Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

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.

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.

Enhanced Activation of HCN Channels Reduces Excitability and Spike-Timing Regularity in Maturing Vestibular Afferent Neurons.

Vestibular ganglion neurons (VGNs) transmit information along parallel neuronal pathways whose signature distinction is variability in spike-timing; some fire at regular intervals while others fire at irregular intervals. The mechanisms driving timing differences are not fully understood but two opposing (but not mutually exclusive) hypotheses have emerged. In the first, regular-spiking is inversely correlated to the density of low-voltage-gated potassium currents (IKL). In the second, regular spiking is directly correlated to the density of hyperpolarization-activated cyclic nucleotide-sensitive currents (IH). Supporting the idea that variations in ion channel composition shape spike-timing, VGNs from the first postnatal week respond to synaptic-noise-like current injections with irregular-firing patterns if they have IKL and with more regular firing patterns if they do not. However, in vitro firing patterns are not as regular as those in vivo Here we considered whether highly-regular spiking requires IH currents and whether this dependence emerges later in development after channel expression matures. We recorded from rat VGN somata of either sex aged postnatal day (P)9-P21. Counter to expectation, in vitro firing patterns were less diverse, more transient-spiking, and more irregular at older ages than at younger ages. Resting potentials hyperpolarized and resting conductance increased, consistent with developmental upregulation of IKL Activation of IH (by increasing intracellular cAMP) increased spike rates but not spike-timing regularity. In a model, we found that activating IH counter-intuitively suppressed regularity by recruiting IKL Developmental upregulation in IKL appears to overwhelm IH These results counter previous hypotheses about how IH shapes vestibular afferent responses.SIGNIFICANCE STATEMENT Vestibular sensory information is conveyed on parallel neuronal pathways with irregularly-firing neurons encoding information using a temporal code and regularly-firing neurons using a rate code. This is a striking example of spike-timing statistics influencing information coding. Previous studies from immature vestibular ganglion neurons (VGNs) identified hyperpolarization-activated mixed cationic currents (IH) as driving highly-regular spiking and proposed that this influence grows with the current during maturation. We found that IH becomes less influential, likely because maturing VGNs also acquire low-voltage-gated potassium currents (IKL), whose inhibitory influence opposes IH Because efferent activity can partly close IKL, VGN firing patterns may become more receptive to extrinsic control. Spike-timing regularity likely relies on dynamic ion channel properties and complementary specializations in synaptic connectivity.

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.

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.

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.

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

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels are important members of the voltage-gated pore loop channels family. They show unique features: they open at hyperpolarizing potential, carry a mixed Na/K current, and are regulated by cyclic nucleotides. Four different isoforms have been cloned (HCN1-4) that can assemble to form homo- or heterotetramers, characterized by different biophysical properties. These proteins are widely distributed throughout the body and involved in different physiologic processes, the most important being the generation of spontaneous electrical activity in the heart and the regulation of synaptic transmission in the brain. Their role in heart rate, neuronal pacemaking, dendritic integration, learning and memory, and visual and pain perceptions has been extensively studied; these channels have been found also in some peripheral tissues, where their functions still need to be fully elucidated. Genetic defects and altered expression of HCN channels are linked to several pathologies, which makes these proteins attractive targets for translational research; at the moment only one drug (ivabradine), which specifically blocks the hyperpolarization-activated current, is clinically available. This review discusses current knowledge about HCN channels, starting from their biophysical properties, origin, and developmental features, to (patho)physiologic role in different tissues and pharmacological modulation, ending with their present and future relevance as drug targets.

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.

