Hyperpolarization-activated Cyclic Nucleotide-gated Research Articles

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.

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.

The Potential Antidepressant Compound Org 34167 Modulates HCN Channels Via a Novel Mode of Action.

Org 34167 is a small molecule hyperpolarization-activated cyclic nucleotide-gated (HCN) channel modulator that has been trialed in humans for its potential antidepressant activity. The precise action of Org 34167 is not fully understood. Here we use two-electrode voltage clamp recordings and an allosteric model to explore the interaction of Org 34167 with human HCN1 channels. The impact of Org 34167 on channel function included a hyperpolarizing shift in activation voltage dependence and a slowing of activation kinetics. Furthermore, a reduction in the maximum open probability at extreme hyperpolarization argued for an additional voltage-independent mechanism. Org 34167 had a similar impact on a truncated HCN1 channel lacking the C-terminal nucleotide binding domain, thus ruling out an interaction with this domain. Fitting a gating model, derived from a 10-state allosteric scheme, predicted that Org 34167 strongly reduced the equilibrium constant for the voltage-independent pore domain to favor a closed pore, as well as reducing the voltage sensing domain-pore domain coupling and shifting the zero voltage equilibrium constant of the voltage sensing domain to favor the inactive state. SIGNIFICANCE STATEMENT: The brain penetrant small molecule Org 34167 has been reported to have an antidepressant action by targeting HCN channels; however, its mode of action is unknown. We used heterologously expressed human HCN1 channels to show that Org 34167 inhibits channel activity by modulating kinetic parameters associated with the channel pore domain, voltage sensing domain, and interdomain coupling.

Concerted suppressive effects of carisbamate, an anti-epileptic alkyl-carbamate drug, on voltage-gated Na+ and hyperpolarization-activated cation currents.

Carisbamate (CRS, RWJ-333369) is a new anti-seizure medication. It remains unclear whether and how CRS can perturb the magnitude and/or gating kinetics of membrane ionic currents, despite a few reports demonstrating its ability to suppress voltage-gated Na+ currents. In this study, we observed a set of whole-cell current recordings and found that CRS effectively suppressed the voltage-gated Na+ (INa) and hyperpolarization-activated cation currents (Ih) intrinsically in electrically excitable cells (GH3 cells). The effective IC50 values of CRS for the differential suppression of transient (INa(T)) and late INa (INa(L)) were 56.4 and 11.4 μM, respectively. However, CRS strongly decreased the strength (i.e., Δarea) of the nonlinear window component of INa (INa(W)), which was activated by a short ascending ramp voltage (Vramp); the subsequent addition of deltamethrin (DLT, 10 μM) counteracted the ability of CRS (100 μM, continuous exposure) to suppress INa(W). CRS strikingly decreased the decay time constant of INa(T) evoked during pulse train stimulation; however, the addition of telmisartan (10 μM) effectively attenuated the CRS (30 μM, continuous exposure)-mediated decrease in the decay time constant of the current. During continued exposure to deltamethrin (10 μM), known to be a pyrethroid insecticide, the addition of CRS resulted in differential suppression of the amplitudes of INa(T) and INa(L). The amplitude of Ih activated by a 2-s membrane hyperpolarization was diminished by CRS in a concentration-dependent manner, with an IC50 value of 38 μM. For Ih, CRS altered the steady-state I-V relationship and attenuated the strength of voltage-dependent hysteresis (Hys(V)) activated by an inverted isosceles-triangular Vramp. Moreover, the addition of oxaliplatin effectively reversed the CRS-mediated suppression of Hys(V). The predicted docking interaction between CRS and with a model of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel or between CRS and the hNaV1.7 channel reflects the ability of CRS to bind to amino acid residues in HCN or hNaV1.7 channel via hydrogen bonds and hydrophobic interactions. These findings reveal the propensity of CRS to modify INa(T) and INa(L) differentially and to effectively suppress the magnitude of Ih. INa and Ih are thus potential targets of the actions of CRS in terms of modulating cellular excitability.

Antidepressant-like activity of a brain penetrant HCN channel inhibitor in mice.

