Hyperpolarization-activated Cyclic Nucleotide-gated Research Articles

Activation of HCN channels caused by elevated cAMP levels in periaqueductal gray promotes bone cancer pain

The periaqueductal gray (PAG) is an important relay center for the descending pathways that regulate nociceptive information transduction. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play critical roles in the nerve injury-induced pain hypersensitivity. Previous studies have identified that HCN1 and HCN2 channel protein located in the ventral-lateral periaqueductal gray (vlPAG), a region important for pain regulation. However, it is not clear whether the HCN channel in vlPAG is involved in bone cancer pain (BCP). In this study, we assessed the role of HCN channels in BCP by measuring changes of HCN channel expression and activity in vlPAG neurons in bone cancer rats. In the present study, the BCP model was established by injecting SHZ-88 breast cancer cells into the right tibia bone marrow in rats. The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured to evaluate pain behavior in rats. HCN1 and HCN2 channels expression in vlPAG were detected by using Western Blot and immunohistochemistry. In addition, the cAMP level in vlPAG neurons was detected by ELISA, and HCN channel current (Ih) of vlPAG neurons was recorded by whole cell patch-clamp to evaluate HCN channel activity. As a result, decreased MWT and TWL were observed in rats on 7d after SHZ-88 cell inoculation, and the allodynia was sustained until 21d after inoculation. At the same time, HCN1 and HCN2 channels expression and neuronal Ih in vlPAG were significantly increased in BCP rats. In addition, the level of cAMP in vlPAG also increased after SHZ-88 cell inoculation. Furthermore, intravlPAG injection of ZD7288 (HCN channels antagonist) could significantly reduce hyperalgesia and the elevation of cAMP in vlPAG in BCP rats. Our observations suggest that the elevation of cAMP may promote the activation of HCN channels in vlPAG in bone cancer rats, thereby promoting the development of bone cancer pain.

A randomized, double-blinded, placebo-controlled, crossover study of the HCN channel blocker ivabradine in a capsaicin-induced pain model in healthy volunteers

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been focused on as a potential therapeutic target for inflammatory and neuropathic pain in rodent models. However, roles of HCN channels in human pain states have been scarcely investigated. We evaluated analgesic effects of 2-day administration of ivabradine, the only clinically available HCN channel blocker, on a capsaicin pain model in a randomized, double-blinded, placebo-controlled, crossover study. Twenty healthy adult subjects (18 males, 2 females) received ivabradine (5–7.5 mg) or a placebo 3 times in 2 days. Then capsaicin (0.5%) was topically applied on the volar forearm for 30 min. The primary outcome was capsaicin-induced spontaneous pain. The secondary outcomes included heat-pain threshold (HPT), flare size, and areas of secondary punctate mechanical hyperalgesia (PMH) and secondary dynamic mechanical allodynia (DMA). There was no significant difference in spontaneous pain (p = 0.7479), HPT (p = 0.7501), area of PMH (p = 0.1052) or flare size (p = 0.5650) at 30 min after capsaicin application between the groups. In contrast, the area of DMA in the ivabradine group was significantly smaller (p < 0.001) than that in the placebo group. HCN channels may be differentially involved in the various pain signal transmission pathways in humans.

Effect of ivabradine on cognitive functions of rats with scopolamine-induced dementia

Alzheimer’s disease is among the challenging diseases to social and healthcare systems because no treatment has been achieved yet. Although the ambiguous pathological mechanism underlying this disorder, ion channel dysfunction is one of the recently accepted possible mechanism. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play important roles in cellular excitability and synaptic transmission. Ivabradine (Iva), an HCN blocker, is acting on HCN channels, and is clinically used for angina and arrhythmia. The current study aimed to investigate the therapeutic effects of Iva against scopolamine (Sco) induced dementia. To test our hypothesis, Sco and Iva injected rats were tested for behavioural changes, followed by ELISA and histopathological analysis of the hippocampus. Induced dementia was confirmed by behavioural tests, inflammatory cytokines and oxidative stress tests and histopathological signs of neurodegeneration, multifocal deposition of congo red stained amyloid beta plaques and the decreased optical density of HCN1 immunoreactivity. Iva ameliorated the scopolamine-induced dysfunction, the hippocampus restored its normal healthy neurons, the amyloid plaques disappeared and the optical density of HCN1 immunoreactivity increased in hippocampal cells. The results suggested that blockage of HCN1 channels might underly the Iva therapeutic effect. Therefore, Iva might have beneficial effects on neurological disorders linked to HCN channelopathies.

