Hyperpolarization-activated Cyclic Nucleotide Channel Research Articles

Effect of adenosine-1 receptor activation on pacemaker activity of interstitial cells of Cajal from mouse colon.

Adenosine plays an important role on gastrointestinal (GI) motility through adenosine receptors. Interstitial cells of Cajal (ICC) are pacemaker cells that regulate GI smooth muscle activity. The functional role and its signal mechanism of adenosine on the pacemaker activity were investigated using whole-cell patch clamp, RT-PCR, and intracellular Ca2+-imaging with ICC from mouse colon. Adenosine depolarized the membrane potentials and increased the pacemaker potential frequency, which was blocked by a selective A1-receptor antagonist, but not A2a-, A2b, or A3-receptor antagonist. A selective A1 receptor agonist represented similar effects as those of adenosine and mRNA transcript of A1-receptor was expressed in ICC. The adenosine-induced effects were blocked by phospholipase C (PLC) and a Ca2+-ATPase inhibitor. Adenosine increased spontaneous intracellular Ca2+ oscillations, as seen fluo4/AM. Both hyperpolarization-activated cyclic nucleotide (HCN) channel inhibitors and adenylate cyclase inhibitors blocked the adenosine-induced effects. And adenosine increased the basal cellular adenylate cyclase activity in colonic ICC. However, adenosine and adenylate cyclase inhibitors did not show any influence on pacemaker activity in small intestinal ICC for a comparison with that of the small intestine. These results suggest adenosine modulates the pacemaker potentials by acting HCN channels- and intracellular Ca2+- dependent mechanisms through A1-receptor. Therefore, adenosine may be a therapeutic target in colonic motility disorders.

Refining HCN1 channel closed and open state models using cysteine bridge simulations.

The hyperpolarization-activated cyclic nucleotide (HCN) gated channel underlies the control of rhythmic activity in cardiac and neuronal pacemaker cells at heart, brain, and kidney. Unlike most of the Kv channels that are activated by membrane depolarization, HCN channels are activated by membrane hyperpolarization. Knowledge of multiple conformational states of ion channels is a prerequisite to understand their function. Atomistic models of spHCN channels in three different states (up-closed, up-closed, down-open) were created using existing hHCN1 cryo-EM structures and the open hERG cryo-EM structure and equilibrated using 1.5 to 3.0 μs all-atom MD simulations. While all three models were stable, they have not satisfied all known experimental derived structural constraints at open and closed states. In this work, we used non-interacting molecular fragments approach to refine our up-closed and down-open spHCN1 models by integrating multiple experimentally determined cysteine-cysteine Cd2+ bridges simultaneously during MD simulations. A total of 28 cysteine bridges were generated for each model. The force constants for the harmonic restraints were gradually turned on. We found that for most of the cysteine bridges between S4-S5 linker and C-linker, an upward movement of C linker was sufficient to satisfy the distance criteria without large conformational re-arrangement from its starting configuration. These refined models will be further validated using new cysteine bridge experiments. These models will help reveal the mechanism of voltage sensor-to-gate coupling and lead to deeper understanding of disease mutations on HCN channel at the molecular level.

Caveolin-3 and Arrhythmias: Insights into the Molecular Mechanisms.

Caveolin-3 is a muscle-specific protein on the membrane of myocytes correlated with a variety of cardiovascular diseases. It is now clear that the caveolin-3 plays a critical role in the cardiovascular system and a significant role in cardiac protective signaling. Mutations in the gene encoding caveolin-3 cause a broad spectrum of clinical phenotypes, ranging from persistent elevations in the serum levels of creatine kinase in asymptomatic humans to cardiomyopathy. The influence of Caveolin-3(CAV-3) mutations on current density parallels the effect on channel trafficking. For example, mutations in the CAV-3 gene promote ventricular arrhythmogenesis in long QT syndrome 9 by a combined decrease in the loss of the inward rectifier current (IK1) and gain of the late sodium current (INa-L). The functional significance of the caveolin-3 has proved that caveolin-3 overexpression or knockdown contributes to the occurrence and development of arrhythmias. Caveolin-3 overexpression could lead to reduced diastolic spontaneous Ca2+ waves, thus leading to the abnormal L-Type calcium channel current-induced ventricular arrhythmias. Moreover, CAV-3 knockdown resulted in a shift to more negative values in the hyperpolarization-activated cyclic nucleotide channel 4 current (IHCN4) activation curve and a significant decrease in IHCN4 whole-cell current density. Recent evidence indicates that caveolin-3 plays a significant role in adipose tissue and is related to obesity development. The role of caveolin-3 in glucose homeostasis has attracted increasing attention. This review highlights the underlining mechanisms of caveolin-3 in arrhythmia. Progress in this field may contribute to novel therapeutic approaches for patients prone to developing arrhythmia.

