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Morphine is an opioid commonly used to treat pain in clinic, but it also has the potential to be highly addictive, which can lead to abuse. Despite these known risks, the cellular and molecular mechanism of morphine conditioned place preference (CPP) is still unclear. In this study, using a rat model of chronic morphine administration, we found that compared with the control group, the mRNA and protein expression of HCN2 channel in the ventral tegmental area (VTA) were upregulated. Further immunofluorescence analysis showed that the fluorescence intensity of HCN2 channel of VTA dopaminergic neurons in morphine group was significantly enhanced, while the patch clamp recording of brain slices showed that both the magnitude and the density of Ih (HCN channel current) of VTA neurons were significantly increased. Moreover, intra‐VTA infusion of ZD7288, a selective inhibitor of HCN channel, into rats of the morphine group decreased morphine CPP. Taken together, our results show that chronic morphine administration induces an upregulation of HCN2 in VTA dopamine neurons, while HCN inhibition reduces morphine CPP, suggesting that HCN channel may be a potential target for the treatment of morphine addiction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8 µM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors. [ABSTRACT FROM AUTHOR]

Morphine is an opioid commonly used to treat pain in clinic, but it also has the potential to be highly addictive, which can lead to abuse. Despite these known risks, the cellular and molecular mechanism of morphine conditioned place preference (CPP) is still unclear. In this study, using a rat model of chronic morphine administration, we found that compared with the control group, the mRNA and protein expression of HCN2 channel in the ventral tegmental area (VTA) were upregulated. Further immunofluorescence analysis showed that the fluorescence intensity of HCN2 channel of VTA dopaminergic neurons in morphine group was significantly enhanced, while the patch clamp recording of brain slices showed that both the magnitude and the density of Ih (HCN channel current) of VTA neurons were significantly increased. Moreover, intra‐VTA infusion of ZD7288, a selective inhibitor of HCN channel, into rats of the morphine group decreased morphine CPP. Taken together, our results show that chronic morphine administration induces an upregulation of HCN2 in VTA dopamine neurons, while HCN inhibition reduces morphine CPP, suggesting that HCN channel may be a potential target for the treatment of morphine addiction.

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate Ih, may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC50 of 13.9 ± 0.8 μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors.

The ability of monkeys and rats to carry out spatial working memory tasks has been shown to depend on the persistent firing of pyramidal cells in the prefrontal cortex (PFC), arising from recurrent excitatory connections on dendritic spines. These spines express hyperpolarization-activated cyclic nucleotide-gated (HCN) channels whose open state is increased by cAMP signaling, and which markedly alter PFC network connectivity and neuronal firing. In traditional neural circuits, activation of these non-selective cation channels leads to neuronal depolarization and increased firing rate. Paradoxically, cAMP activation of HCN channels in PFC pyramidal cells reduces working memory-related neuronal firing. This suggests that activation of HCN channels may hyperpolarize rather than depolarize these neurons. The current study tested the hypothesis that Na+ influx through HCN channels activates Slack Na+-activated K+ (KNa) channels to hyperpolarize the membrane. We have found that HCN and Slack KNa channels co-immunoprecipitate in cortical extracts and that, by immunoelectron microscopy, they colocalize at postsynaptic spines of PFC pyramidal neurons. A specific blocker of HCN channels, ZD7288, reduces KNa current in pyramidal cells that express both HCN and Slack channels, but has no effect on KNa currents in an HEK cell line expressing Slack without HCN channels, indicating that blockade of HCN channels in neurons reduces K+ current indirectly by lowering Na+ influx. Activation of HCN channels by cAMP in a cell line expressing a Ca2+ reporter results in elevation of cytoplasmic Ca2+, but the effect of cAMP is reversed if the HCN channels are co-expressed with Slack channels. Finally, we used a novel pharmacological blocker of Slack channels to show that inhibition of Slack in rat PFC improves working memory performance, an effect previously demonstrated for blockers of HCN channels. Our results suggest that the regulation of working memory by HCN channels in PFC pyramidal neurons is mediated by an HCN-Slack channel complex that links activation HCN channels to suppression of neuronal excitability. [ABSTRACT FROM AUTHOR]

Stroke is the second leading cause of death globally. Cognitive dysfunction is a common complication of stroke, which seriously affects the patient's quality of life. Previous studies have shown that the expression of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel is closely related to ischemia-reperfusion (IR) injury and subsequent cognitive impairment. We also found that ZD7288, a specific inhibitor of the HCN channel, attenuated IR injury during short-term reperfusion. Since apoptosis can induce cell necrosis and aggravate cognitive impairment after IR, the purpose of this study is to define whether ZD7288 could improve cognitive impairment after prolonged cerebral reperfusion in rats by regulating apoptotic pathways. Our data indicated that ZD7288 can ameliorate spatial cognitive behavior and synaptic plasticity, protect the morphology of hippocampal neurons, and alleviate hippocampal apoptotic cells in IR rats. This effect may be related to down-regulating the expressions of pro-apoptotic proteins such as AIF, p53, Bax, and Caspase-3, and increasing the ratio of Bcl-2/Bax. Taken together, it suggested that inhibition of the HCN channel improves cognitive impairment after IR correlated with its regulation of apoptotic pathways. [ABSTRACT FROM AUTHOR]

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN). To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1−/−) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1−/− hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 μM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1−/− hearts. Reduced HCN4 expression was confirmed in Pak1−/− right atria. The reduced HCN activity in Pak1−/− could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined. Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility. [Display omitted] • The loss of p21-activated kinase (Pak1) results in sinoatrial node (SAN) bradycardia. • Pak1 regulates the SAN pacemaker's membrane clock component by attenuating class II HDAC4 activity. • During atrial fibrillation (AF), downregulation of Pak1 can contribute to SAN bradycardia through ERK1/2 activation. [ABSTRACT FROM AUTHOR]