Suppression of Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Function in Thalamocortical Neurons Prevents Genetically Determined and Pharmacologically Induced Absence Seizures.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and the Ih current they generate contribute to the pathophysiological mechanisms of absence seizures (ASs), but their precise role in neocortical and thalamic neuronal populations, the main components of the network underlying AS generation, remains controversial. In diverse genetic AS models, Ih amplitude is smaller in neocortical neurons and either larger or unchanged in thalamocortical (TC) neurons compared with nonepileptic strains. A lower expression of neocortical HCN subtype 1 channels is present in genetic AS-prone rats, and HCN subtype 2 knock-out mice exhibit ASs. Furthermore, whereas many studies have characterized Ih contribution to "absence-like" paroxysmal activity in vitro, no data are available on the specific role of cortical and thalamic HCN channels in behavioral seizures. Here, we show that the pharmacological block of HCN channels with the antagonist ZD7288 applied via reverse microdialysis in the ventrobasal thalamus (VB) of freely moving male Genetic Absence Epilepsy Rats from Strasbourg decreases TC neuron firing and abolishes spontaneous ASs. A similar effect is observed on γ-hydroxybutyric acid-elicited ASs in normal male Wistar rats. Moreover, thalamic knockdown of HCN channels via virally delivered shRNA into the VB of male Stargazer mice, another genetic AS model, decreases spontaneous ASs and Ih-dependent electrophysiological properties of VB TC neurons. These findings provide the first evidence that block of TC neuron HCN channels prevents ASs and suggest that any potential anti-absence therapy that targets HCN channels should carefully consider the opposite role for cortical and thalamic Ih in the modulation of absence seizures.SIGNIFICANCE STATEMENT Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the fine-tuning of cellular and network excitability and have been suggested to be a key element of the pathophysiological mechanism underlying absence seizures. However, the precise contribution of HCN channels in neocortical and thalamic neuronal populations to these nonconvulsive seizures is still controversial. In the present study, pharmacological block and genetic suppression of HCN channels in thalamocortical neurons in the ventrobasal thalamic nucleus leads to a marked reduction in absence seizures in one pharmacological and two genetic rodent models of absence seizures. These results provide the first evidence that block of TC neuron HCN channels prevents absence seizures.

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.

Long-term treatment with ivabradine in transgenic atrial fibrillation mice counteracts hyperpolarization-activated cyclic nucleotide gated channel overexpression.

INTRODUCTION: Recent studies have demonstrated that ivabradine (IVA), is a selective inhibitor of funny current (If) and exerts antiarrhythmic effects in the settings of various diseases such as heart failure and myocardial ischemia. However, little is known regarding the effects of long-term IVA treatment on If current and hyperpolarization-activated cyclic nucleotide gated (HCN) channel overexpression. METHODS AND RESULTS: We investigated both the If current and HCN channel expression in wild-type (WT) mice and transgenic (TG) atrial fibrillation (AF) mice (heart-specific overexpressing of (pro) renin receptor TG mice) and examined the effects of IVA on the If current and HCN channel expression, and whether those effects were sufficient to prevent an AF episode. Compared with WT mice, the If current density (at -170 mV: TG, -39.6 ± 4.6 pA/pF; WT, -26.9 ± 3.0 pA/pF; P < 0.001) and activation kinetics (V1/2 : TG, -109.45 ± 1.35 mV; WT, -128.20 ± 1.65 mV), as well as HCN2 and HCN4 messenger RNA expression and HCN4 protein expression were significantly increased in the atrial myocytes of TG mice. After 4 months of IVA treatment (7 mg/kg per day orally) the effects of IVA on TG AF mice were accompanied by the inhibition of upregulation of HCN2 and HCN4 protein expression in atrial tissue, and then resulted in a uniform If loss of function. Furthermore, we observed that ivabradine significantly decreased the incidence of AF in the TG mice (41.2% in TG mice, 16.7% in TG + IVA mice; P < 0.01). CONCLUSION: IVA reduced the incidence of AF in mice, and the antiarrhythmic effects of IVA are not limited to heart rate reduction, as they partially counteract HCN overexpression and reverse electrophysiological cardiac remodeling by attenuating If gain-of-function.

