Hyperpolarization-activated Cyclic Nucleotide-gated Channels Research Articles

Propofol rescues HCN1 epilepsy mutants and reveals non-canonical voltage-dependent gating in HCN channels

Propofol rescues HCN1 epilepsy mutants and reveals non-canonical voltage-dependent gating in HCN channels

Targeting the facilitatory effect of cAMP on HCN2 channel opening promotes analgesic actions

Targeting the facilitatory effect of cAMP on HCN2 channel opening promotes analgesic actions

Introduction of α-bungarotoxin binding site into extracellular domains of HCN4 channels

Introduction of α-bungarotoxin binding site into extracellular domains of HCN4 channels

LRMP inhibits the cAMP dependence of HCN4 channels by disrupting intramolecular signal transduction

LRMP inhibits the cAMP dependence of HCN4 channels by disrupting intramolecular signal transduction

New insights into the binding of TRIP8BB protein to the CNBD of HCN channels

New insights into the binding of TRIP8BB protein to the CNBD of HCN channels

A high affinity switch for cAMP in the HCN pacemaker channels

Binding of cAMP to Hyperpolarization activated cyclic nucleotide gated (HCN) channels facilitates pore opening. It is unclear why the isolated cyclic nucleotide binding domain (CNBD) displays in vitro lower affinity for cAMP than the full-length channel in patch experiments. Here we show that HCN are endowed with an affinity switch for cAMP. Alpha helices D and E, downstream of the cyclic nucleotide binding domain (CNBD), bind to and stabilize the holo CNBD in a high affinity state. These helices increase by 30-fold cAMP efficacy and affinity measured in patch clamp and ITC, respectively. We further show that helices D and E regulate affinity by interacting with helix C of the CNBD, similarly to the regulatory protein TRIP8b. Our results uncover an intramolecular mechanism whereby changes in binding affinity, rather than changes in cAMP concentration, can modulate HCN channels, adding another layer to the complex regulation of their activity.

Hyperpolarization-activated cyclic nucleotide-gated channel inhibitor in myocardial infarction: Potential benefits beyond heart rate modulation.

Myocardial infarction (MI) and its associated complications including ventricular arrhythmias and heart failure are responsible for a significant incidence of morbidity and mortality worldwide. The ensuing cardiomyocyte loss results in neurohormone-driven cardiac remodeling, which leads to chronic heart failure in MI survivors. Ivabradine is a heart rate modulation agent currently used in treatment of chronic heart failure with reduced ejection fraction. The canonical target of ivabradine is the hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in cardiac pacemaker cells. However, in post-MI hearts, HCN can also be expressed ectopically in non-pacemaker cardiomyocytes. There is an accumulation of intriguing evidence to suggest that ivabradine also possesses cardioprotective effects that are independent of heart rate reduction. This review aims to summarize and discuss the reported cardioprotective mechanisms of ivabradine beyond heart rate modulation in myocardial infarction through various molecular mechanisms including the prevention of reactive oxygen species-induced mitochondrial damage, improvement of autophagy system, modulation of intracellular calcium cycling, modification of ventricular electrophysiology, and regulation of matrix metalloproteinases.

Suppression of HCN channels in the spinal dorsal horn restores KCC2 expression and attenuates diabetic neuropathic pain

Previous studies have shown that the hyperpolarized cyclic nucleotide gated (HCN) ion channels in the spinal dorsal horn (SDH) might be involved in the development of diabetic neuropathic pain (DNP). Additionally, other studies have shown that the decreased potassium-chloride cotransporter 2 (KCC2) expression in the SDH promotes pain hypersensitivity. Both HCN channels and KCC2 were highly expressed in spinal substantia gelatinosa neurons. However, whether the K+ efflux induced by the activation of HCN channels in DNP modulate KCC2 function and subsequently affect the role of γ-aminobutyric acid (GABA)/GABA-A receptors of neurons in the SDH remains to be clarified. The purpose of this work was to investigate the underlying mechanisms of KCC2 participating in HCN channels to promote DNP. Here, we found that the analgesic role of HCN channels blocker ZD7288 was associated with the up-regulated KCC2 expression and could be prevented by DIOA, a KCC2 blocker. Furthermore, the level of GABA in DNP rats significantly increased, which was decreased by ZD72288. Moreover, DIOA pretreatment could partly block the inhibitory effect of ZD7288 on the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling activation of DNP rats. Finally, inhibition of cAMP-PKA signaling alleviated allodynia and elevated KCC2 expression in DNP rats. Altogether, this study reveals that the role of cAMP-PKA signaling-regulated HCN channels in DNP associated with decreased KCC2 expression in the spinal cord and altered GABA nature.