The treatment of depression - searching for new ideas.

Depression is a severe mental health problem that affects people regardless of social status or education, is associated with changes in mood and behavior, and can result in a suicide attempt. Therapy of depressive disorders is based mainly on drugs discovered in the 1960s and early 1970s. Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are frontline pharmacological strategies for the medical treatment of depression. In addition, approved by FDA in 2019, esketamine [as nasal spray; N-methyl-D-aspartate (NMDA) receptors antagonist with additional effects on α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, L-type voltage-dependent calcium channel (L-VDCC), opioid receptors, and monoaminergic receptors] is an essential compound in suicide and drug-resistant depression. However, the treatment of depression is burdened with severe side effects, and in many cases, it is ineffective. An equally important issue is the choice of antidepressant therapy in people with comorbid somatic diseases, for example, due to possible interactions with the patient's other drugs. Therefore, there is a great need for new antidepressants with different mechanisms of action and the need to refine the search for new substances. The purpose of this review was to discuss new research directions and new trends that dominate laboratories worldwide. We have reviewed the literature to present new points on the pharmacological target of substances with antidepressant activity. In addition, we propose a new perspective on depressive therapies.

Validation of the binding stoichiometry between HCN channels and their neuronal regulator TRIP8b by single molecule measurements.

Tetratricopeptide repeat–containing Rab8b-interacting (TRIP8b) protein is a brain-specific subunit of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels, a class of voltage-gated channels modulated by cyclic nucleotides. While the interaction between TRIP8b and the cytosolic C terminus of the channel has been structurally described, the HCN:TRIP8b stoichiometry is less characterized. We employed single molecule mass photometry (MP) to image HCN4 particles purified in complex with TRIP8b. Our data show that four TRIP8b subunits are bound to the tetrameric HCN4 particle, confirming a 1:1 stoichiometry.

PEX5R/Trip8b-HCN2 channel regulating neuroinflammation involved in perioperative neurocognitive disorders

BackgroundClinical and animal studies demonstrated that neuroinflammation from anesthesia (sevoflurane) is the main contributor to cause perioperative neurocognitive disorders (PND). Recently, it was reported that microglia respond to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which was the target of sevoflurane. Whether HCN channels are involved in the induction of neuroinflammation after sevoflurane exposure is still unclear.ResultsSevoflurane exposure had increased cognitive dysfunction and anxiety-like behaviors in rats. Rats inhaled with sevoflurane had activated microglia and increased neuroinflammation (IL-1β, IL-6, and TNF-α) in the hippocampus. RNA sequencing identified 132 DEGs (86 up-regulated and 46 down-regulated DEGs [differentially expressed genes]) in the hippocampus of PND rats. RNA-sequencing also uncovered that sevoflurane exposure down-regulates HCN2 expression. Pathway and process enrichment analysis suggests DEGs are mainly enriched in regulation of system process, positive regulation of glutamate secretion, secretion, regulation of synaptic transmission, regulation of nervous system process, behavior, negative regulation of sodium ion transport, and learning or memory. We validated that sevoflurane exposure can down-regulate the levels of PEX5R/Trip8b (an interaction partner and auxiliary subunit of HCN channels) and HCN1-4 channels in the hippocampus of PND rats. We used immunofluorescence staining to identify that HCN2 co-labels with neurons (Neun), astrocytes (GFAP), and microglia (iba1). We observed that the co-labeling of HCN2 with neurons or microglia decreased in the hippocampus and cortex after sevoflurane exposure. Blocking HCN2 by ZD7288 treatment further activated microglia and aggravated sevoflurane exposure-induced anxiety-like behavior, cognitive impairment, and neuroinflammation.ConclusionsWe concluded that sevoflurane exposure can induce an increased level of neuroinflammation, microglial activation, cognitive dysfunction, and anxiety-like behaviors in rats. HCN2 channel, as the target of sevoflurane action, mediates this process. HCN2 might be a target for the treatment and prevention of sevoflurane-induced PND.