Schizophrenia is a serious mental disorder, and existing antipsychotic drugs show limited efficacy and cause unwanted side effects. The development of glutamatergic drugs for schizophrenia is currently challenging. Most functions of histamine in the brain are mediated by the histamine H1 receptor; however, the role of the H2 receptor (H2R) is not quite clear, especially in schizophrenia. Here, we found that expression of H2R in glutamatergic neurons of the frontal cortex was decreased in schizophrenia patients. Selective knockout of the H2R gene (Hrh2) in glutamatergic neurons (CaMKIIα-Cre; Hrh2fl/fl) induced schizophrenia-like phenotypes including sensorimotor gating deficits, increased susceptibility to hyperactivity, social withdrawal, anhedonia, and impaired working memory, as well as decreased firing of glutamatergic neurons in the medial prefrontal cortex (mPFC) in in vivo electrophysiological tests. Selective knockdown of H2R in glutamatergic neurons in the mPFC but not those in the hippocampus also mimicked these schizophrenia-like phenotypes. Furthermore, electrophysiology experiments established that H2R deficiency decreased the firing of glutamatergic neurons by enhancing the current through hyperpolarization-activated cyclic nucleotide-gated channels. In addition, either H2R overexpression in glutamatergic neurons or H2R agonism in the mPFC counteracted schizophrenia-like phenotypes in an MK-801-induced mouse model of schizophrenia. Taken together, our results suggest that deficit of H2R in mPFC glutamatergic neurons may be pivotal to the pathogenesis of schizophrenia and that H2R agonists can be regarded as potentially efficacious medications for schizophrenia therapy. The findings also provide evidence for enriching the conventional glutamate hypothesis for the pathogenesis of schizophrenia and improve the understanding of the functional role of H2R in the brain, especially in glutamatergic neurons. [ABSTRACT FROM AUTHOR]

Dexmedetomidine is a selective α2-adrenoceptor agonist and appears to disinhibit endogenous sleep-promoting pathways, as well as to attenuate noradrenergic excitation. Recent evidence suggests that dexmedetomidine might also directly inhibit hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We analyzed the effects of dexmedetomidine on native HCN channel function in thalamocortical relay neurons of the ventrobasal complex of the thalamus from mice, performing whole-cell patch-clamp recordings. Over a clinically relevant range of concentrations (1–10 µM), the effects of dexmedetomidine were modest. At a concentration of 10 µM, dexmedetomidine significantly reduced maximal Ih amplitude (relative reduction: 0.86 [0.78–0.91], n = 10, and p = 0.021), yet changes to the half-maximal activation potential V1/2 occurred exclusively in the presence of the very high concentration of 100 µM (−4,7 [−7.5–−4.0] mV, n = 10, and p = 0.009). Coincidentally, only the very high concentration of 100 µM induced a significant deceleration of the fast component of the HCN activation time course (τfast: +135.1 [+64.7–+151.3] ms, n = 10, and p = 0.002). With the exception of significantly increasing the membrane input resistance (starting at 10 µM), dexmedetomidine did not affect biophysical membrane properties and HCN channel-mediated parameters of neuronal excitability. Hence, the sedative qualities of dexmedetomidine and its effect on the thalamocortical network are not decisively shaped by direct inhibition of HCN channel function. [ABSTRACT FROM AUTHOR]

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the cationic I h 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 Ca 2+ 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

Quetiapine is a second-generation atypical antipsychotic drug that has been commonly prescribed for the treatment of schizophrenia, major depressive disorder (depression), and other psychological disorders. Targeted inhibition of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels, which generate I h , may provide effective resistance against schizophrenia and depression. We investigated if HCN channels could contribute to the therapeutic effect of quetiapine, and its major active metabolite norquetiapine. Two-electrode voltage clamp recordings were used to assess the effects of quetiapine and its active metabolites 7-hydroxyquetiapine and norquetiapine on currents from HCN1 channels expressed in Xenopus laevis oocytes. Norquetiapine, but not quetiapine nor 7-hydroxyquetiapine, has an inhibitory effect on HCN1 channels. Norquetiapine selectively inhibited HCN1 currents by shifting the voltage-dependence of activation to more hyperpolarized potentials in a concentration-dependent manner with an IC 50 of 13.9 ± 0.8 μM for HCN1 and slowing channel opening, without changing the kinetics of closing. Inhibition by norquetiapine primarily occurs from in the closed state. Norquetiapine inhibition is not sensitive to the external potassium concentration, and therefore, likely does not block the pore. Norquetiapine inhibition also does not dependent on the cyclic-nucleotide binding domain. Norquetiapine also inhibited HCN4 channels with reduced efficacy than HCN1 and had no effect on HCN2 channels. Therefore, HCN channels are key targets of norquetiapine, the primary active metabolite of quetiapine. These data help to explain the therapeutic mechanisms by which quetiapine aids in the treatment of anxiety, major depressive disorder, bipolar disorder, and schizophrenia, and may represent a novel structure for future drug design of HCN inhibitors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jean Jacques and D’Avanzo.)

Adenosine is an endogenous substance that regulates sleep homeostasis. It plays an important role in sleep induction under physiological condition. So far, the neural mechanisms underlying sleep-promoting effects of adenosine are not completely clear. Recent studies have shown that glutamatergic neurons in the paraventricular hypothalamic nucleus (PVH) play an important role in wakefulness. Using whole-cell patch-clamp, we found that adenosine can inhibit glutamatergic neurons in PVH. This inhibition is mainly achieved by activating adenosine type 1 receptors, thereby reducing hyperpolarization-activated cyclic nucleotide-gated cation channels. By recording electroencephalogram (EEG) and electromyography (EMG), it was found that local administration of adenosine type 1 receptor blocker in PVH could significantly reduce the NREM sleep. On the contrary, if adenosine was given, it could increase the NREM sleep. These results suggest that adenosine can promote sleep by reducing the excitability of PVH neurons. This findings reveal a novel mechanism of adenosine regulating sleep homeostasis. [ABSTRACT FROM AUTHOR]

