Torsadogenic Potential of HCN Channel Blocker Ivabradine Assessed in the Rabbit Proarrhythmia Model.

Torsadogenic effects of ivabradine, an inhibitor of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, were assessed in an in vivo proarrhythmia model of acute atrioventricular block rabbit. Ivabradine at 0.01, 0.1, and 1 mg/kg was intravenously administered to isoflurane-anesthetized rabbits (n = 5) in the stable idioventricular rhythm. Ivabradine at 0.01 and 0.1 mg/kg hardly affected the atrial and ventricular automaticity, QT interval, or the monophasic action potential duration of the ventricle. Additionally administred ivabradine at 1 mg/kg decreased the atrial and ventricular rate significantly but increased the QT interval and duration of the monophasic action potential. Meanwhile, torsade de pointes arrhythmias were detected in 1 out of 5 animals and in 2 out of 5 animals after the administration of 0.1 and 1 mg/kg, respectively. Importantly, torsade de pointes arrhythmias could be observed only in 2 rabbits showing more potent suppressive effects on ventricular automaticity. These results suggest that the torsadogenic potential of ivabradine may become evident when its expected bradycardic action appears more excessively.

Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice.

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

Ion-channel degeneracy: Multiple ion channels heterogeneously regulate intrinsic physiology of rat hippocampal granule cells.

Degeneracy, the ability of multiple structural components to elicit the same characteristic functional properties, constitutes an elegant mechanism for achieving biological robustness. In this study, we sought electrophysiological signatures for the expression of ion-channel degeneracy in the emergence of intrinsic properties of rat hippocampal granule cells. We measured the impact of four different ion-channel subtypes-hyperpolarization-activated cyclic-nucleotide-gated (HCN), barium-sensitive inward rectifier potassium (Kir ), tertiapin-Q-sensitive inward rectifier potassium, and persistent sodium (NaP) channels-on 21 functional measurements employing pharmacological agents, and report electrophysiological data on two characteristic signatures for the expression of ion-channel degeneracy in granule cells. First, the blockade of a specific ion-channel subtype altered several, but not all, functional measurements. Furthermore, any given functional measurement was altered by the blockade of many, but not all, ion-channel subtypes. Second, the impact of blocking each ion-channel subtype manifested neuron-to-neuron variability in the quantum of changes in the electrophysiological measurements. Specifically, we found that blocking HCN or Ba-sensitive Kir channels enhanced action potential firing rate, but blockade of NaP channels reduced firing rate of granule cells. Subthreshold measures of granule cell intrinsic excitability (input resistance, temporal summation, and impedance amplitude) were enhanced by blockade of HCN or Ba-sensitive Kir channels, but were not significantly altered by NaP channel blockade. We confirmed that the HCN and Ba-sensitive Kir channels independently altered sub- and suprathreshold properties of granule cells through sequential application of pharmacological agents that blocked these channels. Finally, we found that none of the sub- or suprathreshold measurements of granule cells were significantly altered upon treatment with tertiapin-Q. Together, the heterogeneous many-to-many mapping between ion channels and single-neuron intrinsic properties emphasizes the need to account for ion-channel degeneracy in cellular- and network-scale physiology.

Discovery of Novel HCN4 Blockers with Unique Blocking Kinetics and Binding Properties.

The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channel underlies the pacemaker currents, called "If," in sinoatrial nodes (SANs), which regulate heart rhythm. Some HCN4 blockers such as ivabradine have been extensively studied for treating various heart diseases. Studies have shown that these blockers have diverse state dependencies and binding sites, suggesting the existence of potential chemical and functional diversity among HCN4 blockers. Here we report approaches for the identification of novel HCN4 blockers through a random screening campaign among 16,000 small-molecule compounds using an automated patch-clamp system. These molecules exhibited various blockade profiles, and their blocking kinetics and associating amino acids were determined by electrophysiological studies and site-directed mutagenesis analysis, respectively. The profiles of these blockers were distinct from those of the previously reported HCN channel blockers ivabradine and ZD7288. Notably, the mutagenesis analysis showed that blockers with potencies that were increased when the channel was open involved a C478 residue, located at the pore cavity region near the cellular surface of the plasma membrane, while those with potencies that were decreased when the channel was open involved residues Y506 and I510, located at the intracellular region of the pore gate. Thus, this study reported for the first time the discovery of novel HCN4 blockers by screening, and their profiling analysis using an automated patch-clamp system provided chemical tools that will be useful to obtain unique molecular insights into the drug-binding modes of HCN4 and may contribute to the expansion of therapeutic options in the future.