Changes in Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel function have been linked to depressive-like traits, making them potential drug targets. However, there is currently no peer-reviewed data supporting the use of a small molecule modulator of HCN channels in depression treatment. Org 34167, a benzisoxazole derivative, has been patented for the treatment of depression and progressed to Phase I trials. In the current study, we analysed the biophysical effects of Org 34167 on HCN channels in stably transfected human embryonic kidney 293 (HEK293) cells and mouse layer V neurons using patch-clamp electrophysiology, and we utilised three high-throughput screens for depressive-like behaviour to assess the activity of Org 34167 in mice. The impact of Org 34167 on locomotion and coordination were measured by performing rotarod and ledged beam tests. Org 34167 is a broad-spectrum inhibitor of HCN channels, slowing activation and causing a hyperpolarising shift in voltage-dependence of activation. It also reduced I h-mediated sag in mouse neurons. Org 34167 (0.5mg/kg) reduced marble burying and increased the time spent mobile in the Porsolt swim and tail suspension tests in both male and female BALB/c mice, suggesting reduced depressive-like behaviour. Although no adverse effects were seen at 0.5mg/kg, an increase in dose to 1mg/kg resulted in visible tremors and impaired locomotion and coordination. These data support the premise that HCN channels are valid targets for anti-depressive drugs albeit with a narrow therapeutic index. Drugs with higher HCN subtype selectivity are needed to establish if a wider therapeutic window can be obtained.

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Ameliorating parkinsonian motor dysfunction by targeting histamine receptors in entopeduncular nucleus–thalamus circuitry

In Parkinson's disease (PD), reduced dopamine levels in the basal ganglia have been associated with altered neuronal firing and motor dysfunction. It remains unclear whether the altered firing rate or pattern of basal ganglia neurons leads to parkinsonism-associated motor dysfunction. In the present study, we show that increased histaminergic innervation of the entopeduncular nucleus (EPN) in the mouse model of PD leads to activation of EPN parvalbumin (PV) neurons projecting to the thalamic motor nucleus via hyperpolarization-activated cyclic nucleotide-gated (HCN) channels coupled to postsynaptic H2R. Simultaneously, this effect is negatively regulated by presynaptic H3R activation in subthalamic nucleus (STN) glutamatergic neurons projecting to the EPN. Notably, the activation of both types of receptors ameliorates parkinsonism-associated motor dysfunction. Pharmacological activation of H2R or genetic upregulation of HCN2 in EPNPV neurons, which reduce neuronal burst firing, ameliorates parkinsonism-associated motor dysfunction independent of changes in the neuronal firing rate. In addition, optogenetic inhibition of EPNPV neurons and pharmacological activation or genetic upregulation of H3R in EPN-projecting STNGlu neurons ameliorate parkinsonism-associated motor dysfunction by reducing the firing rate rather than altering the firing pattern of EPNPV neurons. Thus, although a reduced firing rate and more regular firing pattern of EPNPV neurons correlate with amelioration in parkinsonism-associated motor dysfunction, the firing pattern appears to be more critical in this context. These results also confirm that targeting H2R and its downstream HCN2 channel in EPNPV neurons and H3R in EPN-projecting STNGlu neurons may represent potential therapeutic strategies for the clinical treatment of parkinsonism-associated motor dysfunction.

Ivabradine monotherapy in pediatric patients with focal atrial tachycardia: a single-center study.

Ivabradine is well-tolerated and may provide early heart rate control in pediatric FAT and can be considered early, especially in the presence of left ventricular dysfunction. Further investigations are deserved to confirm the optimal dose and long-term efficacy in this population. •Focal atrial tachycardia (FAT) is the most common arrhythmia associated with tachycardia-induced cardiomyopathy (TIC) in children, and the efficacy of conventional antiarrhythmic medications in the treatment of FAT is poor. •Ivabradine is currently the only selective hyperpolarization-activated cyclic nucleotide-gated (HCN) inhibitor, which can effectively low HR without negative effect on blood pressure or inotropy. • Ivabradine (0.1-0.2 mg/kg every 12 h) can effectively suppress focal atrial tachycardia in 50% of pediatric patients. •Ivabradine provides early control of heart rate and hemodynamic stabilization in children with severe left ventricular dysfunction due to atrial tachycardia within 48 h.

Hyperexcited limbic neurons represent sexual satiety and reduce mating motivation.

Transient sexual experiences can have long-lasting effects on behavioral decisions, but the neural coding that accounts for this change is unclear. We found that the ejaculation experience selectively activated estrogen receptor 2 (Esr2)-expressing neurons in the bed nucleus of the stria terminalis (BNST)-BNSTEsr2-and led to persistent decreases in firing threshold for days, during which time the mice displayed sexual satiety. Inhibition of hyperexcited BNSTEsr2 elicited fast mating recovery in satiated mice of both sexes. In males, such hyperexcitability reduced mating motivation and was partially mediated by larger HCN (hyperpolarization-activated cyclic nucleotide-gated) currents. Thus, BNSTEsr2 not only encode a specific mating action but also represent a persistent state of sexual satiety, and alterations in a neuronal ion channel contribute to sexual experience-dependent long-term changes to mating drive.