Noradrenergic neurons in the locus coeruleus (LC) release noradrenaline (NA) that acts via volume transmission to activate extrasynaptic G-protein coupled receptors (GPCRs) in target cells throughout the brain. As the closest projection, the dorsal LC laterally adjoins the mesencephalic trigeminal nucleus (MTN), in which proprioceptive primary sensory neurons innervating muscle spindles of jaw-closing muscles are exceptionally located. MTN neurons express α2-adrenergic receptors (α2-ARs) and display hyperpolarization-activated cyclic nucleotide-gated (HCN) currents (Ihs), which is downregulated by α2-AR activation. To quantify the activity-dependent outcome of volume transmission of NA from LC to MTN, we investigated how direct LC activation inhibits Ih in MTN neurons by performing dual whole-cell recordings from LC and MTN neurons. Repetition of 20 Hz spike-train evoked with 1-s current-pulse in LC neurons every 30 s resulted in a gradual decrease in Ih evoked every 30 s, revealing a Hill-type relationship between the number of spike-trains in LC neurons and the degree of Ih inhibition in MTN neurons. On the other hand, when microstimulation was applied in LC every 30 s, an LC neuron repeatedly displayed a transient higher-frequency firing followed by a tonic firing at 5–10 Hz for 30 s. This subsequently caused a similar Hill-type inhibition of Ih in the simultaneously recorded MTN neuron, but with a smaller Hill coefficient, suggesting a lower signal transduction efficacy. In contrast, 20 Hz activity induced by a 1-s pulse applied every 5–10 s caused only a transient facilitation of Ih inhibition followed by a forced termination of Ih inhibition. Thus, the three modes of LC activities modulated the volume transmission to activate α2-adrenergic GPCR to differentially inhibit Ih in MTN neurons. [ABSTRACT FROM AUTHOR]

Cannabinoids are a broad class of molecules that act primarily on neurons, affecting pain sensation, appetite, mood, learning, and memory. In addition to interacting with specific cannabinoid receptors (CBRs), cannabinoids can directly modulate the function of various ion channels. Here, we examine whether cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the most prevalent phytocannabinoids in Cannabis sativa , can regulate the function of hyperpolarization-activated cyclic-nucleotide-gated (HCN1) channels independently of CBRs. HCN1 channels were expressed in Xenopus oocytes since they do not express CBRs, and the effects of cannabinoid treatment on HCN1 currents were examined by a two-electrode voltage clamp. We observe opposing effects of CBD and THC on HCN1 current, with CBD acting to stimulate HCN1 function, while THC inhibited current. These effects persist in HCN1 channels lacking the cyclic-nucleotide binding domain (HCN1ΔCNBD). However, changes to membrane fluidity, examined by treating cells with TX-100, inhibited HCN1 current had more pronounced effects on the voltage-dependence and kinetics of activation than THC, suggesting this is not the primary mechanism of HCN1 regulation by cannabinoids. Our findings may contribute to the overall understanding of how cannabinoids may act as promising therapeutic molecules for the treatment of several neurological disorders in which HCN function is disturbed. [ABSTRACT FROM AUTHOR]

The phencyclidine-derivative ketamine [2-(2-chlorophenyl)-2-(methylamino)cyclohexan-1-one] was added to the World Health Organization's Model List of Essential Medicines in 1985 and is also on the Model List of Essential Medicines for Children due to its efficacy and safety as an intravenous anesthetic. In sub-anesthetic doses, ketamine is an effective analgesic for the treatment of acute pain (such as may occur in the perioperative setting). Additionally, ketamine may have efficacy in relieving some forms of chronic pain. In 2019, Janssen Pharmaceuticals received regulatory-approval in both the United States and Europe for use of the S-enantiomer of ketamine in adults living with treatment-resistant major depressive disorder. Pre-existing anxiety/depression and the severity of postoperative pain are risk factors for development of chronic postsurgical pain. An important question is whether short-term administration of ketamine can prevent the conversion of acute postsurgical pain to chronic postsurgical pain. Here, we have reviewed ketamine's effects on the biopsychological processes underlying pain perception and affective mood disorders, focusing on non-NMDA receptor-mediated effects, with an emphasis on results from human trials where available.

Adenosine is an endogenous substance that regulates sleep homeostasis. It plays an important role in sleep induction under physiological condition. So far, the neural mechanisms underlying sleep-promoting effects of adenosine are not completely clear. Recent studies have shown that glutamatergic neurons in the paraventricular hypothalamic nucleus (PVH) play an important role in wakefulness. Using whole-cell patch-clamp, we found that adenosine can inhibit glutamatergic neurons in PVH. This inhibition is mainly achieved by activating adenosine type 1 receptors, thereby reducing hyperpolarization-activated cyclic nucleotide-gated cation channels. By recording electroencephalogram (EEG) and electromyography (EMG), it was found that local administration of adenosine type 1 receptor blocker in PVH could significantly reduce the NREM sleep. On the contrary, if adenosine was given, it could increase the NREM sleep. These results suggest that adenosine can promote sleep by reducing the excitability of PVH neurons. This findings reveal a novel mechanism of adenosine regulating sleep homeostasis.

Noradrenergic neurons in the locus coeruleus (LC) release noradrenaline (NA) that acts via volume transmission to activate extrasynaptic G-protein coupled receptors (GPCRs) in target cells throughout the brain. As the closest projection, the dorsal LC laterally adjoins the mesencephalic trigeminal nucleus (MTN), in which proprioceptive primary sensory neurons innervating muscle spindles of jaw-closing muscles are exceptionally located. MTN neurons express α2-adrenergic receptors (α2-ARs) and display hyperpolarization-activated cyclic nucleotide-gated (HCN) currents (Ihs), which is downregulated by α2-AR activation. To quantify the activity-dependent outcome of volume transmission of NA from LC to MTN, we investigated how direct LC activation inhibits Ih in MTN neurons by performing dual whole-cell recordings from LC and MTN neurons. Repetition of 20 Hz spike-train evoked with 1-s current-pulse in LC neurons every 30 s resulted in a gradual decrease in Ih evoked every 30 s, revealing a Hill-type relationship between the number of spike-trains in LC neurons and the degree of Ih inhibition in MTN neurons. On the other hand, when microstimulation was applied in LC every 30 s, an LC neuron repeatedly displayed a transient higher-frequency firing followed by a tonic firing at 5–10 Hz for 30 s. This subsequently caused a similar Hill-type inhibition of Ih in the simultaneously recorded MTN neuron, but with a smaller Hill coefficient, suggesting a lower signal transduction efficacy. In contrast, 20 Hz activity induced by a 1-s pulse applied every 5–10 s caused only a transient facilitation of Ih inhibition followed by a forced termination of Ih inhibition. Thus, the three modes of LC activities modulated the volume transmission to activate α2-adrenergic GPCR to differentially inhibit Ih in MTN neurons.