Prolonged-duration pulsed radiofrequency is associated with increased neuronal damage without further antiallodynic effects in neuropathic pain model rats.

Aim of investigationPulsed radiofrequency (PRF) is a safe and effective approach for treating neuropathic pain. However, the optimal treatment conditions and analgesic mechanisms of PRF remain unclear. The aim of our study was to assess the beneficial and adverse effects of prolonged-duration PRF and the analgesic mechanisms of PRF treatment with neuropathic pain rats.MethodsMale Sprague Dawley rats received L5 spinal nerve ligation (SNL) for developing neuropathic pain. Fourteen days after L5 SNL surgery, they were divided into three groups according to duration of PRF current for 6 minutes, 12 minutes, and none. PRF current was delivered via direct visualization adjacent to the L5 dorsal root ganglion (DRG). Pain behavior was evaluated every week after L5 SNL surgery, until day 28. Seven days after PRF treatment, L5 DRG tissue was harvested to detect levels of activating translation factor 3 (ATF3; a marker of neuronal damage) and hyperpolarization-activated cyclic nucleotide (HCN)-gated cation channels (key factors in neuropathic pain) using quantitative PCR.ResultsBefore PRF application, withdrawal thresholds were significantly lower than at baseline and did not differ significantly between the three groups. After PRF application, withdrawal thresholds in the PRF6 and PRF12 groups were significantly increased compared to those in the sham group. However, those in the PRF6 and PRF12 groups did not differ significantly. The expression level of ATF3 mRNA in the PRF12 group was significantly higher than that in the sham group (P<0.01), but the expression of HCN1 and HCN2 channels did not differ significantly between the three groups.ConclusionProlonged PRF exposure, from 6 to 12 minutes, was not only ineffective but also associated with increased neuronal damage. These findings do not support prolonged PRF exposure as a helpful treatment for neuropathic pain. In this study, the involvement of HCN channels in the antiallodynic effects of PRF was uncertain.

Inhibition of hyperpolarization-activated cyclic nucleotide-gated channels by β-blocker carvedilol.

Carvedilol is a clinically effective β-blocker broadly used for treating congestive heart failure (CHF), and several clinical trials have demonstrated that it shows a favourable effect compared with other β-blockers in patients with CHF. The mechanism underlying this beneficial effect of carvedilol compared to other β-blockers is not clearly understood. In addition to β-blockers, inhibitors of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels, which play a critical role in spontaneous rhythmic activity in the heart, have also been proposed to be suitable drugs for reducing heart rate and, therefore, beneficial for treating CHF. In the present study, we investigated the effect of carvedilol on HCN channels. Whole-cell patch-clamp recordings were used to assess the effect of carvedilol on currents from wild-type and mutant HCN1, HCN2 and HCN4 channels expressed in CHO cells. Carvedilol was the only β-blocker tested that showed inhibitory effects on the major sinoatrial HCN channel isoform HCN4. Carvedilol inhibited HCN4 in a concentration-dependent manner with an EC50 of 4.4μM. In addition, carvedilol also inhibited HCN1 and HCN2 channels. Carvedilol blocked HCN channels by decelerating the rate of channel activation and increasing that of deactivation, and shifted the voltage-dependence of activation leftwards. Our data also showed that carvedilol, unlike other inhibitors of this channel (ivabradine and ZD7288), is not an 'open-channel' inhibitor of HCN4. Carvedilol is a negative gating modulator of HCN channels. It represents a novel structure for future drug design of HCN channel inhibitors.

