Hyperpolarization-activated Cyclic Nucleotide-gated Channel Currents Research Articles

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

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

Effects of Oxaliplatin on Facial Sensitivity to Cool Temperatures and TRPM8 Expressing Trigeminal Ganglion Neurons in Mice.

The chemotherapeutic agent oxaliplatin is commonly used to treat colorectal cancer. Although effective as a chemotherapeutic, it frequently produces painful peripheral neuropathies. These neuropathies can be divided into an acute sensitivity to cool temperatures in the mouth and face, and chronic neuropathic pain in the limbs and possible numbness. The chronic neuropathy also includes sensitivity to cool temperatures. Neurons that detect cool temperatures are reported to utilize Transient Receptor Potential Cation Channel, Subfamily M, Member 8 (TRPM8). Therefore, we investigated the effects of oxaliplatin on facial nociception to cool temperatures (18°C) in mice and on TRPM8 expressing trigeminal ganglion (TRG) neurons. Paclitaxel, a chemotherapeutic that is used to treat breast cancer, was included for comparison because it produces neuropathies, but acute cool temperature sensitivity in the oral cavity or face is not typically reported. Behavioral testing of facial sensitivity to 18°C indicated no hypersensitivity either acutely or chronically following either chemotherapeutic agent. However, whole cell voltage clamp experiments in TRPM8 expressing TRG neurons indicated that both oxaliplatin and paclitaxel increased Hyperpolarization-Activated Cyclic Nucleotide-Gated channel (HCN), voltage gated sodium channel (Nav), and menthol evoked TRPM8 currents. Voltage gated potassium channel (Kv) currents were not altered. Histological examination of TRPM8 fibers in the skin of the whisker pads demonstrated that the TRPM8 expressing axons and possible Merkel cell-neurite complexes were damaged by oxaliplatin. These findings indicate that oxaliplatin induces a rapid degeneration of TRG neuron axons that express TRPM8, which prevents evoked activation of the sensitized neurons and likely leads to reduced sensitivity to touch and cool temperatures. The changes in HCN, Nav, and TRPM8 currents suggest that spontaneous firing of action potentials may be increased in the deafferented neurons within the ganglion, possibly producing spontaneously induced cooling or nociceptive sensations.

Effects of Ih and TASK-like shunting current on dendritic impedance in layer 5 pyramidal-tract neurons.

Pyramidal neurons in neocortex have complex input-output relationships that depend on their morphologies, ion channel distributions, and the nature of their inputs, but which cannot be replicated by simple integrate-and-fire models. The impedance properties of their dendritic arbors, such as resonance and phase shift, shape neuronal responses to synaptic inputs and provide intraneuronal functional maps reflecting their intrinsic dynamics and excitability. Experimental studies of dendritic impedance have shown that neocortical pyramidal tract neurons exhibit distance-dependent changes in resonance and impedance phase with respect to the soma. We, therefore, investigated how well several biophysically detailed multicompartment models of neocortical layer 5 pyramidal tract neurons reproduce the location-dependent impedance profiles observed experimentally. Each model tested here exhibited location-dependent impedance profiles, but most captured either the observed impedance amplitude or phase, not both. The only model that captured features from both incorporates hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and a shunting current, such as that produced by Twik-related acid-sensitive K+ (TASK) channels. TASK-like channel density in this model was proportional to local HCN channel density. We found that although this shunting current alone is insufficient to produce resonance or realistic phase response, it modulates all features of dendritic impedance, including resonance frequencies, resonance strength, synchronous frequencies, and total inductive phase. We also explored how the interaction of HCN channel current (Ih) and a TASK-like shunting current shape synaptic potentials and produce degeneracy in dendritic impedance profiles, wherein different combinations of Ih and shunting current can produce the same impedance profile.NEW & NOTEWORTHY We simulated chirp current stimulation in the apical dendrites of 5 biophysically detailed multicompartment models of neocortical pyramidal tract neurons and found that a combination of HCN channels and TASK-like channels produced the best fit to experimental measurements of dendritic impedance. We then explored how HCN and TASK-like channels can shape the dendritic impedance as well as the voltage response to synaptic currents.

