Hyperpolarization-activated Cyclic Nucleotide-gated Cation Channels Research Articles

Pacemaker Channels and the Chronotropic Response in Health and Disease

Loss or dysregulation of the normally precise control of heart rate via the autonomic nervous system plays a critical role during the development and progression of cardiovascular disease—including ischemic heart disease, heart failure, and arrhythmias. While the clinical significance of regulating changes in heart rate, known as the chronotropic effect, is undeniable, the mechanisms controlling these changes remain not fully understood. Heart rate acceleration and deceleration are mediated by increasing or decreasing the spontaneous firing rate of pacemaker cells in the sinoatrial node. During the transition from rest to activity, sympathetic neurons stimulate these cells by activating β-adrenergic receptors and increasing intracellular cyclic adenosine monophosphate. The same signal transduction pathway is targeted by positive chronotropic drugs such as norepinephrine and dobutamine, which are used in the treatment of cardiogenic shock and severe heart failure. The cyclic adenosine monophosphate–sensitive hyperpolarization–activated current (I f ) in pacemaker cells is passed by hyperpolarization-activated cyclic nucleotide-gated cation channels and is critical for generating the autonomous heartbeat. In addition, this current has been suggested to play a central role in the chronotropic effect. Recent studies demonstrate that cyclic adenosine monophosphate–dependent regulation of HCN4 (hyperpolarization-activated cyclic nucleotide-gated cation channel isoform 4) acts to stabilize the heart rate, particularly during rapid rate transitions induced by the autonomic nervous system. The mechanism is based on creating a balance between firing and recently discovered nonfiring pacemaker cells in the sinoatrial node. In this way, hyperpolarization-activated cyclic nucleotide-gated cation channels may protect the heart from sinoatrial node dysfunction, secondary arrhythmia of the atria, and potentially fatal tachyarrhythmia of the ventricles. Here, we review the latest findings on sinoatrial node automaticity and discuss the physiological and pathophysiological role of HCN pacemaker channels in the chronotropic response and beyond.

Alterations in HCN1 expression and distribution during epileptogenesis in rats

BackgroundThe hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN1) is predominantly located in key regions associated with epilepsy, such as the neocortex and hippocampus. Under normal physiological conditions, HCN1 plays a crucial role in the excitatory and inhibitory regulation of neuronal networks. In temporal lobe epilepsy, the expression of HCN1 is decreased in the hippocampi of both animal models and patients. However, whether HCN1 expression changes during epileptogenesis preceding spontaneous seizures remains unclear. ObjectiveThe aim of this study was to determine whether the expression of HCN1 is altered during the epileptic prodromal phase, thereby providing evidence for its role in epileptogenesis. MethodsWe utilized a cobalt wire-induced rat epilepsy model to observe changes in HCN1 during epileptogenesis and epilepsy. Additionally, we also compared HCN1 alterations in epileptogenic tissues between cobalt wire- and pilocarpine-induced epilepsy rat models. Long-term video EEG recordings were used to confirm seizures development. Transcriptional changes, translation, and distribution of HCN1 were assessed using high-throughput transcriptome sequencing, total protein extraction, membrane and cytoplasmic protein fractionation, western blotting, immunohistochemistry, and immunofluorescence techniques. ResultsIn the cobalt wire-induced rat epilepsy model during the epileptogenesis phase, total HCN1 mRNA and protein levels were downregulated. Specifically, the membrane expression of HCN1 was decreased, whereas cytoplasmic HCN1 expression showed no significant change. The distribution of HCN1 in the distal dendrites of neurons decreased. During the epilepsy period, similar HCN1 alterations were observed in the neocortex of rats with cobalt wire-induced epilepsy and hippocampus of rats with lithium pilocarpine-induced epilepsy, including downregulation of mRNA levels, decreased total protein expression, decreased membrane expression, and decreased distal dendrite expression. ConclusionsAlterations in HCN1 expression and distribution are involved in epileptogenesis beyond their association with seizure occurrence. Similarities in HCN1 alterations observed in epileptogenesis-related tissues from different models suggest a shared pathophysiological pathway in epileptogenesis involving HCN1 dysregulation. Therefore, the upregulation of HCN1 expression in neurons, maintenance of the HCN1 membrane, and distal dendrite distribution in neurons may represent promising disease-modifying strategies in epilepsy.