Excitatory and inhibitory effects of HCN channel modulation on excitability of layer V pyramidal cells.

Dendrites of cortical pyramidal cells are densely populated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, a.k.a. Ih channels. Ih channels are targeted by multiple neuromodulatory pathways, and thus are one of the key ion-channel populations regulating the pyramidal cell activity. Previous observations and theories attribute opposing effects of the Ih channels on neuronal excitability due to their mildly hyperpolarized reversal potential. These effects are difficult to measure experimentally due to the fine spatiotemporal landscape of the Ih activity in the dendrites, but computational models provide an efficient tool for studying this question in a reduced but generalizable setting. In this work, we build upon existing biophysically detailed models of thick-tufted layer V pyramidal cells and model the effects of over- and under-expression of Ih channels as well as their neuromodulation. We show that Ih channels facilitate the action potentials of layer V pyramidal cells in response to proximal dendritic stimulus while they hinder the action potentials in response to distal dendritic stimulus at the apical dendrite. We also show that the inhibitory action of the Ih channels in layer V pyramidal cells is due to the interactions between Ih channels and a hot zone of low voltage-activated Ca2+ channels at the apical dendrite. Our simulations suggest that a combination of Ih-enhancing neuromodulation at the proximal part of the apical dendrite and Ih-inhibiting modulation at the distal part of the apical dendrite can increase the layer V pyramidal excitability more than either of the two alone. Our analyses uncover the effects of Ih-channel neuromodulation of layer V pyramidal cells at a single-cell level and shed light on how these neurons integrate information and enable higher-order functions of the brain.

Oxytocin-Modulated Ion Channel Ensemble Controls Depolarization, Integration and Burst Firing in CA2 Pyramidal Neurons.

Oxytocin (OXT) and OXT receptor (OXTR)-mediated signaling control excitability, firing patterns, and plasticity of hippocampal CA2 pyramidal neurons, which are pivotal in generation of brain oscillations and social memory. Nonetheless, the ionic mechanisms underlying OXTR-induced effects in CA2 neurons are not fully understood. Using slice physiology in a reporter mouse line and interleaved current-clamp and voltage-clamp experiments, we systematically identified the ion channels modulated by OXT signaling in CA2 pyramidal cells (PYRs) in mice of both sexes and explored how changes in channel conductance support altered electrical activity. Activation of OXTRs inhibits an outward potassium current mediated by inward rectifier potassium channels (IKir) and thus favoring membrane depolarization. Concomitantly, OXT signaling also diminishes inward current mediated by hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels (Ih), providing a hyperpolarizing drive. The combined reduction in both IKir and Ih synergistically elevate the membrane resistance and favor dendritic integration while the membrane potential is restrained from quickly depolarizing from rest. As a result, the responsiveness of CA2 PYRs to synaptic inputs is highly sharpened during OXTR activation. Unexpectedly, OXTR signaling also strongly enhances a tetrodotoxin-resistant (TTX-R), voltage-gated sodium current that helps drive the membrane potential to spike threshold and thus promote rhythmic firing. This novel array of OXTR-stimulated ionic mechanisms operates in close coordination and underpins OXT-induced burst firing, a key step in CA2 PYRs' contribution to hippocampal information processing and broader influence on brain circuitry. Our study deepens our understanding of underpinnings of OXT-promoted social memory and general neuropeptidergic control of cognitive states.SIGNIFICANCE STATEMENT Oxytocin (OXT) plays key roles in reproduction, parenting and social and emotional behavior, and deficiency in OXT receptor (OXTR) signaling may contribute to neuropsychiatric disorders. We identified a novel array of OXTR-modulated ion channels that operate in close coordination to retune hippocampal CA2 pyramidal neurons, enhancing responsiveness to synaptic inputs and sculpting output. OXTR signaling inhibits both potassium conductance (IKir) and mixed cation conductance (Ih), engaging opposing influences on membrane potential, stabilizing it while synergistically elevating membrane resistance and electrotonic spread. OXT signaling also facilitates a tetrodotoxin-resistant (TTX-R) Na+ current, not previously described in hippocampus (HP), engaged on further depolarization. This TTX-R current lowers the spike threshold and supports rhythmic depolarization and burst firing, a potent driver of downstream circuitry.

Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats

Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. AimEvaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. Main methodsRats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. Key findingsIVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. SignificanceIVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.

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

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

Chronic Cerebral Hypoperfusion-induced Dysregulations of Hyperpolarization- activated Cyclic Nucleotide-gated, KCNQ and G Protein-coupled Inwardly Rectifying Potassium Channels Correlated with Susceptibility and Unsusceptibility to Anxiety Behaviors.

Cerebrovascular lesions could induce affective disorders; however, the depression- and anxiety-related symptoms caused by Chronic Cerebral Hypoperfusion (CCH) and the roles of different Hyperpolarization-activated Cyclic Nucleotide-gated (HCN), KCNQ and G protein-coupled inwardly rectifying potassium (GirK) channel subunits in these pathological processes have been poorly elucidated so far. To investigate the behavioral change and the alteration of HCN, KCNQ, and GirK subunits in amygdale rats suffering from CCH. Permanent bilateral occlusion of the common carotid arteries was used to induce CCH. Anxiety and depression levels were assessed by the elevated plus maze test, sucrose preference test and forced swimming test to classify rats as highly anxious or depressive 'susceptibility' vs. 'unsusceptibility'. The expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrKB), HCN1/2, KCNQ2/3, and GirK1/2/3 were quantified by Western blotting. The main emotional change caused by 4 weeks of CCH is likely to be anxiety-like behavior (50%), accompanied by a down-regulation of BDNF and TrKB expression in amygdale. The increase of HCN1 and decrease of KCNQ3 expression in amygdale may be factors to blame for anxiety- like symptom caused by CCH, and the increase of KCNQ2 and Girk1 expression in amygdale may play a role in resilience to the anxiety induced by CCH. The different subunits of HCN, KCNQ and GirK channels in amygdale may contribute to distinct response to aversive stimuli or stress induced by CCH that evokes divergent influences on anxiety-like behavior in rats.

Electrophysiological characterization of the striatal cholinergic interneurons in Dyt1 ΔGAG knock-in mice.

DYT1 dystonia is an inherited early-onset movement disorder characterized by sustained muscle contractions causing twisting, repetitive movements, and abnormal postures. Most DYT1 patients have a heterozygous trinucleotide GAG deletion mutation (ΔGAG) in DYT1/TOR1A, coding for torsinA. Dyt1 heterozygous ΔGAG knock-in (KI) mice show motor deficits and reduced striatal dopamine receptor 2 (D2R). Striatal cholinergic interneurons (ChIs) are essential in regulating striatal motor circuits. Multiple dystonia rodent models, including KI mice, show altered ChI firing and modulation. However, due to the errors in assigning KI mice, it is essential to replicate these findings in genetically confirmed KI mice. Here, we found irregular and decreased spontaneous firing frequency in the acute brain slices from Dyt1 KI mice. Quinpirole, a D2R agonist, showed less inhibitory effect on the spontaneous ChI firing in Dyt1 KI mice, suggesting decreased D2R function on the striatal ChIs. On the other hand, a muscarinic receptor agonist, muscarine, inhibited the ChI firing in both wild-type (WT) and Dyt1 KI mice. Trihexyphenidyl, a muscarinic acetylcholine receptor M1 antagonist, had no significant effect on the firing. Moreover, the resting membrane property and functions of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, μ-opioid receptors, and large-conductance calcium-activated potassium (BK) channels were unaffected in Dyt1 KI mice. The results suggest that the irregular and low-frequency firing and decreased D2R function are the main alterations of striatal ChIs in Dyt1 KI mice. These results appear consistent with the reduced dopamine release and high striatal acetylcholine tone in the previous reports.

Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

While chronic stress increases hyperpolarization-activated current (Ih) in dorsal hippocampal CA1 neurons, the underlying molecular mechanisms are entirely unknown. Following chronic social defeat stress (CSDS), susceptible mice displayed social avoidance and impaired spatial working memory, which were linked to decreased neuronal excitability, increased perisomatic hyperpolarization-activated cyclic nucleotide-gated (HCN) 1 protein expression, and elevated Ih in dorsal but not ventral CA1 neurons. In control mice, bath application of corticosterone reduced neuronal excitability, increased tetratricopeptide repeat–containing Rab8b-interacting protein (TRIP8b) and HCN1 protein expression, and elevated Ih in dorsal but not ventral CA1 region/neurons. Corticosterone-induced upregulation of functional Ih was mediated by the glucocorticoid receptor (GR), HCN channels, and the protein kinase A (PKA) but not the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Three months after the end of CSDS, susceptible mice displayed persistent social avoidance when exposed to a novel aggressor. The sustained behavioral deficit was associated with lower neuronal excitability and higher functional Ih in dorsal CA1 neurons, both of which were unaffected by corticosterone treatment. Our findings show that corticosterone treatment mimics the pathophysiological effects of dorsal CA1 neurons/region found in susceptible mice. The aberrant expression of HCN1 protein along the somatodendritic axis of the dorsal hippocampal CA1 region might be the molecular mechanism driving susceptibility to social avoidance.

Non-uniform distribution of dendritic nonlinearities differentially engages thalamostriatal and corticostriatal inputs onto cholinergic interneurons.

The tonic activity of striatal cholinergic interneurons (CINs) is modified differentially by their afferent inputs. Although their unitary synaptic currents are identical, in most CINs cortical inputs onto distal dendrites only weakly entrain them, whereas proximal thalamic inputs trigger abrupt pauses in discharge in response to salient external stimuli. To test whether the dendritic expression of the active conductances that drive autonomous discharge contribute to the CINs' capacity to dissociate cortical from thalamic inputs, we used an optogenetics-based method to quantify dendritic excitability in mouse CINs. We found that the persistent sodium (NaP) current gave rise to dendritic boosting, and that the hyperpolarization-activated cyclic nucleotide-gated (HCN) current gave rise to a subhertz membrane resonance. This resonance may underlie our novel finding of an association between CIN pauses and internally-generated slow wave events in sleeping non-human primates. Moreover, our method indicated that dendritic NaP and HCN currents were preferentially expressed in proximal dendrites. We validated the non-uniform distribution of NaP currents: pharmacologically; with two-photon imaging of dendritic back-propagating action potentials; and by demonstrating boosting of thalamic, but not cortical, inputs by NaP currents. Thus, the localization of active dendritic conductances in CIN dendrites mirrors the spatial distribution of afferent terminals and may promote their differential responses to thalamic vs. cortical inputs.

Review: HCN Channels in the Heart.

Pacemaker cells are the basis of rhythm in the heart. Cardiovascular diseases, and in particular, arrhythmias are a leading cause of hospital admissions and have been implicated as a cause of sudden death. The prevalence of people with arrhythmias will increase in the next years due to an increase in the ageing population and risk factors. The current therapies are limited, have a lot of side effects, and thus, are not ideal. Pacemaker channels, also called hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels, are the molecular correlate of the hyperpolarization- activated current, called Ih (from hyperpolarization) or If (from funny), that contribute crucially to the pacemaker activity in cardiac nodal cells and impulse generation and transmission in neurons. HCN channels have emerged as interesting targets for the development of drugs, in particular, to lower the heart rate. Nonetheless, their pharmacology is still rather poorly explored in comparison to many other voltage-gated ion channels or ligand-gated ion channels. Ivabradine is the first and currently the only clinically approved compound that specifically targets HCN channels. The therapeutic indication of ivabradine is the symptomatic treatment of chronic stable angina pectoris in patients with coronary artery disease with a normal sinus rhythm. Several other pharmacological agents have been shown to exert an effect on heart rate, although this effect is not always desired. This review is focused on the pacemaking process taking place in the heart and summarizes the current knowledge on HCN channels.