Identification and characterization of a series of novel HCN channel inhibitors.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in controlling pacemaker activity in both heart and nervous system. Developing HCN channel inhibitors has been proposed to be an important strategy for the treatment of pain, heart failure, arrhythmias, and epilepsy. One HCN channel inhibitor, ivabradine, has been clinically approved for the treatment of angina pectoris and heart failure. In this study, we designed and synthesized eight alkanol amine derivatives, and assessed their effects on HCN channels expressed in COS7 cells using a whole-cell patch clamp method. Among them, compound 4e displayed the most potent inhibitory activity with an IC50 of 2.9 ± 1.2 µM at - 120 mV on HCN2 channel expressed in COS7 cells. Further analysis revealed that application of compound 4e (10 µM) caused a slowing of activation and a hyperpolarizing shift (ΔV1/2 = - 30.2 ± 2.9 mV, n = 5) in the voltage dependence of HCN2 channel activation. The inhibitory effect of compound 4e on HCN1 and HCN4 channel expressed in COS7 cells was less potent with IC50 of 17.2 ± 1.3 and 7.3 ± 1.2 µM, respectively. Besides, we showed that application of compound 4e (10 µM) inhibited Ih and action potential firing in acutely dissociated mouse small dorsal root ganglion neurons. Our study provides a new strategy for the design and development of potent HCN channel inhibitors.

Role of the Funny Current Inhibitor Ivabradine in Cardiac Pharmacotherapy: A Systematic Review.

The pharmacology, pharmacokinetics, efficacy and safety of ivabradine are reviewed. Ivabradine is an oral medication that directly and selectively inhibits the hyperpolarization-activated cyclic-nucleotide gated funny (If) current in the sinoatrial node resulting in heart rate reduction. It has a plasma elimination half-life of 6 hours and is administered twice daily. Ivabradine is extensively metabolized by cytochrome P450 3A4, and its metabolism is affected by inducers and inhibitors of the 3A4 enzyme. Studies in patients with heart failure indicate that ivabradine improves surrogate markers such as exercise tolerance. The results of (1) phase III trial demonstrated ivabradine significantly reduced heart failure hospitalizations but had no effect on mortality. Ivabradine has been extensively evaluated for coronary artery disease wherein (2) large trials was shown to have no mortality benefit. Ivabradine has been associated with improved symptoms in stable chronic angina pectoris. Ivabradine has been evaluated for other cardiovascular conditions including tachycardias of various natures, arrhythmia prevention postcardiac surgery, in acute coronary syndrome, and for heart rate control during coronary computed tomography angiogram. The most common adverse events reported in clinical trials were bradycardia, new-onset atrial fibrillation, and phosphenes. Ivabradine, a novel cardiac medication, has been studied in numerous cardiac conditions. It is only currently approved in the United States to reduce hospitalizations for systolic heart failure. The role of this medication in other conditions has not been fully elucidated.

HCN Channels: New Therapeutic Targets for Pain Treatment.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are highly regulated proteins which respond to different cellular stimuli. The HCN currents (Ih) mediated by HCN1 and HCN2 drive the repetitive firing in nociceptive neurons. The role of HCN channels in pain has been widely investigated as targets for the development of new therapeutic drugs, but the comprehensive design of HCN channel modulators has been restricted due to the lack of crystallographic data. The three-dimensional structure of the human HCN1 channel was recently reported, opening new possibilities for the rational design of highly-selective HCN modulators. In this review, we discuss the structural and functional properties of HCN channels, their pharmacological inhibitors, and the potential strategies for designing new drugs to block the HCN channel function associated with pain perception.

Quantitative Trait Loci and a Novel Genetic Candidate for Fear Learning.