The role of HCN channels on the effects of T-type calcium channels and GABAA receptors in the absence epilepsy model of WAG/Rij rats.

In this study we used ivabradine (IVA), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, to identify its effect on spike-wave discharges (SWDs); and aimed to determine the role of IVA on the effects of T-type calcium channel blocker NNC 55-0396, GABAA receptor agonist muscimol and antagonist bicuculline in male WAG/Rij rats. After tripolar electrodes for electrocorticogram (ECoG) recordings were placed on the WAG/Rij rats' skulls, 5, 10, and 20 mg/kg IVA were intraperitoneally administered for 7 consecutive days and ECoG recordings were obtained on days 0th, 3rd, 6th, and 7th for threehours before and after injections. While acute injection of 5, 10, and 20 mg/kg IVA did not affect the total number and the mean duration of SWDs, subacute administration (7 days) of IVA decreased the SWDs parameters 24 hours after the 7th injection. Interestingly, when IVA was administered again 24 hours after the 6th IVA injection, it increased the SWDs parameters. Western-blot analyses showed that HCN1 and HCN2 expressions decreased and HCN4 increased in the 5-month-old WAG/Rij rats compared to the 1-month-old WAG/Rij and 5-month-old native Wistar rats, while subacute IVA administration increased the levels of HCN1 and HCN2 channels, except HCN4. Subacute administration of IVA reduced the antiepileptic activity of NNC, while the proepileptic activity of muscimol and the antiepileptic activity of bicuculline were abolished. It might be suggested that subacute IVA administration reduces absence seizures by changing the HCN channel expressions in WAG/Rij rats, and this affects the T-type calcium channels and GABAA receptors.

Valproate treatment induces age- and sex-dependent neuronal activity changes according to a patch clamp study.

Autism spectrum disorder is a heterogeneous neurodevelopmental disorder characterized by impaired social interactions, restricted, and stereotyped behaviors. The valproic acid model is one of the most recognized and broadly used models in rats to induce core symptoms of this disorder. Comorbidity of epilepsy and autism occurs frequently, due to similar background mechanisms that include the imbalance of excitation and inhibition. In this series of experiments, treatment was performed on rat dams with a single 500mg/kg dose i.p. valproate injection on embryonic day 12.5. Intracellular whole-cell patch clamp recordings were performed on brain slices prepared from adolescent and adult offspring of both sexes on pyramidal neurons of the medial prefrontal cortex and entorhinal cortex. Current clamp stimulation utilizing conventional current step protocols and dynamic clamp stimulation were applied to assess neuronal excitability. Membrane properties and spiking characteristics of layer II-III pyramidal cells were analyzed in both cortical regions. Significant sex-dependent and age-dependent differences were found in several parameters in the control groups. Considering membrane resistance, rheobase, voltage sag slope, and afterdepolarization slope, we observed notable changes mainly in the female groups. Valproate treatment seemed to enhance these differences and increase network excitability. However, it is possible that compensatory mechanisms took place during the maturation of the network while reaching the age-group of 3 months. Based on the results, the expression of the hyperpolarization-activated cyclic nucleotide-gated channels may be appreciably affected by the valproate treatment, which influences fundamental electrophysiological properties of the neurons such as the voltage sag. Remarkable changes appeared in the prefrontal cortex; however, also the entorhinal cortex shows similar tendencies.

Light-Driven Sodium Pump as a Potential Tool for the Control of Seizures in Epilepsy.

The marine flavobacterium Krokinobactereikastus light-driven sodium pump (KR2) generates an outward sodium ion current under 530nm light stimulation, representing a promising optogenetic tool for seizure control. However, the specifics of KR2 application to suppress epileptic activity have not yet been addressed. In the present study, we investigated the possibility of KR2 photostimulation to suppress epileptiform activity in mouse brain slices using the 4-aminopyrindine (4-AP) model. We injected the adeno-associated viral vector (AAV-PHP.eB-hSyn-KR2-YFP) containing the KR2 sodium pump gene enhanced with appropriate trafficking tags. KR2 expression was observed in the lateral entorhinal cortex and CA1 hippocampus. Using whole-cell patch clamp in mouse brain slices, we show that KR2, when stimulated with LED light, induces a substantial hyperpolarization of entorhinal neurons. However, continuous photostimulation of KR2 does not interrupt ictal discharges in mouse entorhinal cortex slices induced by a solution containing 4-AP. KR2-induced hyperpolarization strongly activates neuronal HCN channels. Consequently, turning off photostimulation resulted in HCN channel-mediated rebound depolarization accompanied by a transient increase in spontaneous network activity. Using low-frequency pulsed photostimulation, we induced the generation of short HCN channel-mediated discharges that occurred in response to the light stimulus being turned off; these discharges reliably interrupt ictal activity. Thus, low-frequency pulsed photostimulation of KR2 can be considered as a potential tool for controlling epileptic seizures.