Cannabinoids are a broad class of molecules that act primarily on neurons, affecting pain sensation, appetite, mood, learning, and memory. In addition to interacting with specific cannabinoid receptors (CBRs), cannabinoids can directly modulate the function of various ion channels. Here, we examine whether cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), the most prevalent phytocannabinoids in Cannabis sativa, can regulate the function of hyperpolarization-activated cyclic-nucleotide-gated (HCN1) channels independently of CBRs. HCN1 channels were expressed in Xenopus oocytes since they do not express CBRs, and the effects of cannabinoid treatment on HCN1 currents were examined by a two-electrode voltage clamp. We observe opposing effects of CBD and THC on HCN1 current, with CBD acting to stimulate HCN1 function, while THC inhibited current. These effects persist in HCN1 channels lacking the cyclic-nucleotide binding domain (HCN1ΔCNBD). However, changes to membrane fluidity, examined by treating cells with TX-100, inhibited HCN1 current had more pronounced effects on the voltage-dependence and kinetics of activation than THC, suggesting this is not the primary mechanism of HCN1 regulation by cannabinoids. Our findings may contribute to the overall understanding of how cannabinoids may act as promising therapeutic molecules for the treatment of several neurological disorders in which HCN function is disturbed.

Protein-lipid interactions are key regulators of ion channel function. Numerous ion channels, including hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have been shown to be regulated by phosphoinositides (PIPs), with important implications in cardiac and neuronal function. Specifically, PIPs have been shown to enhance HCN activation. Using computational approaches, we aim to identify potential binding sites for HCN1-PIP interactions. Computational docking and coarse-grained simulations indicate that PIP binding to HCN1 channels is not well coordinated, but rather occurs over a broad surface of charged residues primarily in the HCN-domain, S2 and S3 helices that can be loosely organized in 2 or 3 overlapping clusters. Thus, PIP-HCN1 interactions are more resembling of electrostatic interactions that occur in myristoylated alanine-rich C kinase substrate (MARCKS) proteins, than the specifically coordinated interactions that occur in pleckstrin homology domains (PH domains) or ion channels such as inward rectifier potassium (Kir) channels. Our results also indicate that phosphatidylinositol (PI) interactions with HCN1 are even lower affinity, explaining why unphosphorylated PI have no effect on HCN1 activation unlike phosphorylated PIPs. [ABSTRACT FROM AUTHOR]

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in neurons in the central nervous system. It has been documented that HCN channels regulate the intrinsic excitability of pyramidal cells in the medial prefrontal cortex (mPFC) of rodents. Here, we report that HCN channels limited GABAergic transmission onto pyramidal cells in rat mPFC. The pharmacological blockade of HCN channels resulted in a significant increase in the frequency of both spontaneous and miniature inhibitory postsynaptic currents (IPSCs) in mPFC pyramidal cells, whereas potentiation of HCN channels reversely decreases the frequency of mIPSCs. Furthermore, such facilitation effect on mIPSC frequency required presynaptic Ca2+ influx. Immunofluorescence staining showed that HCN channels expressed in presynaptic GABAergic terminals, as well as in both soma and neurite of parvalbumin-expressing (PV-expressing) basket cells in mPFC. The present results indicate that HCN channels in GABAergic interneurons, most likely PV-expressing basket cells, constrain inhibitory control over layer 5–6 pyramidal cells by restricting presynaptic Ca2+ entry.

Protein-lipid interactions are key regulators of ion channel function. Numerous ion channels, including hyperpolarization-activated cyclic-nucleotide gated (HCN) channels have been shown to be regulated by phosphoinositides (PIPs), with important implications in cardiac and neuronal function. Specifically, PIPs have been shown to enhance HCN activation. Using computational approaches, we aim to identify potential binding sites for HCN1-PIP interactions. Computational docking and coarse-grained simulations indicate that PIP binding to HCN1 channels is not well coordinated, but rather occurs over a broad surface of charged residues primarily in the HCN-domain, S2 and S3 helices that can be loosely organized in 2 or 3 overlapping clusters. Thus, PIP-HCN1 interactions are more resembling of electrostatic interactions that occur in myristoylated alanine-rich C kinase substrate (MARCKS) proteins, than the specifically coordinated interactions that occur in pleckstrin homology domains (PH domains) or ion channels such as inward rectifier potassium (Kir) channels. Our results also indicate that phosphatidylinositol (PI) interactions with HCN1 are even lower affinity, explaining why unphosphorylated PI have no effect on HCN1 activation unlike phosphorylated PIPs.

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model. [ABSTRACT FROM AUTHOR]

The hippocampus is vulnerable to deterioration in Alzheimer's disease (AD). It is, however, a heterogeneous structure, which may contribute to the differential volumetric changes along its septotemporal axis during AD progression. Here, we investigated amyloid plaque deposition along the dorsoventral axis in two strains of transgenic AD (ADTg) mouse models. We also used patch-clamp physiology in these mice to probe for functional consequences of AD pathogenesis in ventral hippocampus, which we found bears significantly higher plaque burden in the aged ADTg group compared to corresponding dorsal regions. Despite dorsoventral differences in amyloid load, ventral CA1 pyramidal neurons of aged ADTg mice exhibited subthreshold physiological changes similar to those previously reported in dorsal neurons, indicative of an HCN channelopathy, but lacked exacerbated suprathreshold accommodation. Additionally, HCN channel function could be rescued by pharmacological manipulation of the endoplasmic reticulum. These observations suggest that an AD-linked HCN channelopathy emerges in both dorsal and ventral CA1 pyramidal neurons, but that the former encounter an additional integrative obstacle in the form of reduced intrinsic excitability. [ABSTRACT FROM AUTHOR]