Similar voltage-sensor movement in spHCN channels can cause closing, opening, or inactivation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K+ channels, most of which are activated by depolarization. HCN channels, however, are activated by hyperpolarization. The mechanism behind this reversed gating polarity of HCN channels is not clear. We here show that sea urchin HCN (spHCN) channels with mutations in the C-terminal part of the voltage sensor use the same voltage-sensor movement to either close or open in response to hyperpolarizations depending on the absence or presence of cAMP. Our results support that non-covalent interactions at the C-terminal end of the voltage sensor are critical for HCN gating polarity. These interactions are also critical for the proper closing of the channels because these mutations exhibit large constitutive currents. Since a similar voltage-sensor movement can cause both depolarization- and hyperpolarization-activation in the same channel, this suggests that the coupling between the voltage sensor and the pore is changed to create channels opened by different polarities. We also show an identical voltage-sensor movement in activated and inactivated spHCN channels and suggest a model for spHCN activation and inactivation. Our results suggest the possibility that channels open by opposite voltage dependence, such as HCN and the related EAG channels, use the same voltage-sensor movement but different coupling mechanisms between the voltage sensor and the gate.

Specific interactions critical for the gating of HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are named appropriately given their ability to open upon hyperpolarization and are modulated by cyclic nucleotides through their cyclic nucleotide binding domain. HCN channels contain six transmembrane domains with S1-S4 forming the voltage-sensing domain and S5-S6 forming the pore domain. HCN channels are permeable to both potassium and sodium ions, playing a key role in a wide range bodily processes such as maintaining heart rate to neuropathic pain signaling. One key difference within the HCN family is, unlike mammalian HCN channels, sea urchin HCN (spHCN) channels undergo a rapid inactivation in the absence of cAMP. This inactivation process remains unclear, it has been proposed that the C-linker plays a role where binding of cAMP prevents a rotation in the A’ helix that is suggested to lead to the inactivation of the channel. Another study has highlighted a single point mutation, F459L, is able to abolish spHCN inactivation. Here we investigated both the C-linker and F459 residue as well as other sites involved in HCN channel gating using voltage clamp fluorometry and inside-out patch clamp in spHCN channels expressed in Xenopus laevis oocytes. Our results highlight some key areas across the HCN channel that may play a role in stabilizing the gate and the inactivation phenomenon seen in spHCN.

Comparison of cyclic AMP binding activity within HCN channel subfamily using atomistic molecular dynamics simulations of isolated cyclic nucleotide binding domains.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are a subfamily of homotetrameric, voltage gated potassium/sodium channels whose activity is modulated by cyclic AMP (cAMP). The binding of cAMP to an intracellular cyclic nucleotide binding domain (CNBD) promotes an increase in channel activity through a depolarizing shift in the voltage threshold necessary for channel activation, as well as kinetically favoring the active channel conformation, leading to improved conductance. While the precise mechanisms of this change are still being investigated, the shift in the CNDB region which is induced by the binding of cAMP is able to be propagated to the remainder of the channel through an adjacent C-linker, causing an alteration in the channel that affects conductance. Although various static structures of the four HCN isoforms with and without bound cyclic nucleotide have been solved and deposited on the Protein Data Bank, there is still a need to understand the precise binding interactions which lead to different sensitivities and selectivities for cyclic nucleotides within the subfamily. Using atomistic molecular dynamics, we have simulated the isolated CNBD of HCN1-4 bound to cAMP and have used both equilibrium simulations and alchemical free energy perturbation to characterize and compare the binding activities of the different isoforms. These simulations highlight the differential cyclic nucleotide binding behavior between HCN channels.

Structural determinant of the ivabradine-induced block of pacemaker HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are the molecular correlate of the Ih (or If) current, which plays a key role in controlling rhythmic activity in cardiac pacemaker cells and spontaneously firing neurons. We have recently obtained the structure of HCN4 with the pore in the open and closed conformation, thus advancing the understanding of permeation and conductance in HCN channels. Being able to purify HCN4 molecules with the pore in the open state, we are currently characterizing, at atomic details, the action of Ivabradine, an open channel blocker specific to HCN channels and currently approved for clinical use in heart failure. By testing purified HCN4 proteins for Ivabradine-induced shifts in a thermal denaturation assay we obtained the first biochemical demonstration of Ivabradine binding to the open pore of HCN4 and proceeded to solve the structure of the complex by single particle cryo-EM. We can thus describe the pattern of contacts that Ivabradine develops with the residues facing the vestibule of the HCN pore. Strikingly, molecular dynamics simulation experiments uncovered the key role of the tertiary ammine of Ivabradine in the mechanism of block, thus explaining the current dependency of the drug-induced block.