Opioid-Induced Hyperalgesia and Tolerance Are Driven by HCN Ion Channels.

Prolonged exposure to opioids causes an enhanced sensitivity to painful stimuli (opioid-induced hyperalgesia, OIH) and a need for increased opioid doses to maintain analgesia (opioid-induced tolerance, OIT), but the mechanisms underlying both processes remain obscure. We found that pharmacological block or genetic deletion of HCN2 ion channels in primary nociceptive neurons of male mice completely abolished OIH but had no effect on OIT. Conversely, pharmacological inhibition of central HCN channels alleviated OIT but had no effect on OIH. Expression of C-FOS, a marker of neuronal activity, was increased in second-order neurons of the dorsal spinal cord by induction of OIH, and the increase was prevented by peripheral block or genetic deletion of HCN2, but block of OIT by spinal block of HCN channels had no impact on C-FOS expression in dorsal horn neurons. Collectively, these observations show that OIH is driven by HCN2 ion channels in peripheral nociceptors, while OIT is driven by a member of the HCN family located in the CNS. Induction of OIH increased cAMP in nociceptive neurons, and a consequent shift in the activation curve of HCN2 caused an increase in nociceptor firing. The shift in HCN2 was caused by expression of a constitutively active μ-opioid receptor (MOR) and was reversed by MOR antagonists. We identified the opioid-induced MOR as a six-transmembrane splice variant, and we show that it increases cAMP by coupling constitutively to Gs HCN2 ion channels therefore drive OIH, and likely OIT, and may be a novel therapeutic target for the treatment of addiction.

Coexistence within one cell of microvillous and ciliary phototransductions across M1- through M6-IpRGCs.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) serve as primary photoceptors by expressing the photopigment, melanopsin, and also as retinal relay neurons for rod and cone signals en route to the brain, in both cases for the purpose of non-image vision as well as aspects of image vision. So far, six subtypes of ipRGCs (M1 through M6) have been characterized. Regarding their phototransduction mechanisms, we have previously found that, unconventionally, rhabdomeric (microvillous) and ciliary signaling motifs co-exist within a given M1-, M2-, and M4-ipRGC, with the first mechanism involving PLCβ4 and TRPC6,7 channels and the second involving cAMP and HCN channels. We have now examined M3-, M5-, and M6-cells and found that each cell likewise uses both signaling pathways for phototransduction, despite differences in the percentage representation by each pathway in a given ipRGC subtype for bright-flash responses (and saturated except for M6-cells). Generally, M3- and M5-cells show responses quite similar in kinetics to M2-responses, and M6-cell responses resemble broadly those of M1-cells although much lower in absolute sensitivity and amplitude. Therefore, similar to rod and cone subtypes in image vision, ipRGC subtypes possess the same phototransduction mechanism(s) even though they do not show microvilli or cilia morphologically.

The enigmatic HCN channels: A cellular neurophysiology perspective.

What physiological role does a slow hyperpolarization-activated ion channel with mixed cation selectivity play in the fast world of neuronal action potentials that are driven by depolarization? That puzzling question has piqued the curiosity of physiology enthusiasts about the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are widely expressed across the body and especially in neurons. In this review, we emphasize the need to assess HCN channels from the perspective of how they respond to time-varying signals, while also accounting for their interactions with other co-expressing channels and receptors. First, we illustrate how the unique structural and functional characteristics of HCN channels allow them to mediate a slow negative feedback loop in the neurons that they express in. We present the several physiological implications of this negative feedback loop to neuronal response characteristics including neuronal gain, voltage sag and rebound, temporal summation, membrane potential resonance, inductive phase lead, spike triggered average, and coincidence detection. Next, we argue that the overall impact of HCN channels on neuronal physiology critically relies on their interactions with other co-expressing channels and receptors. Interactions with other channels allow HCN channels to mediate intrinsic oscillations, earning them the "pacemaker channel" moniker, and to regulate spike frequency adaptation, plateau potentials, neurotransmitter release from presynaptic terminals, and spike initiation at the axonal initial segment. We also explore the impact of spatially non-homogeneous subcellular distributions of HCN channels in different neuronal subtypes and their interactions with other channels and receptors. Finally, we discuss how plasticity in HCN channels is widely prevalent and can mediate different encoding, homeostatic, and neuroprotective functions in a neuron. In summary, we argue that HCN channels form an important class of channels that mediate a diversity of neuronal functions owing to their unique gating kinetics that made them a puzzle in the first place.