Electromechanical coupling mechanism for activation and inactivation of an HCN channel.

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A' helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that - in contrast to electromechanical coupling for channel activation - the A' helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

cAMP-PKA signaling is involved in regulation of spinal HCN channels function in diabetic neuropathic pain.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling acts a pivotal part in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-mediated neuropathic and inflammatory pain. However, there has been no evidence of cAMP-PKA signaling is involved in regulation of spinal HCN channels function in the occurrence of diabetic neuropathic pain (DNP). The study aimed to elucidate the impact of HCN channels on neuropathic pain in a rat model of diabetes induced by streptozotocin, and whether cAMP-PKA signaling is involved in regulation of HCN channels function. In this report, we evaluated the effect of intrathecal administration of HCN channel blockers ZD7288, cAMP inhibitor SQ22536 and PKA inhibitor H-89 on nociceptive behavior in DNP rats. The mechanical withdrawal threshold (MWT) was measured to evaluate pain behavior in rats. Protein expression levels of HCN2, HCN4 channels and PKA in the spinal dorsal horn of rats were assessed. Furthermore, the levels of cAMP in rat spinal dorsal horn was analyzed. We discovered that DNP rats showed significant mechanical allodynia and are related to the increased HCN2 and HCN4 channels expression, enhanced cAMP production and elevated the expression of PKA protein in the spinal dorsal horn, which were attenuated by intrathecal ZD7288. Furthermore, intrathecal injection of SQ22536 and H-89 significantly reduced the HCN2 and HCN4 channels expression in the spinal dorsal horn of DNP rats. Our findings indicate that HCN channels of the spinal dorsal horn participate in the pathogenesis of allodynia in rats with DNP, which could be regulated by cAMP-PKA signaling. Therefore, HCN channels and cAMP-PKA signaling are potential targets for hyperalgesia treatment in DNP patients.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated channels with natural flavonoid quercetin.

Quercetin is a natural flavonoid which has been reported to be analgesic in different animal models of pain. However, the mechanism underlying the pain-relieving effects is still unclear. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in controlling pacemaker activity in cardiac and nervous systems, making the channel a new target for therapeutic exploration. In this study, we explored a series of flavonoids for their modulation on HCN channels. Among all tested flavonoids, quercetin was the most potent inhibitor for HCN channels with an IC50 value of 27.32 ± 1.19 µM for HCN2. Furthermore, quercetin prominently left shifted the voltage-dependent activation curves of HCN channels and decelerated deactivation process. The results presented herein firstly characterize quercetin as a novel and potent inhibitor for HCN channels, which represents a novel structure for future drug design of HCN channel inhibitors.

Involvement of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 3 in Oxytocin Neuronal Activity in Lactating Rats With Pup Deprivation.

Oxytocin, a hypothalamic neuropeptide essential for breastfeeding, is mainly produced in oxytocin neurons in the supraoptic nucleus (SON) and paraventricular nucleus. However, mechanisms underlying oxytocin secretion, specifically the involvement of hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) in oxytocin neuronal activity, remain unclear. Using a rat model of intermittent and continuous pup deprivation (PD) at the middle stage of lactation, we analyzed the contribution of HCN3 in oxytocin receptor (OTR)-associated signaling cascade to oxytocin neuronal activity in the SON. PD caused maternal depression, anxiety, milk shortage, involution of the mammary glands, and delays in uterine recovery, particularly in continuous PD. PD increased hypothalamic but not plasma oxytocin levels in enzyme-linked immunosorbent assay. In the SON, PD increased c-Fos expression but reduced expressions of cyclooxygenase-2 and HCN3 in Western blots and/or immunohistochemistry. Moreover, PD significantly increased the molecular association of OTR with HCN3 in coimmunoprecipitation. In brain slices, inhibition of HCN3 activity with DK-AH269 blocked prostaglandin E2-evoked increase in the firing activity and burst discharge in oxytocin neurons in patch-clamp recordings. In addition, oxytocin-evoked increase in the molecular association between OTR and HCN3 in brain slices of the SON was blocked by pretreatment with indomethacin, an inhibitor of cyclooxygenase-2. These results indicate that normal activity of oxytocin neurons is under the regulation of an oxytocin receptor-cyclooxygenase-2-HCN3 pathway and that PD disrupts maternal behavior through increasing intranuclear oxytocin secretion in the SON but likely reducing bolus oxytocin release into the blood through inhibition of HCN3 activity.