BackgroundPsychiatric disorders such as schizophrenia are worsened by stress, and working memory deficits are often a central feature of illness. Working memory is mediated by the persistent firing of prefrontal cortical (PFC) pyramidal neurons. Stress impairs working memory via high levels of dopamine D1 receptor (D1R) activation of cyclic adenosine monophosphate signaling, which reduces PFC neuronal firing. The current study examined whether D1R-cyclic adenosine monophosphate signaling reduces neuronal firing and impairs working memory by increasing the open state of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, which are concentrated on dendritic spines where PFC pyramidal neurons interconnect.MethodsA variety of methods were employed to test this hypothesis: dual immunoelectron microscopy localized D1R and HCN channels, in vitro recordings tested for D1R actions on HCN channel current, while recordings in monkeys performing a working memory task tested for D1R-HCN channel interactions in vivo. Finally, cognitive assessments following intra-PFC infusions of drugs examined D1R-HCN channel interactions on working memory performance.ResultsImmunoelectron microscopy confirmed D1R colocalization with HCN channels near excitatory-like synapses on dendritic spines in primate PFC. Mouse PFC slice recordings demonstrated that D1R stimulation increased HCN channel current, while local HCN channel blockade in primate PFC protected task-related firing from D1R-mediated suppression. D1R stimulation in rat or monkey PFC impaired working memory performance, while HCN channel blockade in PFC prevented this impairment in rats exposed to either stress or D1R stimulation.ConclusionsThese findings suggest that D1R stimulation or stress weakens PFC function via opening of HCN channels at network synapses.

Sensory hypersensitivity and somatosensory deficits represent the core symptoms of Fragile X syndrome (FXS). These alterations are believed to arise from changes in cortical sensory processing, while potential deficits in the function of peripheral sensory neurons residing in dorsal root ganglia remain unexplored. We found that peripheral sensory neurons exhibit pronounced hyperexcitability in Fmr1 KO mice, manifested by markedly increased action potential (AP) firing rate and decreased threshold. Unlike excitability changes found in many central neurons, no significant changes were observed in AP rising and falling time, peak potential, amplitude, or duration. Sensory neuron hyperexcitability was caused primarily by increased input resistance, without changes in cell capacitance or resting membrane potential. Analyses of the underlying mechanisms revealed reduced activity of HCN channels and reduced expression of HCN1 and HCN4 in Fmr1 KO compared to WT. A selective HCN channel blocker abolished differences in all measures of sensory neuron excitability between WT and Fmr1 KO neurons. These results reveal a hyperexcitable state of peripheral sensory neurons in Fmr1 KO mice caused by dysfunction of HCN channels. In addition to the intrinsic neuronal dysfunction, the accompanying paper examines deficits in sensory neuron association/communication with their enveloping satellite glial cells, suggesting contributions from both neuronal intrinsic and extrinsic mechanisms to sensory dysfunction in the FXS mouse model.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of -30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that ß-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If. Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions. [ABSTRACT FROM AUTHOR]

Objective(s): Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play essential roles in various hippocampal functions, including regulation of long-term potentiation, synaptic plasticity, and hippocampal-dependent cognitive process. The objective of this study was to investigate age-related changes in HCN1 and HCN2 protein expressions in gerbil hippocampus at various ages. Materials and Methods: In this study, the protein expressions of HCN1 and HCN2 were compared in the hippocampus at the ages of 1, 3, 12, and 24 months using Western blot analysis and immunohistochemistry.Results: Immunoreactivity of both HCN1 and HCN2 was shown primarily in cells of the pyramidal cell layer in the hippocampus proper and in cells of the granule cell layer in the dentate gyrus. HCN1 and HCN2 protein expression levels and immunoreactivity were significantly increased at three months (3 M) of age compared with those at 1 M of age. After that, both HCN1 and HCN2 expression levels in the hippocampus were gradually decreased with age. Conclusion: Our results show that the normal aging process affects the expression levels of HCN1 and HCN2 in hippocampal cells in gerbils. There are marked reductions in HCN1 and HCN2 expressions in the aged hippocampus compared to the young hippocampus. Such reductions might be related to aging in the hippocampus.

Subtypes (1–4) of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are widely expressed in the central and peripheral nervous systems, as well as the cells of smooth muscles in many organs. They mainly serve to regulate cellular excitability in these tissues. The HCN channel blocker ZD7288 has been shown to reduce apomorphine-induced conditioned taste aversion on saccharin preference in rats suggesting potential antinausea/antiemetic effects. Currently, in the least shew model of emesis we find that ZD7288 induces vomiting in a dose-dependent manner, with maximal efficacies of 100% at 1 mg/kg (i.p.) and 83.3% at 10 µg (i.c.v.). HCN channel subtype (1–4) expression was assessed using immunohistochemistry in the least shrew brainstem dorsal vagal complex (DVC) containing the emetic nuclei (area postrema (AP), nucleus tractus solitarius and dorsal motor nucleus of the vagus). Highly enriched HCN1 and HCN4 subtypes are present in the AP. A 1 mg/kg (i.p.) dose of ZD7288 strongly evoked c-Fos expression and ERK1/2 phosphorylation in the shrew brainstem DVC, but not in the in the enteric nervous system in the jejunum, suggesting a central contribution to the evoked vomiting. The ZD7288-evoked c-Fos expression exclusively occurred in tryptophan hydroxylase 2-positive serotonin neurons of the dorsal vagal complex, indicating activation of serotonin neurons may contribute to ZD7288-induced vomiting. To reveal its mechanism(s) of emetic action, we evaluated the efficacy of diverse antiemetics against ZD7288-evoked vomiting including the antagonists/inhibitors of: ERK1/2 (U0126), L-type Ca2+ channel (nifedipine); store-operated Ca2+ entry (MRS 1845); T-type Ca2+ channel (Z944), IP3R (2-APB), RyR receptor (dantrolene); the serotoninergic type 3 receptor (palonosetron); neurokinin 1 receptor (netupitant), dopamine type 2 receptor (sulpride), and the transient receptor potential vanilloid 1 receptor agonist, resiniferatoxin. All tested antiemetics except sulpride attenuated ZD7288-evoked vomiting to varying degrees. In sum, ZD7288 has emetic potential mainly via central mechanisms, a process which involves Ca2+ signaling and several emetic receptors. HCN channel blockers have been reported to have emetic potential in the clinic since they are currently used/investigated as therapeutic candidates for cancer therapy related- or unrelated-heart failure, pain, and cognitive impairment.