Allostery between two binding sites in the ion channel subunit TRIP8b confers binding specificity to HCN channels

Tetratricopeptide repeat (TPR) domains are ubiquitous structural motifs that mediate protein-protein interactions. For example, the TPR domains in the peroxisomal import receptor PEX5 enable binding to a range of type 1 peroxisomal targeting signal motifs. A homolog of PEX5, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), binds to and functions as an auxiliary subunit of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels. Given the similarity between TRIP8b and PEX5, this difference in function raises the question of what mechanism accounts for their binding specificity. In this report, we found that the cyclic nucleotide-binding domain and the C terminus of the HCN channel are critical for conferring specificity to TRIP8b binding. We show that TRIP8b binds the HCN cyclic nucleotide-binding domain through a 37-residue domain and the HCN C terminus through the TPR domains. Using a combination of fluorescence polarization- and co-immunoprecipitation-based assays, we establish that binding at either site increases affinity at the other. Thus, allosteric coupling of the TRIP8b TPR domains both promotes binding to HCN channels and limits binding to type 1 peroxisomal targeting signal substrates. These results raise the possibility that other TPR domains may be similarly influenced by allosteric mechanisms as a general feature of protein-protein interactions.

Long-QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-activated cyclic nucleotide gated channel 4.

Background Caveolin-3 (cav-3) mutations are linked to the long-QT syndrome (LQTS) causing distinct clinical symptoms. Hyperpolarization-activated cyclic nucleotide channel 4 (HCN4) underlies the pacemaker current If. It associates with cav-3 and both form a macromolecular complex. Methods To examine the effects of human LQTS-associated cav-3 mutations on HCN4-channel function, HEK293-cells were cotransfected with HCN4 and wild-type (WT) cav-3 or a LQTS-associated cav-3 mutant (T78M, A85T, S141R, or F97C). HCN4 currents were recorded using the whole-cell patch-clamp technique. Results WT cav-3 significantly decreased HCN4 current density and shifted midpoint of activation into negative direction. HCN4 current properties were differentially modulated by LQTS-associated cav-3 mutations. When compared with WT cav-3, A85T, F97C, and T78M did not alter the specific effect of cav-3, but S141R significantly increased HCN4 current density. Compared with WT cav-3, no significant modifications of voltage dependence of steady-state activation curves were observed. However, while WT cav-3 alone had no significant effect on HCN4 current activation, all LQTS-associated cav-3 mutations significantly accelerated HCN4 activation kinetics. Conclusions Our results indicate that HCN4 channel function is modulated by cav-3. LQTS-associated mutations of cav-3 differentially influence pacemaker current properties indicating a pathophysiological role in clinical manifestations.

Lidocaine Inhibits HCN Currents in Rat Spinal Substantia Gelatinosa Neurons

Lidocaine, which blocks voltage-gated sodium channels, is widely used in surgical anesthesia and pain management. Recently, it has been proposed that the hyperpolarization-activated cyclic nucleotide (HCN) channel is one of the other novel targets of lidocaine. Substantia gelatinosa in the spinal dorsal horn, which plays key roles in modulating nociceptive information from primary afferents, comprises heterogeneous interneurons that can be electrophysiologically categorized by firing pattern. Our previous study demonstrated that a substantial proportion of substantia gelatinosa neurons reveal the presence of HCN current (Ih); however, the roles of lidocaine and HCN channel expression in different types of substantia gelatinosa neurons remain unclear. By using the whole-cell patch-clamp technique, we investigated the effect of lidocaine on Ih in rat substantia gelatinosa neurons of acute dissociated spinal cord slices. We found that lidocaine rapidly decreased the peak Ih amplitude with an IC50 of 80 μM. The inhibition rate on Ih was not significantly different with a second application of lidocaine in the same neuron. Tetrodotoxin, a sodium channel blocker, did not affect lidocaine's effect on Ih. In addition, lidocaine shifted the half-activation potential of Ih from -109.7 to -114.9 mV and slowed activation. Moreover, the reversal potential of Ih was shifted by -7.5 mV by lidocaine. In the current clamp, lidocaine decreased the resting membrane potential, increased membrane resistance, delayed rebound depolarization latency, and reduced the rebound spike frequency. We further found that approximately 58% of substantia gelatinosa neurons examined expressed Ih, in which most of them were tonically firing. Our studies demonstrate that lidocaine strongly inhibits Ih in a reversible and concentration-dependent manner in substantia gelatinosa neurons, independent of tetrodotoxin-sensitive sodium channels. Thus, our study provides new insight into the mechanism underlying the central analgesic effect of the systemic administration of lidocaine.