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.

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.

The impact of HCN4 channels on CNS brain networks as a new target in pain development.

While it is well established that the isoform 2 of the hyperpolarization-activated cyclic nucleotide-gated cation channel (HCN2) plays an important role in the development and maintenance of pain, the role of the closely related HCN4 isoform in the processing of nociceptive signals is not known. HCN4 channels are highly expressed in the thalamus, a region important for stimulus transmission and information processing. We used a brain-specific HCN4-knockout mouse line (HCN4-KO) to explore the role of HCN4 channels in acute nociceptive processing using several behavioral tests as well as a multimodal magnetic resonance imaging (MRI) approach. Functional MRI (fMRI) brain responses were measured during acute peripheral thermal stimulation complemented by resting state (RS) before and after stimulation. The data were analyzed by conventional and graph-theoretical approaches. Finally, high-resolution anatomical brain data were acquired. HCN4-KO animals showed a central thermal, but not a mechanical hypersensitivity in behavioral experiments. The open field analysis showed no significant differences in motor readouts between HCN4-KO and controls but uncovered increased anxiety in the HCN4-KO mice. Thermal stimulus-driven fMRI (s-fMRI) data revealed increased response volumes and response amplitudes for HCN4-KO, most pronounced at lower stimulation temperatures in the subcortical input, the amygdala as well as in limbic/hippocampal regions, and in the cerebellum. These findings could be cross-validated by graph-theoretical analyses. Assessment of short-term RS before and after thermal stimulation revealed that stimulation-related modulations of the functional connectivity only occurred in control animals. This was consistent with the finding that the hippocampus was found to be smaller in HCN4-KO. In summary, the deletion of HCN4 channels impacts on processing of acute nociception, which is remarkably manifested as a thermal hypersensitive phenotype. This was mediated by the key regions hypothalamus, somatosensory cortex, cerebellum and the amygdala. As consequence, HCN4-KO mice were more anxious, and their brain-wide RS functional connectivity could not be modulated by thermal nociceptive stimulation.

Activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in a chronic migraine rat model

BackgroundCentral sensitization has been widely accepted as an underlying pathophysiological mechanism of chronic migraine (CM), activation of cannabinoid type-1 receptor (CB1R) exerts antinociceptive effects by relieving central sensitization in many pain models. However, the role of CB1R in the central sensitization of CM is still unclear.MethodsA CM model was established by infusing inflammatory soup (IS) into the dura of male Wistar rats for 7 days, and hyperalgesia was assessed by the mechanical and thermal thresholds. In the periaqueductal gray (PAG), the mRNA and protein levels of CB1R and hyperpolarization-activated cyclic nucleotide-gated cation channel 2 (HCN2) were measured by qRT–PCR and western blotting. After intraventricular injection of Noladin ether (NE) (a CB1R agonist), ZD 7288 (an HCN2 blocker), and AM 251 (a CB1R antagonist), the expression of tyrosine phosphorylation of N-methyl-D-aspartate receptor subtype 2B (pNR2B), calcium-calmodulin-dependent kinase II (CaMKII), and phosphorylated cAMP-responsive element binding protein (pCREB) was detected, and central sensitization was evaluated by the expression of calcitonin gene-related peptide (CGRP), c-Fos, and substance P (SP). Synaptic-associated protein (postsynaptic density protein 95 (PSD95) and synaptophysin (Syp)) and synaptic ultrastructure were detected to explore synaptic plasticity in central sensitization.ResultsWe observed that the mRNA and protein levels of CB1R and HCN2 were both significantly increased in the PAG of CM rats. The application of NE or ZD 7288 ameliorated IS-induced hyperalgesia; repressed the pNR2B/CaMKII/pCREB pathway; reduced CGRP, c-Fos, SP, PSD95, and Syp expression; and inhibited synaptic transmission. Strikingly, the application of ZD 7288 relieved AM 251-evoked elevation of pNR2B, CGRP, and c-Fos expression.ConclusionsThese data reveal that activation of CB1R alleviates central sensitization by regulating HCN2-pNR2B signaling in CM rats. The activation of CB1R might have a positive influence on the prevention of CM by mitigating central sensitization.