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach.

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine-an open channel blocker of HCNs-for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).

Blockade of HCN2 Channels Provides Neuroprotection Against Ischemic Injury via Accelerating Autophagic Degradation in Hippocampal Neurons.

In the central nervous system, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential to maintain normal neuronal function. Recent studies have shown that HCN channels may be involved in the pathological process of ischemic brain injury, but the mechanisms remain unclear. Autophagy is activated in cerebral ischemia, but its role in cell death/survival remains controversial. In this study, our results showed that the HCN channel blocker ZD7288 remarkably decreased the percentage of apoptotic neurons and corrected the excessive autophagy induced by oxygen-glucose deprivation followed by reperfusion (OGD/R) in hippocampal HT22 neurons. Furthermore, in the OGD/R group, p-mTOR, p-ULK1 (Ser757), and p62 were significantly decreased, while p-ULK1 (Ser317), atg5, and beclin1 were remarkably increased. ZD7288 did not change the expression of p-ULK1 (Ser757), ULK1 (Ser317), p62, Beclin1, and atg5, which are involved in regulating autophagosome formation. Besides, we found that OGD/R induced a significant increase in Cathepsin D expression, but not LAMP-1. Treatment with ZD7288 at 10 µmol/L in the OGD/R group did not change the expression of cathepsin D and LAMP-1. However, chloroquine (CQ), which decreases autophagosome-lysosome fusion, eliminated the correction of excessive autophagy and neuroprotection by ZD7288. Besides, shRNA knockdown of HCN2 channels significantly reduced the accumulation of LC3-II and increased neuron survival in the OGD/R and transient global cerebral ischemia (TGCI) models, and CQ also eliminated the effects of HCN2-shRNA. Furthermore, we found that the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes decreased in Con-shRNA-transfected HT22 neurons exposed to OGD/R or CQ. In HCN2-shRNA-transfected HT22 neurons, the percentage of LC3-positive puncta that co-localized with LAMP-1-positive lysosomes increased under OGD/R; however, the percentage was significantly decreased by the addition of CQ to HCN2-shRNA-transfected HT22 neurons. The present results demonstrated that blockade of HCN2 channels provides neuroprotection against OGD/R and TGCI by accelerating autophagic degradation attributable to the promotion of autophagosome and lysosome fusion.

The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function.

BACKGROUND: Microglia are essential to maintain cell homeostasis in the healthy brain and are activated after brain injury. Upon activation, microglia polarize towards different phenotypes. The course of microglia activation is complex and depends on signals in the surrounding milieu. Recently, it has been suggested that microglia respond to ion currents, as a way of regulating their activity and function. METHODS AND RESULTS: Under the hypothesis that HCN and KCNQ/Kv7 channels impact on microglia, we studied primary rat microglia in the presence or absence of specific pharmacological blockade or RNA silencing. Primary microglia expressed the subunits HCN1-4, Kv7.2, Kv7.3, and Kv7.5. The expression of HCN2, as well as Kv7.2 and Kv7.3, varied among different microglia phenotypes. The pharmacological blockade of HCN channels by ZD7288 resulted in cell depolarization with slowly rising intracellular calcium levels, leading to enhanced survival and reduced proliferation rates of resting microglia. Furthermore, ZD7288 treatment, as well as knockdown of HCN2 RNA by small interfering RNA, resulted in an attenuation of later microglia activation-both towards the anti- and pro-inflammatory phenotype. However, HCN channel inhibition enhanced the phagocytic capacity of IL4-stimulated microglia. Blockade of Kv7/KCNQ channel by XE-991 exclusively inhibited the migratory capacity of resting microglia. CONCLUSION: These observations suggest that the HCN current contributes to various microglia functions and impacts on the course of microglia activation, while the Kv7/KCNQ channels affect microglia migration. Characterizing the role of HCN channels in microglial functioning may offer new therapeutic approaches for targeted modulation of neuroinflammation as a hallmark of various neurological disorders.