The Symptom Complex of Familial Sinus Node Dysfunction and Myocardial Noncompaction Is Associated With Mutations in the HCN4 Channel

BackgroundInherited arrhythmias were originally considered isolated electrical defects. There is growing evidence that ion channel dysfunction also contributes to myocardial disorders, but genetic overlap has not been reported for sinus node dysfunction (SND) and noncompaction cardiomyopathy (NCCM). ObjectivesThe study sought to investigate a familial electromechanical disorder characterized by SND and NCCM, and to identify the underlying genetic basis. MethodsThe index family and a cohort of unrelated probands with sinus bradycardia were examined by electrocardiography, Holter recording, exercise stress test, echocardiography, and/or cardiac magnetic resonance imaging. Targeted next-generation and direct sequencing were used for candidate gene analysis and mutation scanning. Ion channels were expressed in HEK293 cells and studied using patch-clamp recordings. ResultsSND and biventricular NCCM were diagnosed in multiple members of a German family. Segregation analysis suggested autosomal-dominant inheritance of the combined phenotype. When looking for potentially disease-causing gene variants with cosegregation, a novel hyperpolarization-activated cyclic nucleotide channel 4 (HCN4)-G482R mutation and a common cysteine and glycine-rich protein 3 (CSRP3)-W4R variant were identified. HCN4-G482R is located in the highly conserved channel pore domain. Mutant subunits were nonfunctional and exerted dominant-negative effects on wild-type current. CSRP3-W4R has previously been linked to dilated and hypertrophic cardiomyopathy, but was also found in healthy subjects. Moreover, different truncation (695X) and missense (P883R) HCN4 mutations segregated with a similar combined phenotype in an additional, unrelated family and a single unrelated proband respectively, which both lacked CSRP3-W4R. ConclusionsThe symptom complex of SND and NCCM is associated with heritable HCN4 defects. The NCCM phenotype may be aggravated by a common CSRP3 variant in one of the families.

Postsynaptic NO/cGMP Increases NMDA Receptor Currents via Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in the Hippocampus

The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling cascade participates in the modulation of synaptic transmission. The effects of NO are mediated by the NO-sensitive cGMP-forming guanylyl cyclases (NO-GCs), which exist in 2 isoforms with indistinguishable regulatory properties. The lack of long-term potentiation (LTP) in knock-out (KO) mice deficient in either one of the NO-GC isoforms indicates the contribution of both NO-GCs to LTP. Recently, we showed that the NO-GC1 isoform is located presynaptically in glutamatergic neurons and increases the glutamate release via hyperpolarization-activated cyclic nucleotide (HCN)-gated channels in the hippocampus. Electrophysiological analysis of hippocampal CA1 neurons in whole-cell recordings revealed a reduction of HCN currents and a hyperpolarizing shift of the activation curve in the NO-GC2 KOs associated with reduced resting membrane potentials. These features were mimicked in wild-type (WT) neurons with an NO-GC inhibitor. Analysis of glutamate receptors revealed a cGMP-dependent reduction of NMDA receptor currents in the NO-GC2 KO mice, which was mimicked in WT by HCN channel inhibition. Lowering extracellular Mg(2+) increased NMDA receptor currents in the NO-GC2 KO and allowed the induction of LTP that was absent at physiological Mg(2+). In sum, our data indicate that postsynaptic cGMP increases the N-methyl-D-aspartate (NMDA) receptor current by gating HCN channels and thereby is required for LTP.