Characterization of Kv1.2-mediated outward current in TRIP8b-deficient mice.

Tonic current through hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels is influencing neuronal firing properties and channel function is strongly influenced by the brain-specific auxiliary subunit tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). Since Kv1.2 channels and TRIP8b were also suggested to interact, we assessed brain Kv1.2 mRNA and protein expression as well as the reduction of K+ outward currents by Kv1.2-blocking compounds (Psora-4; tityustoxin-Kα, TsTX-Kα) in different brain areas of TRIP8b-deficient (TRIP8b -/- ) compared to wildtype (WT) mice. We found that transcription levels of Kv1.2 channels were not different between genotypes. Furthermore, Kv1.2 current amplitude was not affected upon co-expression with TRIP8b in oocytes. However, Kv1.2 immunofluorescence was stronger in dendritic areas of cortical and hippocampal neurons. Furthermore, the peak net outward current was increased and the inactivation of the Psora-4-sensitive current component was less pronounced in cortical neurons in TRIP8b -/- mice. In current clamp recordings, application of TsTX increased the excitability of thalamocortical (TC) neurons with increased number of elicited action potentials upon step depolarization. We conclude that TRIP8b may not preferentially influence the amplitude of current through Kv1.2 channels but seems to affect current inactivation and channel localization. In TRIP8b -/- a compensatory upregulation of other Kv channels was observed.

HCN channels at the cell soma ensure the rapid electrical reactivity of fast-spiking interneurons in human neocortex

Accumulating evidence indicates that there are substantial species differences in the properties of mammalian neurons, yet theories on circuit activity and information processing in the human brain are based heavily on results obtained from rodents and other experimental animals. This knowledge gap may be particularly important for understanding the neocortex, the brain area responsible for the most complex neuronal operations and showing the greatest evolutionary divergence. Here, we examined differences in the electrophysiological properties of human and mouse fast-spiking GABAergic basket cells, among the most abundant inhibitory interneurons in cortex. Analyses of membrane potential responses to current input, pharmacologically isolated somatic leak currents, isolated soma outside-out patch recordings, and immunohistochemical staining revealed that human neocortical basket cells abundantly express hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel isoforms HCN1 and HCN2 at the cell soma membrane, whereas these channels are sparse at the rodent basket cell soma membrane. Antagonist experiments showed that HCN channels in human neurons contribute to the resting membrane potential and cell excitability at the cell soma, accelerate somatic membrane potential kinetics, and shorten the lag between excitatory postsynaptic potentials and action potential generation. These effects are important because the soma of human fast-spiking neurons without HCN channels exhibit low persistent ion leak and slow membrane potential kinetics, compared with mouse fast-spiking neurons. HCN channels speed up human cell membrane potential kinetics and help attain an input-output rate close to that of rodent cells. Computational modeling demonstrated that HCN channel activity at the human fast-spiking cell soma membrane is sufficient to accelerate the input-output function as observed in cell recordings. Thus, human and mouse fast-spiking neurons exhibit functionally significant differences in ion channel composition at the cell soma membrane to set the speed and fidelity of their input-output function. These HCN channels ensure fast electrical reactivity of fast-spiking cells in human neocortex.

