Hyperpolarization-activated Cyclic Nucleotide-gated Channels Research Articles

Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.

Ih current stabilizes excitability in rodent DRG neurons and reverses hyperexcitability in a nociceptive neuron model of inherited neuropathic pain.

We show here that hyperpolarization-activated current (Ih ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of Ih and used it, in combination with dynamic-clamp, to study Ih function in DRG neurons. We show, for the first time, that Ih increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . Our results show that Ih , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that Ih reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that Ih unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, Ih reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of Ih direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of Ih limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (Ih ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that Ih increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . We also demonstrate that Ih reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, Ih acts to stabilize DRG neuron excitability and prevents excessive firing.

Glia instruct axon regeneration via a ternary modulation of neuronal calcium channels in Drosophila

A neuron’s regenerative capacity is governed by its intrinsic and extrinsic environment. Both peripheral and central neurons exhibit cell-type-dependent axon regeneration, but the underlying mechanism is unclear. Glia provide a milieu essential for regeneration. However, the routes of glia-neuron signaling remain underexplored. Here, we show that regeneration specificity is determined by the axotomy-induced Ca2+ transients only in the fly regenerative neurons, which is mediated by L-type calcium channels, constituting the core intrinsic machinery. Peripheral glia regulate axon regeneration via a three-layered and balanced modulation. Glia-derived tumor necrosis factor acts through its neuronal receptor to maintain calcium channel expression after injury. Glia sustain calcium channel opening by enhancing membrane hyperpolarization via the inwardly-rectifying potassium channel (Irk1). Glia also release adenosine which signals through neuronal adenosine receptor (AdoR) to activate HCN channels (Ih) and dampen Ca2+ transients. Together, we identify a multifaceted glia-neuron coupling which can be hijacked to promote neural repair.

A novel de novo HCN2 loss-of-function variant causing developmental and epileptic encephalopathy treated with ketogenic diet.

Missense variants of HCN ion channels cause variable phenotypes, ranging from mild generalized epilepsy to developmental and epileptic encephalopathy (DEE). While variants of HCN1 are an established cause of DEE, those of HCN2 have been reported in generalized epilepsies. Here, we describe the first case of DEE caused by the novel de novo heterozygous missense variant c.1379G>A (p.G460D) of HCN2. Functional characterization in transfected HEK293 cells and neonatal rat cortical neurons revealed that HCN2 p.G460D currents were strongly reduced compared to wild-type, consistent with a dominant negative loss-of-function effect. Immunofluorescence staining showed that mutant channels are retained within the cell and do not reach the membrane. Moreover, mutant HCN2 also affect HCN1 channels, by reducing the Ih current expressed by the HCN1-HCN2 heteromers. Due to the persistence of frequent seizures despite pharmacological polytherapy, the patient was treated with ketogenic diet, reporting a significant and long-lasting reduction of seizures. In vitro experiments conducted in a ketogenic environment demonstrated that the clinical improvement observed with this dietary regimen was not mediated by a direct action on HCN2 activity. These results expand the clinical spectrum related to HCN2 channelopathies, further broadening our understanding of the pathogenesis of DEE.

Ion Channel Genes in Painful Neuropathies.

Neuropathic pain (NP) is a typical symptom of peripheral nerve disorders, including painful neuropathy. The biological mechanisms that control ion channels are important for many cell activities and are also therapeutic targets. Disruption of the cellular mechanisms that govern ion channel activity can contribute to pain pathophysiology. The voltage-gated sodium channel (VGSC) is the most researched ion channel in terms of NP; however, VGSC impairment is detected in only <20% of painful neuropathy patients. Here, we discuss the potential role of the other peripheral ion channels involved in sensory signaling (transient receptor potential cation channels), neuronal excitation regulation (potassium channels), involuntary action potential generation (hyperpolarization-activated cyclic nucleotide-gated channels), thermal pain (anoctamins), pH modulation (acid sensing ion channels), and neurotransmitter release (calcium channels) related to pain and their prospective role as therapeutic targets for painful neuropathy.