CNSC-43. TARGETING NEURONAL EXCITABILITY FOR NF1 LOW-GRADE GLIOMA THERAPY

Abstract While neuronal activity has previously been implicated in malignant glioma growth control, we recently demonstrated that neuron excitability can also regulate low-grade (optic) glioma initiation and progression. In this prior study, we showed that NF1 germline mutation-dependent neuronal excitability sustains optic glioma growth through HCN channel-mediated midkine activation of a T cell-microglia stromal circuit. Relevant to clinical translation, lamotrigine modulation of HCN channel function reduced neuronal hyperexcitability, midkine production and optic glioma (OPG) growth. To establish an obligate role for neuronal activity and midkine (Mdk) production in OPG growth, we leveraged a series of unique genetically engineered mouse models. First, we demonstrate that midkine is necessary for gliomagenesis, as Nf1-OPG; Mdk-/- mice do not develop tumors and have restored retinal pathology, as evidenced by increased retinal ganglion cell (RGC) numbers and retinal nerve fiber layer thickness. Second, we show that midkine production and neuronal hyperactivity are interdependent, such that only tumor-associated germline Nf1 mutations result in increased RGC excitability and midkine production, whereas those mutations that do not cause optic gliomas lack RGC hyperexcitability or elevated midkine expression. Third, lamotrigine treatment both during and after Nf1-OPG development attenuates midkine levels and tumor proliferation. Moreover, this anti-tumoral effect is durable for months after the cessation of treatment. Fourth, oral treatment of Nf1-OPG-bearing mice with clinically relevant doses of lamotrigine (2.5-7.5mg/kg/day) during OPG development blocks tumor formation. Together, these data establish midkine and neuronal hyperexcitability as key drivers of Nf1 optic glioma growth, relevant to planned clinical trials of this anti-seizure medication for children with NF1-OPG.

Melanopsin activates divergent phototransduction pathways in intrinsically photosensitive retinal ganglion cell subtypes.

Melanopsin signaling within intrinsically photosensitive retinal ganglion cell (ipRGC) subtypes impacts a broad range of behaviors from circadian photoentrainment to conscious visual perception. Yet, how melanopsin phototransduction within M1-M6 ipRGC subtypes impacts cellular signaling to drive diverse behaviors is still largely unresolved. The identity of the phototransduction channels in each subtype is key to understanding this central question but has remained controversial. In this study, we resolve two opposing models of M4 phototransduction, demonstrating that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dispensable for this process and providing support for a pathway involving melanopsin-dependent potassium channel closure and canonical transient receptor potential (TRPC) channel opening. Surprisingly, we find that HCN channels are likewise dispensable for M2 phototransduction, contradicting the current model. We instead show that M2 phototransduction requires TRPC channels in conjunction with T-type voltage-gated calcium channels, identifying a novel melanopsin phototransduction target. Collectively, this work resolves key discrepancies in our understanding of ipRGC phototransduction pathways in multiple subtypes and adds to mounting evidence that ipRGC subtypes employ diverse phototransduction cascades to fine-tune cellular responses for downstream behaviors.

Interaction Between HCN and Slack Channels Regulates mPFC Pyramidal Cell Excitability in Working Memory Circuits.

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.

Localization of PDE4D, HCN1 channels, and mGluR3 in rhesus macaque entorhinal cortex may confer vulnerability in Alzheimer's disease.

Alzheimer's disease cortical tau pathology initiates in the layer II cell clusters of entorhinal cortex, but it is not known why these specific neurons are so vulnerable. Aging macaques exhibit the same qualitative pattern of tau pathology as humans, including initial pathology in layer II entorhinal cortex clusters, and thus can inform etiological factors driving selective vulnerability. Macaque data have already shown that susceptible neurons in dorsolateral prefrontal cortex express a "signature of flexibility" near glutamate synapses on spines, where cAMP-PKA magnification of calcium signaling opens nearby potassium and hyperpolarization-activated cyclic nucleotide-gated channels to dynamically alter synapse strength. This process is regulated by PDE4A/D, mGluR3, and calbindin, to prevent toxic calcium actions; regulatory actions that are lost with age/inflammation, leading to tau phosphorylation. The current study examined whether a similar "signature of flexibility" expresses in layer II entorhinal cortex, investigating the localization of PDE4D, mGluR3, and HCN1 channels. Results showed a similar pattern to dorsolateral prefrontal cortex, with PDE4D and mGluR3 positioned to regulate internal calcium release near glutamate synapses, and HCN1 channels concentrated on spines. As layer II entorhinal cortex stellate cells do not express calbindin, even when young, they may be particularly vulnerable to magnified calcium actions and ensuing tau pathology.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated cation channel attenuates cerebral ischemia reperfusion-induced impairment of learning and memory by regulating apoptotic pathway.

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.

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.