Bi-directional modulation of hyperpolarization-activated cation currents (Ih) by ethanol in rat hippocampal CA3 pyramidal neurons

It is widely acknowledged that ethanol (EtOH) can alter many neuronal functions, including synaptic signaling, firing discharge, and membrane excitability, through its interaction with multiple membrane proteins and intracellular pathways. Previous work has demonstrated that EtOH enhances the firing rate of hippocampal GABAergic interneurons and thus the presynaptic GABA release at CA1 and CA3 inhibitory synapses through a positive modulation of the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels. Activation of HCN channels produce an inward current, commonly called Ih, which plays an essential role in generating/regulating specific neuronal activities in GABAergic interneurons and principal glutamatergic pyramidal neurons such as those in the CA3 subregion. Since the direct effect of EtOH on HCN channels expressed in CA3 pyramidal neurons was not thoroughly elucidated, we investigated the possible interaction between EtOH and HCN channels and the impact on excitability and postsynaptic integration of these neurons. Patch-clamp recordings were performed in single CA3 pyramidal neurons from acute male rat coronal hippocampal slices. Our results show that EtOH modulates HCN-mediated Ih in a concentration-dependent and bi-directional manner, with a positive modulation at lower (20 mM) and an inhibitory action at higher (60–80 mM) concentrations. The modulation of Ih by EtOH was mimicked by forskolin, antagonized by different drugs that selectively interfere with the AC/cAMP/PKA intracellular pathway, as well as by the selective HCN inhibitor ZD7288. Altogether, these data further support the evidence that HCN channels may represent an important molecular target through which EtOH may regulate neuronal activity.

Activation of metabotropic glutamate receptor 1 regulates hippocampal CA1 region excitability in rats with status epilepticus by suppressing the HCN1 channel.

Dysregulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels alters neuronal excitability. However, the role of HCN channels in status epilepticus is not fully understood. In this study, we established rat models of pentylenetetrazole-induced status epilepticus. We performed western blot assays and immunofluorescence staining. Our results showed that HCN1 channel protein expression, particularly HCN1 surface protein, was significantly decreased in the hippocampal CA1 region, whereas the expression of HCN2 channel protein was unchanged. Moreover, metabolic glutamate receptor 1 (mGluR1) protein expression was increased after status epilepticus. The mGluR1 agonist (RS)-3,5-dihydroxyphenylglycine injected intracerebroventricularly increased the sensitivity and severity of pentylenetetrazole-induced status epilepticus, whereas application of the mGluR1 antagonist (+)-2-methyl-4-carboxyphenylglycine (LY367385) alleviated the severity of pentylenetetrazole-induced status epilepticus. The results from double immunofluorescence labeling revealed that mGluR1 and HCN1 were co-localized in the CA1 region. Subsequently, a protein kinase A inhibitor (H89) administered intraperitoneally successfully reversed HCN1 channel inhibition, thereby suppressing the severity and prolonging the latency of pentylenetetrazole-induced status epilepticus. Furthermore, H89 reduced the level of mGluR1, downregulated cyclic adenosine monophosphate (cAMP)/protein kinase A expression, significantly increased tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) (1a-4) expression, and restored TRIP8b (1b-2) levels. TRIP8b (1a-4) and TRIP8b (1b-2) are subunits of Rab8b interacting protein that regulate HCN1 surface protein.

SHOX2 refines the identification of human sinoatrial nodal cell population in the invitro cardiac differentiation.

IntroductionDysfunction of the sinoatrial node (SAN) cells causes arrhythmias, and many patients require artificial cardiac pacemaker implantation. However, the mechanism of impaired SAN automaticity remains unknown, and the generation of human SAN cells in vitro may provide a platform for understanding the pathogenesis of SAN dysfunction. The short stature homeobox 2 (SHOX2) and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) genes are specifically expressed in SAN cells and are important for SAN development and automaticity. In this study, we aimed to purify and characterize human SAN-like cells in vitro, using HCN4 and SHOX2 as SAN markers.MethodsWe developed an HCN4-EGFP/SHOX2-mCherry dual reporter cell line derived from human induced pluripotent stem cells (hiPSCs), and HCN4 and SHOX2 gene expressions were visualized using the fluorescent proteins EGFP and mCherry, respectively. The dual reporter cell line was established using an HCN4-EGFP bacterial artificial chromosome-based semi-knock-in system and a CRISPR-Cas9-dependent knock-in system with a SHOX2-mCherry targeting vector. Flow cytometry, RT-PCR, and whole-cell patch-clamp analyses were performed to identify SAN-like cells.ResultsFlow cytometry analysis and cell sorting isolated HCN4-EGFP single-positive (HCN4+/SHOX2-) and HCN4-EGFP/SHOX2-mCherry double-positive (HCN4+/SHOX2+) cells. RT-PCR analyses showed that SAN-related genes were enriched within the HCN4+/SHOX2+ cells. Further, electrophysiological analyses showed that approximately 70% of the HCN4+/SHOX2+ cells exhibited SAN-like electrophysiological characteristics, as defined by the action potential parameters of the maximum upstroke velocity and action potential duration.ConclusionsThe HCN4-EGFP/SHOX2-mCherry dual reporter hiPSC system developed in this study enabled the enrichment of SAN-like cells within a mixed HCN4+/SHOX2+ population of differentiating cardiac cells. This novel cell line is useful for the further enrichment of human SAN-like cells. It may contribute to regenerative medicine, for example, biological pacemakers, as well as testing for cardiotoxic and chronotropic actions of novel drug candidates.