Hyperpolarization-activated cyclic nucleotide-gated cation channel 3 promotes HCC development in a female-biased manner

Sex differences in hepatocellular carcinoma (HCC) development are regulated by sex and non-sex chromosomes, sex hormones, and environmental factors. We previously reported that Ncoa5+/- mice develop HCC in a male-biased manner. Here we show that NCOA5 expression is reduced in male patient HCCs while the expression of an NCOA5-interacting tumor suppressor, TIP30, is lower in female HCCs. Tip30 heterozygous deletion does not change HCC incidence in Ncoa5+/- male mice but dramatically increases HCC incidence in Ncoa5+/- female mice, accompanied by hepatic hyperpolarization-activated cyclic nucleotide-gated cation channel 3 (HCN3) overexpression. HCN3 overexpression cooperates with MYC to promote mouse HCC development, whereas Hcn3 knockout preferentially hinders HCC development in female mice. Furthermore, HCN3 amplification and overexpression occur in human HCCs and correlate with a poorer prognosis of patients in a female-biased manner. Our results suggest that TIP30 and NCOA5 protect against female liver oncogenesis and that HCN3 is a female-biased HCC driver.

Multiple neuron clusters on Micro-Electrode Arrays as an in vitro model of brain network

Understanding the brain functioning is essential for governing brain processes with the aim of managing pathological network dysfunctions. Due to the morphological and biochemical complexity of the central nervous system, the development of general models with predictive power must start from in vitro brain network engineering. In the present work, we realized a micro-electrode array (MEA)-based in vitro brain network and studied its emerging dynamical properties. We obtained four-neuron-clusters (4N) assemblies by plating rat embryo cortical neurons on 60-electrode MEA with cross-shaped polymeric masks and compared the emerging dynamics with those of sister single networks (1N). Both 1N and 4N assemblies exhibited spontaneous electrical activity characterized by spiking and bursting signals up to global activation by means of network bursts. Data revealed distinct patterns of network activity with differences between 1 and 4N. Rhythmic network bursts and dominant initiator clusters suggested pacemaker activities in both assembly types, but the propagation of activation sequences was statistically influenced by the assembly topology. We proved that this rhythmic activity was ivabradine sensitive, suggesting the involvement of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and propagated across the real clusters of 4N, or corresponding virtual clusters of 1N, with dominant initiator clusters, and nonrandom cluster activation sequences. The occurrence of nonrandom series of identical activation sequences in 4N revealed processes possibly ascribable to neuroplasticity. Hence, our multi-network dissociated cortical assemblies suggest the relevance of pacemaker neurons as essential elements for generating brain network electrophysiological patterns; indeed, such evidence should be considered in the development of computational models for envisaging network behavior both in physiological and pathological conditions.

HCN2 Channels in the Ventral Hippocampal CA1 Regulate Nociceptive Hypersensitivity in Mice.

Chronic pain is a significant health problem worldwide. Recent evidence has suggested that the ventral hippocampus is dysfunctional in humans and rodents, with decreased neuronal excitability and connectivity with other brain regions, parallel pain chronicity, and persistent nociceptive hypersensitivity. But the molecular mechanisms underlying hippocampal modulation of pain remain poorly elucidated. In this study, we used ex vivo whole-cell patch-clamp recording, immunofluorescence staining, and behavioral tests to examine whether hyperpolarization-activated cyclic nucleotide-gated channels 2 (HCN2) in the ventral hippocampal CA1 (vCA1) were involved in regulating nociceptive perception and CFA-induced inflammatory pain in mice. Reduced sag potential and firing rate of action potentials were observed in vCA1 pyramidal neurons from CFA-injected mice. Moreover, the expression of HCN2, but not HCN1, in vCA1 decreased in mice injected with CFA. HCN2 knockdown in vCA1 pyramidal neurons induced thermal hypersensitivity, whereas overexpression of HCN2 alleviated thermal hyperalgesia induced by intraplantar injection of CFA in mice. Our findings suggest that HCN2 in the vCA1 plays an active role in pain modulation and could be a promising target for the treatment of chronic pain.

Volatile anesthetics inhibit presynaptic cGMP signaling to depress presynaptic excitability in rat hippocampal neurons

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.