Novel HCN1 Mutations Associated With Epilepsy and Impacts on Neuronal Excitability.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel plays a critical role in regulating the resting membrane potential and integrating synaptic transmission. Variants of HCN1 have been recognized as causes of epilepsy, and mutant HCN1 channels could act with loss-of-function (LOF), loss- and gain-of-function (LOF and GOF) and gain-of-function (GOF) mechanisms. However, phenotypes and pathogenesis of HCN1-related epilepsy are still poorly understood. This study enrolled five epileptic cases carrying five different HCN1 variants: two pathogenic variants (I380F and S710Rfs*71), two likely pathogenic variants (E240G and A395G), and a paternally inherited variant (V572A). Four variants were novel. Electrophysiological experiments revealed impaired biophysical properties of the identified mutants, including current densities and activation/deactivation kinetics. Moreover, three variants exerted effects on the biophysical properties of wild-type HCN1 channels in heterozygous conditions. Immunofluorescence experiments showed that two variants reduced the protein expression of HCN1channels in neurons. Neurons expressing E240G (GOF) variant showed increased input resistance. However, the variant of I380F (LOF) increased the neuronal firing rate, thus leading to neuronal hyperexcitability. In conclusion, the present study expands the genotypic and phenotypic spectrum of patients with HCN1-related epilepsy and clarifies the underlying mechanisms. We reported five new cases including four unreported likely/pathogenic variants. We provided assessments of biophysical function for each variant, which could help patients to receive individual therapy in the future. We confirmed that HCN1 variants contributed to neuronal hyperexcitability by regulating input resistance and the action potential firing rate, and we have shown that they can affect protein expression in neurons for the first time.

Effects of Axonal Demyelination, Inflammatory Cytokines and Divalent Cation Chelators on Thalamic HCN Channels and Oscillatory Bursting.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system that is characterized by the progressive loss of oligodendrocytes and myelin and is associated with thalamic dysfunction. Cuprizone (CPZ)-induced general demyelination in rodents is a valuable model for studying different aspects of MS pathology. CPZ feeding is associated with the altered distribution and expression of different ion channels along neuronal somata and axons. However, it is largely unknown whether the copper chelator CPZ directly influences ion channels. Therefore, we assessed the effects of different divalent cations (copper; zinc) and trace metal chelators (EDTA; Tricine; the water-soluble derivative of CPZ, BiMPi) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that are major mediators of thalamic function and pathology. In addition, alterations of HCN channels induced by CPZ treatment and MS-related proinflammatory cytokines (IL-1β; IL-6; INF-α; INF-β) were characterized in C57Bl/6J mice. Thus, the hyperpolarization-activated inward current (Ih) was recorded in thalamocortical (TC) neurons and heterologous expression systems (mHCN2 expressing HEK cells; hHCN4 expressing oocytes). A number of electrophysiological characteristics of Ih (potential of half-maximal activation (V0.5); current density; activation kinetics) were unchanged following the extracellular application of trace metals and divalent cation chelators to native neurons, cell cultures or oocytes. Mice were fed a diet containing 0.2% CPZ for 35 days, resulting in general demyelination in the brain. Withdrawal of CPZ from the diet resulted in rapid remyelination, the effects of which were assessed at three time points after stopping CPZ feeding (Day1, Day7, Day25). In TC neurons, Ih was decreased on Day1 and Day25 and revealed a transient increased availability on Day7. In addition, we challenged naive TC neurons with INF-α and IL-1β. It was found that Ih parameters were differentially altered by the application of the two cytokines to thalamic cells, while IL-1β increased the availability of HCN channels (depolarized V0.5; increased current density) and the excitability of TC neurons (depolarized resting membrane potential (RMP); increased the number of action potentials (APs); produced a larger voltage sag; promoted higher input resistance; increased the number of burst spikes; hyperpolarized the AP threshold), INF-α mediated contrary effects. The effect of cytokine modulation on thalamic bursting was further assessed in horizontal slices and a computational model of slow thalamic oscillations. Here, IL-1β and INF-α increased and reduced oscillatory bursting, respectively. We conclude that HCN channels are not directly modulated by trace metals and divalent cation chelators but are subject to modulation by different MS-related cytokines.