Perturbation of HCN1 response to small molecule modulation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are proteins with essential roles in pacemaking activity, neural signaling, and the development of neuropathic pain. Several allosteric ligands have been shown to inhibit HCN1; however, the structural basis of their action has yet to be defined. We uncovered putative binding sites for the intravenous general anesthetic propofol (2,6-di-isopropylphenol), a known HCN1 small molecule allosteric modulator, by cryoEM. Location of the sites correlate well with computational models. Moreover, these sites align with our bioinformatically derived allosteric network for channel activation, suggesting a mode of inhibition. Perturbation of one of the structurally identified binding sites resulted in differential inhibitor response compared to wildtype HCN1, as measured by Xenopus laevis oocyte two electrode voltage clamp. Future studies will further characterize these inhibitor binding sites and mechanistically investigate the source of isoform specificity.

S5-S6 interactions important for the electromechanical coupling in HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to the rhythmic firing of pacemaker neurons and cardiomyocytes. Mutations in HCN channels are associated with cardiac arrhythmia and epilepsy. HCN channels belong to the superfamily of voltage-gated K+ channels, most of which are activated by depolarizations. HCN channels, however, are activated by hyperpolarizations and the mechanism underlying the electromechanical coupling in HCN channels is not fully clear yet. The electromechanical coupling is thought to be mainly contributed by non-covalent interactions between S4 in the voltage-sensing domain (VSD) and S5 in the pore domain (PD). Little attention has been paid to the interactions between S5 and S6 within the PD. This study seeks to identify the role of the S5-S6 interactions in the electromechanical coupling of HCN channels. Voltage clamp fluorometry, patch clamp, and molecular modeling were conducted on mutations in S5 and S6 in the PD. Here we show that some mutations in S5 and S6 change the gating of HCN channels without affecting the S4 movement. These interactions between S5 and S6 are important for closing HCN channels. Our results suggest that in addition to canonical S4-S5 interactions, S5-S6 interactions are crucial for the electromechanical coupling in HCN channels.

Visualizing ion channels in lipid nanodomains using live-cell fluorescence lifetime imaging.

Ion channels function in a lipid-membrane nano-environment where the lipid composition and membrane compartmentalization can undergo rapid changes. How transmembrane potential activates voltage-gated ion channels differently in liquid-ordered, cholesterol-rich (Lo, or called “lipid raft”) versus in liquid-disordered, cholesterol-poor membrane (Ld) nanodomains remains less explored in intact cells. Using phasor plot fluorescence-lifetime imaging microscopy (phasor FLIM), combined with Förster resonance energy transfer (FRET) between donor- and acceptor-fluorophores conjugated to cholera toxin subunit B (CTxB) or to short lipidated peptides that specifically recognize Lo and Ld domains, we visualized the membrane domains in different cells, including Xenopus oocytes, cultured primary hepatocytes and dorsal root ganglion (DRG) sensory neurons. We postulate that a large percentage of the plasma membrane could be Lo domain. Extracting cholesterols using β-cyclodextrin was able to decrease the FRET efficiency indicating a reduced size of Lo domains. Interestingly, the measured apparent FRET was low in DRG neurons, consistent with the unique lipid organization of neuronal membranes, in comparison to other cell types. Furthermore, the localization of voltage-gated ion channels in proximity to lipid nanodomains could be examined by either FLIM-FRET or spectral FRET using these fluorescent markers. Moreover, fluorescent noncanonical amino acids (ncAAs) incorporated near the hydrophobic constriction site of voltage sensors of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels showed a similar lifetime as when their free forms were dissolved in hydrophobic solvents; lifetime of the incorporated ncAAs changed upon membrane hyperpolarization. In addition, using the stop-codon suppression strategies and click chemistry via the inverse-electron demand Diels-Alder cycloaddition, a photostable dye was tagged site-specifically to the extracellular region of the HCN voltage sensor. These labeling strategies will facilitate measuring the voltage-sensor conformational dynamics of ion channels in distinct lipid nanodomains using time-resolved phasor FLIM-FRET or single-molecule FRET.