CAMP-mediated upregulation of HCN channels in VTA dopamine neurons promotes cocaine reinforcement

Chronic cocaine exposure induces enduring neuroadaptations that facilitate motivated drug taking. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to modulate neuronal firing and pacemaker activity in ventral tegmental area (VTA) dopamine neurons. However, it remained unknown whether cocaine self-administration affects HCN channel function and whether HCN channel activity modulates motivated drug taking. We report that rat VTA dopamine neurons predominantly express Hcn3-4 mRNA, while VTA GABA neurons express Hcn1–4 mRNA. Both neuronal types display similar hyperpolarization-activated currents (Ih), which are facilitated by acute increases in cAMP. Acute cocaine application decreases voltage-dependent activation of Ih in VTA dopamine neurons, but not in GABA neurons. Unexpectedly, chronic cocaine self-administration results in enhanced Ih selectively in VTA dopamine neurons. This differential modulation of Ih currents is likely mediated by a D2 autoreceptor-induced decrease in cAMP as D2 (Drd2) mRNA is predominantly expressed in dopamine neurons, whereas D1 (Drd1) mRNA is barely detectable in the VTA. Moreover, chronically decreased cAMP via Gi-DREADD stimulation leads to an increase in Ih in VTA dopamine neurons and enhanced binding of HCN3/HCN4 with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), an auxiliary subunit that is known to facilitate HCN channel surface trafficking. Finally, we show that systemic injection and intra-VTA infusion of the HCN blocker ivabradine reduces cocaine self-administration under a progressive ratio schedule and produces a downward shift of the cocaine dose-response curve. Our results suggest that cocaine self-administration induces an upregulation of Ih in VTA dopamine neurons, while HCN inhibition reduces the motivation for cocaine intake.

A pilot study evaluating the role of ivabradine for rate control in patients with rheumatic atrial fibrillation

ObjectivesIvabradine may have a role in rate control of atrial fibrillation (AF) due to effects on HCN channels in AV node. We studied role of Ivabradine in rate control of rheumatic AF. Methods80 patients, rheumatic AF, HR > 100 bpm (age 47 ± 11 yrs, AF duration 6.8 ± 2.9 years, rate 131 ± 16 bpm) on maximally tolerated ββ or CCB's, randomized to Ivabradine or escalated ββ/CCB. Ivabradine started @ 2.5 mg BD; increased to 5 mg BD if inadequate response at 1 week (failure to decrease HR < 10% vs baseline). After Holter at 1 month, dose escalated to 7.5 mg BD if needed. ResultsIvabradine resulted in significantly lower HR (81 ± 10 vs 99 ± 9) at 3 months and 6 months (79 ± 8 vs 94 ± 8, p < 0.001). Absolute reduction in HR: 56 ± 15 vs 31 ± 14 bpm and % change in HR: 41 ± 7 vs 24 ± 9%, both p < 0.00001).At 6 months, Ivabradine group had.1Significantly lower NT Pro BNP (1168 vs 1314 pg/ml), higher 6 min walk distance (410 ± 47 vs 349 ± 54 m, all p < 0.001)2Better symptom class (EHRA score 1: asymptomatic 84% vs 40%), improvement >1 EHRA class; baseline 60% vs 17%3Better LA Strain (22.8 ± 2.8% vs 20.6 ± 2.5%)Ivabradine was well tolerated and there was no drug withdrawal. ConclusionOur data suggest that Ivabradine can be an option for rate control in rheumatic AF.

Cerebellar interneurons control fear memory consolidation via learning-induced HCN plasticity

While synaptic plasticity is considered the basis of learning and memory, modifications of the intrinsic excitability of neurons can amplify the output of neuronal circuits and consequently change behavior. However, the mechanisms that underlie learning-induced changes in intrinsic excitability during memory formation are poorly understood. In the cerebellum, we find that silencing molecular layer interneurons completely abolishes fear memory, revealing their critical role in memory consolidation. The fear conditioning paradigm produces a lasting reduction in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in these interneurons. This change increases intrinsic membrane excitability and enhances the response to synaptic stimuli. HCN loss is driven by a decrease in endocannabinoid levels via altered cGMP signaling. In contrast, an increase in release of cerebellar endocannabinoids during memory consolidation abolishes HCN plasticity. Thus, activity in cerebellar interneurons drives fear memory formation via a learning-specific increase in intrinsic excitability, and this process requires the loss of endocannabinoid-HCN signaling.