Cytoplasmic Autoinhibition in HCN Channels is Regulated by the Transmembrane Region.

Hyperpolarization-activated cation-nonselective (HCN) channels regulate electrical activity in the brain and heart in a cAMP-dependent manner. The voltage-gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide-binding (CNB) fold in the cytoplasmic C-terminal region. Cyclic AMP potentiation has been explained by an autoinhibition model which views the unliganded CNB fold as an inhibitory module whose influence is disrupted by cAMP binding. However, the HCN2 subtype uses two other CNB fold-mediated mechanisms called open-state trapping and Quick-Activation to respectively slow the deactivation kinetics and speed the activation kinetics, against predictions of an autoinhibition model. To test how these multiple mechanisms are influenced by the TM region, we replaced the TM region of HCN2 with that of HCN4. This HCN4 TM-replacement preserved cAMP potentiation but augmented the magnitude of autoinhibition by the unliganded CNB fold; it moreover disrupted open-state trapping and Quick-Activation so that autoinhibition became the dominant mechanism contributed by the C-terminal region to determine kinetics. Truncation within the CNB fold partially relieved this augmented autoinhibition. This argues against the C-terminal region acting like a portable module with consistent effects on TM regions of different subtypes. Our findings provide evidence that functional interactions between the HCN2 TM region and C-terminal region govern multiple CNB fold-mediated mechanisms, implying that the molecular mechanisms of autoinhibition, open-state trapping, and Quick-Activation include participation of TM region structures.

Gastrointestinal Neurons Expressing HCN4 Regulate Retrograde Peristalsis.

Peristalsis is indispensable for physiological function of the gut. The enteric nervous system (ENS) plays an important role in regulating peristalsis. While the neural network regulating anterograde peristalsis, which migrates from the oral end to the anal end, is characterized to some extent, retrograde peristalsis remains unresolved with regards to its neural regulation. Using forward genetics in zebrafish, we reveal that a population of neurons expressing a hyperpolarization-activated nucleotide-gated channel HCN4 specifically regulates retrograde peristalsis. When HCN4 channels are blocked by an HCN channel inhibitor or morpholinos blocking the protein expression, retrograde peristalsis is specifically attenuated. Conversely, when HCN4(+) neurons expressing channelrhodopsin are activated by illumination, retrograde peristalsis is enhanced while anterograde peristalsis remains unchanged. We propose that HCN4(+) neurons in the ENS forward activating signals toward the oral end and simultaneously stimulate local circuits regulating the circular muscle.

Characterization of Inhibitory Effectiveness in Hyperpolarization-Activated Cation Currents by a Group of ent-Kaurane-Type Diterpenoids from Croton tonkinensis.

Croton is an extensive flowering plant genus in the spurge family, Euphorbiaceae. Three croton compounds with the common ent-kaurane skeleton have been purified from Croton tonkinensis. METHODS: We examined any modifications of croton components (i.e., croton-01 [ent-18-acetoxy-7α-hydroxykaur-16-en-15-one], croton-02 [ent-7α,14ß-dihydroxykaur-16-en-15-one] and croton-03 [ent-1ß-acetoxy-7α,14ß-dihydroxykaur-16-en-15-one] on either hyperpolarization-activated cation current (Ih) or erg-mediated K+ current identified in pituitary tumor (GH3) cells and in rat insulin-secreting (INS-1) cells via patch-clamp methods. RESULTS: Addition of croton-01, croton-02, or croton-03 effectively and differentially depressed Ih amplitude. Croton-03 (3 µM) shifted the activation curve of Ih to a more negative potential by approximately 11 mV. The voltage-dependent hysteresis of Ih was also diminished by croton-03 administration. Croton-03-induced depression of Ih could not be attenuated by SQ-22536 (10 µM), an inhibitor of adenylate cyclase, but indeed reversed by oxaliplatin (10 µM). The Ih in INS-1 cells was also depressed effectively by croton-03. CONCLUSION: Our study highlights the evidence that these ent-kaurane diterpenoids might conceivably perturb these ionic currents through which they have high influence on the functional activities of endocrine or neuroendocrine cells.