Adenosine Downregulates the Activities of Glutamatergic Neurons in the Paraventricular Hypothalamic Nucleus Required for Sleep.

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

The Bradycardic Agent Ivabradine Acts as an Atypical Inhibitor of Voltage-Gated Sodium Channels.

Background and purpose: Ivabradine is clinically administered to lower the heart rate, proposedly by inhibiting hyperpolarization-activated cyclic nucleotide-gated cation channels in the sinoatrial node. Recent evidence suggests that voltage-gated sodium channels (VGSC) are inhibited within the same concentration range. VGSCs are expressed within the sinoatrial node and throughout the conduction system of the heart. A block of these channels thus likely contributes to the established and newly raised clinical indications of ivabradine. We, therefore, investigated the pharmacological action of ivabradine on VGSCs in sufficient detail in order to gain a better understanding of the pro- and anti-arrhythmic effects associated with the administration of this drug. Experimental Approach: Ivabradine was tested on VGSCs in native cardiomyocytes isolated from mouse ventricles and the His-Purkinje system and on human Nav1.5 in a heterologous expression system. We investigated the mechanism of channel inhibition by determining its voltage-, frequency-, state-, and temperature-dependence, complemented by a molecular drug docking to the recent Nav1.5 cryoEM structure. Automated patch-clamp experiments were used to investigate ivabradine-mediated changes in Nav1.5 inactivation parameters and inhibition of different VGSC isoforms. Key results: Ivabradine inhibited VGSCs in a voltage- and frequency-dependent manner, but did not alter voltage-dependence of activation and fast inactivation, nor recovery from fast inactivation. Cardiac (Nav1.5), neuronal (Nav1.2), and skeletal muscle (Nav1.4) VGSC isoforms were inhibited by ivabradine within the same concentration range, as were sodium currents in native cardiomyocytes isolated from the ventricles and the His-Purkinje system. Molecular drug docking suggested an interaction of ivabradine with the classical local anesthetic binding site. Conclusion and Implications: Ivabradine acts as an atypical inhibitor of VGSCs. Inhibition of VGSCs likely contributes to the heart rate lowering effect of ivabradine, in particular at higher stimulation frequencies and depolarized membrane potentials, and to the observed slowing of intra-cardiac conduction. Inhibition of VGSCs in native cardiomyocytes and across channel isoforms may provide a potential basis for the anti-arrhythmic potential as observed upon administration of ivabradine.

Role of the Dopamine Subtype 1 Receptor (D1R) Modulation of the Ih Current in Rhythmic Spinal Mammalian Motor Networks