As thalamocortical relay neurons are ascribed a crucial role in signal propagation and information processing, they have attracted considerable attention as potential targets for anesthetic modulation. In this study, we analyzed the effects of different concentrations of sevoflurane on the excitability of thalamocortical relay neurons and hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels, which play a decisive role in regulating membrane properties and rhythmic oscillatory activity. The effects of sevoflurane on single-cell excitability and native HCN channels were investigated in acutely prepared brain slices from adult wild-type mice with the whole-cell patch-clamp technique, using voltage-clamp and current-clamp protocols. Sevoflurane dose-dependently depressed membrane biophysics and HCN-mediated parameters of neuronal excitability. Respective half-maximal inhibitory and effective concentrations ranged between 0.30 (95% CI, 0.18–0.50) mM and 0.88 (95% CI, 0.40–2.20) mM. We witnessed a pronounced reduction of HCN dependent Ih current amplitude starting at a concentration of 0.45 mM [relative change at −133 mV; 0.45 mM sevoflurane: 0.85 (interquartile range, 0.79–0.92), n = 12, p = 0.011; 1.47 mM sevoflurane: 0.37 (interquartile range, 0.34–0.62), n = 5, p < 0.001] with a half-maximal inhibitory concentration of 0.88 (95% CI, 0.40–2.20) mM. In contrast, effects on voltage-dependent channel gating were modest with significant changes only occurring at 1.47 mM [absolute change of half-maximal activation potential; 1.47 mM: −7.2 (interquartile range, −10.3 to −5.8) mV, n = 5, p = 0.020]. In this study, we demonstrate that sevoflurane inhibits the excitability of thalamocortical relay neurons in a concentration-dependent manner within a clinically relevant range. Especially concerning its effects on native HCN channel function, our findings indicate substance-specific differences in comparison to other anesthetic agents. Considering the importance of HCN channels, the observed effects might mechanistically contribute to the hypnotic properties of sevoflurane.

As thalamocortical relay neurons are ascribed a crucial role in signal propagation and information processing, they have attracted considerable attention as potential targets for anesthetic modulation. In this study, we analyzed the effects of different concentrations of sevoflurane on the excitability of thalamocortical relay neurons and hyperpolarization-activated, cyclic-nucleotide gated (HCN) channels, which play a decisive role in regulating membrane properties and rhythmic oscillatory activity. The effects of sevoflurane on single-cell excitability and native HCN channels were investigated in acutely prepared brain slices from adult wild-type mice with the whole-cell patch-clamp technique, using voltage-clamp and current-clamp protocols. Sevoflurane dose-dependently depressed membrane biophysics and HCN-mediated parameters of neuronal excitability. Respective half-maximal inhibitory and effective concentrations ranged between 0.30 (95% CI, 0.18–0.50) mM and 0.88 (95% CI, 0.40–2.20) mM. We witnessed a pronounced reduction of HCN dependent Ih current amplitude starting at a concentration of 0.45 mM [relative change at −133 mV; 0.45 mM sevoflurane: 0.85 (interquartile range, 0.79–0.92), n = 12, p = 0.011; 1.47 mM sevoflurane: 0.37 (interquartile range, 0.34–0.62), n = 5, p < 0.001] with a half-maximal inhibitory concentration of 0.88 (95% CI, 0.40–2.20) mM. In contrast, effects on voltage-dependent channel gating were modest with significant changes only occurring at 1.47 mM [absolute change of half-maximal activation potential; 1.47 mM: −7.2 (interquartile range, −10.3 to −5.8) mV, n = 5, p = 0.020]. In this study, we demonstrate that sevoflurane inhibits the excitability of thalamocortical relay neurons in a concentration-dependent manner within a clinically relevant range. Especially concerning its effects on native HCN channel function, our findings indicate substance-specific differences in comparison to other anesthetic agents. Considering the importance of HCN channels, the observed effects might mechanistically contribute to the hypnotic properties of sevoflurane. [ABSTRACT FROM AUTHOR]

The hyperpolarization-activated cation current If is a key determinant for cardiac pacemaker activity. It is conducted by subunits of the hyperpolarization-activated cyclic nucleotide–gated (HCN) channel family, of which HCN4 is predominant in mammalian heart. Both loss-of-function and gain-of-function mutations of the HCN4 gene are associated with sinus node dysfunction in humans; however, their functional impact is not fully understood yet. Here, we sought to characterize a HCN4 V759I variant detected in a patient with a family history of sick sinus syndrome. The genomic analysis yielded a mono-allelic HCN4 V759I variant in a 49-year-old woman presenting with a family history of sick sinus syndrome. This HCN4 variant was previously classified as putatively pathogenic because genetically linked to sudden infant death syndrome and malignant epilepsy. However, detailed electrophysiological and cell biological characterization of HCN4 V759I in Xenopus laevis oocytes and embryonic rat cardiomyocytes, respectively, did not reveal any obvious abnormality. Voltage dependence and kinetics of mutant channel activation, modulation of cAMP-gating by the neuronal HCN channel auxiliary subunit PEX5R, and cell surface expression were indistinguishable from wild-type HCN4. In good agreement, the clinically likewise affected mother of the patient does not exhibit the reported HCN4 variance. HCN4 V759I resembles an innocuous genetic HCN channel variant, which is not sufficient to disturb cardiac pacemaking. Once more, our work emphasizes the importance of careful functional interpretation of genetic findings not only in the context of hereditary cardiac arrhythmias. [ABSTRACT FROM AUTHOR]

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteinsmodulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways. [ABSTRACT FROM AUTHOR]

Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated "pacemaker" channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel's voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+ freshwater environment to conserve sperm's ability to depolarize. [ABSTRACT FROM AUTHOR]

