most frequent adverse effect of pharmacological cancer treatments. The occurrence of neuropathy prevents the administration of fully-effective drug regimen, affects negatively the quality of life of patients, and may lead to therapy discontinuation. CIPN is currently treated with anticonvulsants, antidepressants, opioids and non-opioid analgesics, all of which are flawed by insufficient anti-hyperalgesic efficacy or addictive potential. Understandably, developing new drugs targeting CIPN-specific pathogenic mechanisms would dramatically improve efficacy and tolerability of anti-neuropathic therapies. Neuropathies are associated to aberrant excitability of DRG neurons due to the alteration in the expression or function of a variety of ion channels. In this regard, Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are overexpressed in inflammatory and neuropathic pain states, and HCN blockers have been shown to reduce neuronal excitability and to ameliorate painful states in animal models. However, HCN channels are critical in cardiac action potential, and HCN blockers used so far in pre-clinical models do not discriminate between cardiac and non-cardiac HCN isoforms. In this work, we show an HCN current gain of function in DRG neurons from oxaliplatin-treated rats. Biochemically, we observed a downregulation of HCN2 expression and an upregulation of the HCN regulatory beta-subunit MirP1. Finally, we report the efficacy of the selective HCN1 inhibitor MEL57A in reducing hyperalgesia and allodynia in oxaliplatin-treated rats without cardiac effects. In conclusion, this study strengthens the evidence for a disease-specific role of HCN1 in CIPN, and proposes HCN1-selective inhibitors as new-generation pain medications with the desired efficacy and safety profile.

Hyperpolarization-activated cyclic-nucleotide-gated channels potentially modulate axonal excitability at different thresholds.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels mediate differences in sensory and motor axonal excitability at different thresholds in animal models. Importantly, HCN channels are responsible for voltage-gated inward rectifying (Ih) currents activated during hyperpolarization. The Ih currents exert a crucial role in determining the resting membrane potential and have been implicated in a variety of neurological disorders, including neuropathic pain. In humans, differences in biophysical properties of motor and sensory axons at different thresholds remain to be elucidated and could provide crucial pathophysiological insights in peripheral neurological diseases. Consequently, the aim of this study was to characterize sensory and motor axonal function at different threshold. Median nerve motor and sensory axonal excitability studies were undertaken in 15 healthy subjects (45 studies in total). Tracking targets were set to 20, 40, and 60% of maximum for sensory and motor axons. Hyperpolarizing threshold electrotonus (TEh) at 90-100 ms was significantly increased in lower threshold sensory axons times (F = 11.195, P < 0.001). In motor axons, the hyperpolarizing current/threshold (I/V) gradient was significantly increased in lower threshold axons (F = 3.191, P < 0.05). The minimum I/V gradient was increased in lower threshold motor and sensory axons. In conclusion, variation in the kinetics of HCN isoforms could account for the findings in motor and sensory axons. Importantly, assessing the function of HCN channels in sensory and motor axons of different thresholds may provide insights into the pathophysiological processes underlying peripheral neurological diseases in humans, particularly focusing on the role of HCN channels with the potential of identifying novel treatment targets.NEW & NOTEWORTHY Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels, which underlie inward rectifying currents (Ih), appear to mediate differences in sensory and motor axonal properties. Inward rectifying currents are increased in lower threshold motor and sensory axons, although different HCN channel isoforms appear to underlie these changes. While faster activating HCN channels seem to underlie Ih changes in sensory axons, slower activating HCN isoforms appear to be mediating the differences in Ih conductances in motor axons of different thresholds. The differences in HCN gating properties could explain the predilection for dysfunction of sensory and motor axons in specific neurological diseases.

Coordinating heart morphogenesis: A novel role for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels during cardiogenesis in Xenopus laevis

ABSTRACTHyperpolarization-activated cyclic-nucleotide gated channel (HCN) proteins are important regulators of both neuronal and cardiac excitability. Among the 4 HCN isoforms, HCN4 is known as a pacemaker channel, because it helps control the periodicity of contractions in vertebrate hearts. Although the physiological role of HCN4 channel has been studied in adult mammalian hearts, an earlier role during embryogenesis has not been clearly established. Here, we probe the embryonic roles of HCN4 channels, providing the first characterization of the expression profile of any of the HCN isoforms during Xenopus laevis development and investigate the consequences of altering HCN4 function on embryonic pattern formation. We demonstrate that both overexpression of HCN4 and injection of dominant-negative HCN4 mRNA during early embryogenesis results in improper expression of key patterning genes and severely malformed hearts. Our results suggest that HCN4 serves to coordinate morphogenetic control factors that provide positional information during heart morphogenesis in Xenopus.

Molecular Mapping of Sinoatrial Node HCN Channel Expression in the Human Heart.

The hyperpolarization-activated current, If, plays an important role in sinoatrial node (SAN) pacemaking. Surprisingly, the distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in human SAN has only been investigated at the mRNA level. Our aim was to define the expression pattern of HCN proteins in human SAN and different atrial regions. Entire SAN complexes were isolated from failing (n=5) and nonfailing (n=9) human hearts cardioplegically arrested in the operating room. Three-dimensional intramural SAN structure was identified as the fibrotic compact region around the SAN artery with Connexin 43-negative pacemaker cardiomyocytes visualized in Masson's trichrome and immunostained cryosections. SAN protein was precisely isolated from the adjacent frozen SAN tissue blocks using a 16G biopsy needle. The purity of the SAN protein was confirmed by Connexin 43 immunoblot. All 3 HCN isoform proteins were detected in SAN. HCN1 was predominantly distributed in the human SAN with a 125.1±40.2 (n=12) expression ratio of SAN to right atrium. HCN2 and HCN4 expression levels were higher in SAN than in atria, with SAN to right atrium ratios of 6.1±0.9 and 4.6±0.6 (n=12), respectively. This is the first study to conduct precise 3D molecular mapping of the human SAN by isolating pure pacemaker SAN tissue. All 3 cardiac HCN isoforms had higher expression in the SAN than in the atria. HCN1 was almost exclusively expressed in SAN, emphasizing its utility as a new specific molecular marker of the human SAN and as a potential target of specific treatments intended to modify sinus rhythm.