UNLABELLED: Trauma- and stress-related disorders are clinically heterogeneous and associated with substantial genetic risk. Understanding the biological origins of heterogeneity of key intermediate phenotypes such as cognition and emotion can provide novel mechanistic insights into disorder pathogenesis. Performing quantitative genetics in animal models is a tractable strategy for examining both the genetic basis of intermediate phenotypes and functional testing of candidate quantitative traits genes (QTGs). Here, existing and newly collected data were used for collaborative genome-wide mapping of cued fear acquisition and expression in 65 mouse strains from the BXD genetic reference panel. For fear acquisition, we identified a significant locus on chromosome (Chr) 10 and eight suggestive loci on Chr 2, 4, 5, 11, 13, and 15. For fear expression, we identified one significant and another highly suggestive locus on Chr 13, as well as four suggestive loci on Chr 10, 11, and X. Across these loci, 60 putative QTGs were identified. The quantitative trait locus on distal Chr 13 contained a single, highly promising gene at the location of the peak likelihood ratio statistic score. The gene, hyperpolarization-activated cyclic nucleotide-gated channel 1 (Hcn1), regulates neuronal excitability. Validation experiments using behavioral pharmacology revealed that functional Hcn channels in the basolateral amygdala are necessary for conditioned fear acquisition and expression. Hcn1, together with the other candidate QTGs, thus provide new targets for neurobiological and treatment studies of fear learning and trauma- and stress-related disorders. SIGNIFICANCE STATEMENT: There is a knowledge gap in understanding the genetic contributions to behavioral heterogeneity in typical and atypical populations. Mouse genetic reference panels (GRPs) provide one approach for identifying genetic sources of variation. Here, we identified three loci for conditioned fear acquisition and expression in a mouse GRP. Each locus contained candidate quantitative trait genes (QTGs). One locus had a single QTG, Hcn1 (hyperpolarization-activated cyclic nucleotide-gated channel 1), which has been implicated in neuronal excitability and learning. This discovery was validated using behavioral pharmacology, revealing that Hcn channels in the basolateral amygdala are required for fear acquisition and expression. The study thus identifies novel candidate QTGs that may contribute to variation in emotional learning and highlight the utility of mouse GRPs for the identification of genes underlying complex traits.

A Functional Assay for Sick Sinus Syndrome Genetic Variants.

BACKGROUND/AIMS: Congenital Sick Sinus Syndrome (SSS) is a disorder associated with sudden cardiac death due to severe bradycardia and prolonged pauses. Mutations in HCN4, the gene encoding inward Na+/K+ current (If), have been described as a cause of congenital SSS. The objective of this study is to develop an SSS model in embryonic zebrafish, and use zebrafish as a moderate-throughput assay to functionally characterize HCN4 variants. METHODS: To determine the function of hcn4 in zebrafish, embryos were either bathed in the If -specific blocker (ZD-7288), or endogenous hcn4 expression was knocked down using splice-blocking morpholinos. To assess whether the zebrafish model discriminates benign from pathogenic variants, we tested four HCN4 mutations known to cause human SSS and four variants of unknown significance (VUS). RESULTS: Pharmacological blockade and knockdown of hcn4 in zebrafish phenocopied human SSS, displaying bradycardia and cardiac pauses in intact embryos and explanted hearts. The zebrafish assay correctly identified all disease-causing variants. Of the VUS, the assay predicted 2 as benign and 2 as hypomorphic variants. CONCLUSIONS: We conclude that our embryonic zebrafish assay is a novel and effective tool to functionally characterize human HCN4 variants, which can be translated into important clinical prognostic information.

EP3 activation facilitates bladder excitability via HCN channels on ICCs.

EP3 is a receptor for prostaglandin E2 (PGE2), and although its effect on bladder excitability has attracted considerable attention, the underlying mechanism remains unclear. To investigate whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the interstitial cells of Cajal (ICCs) of the bladder are involved in the effect of EP3 activation on bladder excitability, wild-type mice, HCN1 knockout (HCN1-/-) mice and rats were used in our study. Double immunofluorescence staining and immunoprecipitation assays demonstrated the interaction between EP3 and the HCN channels. Sulprostone is a selective agonist of EP3. The current density of HCN channels was enhanced by sulprostone or PGE2 using whole-cell patch clamping. Western blot analyses showed that the expression levels of HCN1 and HCN4 were higher in bladders that had undergone intravesical instillation with sulprostone than in bladders treated with normal saline (NS). Both PGE2 and sulprostone increased the calcium concentration of the ICCs, and their effects were inhibited by ZD7288 (antagonist of HCN channels) treatment. In bladder detrusor strip testing, both PGE2 and sulprostone enhanced the amplitude of the bladder detrusor in HCN1-/- mice; however, these effects were less than those in the wild-type mice. Furthermore, the effects of PGE2 and sulprostone were inhibited by ZD7288. Taken together, our results indicate that EP3 is expressed in bladder ICCs and facilitates bladder excitability via HCN channels. This study provides more comprehensive insights into the mechanism between inflammation and bladder excitability and highlights methods that can resolve bladder hyperactivity.