Neurons located in dorsal root ganglia (DRG) are crucial for transmitting peripheral sensations such as proprioception, touch, temperature, and nociception to the spinal cord before propagating these signals to higher brain structures. To date, difficulty in identifying modality-specific DRG neurons has limited our ability to study specific populations in detail. As the calcium-binding protein parvalbumin (PV) is a neurochemical marker for proprioceptive DRG cells we used a transgenic mouse line expressing green fluorescent protein (GFP) in PV positive DRGs, to study the functional and molecular properties of putative proprioceptive neurons. Immunolabeled DRGs showed a 100% overlap between GFP positive (GFP+) and PV positive cells, confirming the PVeGFP mouse accurately labeled PV neurons. Targeted patch-clamp recording from isolated GFP+ and GFP negative (GFP−) neurons showed the passive membrane properties of the two groups were similar, however, their active properties differed markedly. All GFP+ neurons fired a single spike in response to sustained current injection and their action potentials (APs) had faster rise times, lower thresholds and shorter half widths. A hyperpolarization-activated current (Ih) was observed in all GFP+ neurons but was infrequently noted in the GFP− population (100% vs. 11%). For GFP+ neurons, Ih activation rates varied markedly, suggesting differences in the underlying hyperpolarization-activated cyclic nucleotide-gated channel (HCN) subunit expression responsible for the current kinetics. Furthermore, quantitative polymerase chain reaction (qPCR) showed the HCN subunits 2, 1, and 4 mRNA (in that order) was more abundant in GFP+ neurons, while HCN 3 was more highly expressed in GFP− neurons. Likewise, immunolabeling confirmed HCN 1, 2, and 4 protein expression in GFP+ neurons. In summary, certain functional properties of GFP+ and GFP− cells differ markedly, providing evidence for modality-specific signaling between the two groups. However, the GFP+ DRG population demonstrates considerable internal heterogeneity when hyperpolarization-activated cyclic nucleotide-gated channel (HCN channel) properties and subunit expression are considered. We propose this heterogeneity reflects the existence of different peripheral receptors such as tendon organs, muscle spindles or mechanoreceptors in the putative proprioceptive neuron population. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Epilepsy may result from abnormal function of ion channels, such as those caused by genetic mutations. Recently, pathological alterations of the expression or localization of normal channels have been implicated in epilepsy generation, and termed 'acquired channelopathies'. Altered expression levels of the HCN channels - that conduct the hyperpolarization-activated current, I(h) - have been demonstrated in hippocampus of patients with severe temporal lobe epilepsy as well as in animal models of temporal lobe and absence epilepsies. Here we probe the mechanisms for the altered expression of HCN channels which is provoked by seizures. In organotypic hippocampal slice cultures, seizure-like events selectively reduced HCN type 1 channel expression and increased HCN2 mRNA levels, as occurs in vivo. The mechanisms for HCN1 reduction involved Ca(2+)-permeable AMPA receptor-mediated Ca(2+) influx, and subsequent activation of Ca(2+)/calmodulin-dependent protein kinase II. In contrast, upregulation of HCN2 expression was independent of these processes. The data demonstrate an orchestrated program for seizure-evoked transcriptional channelopathy involving the HCN channels that may contribute to certain epilepsies.

Cuprizone (CPZ)-induced alterations in axonal myelination are associated with a period of neuronal hyperexcitability and increased activity of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in the thalamocortical (TC) system. Substances used for the treatment of multiple sclerosis (MS) have been shown to normalize neuronal excitability in CPZ-treated mice. Therefore, we aimed to examine the effects of diroximel fumarate (DRF) and the sphingosine 1-phospate receptor (S1PR) modulator siponimod on action potential firing and the inward current (I h) carried by HCN ion channels in naive conditions and during different stages of de- and remyelination. Here, DRF application reduced I h current density in ex vivo patch clamp recordings from TC neurons of the ventrobasal thalamic complex (VB), thereby counteracting the increase of I h during early remyelination. Siponimod reduced I h in VB neurons under control conditions but had no effect in neurons of the auditory cortex (AU). Furthermore, siponimod increased and decreased AP firing properties of neurons in VB and AU, respectively. Computational modeling revealed that both DRF and siponimod influenced thalamic bursting during early remyelination by delaying the onset and decreasing the interburst frequency. Thus, substances used in MS treatment normalize excitability in the TC system by influencing AP firing and I h. • Axonal de- and remyelination influence neuronal excitability and Ih currents. • The cuprizone model is a powerful tool to study the myelination process in vitro. • Drugs used in MS therapy normalize hyperexcitability in distinct brain regions. • Diroximel fumarate (DRF) impacts thalamic bursting only during early remyelination. • Siponimod acts opposingly on thalamic and cortical neurons in all conditions. [ABSTRACT FROM AUTHOR]

Hyperpolarization-activated cyclic nucleotide-gated ion channels (HCN channels) are widely expressed in the central and peripheral nervous systems and organs, while their functions are not well elucidated especially in the sympathetic nerve. The present study aimed to investigate the roles of HCN channel isoforms in the differentiation of sympathetic neurons using PC12 cell as a model. PC12 cells derived from rat pheochromocytoma were cultured and induced by nerve growth factor (NGF) (25 ng/ml) to differentiate to sympathetic neuron-like cells. Sympathetic directional differentiation of PC12 cells were evaluated by expressions of growth-associated protein 43 (GAP-43) (a growth cone marker), tyrosine hydroxylase (TH) (a sympathetic neuron marker) and neurite outgrowth. Results show that the HCN channel isoforms (HCN1-4) were all expressed in PC12 cells; blocking HCN channels with ivabradine suppressed NGF-induced GAP-43 expression and neurite outgrowth; silencing the expression of HCN2 and HCN4 using silenced using small interfering RNAs (siRNA), rather than HCN1 and HCN3, restrained GAP-43 expression and neurite outgrowth, while overexpression of HCN2 and HCN4 channels with gene transfer promoted GAP-43 expression and neurite outgrowth. Patch clamp experiments show that PC12 cells exhibited resting potentials (RP) of about −65 to −70 mV, and also presented inward HCN channel currents and outward (K+) currents, but no inward voltage-gated Na+ current was induced; NGF did not significantly affect the RP but promoted the establishment of excitability as indicated by the increased ability to depolarize and repolarize in the evoked suspicious action potentials (AP). We conclude that HCN2 and HCN4 channel isoforms, but not HCN1 and HCN3, promote the differentiation of PC12 cells toward sympathetic neurons. NGF potentiates the establishment of excitability during PC12 cell differentiation.