Restless Legs Syndrome (RLS) is a chronic sensorimotor disorder that has been recently associated with decreased expression of inhibitory Gi‐coupled adenosine A1 receptors (A1Rs), leading to increased dopamine release (hyperdopaminergic signaling) within the central nervous system. Previous studies have shown that A1Rs specifically inhibit the Gs‐coupled dopamine D1 receptor (D1R) subtype through a functional A1R‐D1R heteromer receptor complex. Furthermore, D1Rs control spinal motor neuron (MN) excitability by increasing cyclic adenosine monophosphate (cAMP) concentration and opening hyperpolarization‐activated cyclic nucleotide‐gated non‐selective cation channel (HCN). This finding raises the possibility of increased HCN activity underlying some of the motor dysfunctions associated with periodic leg movements (PLMS) during sleep in RLS. Thus, this study aims to elucidate the importance of the D1 receptor subtype on modulating the hyperpolarization‐activated cation current (Ih) in spinal mammalian motor networks. I hypothesized that blocking Ih in the presence of a D1R agonist (enhanced dopaminergic modulation) would slow locomotor activity and disrupt MN bursting stability. Neonatal (P0‐P5; n = 7) mouse spinal cord preparations were used to measure MN bursting activity patterns including amplitude, duration, and cycle period of extracellular ventral root (L2 and L5) recordings via suction electrodes. Treatment conditions include serotonin (9‐15 μM 5‐HT) and N‐methyl‐D‐aspartate (6 μM NMDA) with and without a D1R agonist (10 μM SKF38393) to assess the effect of D1R on rhythmic bursting. Subsequently, an Ih blocker (1 μM ZD7288) was added to observe the importance of Ih in modulating MN burst activity. For both L2 and L5 roots, blocking the Ih current led to increases in burst amplitude, duration, and cycle period in respect to the control 5‐HT/NMDA condition. Our results propose that D1Rs play a significant role in modulating Ih by increasing cAMP levels and opening HCN1 channels at a more depolarized membrane potential. Future experiments involve: 1) using TM5 transmembrane disrupting peptide to disrupt A1R‐D1R heteromers in spinal MNs (and use TM7 as a negative control) to confirm if dopamine modulation of Ih activity is linked to the A1R‐D1R heteromer complexes and 2) applying adenyl cyclase activators and blockers to verify that the modulation of the Ih current is through the D1R‐mediated Gs second messenger pathway.

Probability that there is a mammalian counterpart of cardiac clock in insects.

Whether or not the hyperpolarization-activated cyclic nucleotide-gated nonselective cation channel (HCN or funny current If ) is involved in pacemaking - recurrent heartbeat, it is attributed to electrical activities in all excitable cells, including those of invertebrates. In latter group of animals prevailingly the electrical signals and function of heart in terms of chrono- and inotropy are elucidated. Although in simpler models including insects experimental outcomes are reproducible and robust, involvement of "cardiac clock" mechanism in pacemaking is not conclusive. In this assay, the mechanisms of heartbeat are synthesized by focused comparisons between insect and mammalian hearts.

Analgesic effect of ivabradine against inflammatory pain mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels expressed on primary afferent terminals in the spinal dorsal horn.

Ivabradine, a hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel blocker and clinically approved bradycardic agent, has analgesic effects against neuropathic pain. Although the expression of HCN channels in the spinal dorsal horn (SDH) is augmented under inflammatory pain, spinal responses to centrally and peripherally applied ivabradine remain poorly understood. We investigated the spinal action and cellular mechanisms underlying the drug's analgesic effects against inflammatory pain using inflammatory pain model rats. Intraperitoneal and intrathecal injections of ivabradine inhibited mechanical allodynia (6 rats/dose; P < 0.05), and immunohistochemical staining showed that ivabradine suppresses the phosphorylated extracellular signal-regulated kinase activation in the SDH (6 rats/group, P < 0.01). In vitro whole-cell patch-clamp and in vivo extracellular recordings showed that direct application of ivabradine to the spinal cord decreases the mean miniature excitatory postsynaptic currents' frequency (13 rats; P < 0.01), and direct and peripheral application of ivabradine suppresses the spinal response to mechanical stimulation-evoked firing (8 rats/group, P < 0.01). Moreover, ivabradine reduces the amplitudes of monosynaptic excitatory postsynaptic currents evoked by Aδ-fiber and C-fiber stimulation (6 rats; P < 0.01) and induces a stronger inhibition of those evoked by C-fiber stimulation. These phenomena were inhibited by forskolin, an activator of HCN channels. In conclusion, spinal responses mediated by HCN channels on primary afferent terminals are suppressed by central and peripheral administration of ivabradine; the drug also exhibits analgesic effects against inflammatory pain. In addition, ivabradine preferentially acts on C-fiber terminals of SDH neurons and induces a stronger inhibition of neuronal excitability in inflammatory pain.