Investigation of cardiac pacemaker channel HCN4 leads to new therapeutic approaches in the treatment of viral diseases associated with bradycardia or cardiac arrest

Investigation of cardiac pacemaker channel HCN4 leads to new therapeutic approaches in the treatment of viral diseases associated with bradycardia or cardiac arrest

HCN channel inhibitor induces ketamine-like rapid and sustained antidepressant effects in chronic social defeat stress model

Repeated, long-term (weeks to months) exposure to standard antidepressant medications is required to achieve treatment efficacy. In contrast, acute ketamine quickly improves mood for an extended time. Recent work implicates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are involved in mediating ketamine's antidepressant effects. In this study, we directly targeted HCN channels and achieved ketamine-like rapid and sustained antidepressant efficacy. Our in vitro electrophysiological recordings first showed that HCN inhibitor DK-AH 269 (also called cilobradine) decreased the pathological HCN-mediated current (Ih) and abnormal hyperactivity of ventral tegmental area (VTA) dopamine (DA) neurons in a depressive-like model produced by chronic social defeat stress (CSDS). Our in vivo studies further showed that acute intra-VTA or acute systemic administration of DK-AH 269 normalized social behavior and rescued sucrose preference in CSDS-susceptible mice. The single-dose of DK-AH 269, both by intra-VTA microinfusion and intraperitoneal (ip) approaches, could produce an extended 13-day duration of antidepressant-like efficacy. Animals treated with acute DK-AH 269 spent less time immobile than vehicle-treated mice during forced swim test. A social behavioral reversal lasted up to 13 days following the acute DK-AH 269 ip injection, and this rapid and sustained antidepressant-like response is paralleled with a single-dose treatment of ketamine. This study provides a novel ion channel target for acutely acting, long-lasting antidepressant-like effects.

New Therapeutics for Heart Failure: Focusing on cGMP Signaling.

Current drugs for treating heart failure (HF), for example, angiotensin II receptor blockers and β-blockers, possess specific target molecules involved in the regulation of the cardiac circulatory system. However, most clinically approved drugs are effective in the treatment of HF with reduced ejection fraction (HFrEF). Novel drug classes, including angiotensin receptor blocker/neprilysin inhibitor (ARNI), sodium-glucose co-transporter-2 (SGLT2) inhibitor, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, soluble guanylyl cyclase (sGC) stimulator/activator, and cardiac myosin activator, have recently been introduced for HF intervention based on their proposed novel mechanisms. SGLT2 inhibitors have been shown to be effective not only for HFrEF but also for HF with preserved ejection fraction (HFpEF). In the myocardium, excess cyclic adenosine monophosphate (cAMP) stimulation has detrimental effects on HFrEF, whereas cyclic guanosine monophosphate (cGMP) signaling inhibits cAMP-mediated responses. Thus, molecules participating in cGMP signaling are promising targets of novel drugs for HF. In this review, we summarize molecular pathways of cGMP signaling and clinical trials of emerging drug classes targeting cGMP signaling in the treatment of HF.

Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral Tegmental Area (VTA) DA Neurons.

The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders, including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the VTA DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.SIGNIFICANCE STATEMENT This study provides evidence that Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with autism spectrum disorder and schizophrenia, controls the excitability of dopamine (DA) neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3's role in DA neurons and offer insights into the pathogenic mechanisms of autism spectrum disorder and schizophrenia.