The hyperpolarization-activated cyclic nucleotide-gated channel currents contribute to oxaliplatin-induced hyperexcitability of DRG neurons

Humans are likely to experience mechanical allodynia and cold hyperalgesia after oxaliplatin intravenous injection. The mechanism by which oxaliplatin leads to these side effects is unknown. Since the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are involved in the automatic depolarization of action potentials, we speculated that HCN channels are involved in oxaliplatin-induced hyperalgesia through action potentials. Our results showed that the density of HCN channel currents and the excitability of dorsal root ganglion neurons both increased after oxaliplatin perfusion at the cellular level. The neuronal hyperexcitability could be alleviated by ivabradine. Ivabradine inhibited oxaliplatin-induced mechanical allodynia and cold hyperalgesia at the individual rat level. Oxaliplatin enhanced the function of HCN channels, which in turn promoted the automatic depolarization of action potentials. The acceleration of automatic depolarization excited the neurons and caused more rapid firing of action potentials. Therefore, the HCN channel is a potential therapeutic target for the hyperalgesia induced by oxaliplatin.

Effects of ghrelin on the electrical activities of substantia nigra dopaminergic neurons treated with MPP+

Ghrelin, a 28 amino acid brain-gut peptide, has attracted increasing attention for its neuroprotective effect in Parkinson's disease (PD). In view of the pivotal role of excitability of dopaminergic neurons in substantia nigra pars compacta (SNc) in the function of nigrostriatal system, it is of great significance to elucidate the regulation of electrical activity of dopaminergic neurons by ghrelin, especially in PD pathogenesis. In this study, we tackled this issue by probing the effects of ghrelin on the electrophysiological properties of dopaminergic neurons in acute application of Methyl-4-phenylpyridinium (MPP+), a potent parkinsonizing agent in primates and rodents, with whole cell patch clamp technique. We first observed that MPP+ (10, 20 and 50 μM) inhibited the spontaneous firing activity of dopaminergic neurons with dose-dependent and time-dependent properties. MPP+ also hyperpolarized the membrane potential, inhibited the evoked firing activity and reduced the amplitude of the inward rectification characteristic (Sag) in dopaminergic neurons. Importantly, ghrelin (100 nM) could improve the above effects of MPP+ on the electrical activities of dopaminergic neurons. The potential mechanism of this phenomenon may be that ghrelin upregulated hyperpolarization-activated cyclic nucleotide-gated channel current (Ih) to antagonize the inhibition of MPP+ on Ih, thereby improving the electrical activities of dopaminergic neurons.

Age-Dependent Up-Regulation of HCN Channels in Spiral Ganglion Neurons Coincide With Hearing Loss in Mice.

Age-related hearing loss (AHL) is the most common sensory disorder in the elderly population, and the etiologies are diverse. To understand the underlying mechanisms of AHL, one strategy is to identify correlates of the disease for comprehensive evaluation of treatment approaches. Dysfunction and degeneration of spiral ganglion neurons (SGNs) are major contributors to AHL. Previously, we showed that one of the changes in the aging auditory system is SGN excitability increase in mice. Since hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play important roles in determining neuronal excitability, we predicted that HCN channels in SGNs are involved in AHL. To investigate the contribution of HCN channels to AHL, we examined the expression and biophysical properties of HCN channels in SGNs from adult (2–3 months) and 11–12-month-old mice. We report a dramatic increase of HCN channel current (Ih) in SGNs in old mice (11–12 months old). The results matched well with increased expression of HCN1 and HCN2 subunits, suggesting that upregulation of HCN channels in SGNs is one of the important facets of the aging SGNs. Moreover, the activity of Ih produced a major impact on the firing properties of SGNs in older mice. The upregulation of Ih may contribute to AHL by regulating SGN excitability. We assessed whether increased SGNs excitability dovetail with neurodegeneration. Apoptosis-inducing factor (AIF)-mediated apoptosis in SGNs was observed in old mice and activation of HCN channels mediates AIF activation. Thus, these findings demonstrate stark correlation between age-dependent increased expression of HCN channels and Ih, and apoptosis in SGNs, which may contribute towards the varied mechanisms of AHL.

Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Currents in Neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are voltage-gated ion channels that activate at potentials more negative than -50 mV and are predominantly permeable to Na(+) and K(+) ions. Four HCN subunits (HCN1-4) have been cloned. These subunits have distinct expression patterns and biophysical properties. In addition, cyclic nucleotides as well as multiple intracellular substances including various kinases and phosphatases modulate the expression and function of the subunits. Hence, the characteristics of the current, Ih, are likely to vary among neuronal subtypes. In many neuronal subtypes, Ih is present postsynaptically, where it plays a critical role in setting the resting membrane potential and the membrane resistance. By influencing these intrinsic properties, Ih will affect synaptic potential shapes and summation and thereby affect neuronal excitability. Additionally, Ih can have an effect on resonance properties and intrinsic neuronal oscillations. In some neurons, Ih may also be present presynaptically in axons and synaptic terminals, where it modulates neuronal transmitter release. Hence the effects of Ih on neuronal excitability are complex. It is, however, necessary to fully understand these as Ih has a significant impact on physiological conditions such as learning as well as pathophysiological states such as epilepsy.