TrkA inhibition alleviates bladder overactivity in cyclophosphamide-induced cystitis by targeting hyperpolarization-activated cyclic nucleotide-gated channels.

To investigate the potential of Tropomyosin receptor kinase A (TrkA) for the treatment of interstitial cystitis/ bladder pain syndrome (IC/BPS). Sixty-four female rats were randomly assigned to the control and cyclophosphamide (CYP) groups. Quantitative reverse transcription polymerase chain reaction was utilized to detect the mRNA level of TrkA. Western blot analysis was used to measure the protein levels of TNF-α, IL-6, and TrkA. Immunostaining was used to detect the expression of TrkA in bladder sections. Contractility studies and urodynamic measurements were utilized to test the spontaneous contractions of detrusor muscle strips and the global bladder activity, respectively. Rat models of chronic cystitis were successfully established. The mRNA and protein levels of TrkA were significantly increased in the bladders of CYP-treated rats. Also, results of immunohistochemical staining and immunofluorescence staining showed that increased TrkA expression in the CYP group was mainly observed in the urothelium layer and bladder interstitial Cajal-like cells (ICC-LCs) but not in the detrusor smooth muscle cells. The specific inhibitor of TrkA, GW441756 (10 μM), significantly suppressed the robust spontaneous contractions of detrusor muscle strips in the CYP group and alleviated the overall bladder overactivity of CYP-treated rats. However, the inhibitory effects of GW441756 (10 μM) on the spontaneous contractions of detrusor muscle strips and the overall bladder activity were eliminated after pretreatments with the specific blocker of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, ZD7288 (50 μM). Our results suggested that increased TrkA expression during chronic cystitis promotes the development of bladder overactivity by targeting the HCN channels.

STABILIZING ROLE OF HCN CHANNELS ON POST-CAMP MECHANISMS OF DETRUSOR MYOCYTE CONTROL

Abstract Aging is associated with an increased incidence of co-morbidities, including detrusor underactivity (DU). DU is defined as the failure to create sufficient and durable expulsive force to adequately empty the urinary bladder during a normal voiding timespan. DU is prevalent in older adults, as evidenced by its prevalence in nearly two-thirds of nursing home residents. Current treatments are mostly palliative or come with many side effects. β-adrenoceptor-mediated relaxation is the primary mechanism of detrusor relaxation, and Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels have previously been identified by us and others as a very important mediator of this relaxation, however the role of HCN in detrusor relaxation has not been elucidated. Hence, we seek to characterize its role in adrenergic relaxation mechanisms and spontaneous myocyte activity. Male and female 10–12-month-old C57Bl/6 mice were used for this study. Pharmacomyography studies were performed to assess the effect of different drugs that act at various steps along the adrenergic relaxation pathway, +/- CsCl, an HCN blockade at [5mM]. As expected, we saw that increasing HCN opening probability by isoproterenol or forskolin (adenylyl cyclase/cAMP-agonist) or lamotrigine (HCN-activator) resulted in decreases in tonic tension, but were diminished in the presence of CsCl. Mechanisms modulated by H89 (PKA-inhibitor) and NS1619 (BK-channel-agonist) show no change in tonic tension, however spontaneous phasic activity significantly increases. These data support increased cAMP, not hyperpolarization, as the key inductor of HCN in adrenergic relaxation.

Thalamocortical Circuit Controls Neuropathic Pain via Up-regulation of HCN2 in the Ventral Posterolateral Thalamus

The thalamocortical (TC) circuit is closely associated with pain processing. The hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 channel is predominantly expressed in the ventral posterolateral thalamus (VPL) that has been shown to mediate neuropathic pain. However, the role of VPL HCN2 in modulating TC circuit activity is largely unknown. Here, by using optogenetics, neuronal tracing, electrophysiological recordings, and virus knockdown strategies, we showed that the activation of VPL TC neurons potentiates excitatory synaptic transmission to the hindlimb region of the primary somatosensory cortex (S1HL) as well as mechanical hypersensitivity following spared nerve injury (SNI)-induced neuropathic pain in mice. Either pharmacological blockade or virus knockdown of HCN2 (shRNA-Hcn2) in the VPL was sufficient to alleviate SNI-induced hyperalgesia. Moreover, shRNA-Hcn2 decreased the excitability of TC neurons and synaptic transmission of the VPL–S1HL circuit. Together, our studies provide a novel mechanism by which HCN2 enhances the excitability of the TC circuit to facilitate neuropathic pain.

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.