Functional Characterization of the A414G Loss-of-Function Mutation in HCN4 Associated with Sinus Bradycardia

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p &lt; 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p &lt; 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

Probucol is anti-hyperalgesic in a mouse peripheral nerve injury model of neuropathic pain

2,6-di-tert-butylphenol (2,6-DTBP) ameliorates mechanical allodynia and thermal hyperalgesia produced by partial sciatic nerve ligation in mice, and selectively inhibits HCN1 channel gating. We hypothesized that the clinically utilized non-anesthetic dimerized congener of 2,6-DTBP, probucol (2,6-di-tert-butyl-4-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)sulfanylpropan-2-ylsulfanyl]phenol), would relieve the neuropathic phenotype that results from peripheral nerve damage, and that the anti-hyperalgesic efficacy in vivo would correlate with HCN1 channel inhibition in vitro. A single oral dose of probucol (800 mg/kg) relieved mechanical allodynia and thermal hyperalgesia in a mouse spared-nerve injury neuropathic pain model. While the low aqueous solubility of probucol precluded assessment of its possible interaction with HCN1 channels, our results, in conjunction with recent data demonstrating that probucol reduces lipopolysaccharide-induced mechanical allodynia and thermal hyperalgesia, support the testing/development of probucol as a non-opioid, oral antihyperalgesic albeit one of unknown mechanistic action.

Alkali metal cations modulate the geometry of different binding sites in HCN4 selectivity filter for permeation or block.

Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels are important for timing biological processes like heartbeat and neuronal firing. Their weak cation selectivity is determined by a filter domain with only two binding sites for K+ and one for Na+. The latter acts as a weak blocker, which is released in combination with a dynamic widening of the filter by K+ ions, giving rise to a mixed K+/Na+ current. Here, we apply molecular dynamics simulations to systematically investigate the interactions of five alkali metal cations with the filter of the open HCN4 pore. Simulations recapitulate experimental data like a low Li+ permeability, considerable Rb+ conductance, a block by Cs+ as well as a punch through of Cs+ ions at high negative voltages. Differential binding of the cation species in specific filter sites is associated with structural adaptations of filter residues. This gives rise to ion coordination by a cation-characteristic number of oxygen atoms from the filter backbone and solvent. This ion/protein interplay prevents Li+, but not Na+, from entry into and further passage through the filter. The site equivalent to S3 in K+ channels emerges as a preferential binding and presumably blocking site for Cs+. Collectively, the data suggest that the weak cation selectivity of HCN channels and their block by Cs+ are determined by restrained cation-generated rearrangements of flexible filter residues.

Investigation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in vitro inflammation model at molecular level.

In our study, we aimed to create an inflammation model in endothelial and macrophage cell lines and to examine the changes in the expression of hyperpolarization activated cyclic nucleotide gated (HCN) channels at the molecular level. HUVEC and RAW cell lines were used in our study. 1µg/mL LPS was applied to the cells. Cell media were taken 6h later. TNF-α, IL-1, IL-2, IL-4, IL-10 concentrations were measured by ELISA method. Cell media were cross-applied to cells for 24h after LPS. HCN1/HCN2 protein levels were determined by Western-Blot method. HCN-1/HCN-2 gene expressions were determined by qRT-PCR method. In the inflammation model, a significant increase in TNF-α, IL-1, and IL-2 levels was observed in RAW cell media compared to the control. While no significant difference was observed in IL-4 level, a significant decrease was observed in IL-10 level. While a significant increase in TNF-α level was observed in HUVEC cell medium, no difference was observed in other cytokines. In our inflammation model, an 8.44-fold increase in HCN1 gene expression was observed in HUVEC cells compared to the control group. No significant change was observed in HCN2 gene expression. 6.71-fold increase in HCN1 gene expression was observed in RAW cells compared to the control. The change in HCN2 expression was not statistically significant. In the Western-Blot analysis, a statistically significant increase in HCN1 level was observed in the LPS group in HUVEC cells compared to the control; no significant increase in HCN2 level was observed. While a statistically significant increase in HCN1 level was observed in the LPS group in RAW cells compared to the control; no significant increase in HCN2 level was observed. In immunofluorescence examination, it was observed that the level of HCN1 and HCN2 proteins in the cell membrane of HUVEC and RAW cells increased in the LPS group compared to the control group. While HCN1 gene/protein levels were increased in RAW and HUVEC cells in the inflammation model, no significant change was observed in HCN2 gene/protein levels. Our data suggest that the HCN1 subtype is dominant in endothelium and macrophages and may play a critical role in inflammation.