Mechanisms and functional impact of Group I metabotropic glutamate receptor modulation of excitability in mouse MNTB neurons.

We examined effects of Group I metabotropic glutamate receptors on the excitability of mouse medial nucleus of the trapezoid body (MNTB) neurons. The selective agonist, S-3,5-dihydroxyphenylglycine (DHPG), evoked a dose-dependent depolarization of the resting potential, increased membrane resistance, increased sag depolarization, and promoted rebound action potential firing. Under voltage-clamp, DHPG evoked an inward current, referred to as IDHPG , which was developmentally stable through postnatal day P56. IDHPG had low temperature dependence in the range 25-34°C, consistent with a channel mechanism. However, the I-V relationship took the form of an inverted U that did not reverse at the calculated Nernst potential for K+ or Cl- . Thus, it is likely that more than one ion type contributes to IDHPG and the mix may be voltage dependent. IDHPG was resistant to the Na+ channel blockers tetrodotoxin and amiloride, and to inhibitors of iGluR (CNQX and MK801). IDHPG was inhibited 21% by Ba2+ (500 µM), 60% by ZD7288 (100 µM) and 73% when the two antagonists were applied together, suggesting that KIR channels and HCN channels contribute to the current. Voltage clamp measurements of IH indicated a small (6%) increase in Gmax by DHPG with no change in the voltage dependence. DHPG reduced action potential rheobase and reduced the number of post-synaptic AP failures during high frequency stimulation of the calyx of Held. Thus, activation of post-synaptic Group I mGlu receptors modifies the excitability of MNTB neurons and contributes to the reliability of high frequency firing in this auditory relay nucleus.

Active membrane conductances and morphology of a collision detection neuron broaden its impedance profile and improve discrimination of input synchrony.

How neurons filter and integrate their complex patterns of synaptic inputs is central to their role in neural information processing. Synaptic filtering and integration are shaped by the frequency-dependent neuronal membrane impedance. Using single and dual dendritic recordings in vivo, pharmacology, and computational modeling, we characterized the membrane impedance of a collision detection neuron in the grasshopper Schistocerca americana. This neuron, the lobula giant movement detector (LGMD), exhibits consistent impedance properties across frequencies and membrane potentials. Two common active conductances gH and gM, mediated respectively by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and by muscarine-sensitive M-type K+ channels, promote broadband integration with high temporal precision over the LGMD's natural range of membrane potentials and synaptic input frequencies. Additionally, we found that a model based on the LGMD's branching morphology increased the gain and decreased the delay associated with the mapping of synaptic input currents to membrane potential. More generally, this was true for a wide range of model neuron morphologies, including those of neocortical pyramidal neurons and cerebellar Purkinje cells. These findings show the unexpected role played by two widespread active conductances and by dendritic morphology in shaping synaptic integration.NEW & NOTEWORTHY Neuronal filtering and integration of synaptic input patterns depend on the electrochemical properties of dendrites. We used an identified collision detection neuron in grasshoppers to examine how its morphology and two conductances affect its membrane impedance in relation to the computations it performs. The neuronal properties examined are ubiquitous and therefore promote a general understanding of neuronal computations, including those in the human brain.

New drugs: big changes in conservative heart failure therapy?

During the last 20 years, the prognosis for heart failure (HF) with reduced ejection fraction has steadily improved due to advances in drug treatment and the consistent implementation of guideline-recommended evidence-based drug therapy. Nevertheless, the morbidity and mortality rates of patients with HF can still be improved. The prevalence of HF is high and continues to increase steadily. Thus, timely and efficient drug treatment plays a central role in improving the quality of life and prognosis for patients with HF. Current therapeutic concepts combine inhibition of the renin-angiotensin-aldosterone system with blockage of the sympathetic system. New therapeutic approaches such as selective heart rate reduction, attenuation of the degradation of natriuretic peptides by neutral endopeptidase inhibition and treatment of comorbidities (e.g. iron deficiency, diabetes mellitus, hyperkalaemia) have led to a further improvement in the survival, time-out-of hospital and quality of life of affected patients. The goal of this article was to give an overview of the current standard drug therapy for HF and the value of new therapeutic approaches implemented in recent years.