Moxibustion Regulates Gastrointestinal Motility via HCN1 in Functional Dyspepsia Rats.

BackgroundMoxibustion therapy has been found to ameliorate clinical symptoms of functional dyspepsia (FD). We aimed to examine the regulatory effect of moxibustion on the gastrointestinal (GI) motility in FD and explore the underlying mechanism based on the hyperpolarization-activated cyclic nucleotide-gated cation channel 1 (HCN1).Material/MethodsMoxibustion therapy was used in FD rats induced by using classic tail-pinch and irregular feeding. Weight gain and food intake were recorded weekly, followed by detecting gastric residual rate (GRR) and small intestine propulsion rate (IPR). Next, western blotting was performed to determine the expression levels of HCN1 in the gastric antrum. qRT-PCR was used to detect HCN1 in the small intestine and hypothalamic satiety center. Double immunolabeling was used for HCN1 and ICCs in gastric antrum and small intestine.ResultsThe obtained results suggested that moxibustion treatment could increase weight gain and food intake in FD rats. The GRR and IPR were compared among the groups, which showed that moxibustion treatment could decrease GRR and increase IPR. Moxibustion increased the expression of HCN1 in the gastric antrum, small intestine, and hypothalamic satiety center. Histologically, the co-expressions of HCN1 and ICCs tended to increase in gastric antrum and small intestine. Meanwhile, HCN channel inhibitor ZD7288 prevented the above-mentioned therapeutic effects of moxibustion.ConclusionsThe results of the present study suggest that moxibustion can effectively improve the GI motility of FD rats, which may be related to the upregulation of HCN1 expression in gastric antrum, small intestine, and satiety center.

Hypoxia with inflammation and reperfusion alters membrane resistance by dynamically regulating voltage-gated potassium channels in hippocampal CA1 neurons

Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (IDR) can explain the input resistance variability. Furthermore, voltage-dependent inactivation of A-type potassium (IA) channels shifted in the depolarizing direction during Hypo-Inf and reverted to normal upon reperfusion without a significant alteration in the maximum current density. Our results indicate that changes in the input resistance, and consequently excitability, caused by Hypo-Inf and reperfusion are at least partially regulated by the availability and voltage dependence of KV channels. Moreover, these results suggest that selective KV channel modulators can be used as potential neuroprotective drugs to minimize hypoxia- and reperfusion-induced neuronal damage.

Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

Speeding Up the Heart? Traditional and New Perspectives on HCN4 Function.

The sinoatrial node (SAN) is the primary pacemaker of the heart and is responsible for generating the intrinsic heartbeat. Within the SAN, spontaneously active pacemaker cells initiate the electrical activity that causes the contraction of all cardiomyocytes. The firing rate of pacemaker cells depends on the slow diastolic depolarization (SDD) and determines the intrinsic heart rate (HR). To adapt cardiac output to varying physical demands, HR is regulated by the autonomic nervous system (ANS). The sympathetic and parasympathetic branches of the ANS innervate the SAN and regulate the firing rate of pacemaker cells by accelerating or decelerating SDD–a process well-known as the chronotropic effect. Although this process is of fundamental physiological relevance, it is still incompletely understood how it is mediated at the subcellular level. Over the past 20 years, most of the work to resolve the underlying cellular mechanisms has made use of genetically engineered mouse models. In this review, we focus on the findings from these mouse studies regarding the cellular mechanisms involved in the generation and regulation of the heartbeat, with particular focus on the highly debated role of the hyperpolarization-activated cyclic nucleotide-gated cation channel HCN4 in mediating the chronotropic effect. By focusing on experimental data obtained in mice and humans, but not in other species, we outline how findings obtained in mice relate to human physiology and pathophysiology and provide specific information on how dysfunction or loss of HCN4 channels leads to human SAN disease.