Reduced Hyperpolarization-Activated Current Contributes to Enhanced Intrinsic Excitability in Cultured Hippocampal Neurons from PrP(-/-) Mice.

Genetic ablation of cellular prion protein (PrPC) has been linked to increased neuronal excitability and synaptic activity in the hippocampus. We have previously shown that synaptic activity in hippocampi of PrP-null mice is increased due to enhanced N-methyl-D-aspartate receptor (NMDAR) function. Here, we focused on the effect of PRNP gene knock-out (KO) on intrinsic neuronal excitability, and in particular, the underlying ionic mechanism in hippocampal neurons cultured from P0 mouse pups. We found that the absence of PrPC profoundly affected the firing properties of cultured hippocampal neurons in the presence of synaptic blockers. The membrane impedance was greater in PrP-null neurons, and this difference was abolished by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker ZD7288 (100 μM). HCN channel activity appeared to be functionally regulated by PrPC. The amplitude of voltage sag, a characteristic of activating HCN channel current (Ih), was decreased in null mice. Moreover, Ih peak current was reduced, along with a hyperpolarizing shift in activation gating and slower kinetics. However, neither HCN1 nor HCN2 formed a biochemical complex with PrPC. These results suggest that the absence of PrP downregulates the activity of HCN channels through activation of a cell signaling pathway rather than through direct interactions. This in turn contributes to an increase in membrane impedance to potentiate neuronal excitability.

Increased excitability of medium-sized dorsal root ganglion neurons by prolonged interleukin-1β exposure is K(+) channel dependent and reversible.

Neuropathic pain resulting from peripheral nerve injury is initiated and maintained by persistent ectopic activity in primary afferent neurons. Sciatic nerve injury increases the excitability of medium-sized dorsal root ganglion (DRG) neurons. Levels of the inflammatory cytokine interleukin 1β (IL-1β) increase and peak after 7days. Five to six days of exposure of medium sized DRG neurons to 100pm IL-1β promotes persistent increases in excitability which abate within 3-4days of cytokine removal. This is associated with a profound attenuation of K(+) channel currents but only modest increases in function of cyclic nucleotide-sensitive hyperpolarization-activated channels (HCNs) and of voltage-gated Na(+) and Ca(2+) channel currents. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons is capable of initiating an enduring phenotypic shift in their electrophysiological properties that follows sciatic nerve injury. The findings also underline the importance of K(+) channel modulation in the actions of inflammatory mediators on peripheral neurons. Chronic constriction injury of rat sciatic nerve promotes signs of neuropathic pain. This is associated with an increase in the level of interleukin 1β (IL-1β) in primary afferents that peaks at 7days. This initial cytokine exposure has been proposed to trigger an enduring alteration in neuronal phenotype that underlies chronic hyper-excitability in sensory nerves, which initiates and maintains chronic neuropathic pain. We have shown previously that 5-6days of exposure of rat dorsal root ganglia (DRGs) to 100pm IL-1β increases the excitability of medium-sized neurons. We have now found using whole-cell recording that this increased excitability reverts to control levels within 3-4days of cytokine removal. The effects of IL-1β were dominated by changes in K(+) currents. Thus, the amplitudes of A-current, delayed rectifier and Ca(2+) -sensitive K(+) currents were reduced by ∼68%, ∼64% and ∼36%, respectively. Effects of IL-1β on other cation currents were modest by comparison. There was thus a slight decrease in availability of high voltage-activated Ca(2+) channel current, a small increase in rates of activation of hyperpolarization-activated cyclic nucleotide-gated channel current (IH ), and a shift in the voltage dependence of activation of tetrodotoxin-sensitive sodium current (TTX-S INa ) to more negative potentials. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons initiates an enduring phenotypic shift in their electrophysiological properties following sciatic nerve injury. Persistent increases in primary afferent excitability following nerve injury may instead depend on altered K(+) channel function and on the continued presence of slightly elevated levels IL-1β and other cytokines.

Hyperpolarization-activated cyclic nucleotide-gated channels may contribute to regional anesthetic effects of lidocaine.