Alpha 2 adrenoceptor agonist guanabenz directly inhibits hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in mesencephalic trigeminal nucleus neurons.

Alpha 2 (α2-) adrenoceptor agonists, such as clonidine or dexmedetomidine, have been found to inhibit hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, not only by reducing intracellular cyclic AMP levels but also by directly blocking HCN channels. In this study, we examined the inhibitory effect of guanabenz, a centrally acting α2-adrenoceptor agonist with high specificity for α2A-subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α2A-adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (Ih) was examined during guanabenz treatment. Guanabenz inhibited Ih in a dose-dependent manner, which was likely to be ZD7288-sensitive HCN current as it did not affect barium-sensitive inward rectifying potassium current. Guanabenz not only inhibited Ih but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, Ih inhibition by guanabenz was not reversed by α2-adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α2A-adrenoceptor signalling pathway but from direct inhibition of HCN channels. Coherent to our electrophysiological results, single-cell RT-PCR revealed that most MTN neurons lack α2A-adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α2A-adrenoceptors.

HCN channel antagonist ZD7288 ameliorates neuropathic pain and associated depression.

Chronic neuropathic pain has demonstrated that coexisting psychiatric disorders are associated with disability and poorer treatment outcomes. Hyperpolarization-activated cyclic nucleotide-gated (HCN, Ih) channels play a major role in pain via hyperexcitability and facilitation of ectopic firing in neurons. Neuronal hyperexcitability contributes to pain maintenance and anxiety/depression. GABA-mediated inhibitory postsynaptic neurotransmission in the brain is impaired in the pathophysiology of chronic neuropathic pain with comorbidity mood disorders. Currently, interaction of HCN channels and GABAergic synaptic transmission inhibition in neuropathic pain and the associated comorbidity anxiety/depression mechanism remains relatively unknown. To address this, the HCN channel inhibitor, ZD7288, was administrated to Wistar Kyoto (WKY) rats after spared nerve injury (SNI). Our findings show that intracerebroventricular injection of ZD7288 concurrently attenuates co-existing nociceptive and depression-like behaviors, and increases glutamicacid decarboxylase (GAD67/65) expression and GABA levels in the hippocampus and thalamus with High-performance liquid chromatography technique. It suggests that inhibition of HCN channels is likely to decrease the hyperexcitability of neurons in rat SNI and improve the level of GABA. Further, HCN channel may offer a new strategy to alleviate both neuropathic pain and comorbidity for depression.

Hyperpolarization-activated and cyclic nucleotide-gated channel proteins as emerging new targets in neuropathic pain.

Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are activated during hyperpolarization, and there is an inward flow of current, which is termed as hyperpolarization-activated current, Ih. Initially, these channels were identified on the pacemaker cells of the heart. Nowadays, these are identified on different regions of the nervous system, including peripheral nerves, dorsal root ganglia, dorsal horns, and different parts of the brain. There are four different types of HCN channels (HCN1-HCN4); however, HCN1 and HCN2 are more prominent. A large number of studies have shown that peripheral nerve injury increases the amplitude of Ih current in the neurons of the spinal cord and the brain. Moreover, there is an increase in the expression of HCN1 and HCN2 protein channels in peripheral axons and the spinal cord and brain regions in experimental models of nerve injury. Studies have also documented the pain-attenuating actions of selective HCN inhibitors, such as ivabradine and ZD7288. Moreover, certain drugs with additional HCN-blocking activities have also shown pain-attenuating actions in different pain models. There have been few studies documenting the relationship of HCN channels with other mediators of pain. Nevertheless, it may be proposed that the HCN channel activity is modulated by endogenous opioids and cyclo-oxygenase-2, whereas the activation of these channels may modulate the actions of substance P and the expression of spinal N-methyl-D-aspartate receptor subunit 2B to modulate pain. The present review describes the role and mechanisms of HCN ion channels in the development of neuropathic pain.