Liraglutide increases islet Ca2+ oscillation frequency and insulin secretion by activating hyperpolarization-activated cyclic nucleotide-gated channels.

To determine whether hyperpolarization-activated cyclic nucleotide-gated (HCN) channels impact glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) modulation of islet Ca2+ handling and insulin secretion. The impact of liraglutide (GLP-1 analogue) on islet Ca2+ handling, HCN currents and insulin secretion was monitored with fluorescence microscopy, electrophysiology and enzyme immunoassays, respectively. Furthermore, liraglutide-mediated β-to-δ-cell cross-communication was assessed following selective ablation of either mouse islet δ or β cells. Liraglutide increased β-cell Ca2+ oscillation frequency in mouse and human islets under stimulatory glucose conditions. This was dependent in part on liraglutide activation of HCN channels, which also enhanced insulin secretion. Similarly, liraglutide activation of HCN channels also increased β-cell Ca2+ oscillation frequency in islets from rodents exposed to a diabetogenic diet. Interestingly, liraglutide accelerated Ca2+ oscillations in a majority of islet δ cells, which showed synchronized Ca2+ oscillations equivalent to β cells; therefore, we assessed if either cell type was driving this liraglutide-mediated islet Ca2+ response. Although δ-cell loss did not impact liraglutide-mediated increase in β-cell Ca2+ oscillation frequency, β-cell ablation attenuated liraglutide-facilitated acceleration of δ-cell Ca2+ oscillations. The data presented here show that liraglutide-induced stimulation of islet HCN channels augments Ca2+ oscillation frequency. As insulin secretion oscillates with β-cell Ca2+ , these findings have important implications for pulsatile insulin secretion that is probably enhanced by liraglutide activation of HCN channels and therapeutics that target GLP-1Rs for treating diabetes. Furthermore, these studies suggest that liraglutide as well as GLP-1-based therapies enhance δ-cell Ca2+ oscillation frequency and somatostatin secretion kinetics in a β-cell-dependent manner.

Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

Neuronal activity is emerging as a driver of central and peripheral nervous system cancers. Here, we examined neuronal physiology in mouse models of the tumor predisposition syndrome Neurofibromatosis-1 (NF1), with different propensities to develop nervous system cancers. We show that central and peripheral nervous system neurons from mice with tumor-causing Nf1 gene mutations exhibit hyperexcitability and increased secretion of activity-dependent tumor-promoting paracrine factors. We discovered a neurofibroma mitogen (COL1A2) produced by peripheral neurons in an activity-regulated manner, which increases NF1-deficient Schwann cell proliferation, establishing that neurofibromas are regulated by neuronal activity. In contrast, mice with the Arg1809Cys Nf1 mutation, found in NF1 patients lacking neurofibromas or optic gliomas, do not exhibit neuronal hyperexcitability or develop these NF1-associated tumors. The hyperexcitability of tumor-prone Nf1-mutant neurons results from reduced NF1-regulated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel function, such that neuronal excitability, activity-regulated paracrine factor production, and tumor progression are attenuated by HCN channel activation. Collectively, these findings reveal that NF1 mutations act at the level of neurons to modify tumor predisposition by increasing neuronal excitability and activity-regulated paracrine factor production.

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

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