Local anesthetics (e.g., lidocaine) have been found to inhibit hyperpolarization-activated cyclic nucleotide-gated (HCN) channels besides sodium channels. However, the exact role of HCN channels in regional anesthesia in vivo is still elusive. Sciatic nerve block and intrathecal anesthesia were performed using lidocaine in wild-type and HCN1 channel knockout (HCN1) mice. EC50 of lidocaine and durations of 1% lidocaine were determined. In electrophysiologic recordings, effects of lidocaine on HCN channel currents, voltage-gated sodium channel currents, and neural membrane properties were recorded on dorsal root ganglia neurons. In both sciatic nerve block and intrathecal anesthesia, EC50 of lidocaine for tactile sensory blockade (2 g von Frey fiber) was significantly increased in HCN1 mice, whereas EC50 of lidocaine for pinprick blockade was unaffected. Durations of 1% lidocaine were significantly shorter in HCN1 mice for both sciatic nerve block and intrathecal anesthesia (n = 10). ZD7288 (HCN blocker) could significantly prolong durations of 1% lidocaine including pinprick blockade in sciatic nerve block (n = 10). Forskolin (raising cyclic adenosine monophosphate to enhance HCN2) could significantly shorten duration of pinprick blockade of 1% lidocaine in sciatic nerve block (n = 10). In electrophysiologic recordings, lidocaine could nonselectively inhibit HCN channel and sodium channel currents both in large and in small dorsal root ganglia neurons (n = 5 to 6). Meanwhile, lidocaine caused neural membrane hyperpolarization and increased input resistance of dorsal root ganglia neurons but not in large dorsal root ganglia neurons from HCN1 mice (n = 5-7). These data indicate that HCN channels may contribute to regional anesthetic effects of lidocaine. By inhibiting HCN channels, lidocaine could alter membrane properties of neurons.

Stress Impairs Prefrontal Cortical Function via D1 Dopamine Receptor Interactions With Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels

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

Novel Role of KT5720 on Regulating Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel Activity and Dorsal Root Ganglion Neuron Excitability

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in dorsal root ganglion (DRG) neurons, which are involved in diverse mechanisms that regulate DRG functions. Protein kinase A (PKA) is an essential kinase that plays a key role in almost all types of cells; it regulates the ion channel activity, the intracellular Ca(2+) concentration, as well as modulates cellular signals transduction. Nevertheless, the effect of PKA inhibition on the HCN channel activity in DRG neuron remains to be elucidated. Here we investigated the impact of PKA inhibition on the HCN channel activity and DRG neurons excitability. Our patch-clamp experiments both under whole-cell and single-channel conditions demonstrated that PKA inhibition with KT5720, a cell membrane permeable PKA-specific inhibitor, significantly attenuated HCN channel currents. Current clamp recording on freshly isolated DRG neurons showed KT5720 reduced overshoot amplitude and enhanced the threshold of the action potential. Moreover, our live-cell Ca(2+) imaging experiments illustrated KT5720 markedly reduced the intracellular Ca(2+) level. Collectively, this is the first report that addresses KT5720 attenuates the HCN channel activity and intracellular Ca(2+), thus reducing DRG neurons excitability. Therefore, our data strongly suggest that PKA is a potential target for curing HCN and DRG neuron relevant diseases.

Is ZD7288 a selective blocker of hyperpolarization-activated cyclic nucleotide-gated channel currents?

ZD7288 has been widely used as a tool in the study of hyperpolarization-activated cyclic nucleotide-gated channels (HCN channels), and to test the relationships between HCN channels and heart and brain function. ZD7288 is widely considered a selective blocker of HCN currents. Here we show that ZD7288 inhibits not only HCN channel currents, but also Na+ currents in DRG neurons and ZD7288 was confirmed to inhibit Na+ current in HEK293 cells transfected with Nav1.4 plasmids. Thus our findings challenge the view that ZD7288 is a selective blocker of HCN channels. Conclusions about the role of NCN channels in neuronal function should be re-evaluated if based exclusively on the effect of ZD7288.

A Novel Carbamoyloxy Arylalkanoyl Arylpiperazine Compound (SKL-NP) Inhibits Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channel Currents in Rat Dorsal Root Ganglion Neurons

In this study, we determined mode of action of a novel carbamoyloxy arylalkanoyl arylpiperazine compound (SKL-NP) on hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents (Ih) that plays important roles in neuropathic pain. In small or medium-sized dorsal root ganglion (DRG) neurons (<40 µm in diameter) exhibiting tonic firing and prominent Ih, SKL-NP inhibited Ih and spike firings in a concentration dependent manner (IC50=7.85 µM). SKL-NP-induced inhibition of Ih was blocked by pretreatment of pertussis toxin (PTX) and N-ethylmaleimide (NEM) as well as 8-Br-cAMP, a membrane permeable cAMP analogue. These results suggest that SKL-NP modulates Ih in indirect manner by the activation of a Gi-protein coupled receptor that decreases intracellular cAMP concentration. Taken together, SKL-NP has the inhibitory effect on HCN channel currents (Ih) in DRG neurons of rats.