Ivabradine (IVA) reduces heart rate by inhibiting hyperpolarization-activated cyclic nucleotide-gated channels (HCNs), which play a role in the promotion of pacemaker activity in cardiac sinoatrial node cells. HCNs are highly expressed in neural and myocardial tissues and are involved in the modulation of inflammatory neuropathic pain. However, whether IVA exerts any effect on myocardial inflammation in the pathogenesis of heart failure is unclear. We employed single-cell RNA sequencing (scRNA-seq) in porcine cardiac myosin-induced experimental autoimmune myocarditis rat model to determine the effects and mechanisms of IVA. Lewis rats (n = 32) were randomly divided into the normal, control, high-dose-IVA, and low-dose-IVA groups. Heart rate and blood pressure were measured on days 0 and 21, respectively. Echocardiography was performed on day 22, and inflammation of the myocardium was evaluated via histopathological examination. Western blot was employed to detect the expression of HCN1-4, MinK-related protein 1 (MiRP1), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor-β (TGF-β). Furthermore, enzyme-linked immunosorbent assay was performed to measure serum IL-1, IL-6, and TNF-α. The relative mRNA levels of collagen I, collagen III, and α-smooth muscle actin (α-SMA) were determined via qRT-PCR. We found that IVA reduced the total number of cells infiltrated into the myocardium, particularly in the subset of fibroblasts, endocardia, and monocytes. IVA administration ameliorated cardiac inflammation and reduced collagen production. Results of the echocardiography indicated that left ventricular internal diameter at end-systole LVIDs increased whereas left ventricular ejection fraction and left ventricular fractional shortening decreased in the control group. IVA improved cardiac performance. The expression of HCN4 and MiRP1 protein and the level of serum IL-1, IL-6, and TNF-α were decreased by IVA treatment. In conclusion, HCNs and the helper proteins were increased in the profile of myocardial inflammation. HCNs may be involved in the regulation of myocardial inflammation by inhibiting immune cell infiltration. Our findings can contribute to the development of IVA-based combination therapies for the future treatment of cardiac inflammation and heart failure.

• Inhibiting HCN channel attenuates nociceptive and depression-like behaviors. • HCN channel and GABA are likely to exert a role in both pain and comorbidity depression. • Expression levels of TRIP8b in hippocampus is down-regulated in SNI. 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. [ABSTRACT FROM AUTHOR]

Alpha 2 (α 2 -) 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 α 2 -adrenoceptor agonist with high specificity for α 2A -subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α 2A -adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (I h) was examined during guanabenz treatment. Guanabenz inhibited I h 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 I h but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, I h inhibition by guanabenz was not reversed by α 2 -adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α 2A -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 α 2A -adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α 2A -adrenoceptors. [ABSTRACT FROM AUTHOR]

Volatile anesthetics alter presynaptic function through effects on Ca2+ influx and neurotransmitter release. These actions are proposed to play important roles in their pleiotropic neurophysiological effects including immobility, unconsciousness and amnesia. Nitric oxide and cyclic guanosine monophosphate (NO/cGMP) signaling has been implicated in presynaptic mechanisms, and disruption of NO/cGMP signaling has been shown to alter sensitivity to volatile anesthetics in vivo. We investigated volatile anesthetic actions NO/cGMP signaling in relation to presynaptic function in cultured rat hippocampal neurons using pharmacological tools and genetically encoded biosensors and sequestering probes of cGMP levels. Using the fluorescent cGMP biosensor cGull, we found that electrical stimulation-evoked NMDA-type glutamate receptor-independent presynaptic cGMP transients were inhibited 33.2% by isoflurane (0.51 mM) and 26.4% by sevoflurane (0.57 mM) (p < 0.0001) compared to control stimulation without anesthetic. Stimulation-evoked cGMP transients were blocked by the nonselective inhibitor of nitric oxide synthase N-ω-nitro- l -arginine, but not by the selective neuronal nitric oxide synthase inhibitor N5-(1-imino-3-butenyl)- l -ornithine. Isoflurane and sevoflurane inhibition of stimulation-evoked increases in presynaptic Ca2+ concentration, measured with synaptophysin-GCaMP6f, and of synaptic vesicle exocytosis, measured with synaptophysin-pHlourin, was attenuated in neurons expressing the cGMP scavenger protein sponge (inhibition of exocytosis reduced by 54% for isoflurane and by 53% for sevoflurane). The anesthetic-induced reduction in presynaptic excitability was partially occluded by inhibition of HCN channels, a cGMP-modulated excitatory ion channel that can facilitate glutamate release. We propose that volatile anesthetics depress presynaptic cGMP signaling and downstream effectors like HCN channels that are essential to presynaptic function and excitability. These findings identify novel mechanisms by which volatile anesthetics depress synaptic transmission via second messenger signaling involving the NO/cGMP pathway in hippocampal neurons. • A presynaptic role of NO/cGMP signaling in volatile anesthetics effects is identified. • Novel biosensors were used to quantify and modulate cGMP in hippocampal neurons. • A role for HCN channels as targets of volatile anesthetic actions was implicated. [ABSTRACT FROM AUTHOR]

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, which mediates the influx of cations, has an important role in action potential generation. In this article, we describe the contribution of the HCN channel to action potential generation. We simulated several common ion channels in neuron membranes based on data from rat dorsal root ganglion cells and modeled the action potential. The ion channel models employed in this paper were based on the Markov model. After modifying and calibrating these models, we compared the simulated action potential curves under the presence and absence of an HCN channel and calculated that the proportional contribution of the HCN channel in the potential recovery phase was 33.39%. This result indicates that the HCN channel is critical in assisting membrane potential recovery from a hyperpolarized state to a resting state. Furthermore, we showed how the HCN channel modifies the firing of the action potential using mathematic modeling. Our results indicated that although the loss of the HCN channel made recovery of the membrane potential more difficult from the most negative point to resting in comparison with the control, the firing rate of the action potential increased in certain circumstances. We present a novel explanation for the HCN channels’ mechanism in neuron action potential generation using mathematical models.