Asymmetric Divergence in Structure and Function of HCN Channel Duplicates in Ciona intestinalis

Hyperpolarization-activated Cyclic Nucleotide (HCN) channels are voltage-gated cation channels and are critical for regulation of membrane potential in electrically active cells. To understand the evolution of these channels at the molecular level, we cloned and examined two of three HCN homologs of the urochordate Ciona intestinalis (ciHCNa and ciHCNb). ciHCNa is like mammalian HCNs in that it possesses similar electrical function and undergoes N-glycosylation of a sequon near the pore. ciHCNb lacks the pore-associated N-glycosylation sequon and is predictably not N-glycosylated, and it also has an unusual gating phenotype in which the channel's voltage-sensitive gate appears to close incompletely. Together with previous findings, the data support an evolutionary trajectory in which an HCN ancestor underwent lineage-specific duplication in Ciona, to yield one HCN with most features that are conserved with the mammalian HCNs and another HCN that has been uniquely altered.

Charge movement in gating-locked HCN channels reveals weak coupling of voltage sensors and gate.

HCN (hyperpolarization-activated cyclic nucleotide gated) pacemaker channels have an architecture similar to that of voltage-gated K+ channels, but they open with the opposite voltage dependence. HCN channels use essentially the same positively charged voltage sensors and intracellular activation gates as K+ channels, but apparently these two components are coupled differently. In this study, we examine the energetics of coupling between the voltage sensor and the pore by using cysteine mutant channels for which low concentrations of Cd2+ ions freeze the open–closed gating machinery but still allow the sensors to move. We were able to lock mutant channels either into open or into closed states by the application of Cd2+ and measure the effect on voltage sensor movement. Cd2+ did not immobilize the gating charge, as expected for strict coupling, but rather it produced shifts in the voltage dependence of voltage sensor charge movement, consistent with its effect of confining transitions to either closed or open states. From the magnitude of the Cd2+-induced shifts, we estimate that each voltage sensor produces a roughly three- to sevenfold effect on the open–closed equilibrium, corresponding to a coupling energy of ∼1.3–2 kT per sensor. Such coupling is not only opposite in sign to the coupling in K+ channels, but also much weaker.

The Modulation of Orexin A on HCN Currents of Pyramidal Neurons in Mouse Prelimbic Cortex

The hyperpolarization-activated/cyclic nucleotide (HCN)-gated channels make important contributions to neural excitability. In prefrontal cortex, HCN channels are localized on the distal dendrites of layer V pyramidal neurons and decrease neural excitability when they are open. In the present study, using whole-cell voltage clamp recordings, the effect of an arousal peptide, orexin A, on HCN currents in layer V pyramidal neurons from mouse prelimbic cortex (PL), the homolog of the prefrontal cortex was investigated. The results demonstrated that orexin A suppressed HCN currents and shifted their activation curve to a more negative direction. This action of orexin A was blocked by SB334867, an orexin receptor 1 (OXR1) blocker and bisindolylmaleimide, a protein kinase C (PKC) inhibitor, indicating the involvement of OXR1 and PKC. The excitatory effect of orexin A on PL pyramidal neurons was enhanced when HCN currents were diminished, while attenuated when HCN currents were enlarged. In summary, orexin A inhibits HCN currents and enhances excitability of pyramidal neurons in PL, which may contribute to arousal and cognition.

Postnatal Development of Dendritic Synaptic Integration in Rat Neocortical Pyramidal Neurons

The dendritic tree of layer 5 (L5) pyramidal neurons spans the neocortical layers, allowing the integration of intra- and extracortical synaptic inputs. Here we investigate the postnatal development of the integrative properties of rat L5 pyramidal neurons using simultaneous whole cell recording from the soma and distal apical dendrite. In young (P9-10) neurons, apical dendritic excitatory synaptic input powerfully drove action potential output by efficiently summating at the axonal site of action potential generation. In contrast, in mature (P25-29) neurons, apical dendritic excitatory input provided little direct depolarization at the site of action potential generation but was integrated locally in the apical dendritic tree leading to the generation of dendritic spikes. Consequently, over the first postnatal month the fraction of action potentials driven by apical dendritic spikes increased dramatically. This developmental remodeling of the integrative operations of L5 pyramidal neurons was controlled by a >10-fold increase in the density of apical dendritic Hyperpolarization-activated cyclic nucleotide (HCN)-gated channels found in cell-attached patches or by immunostaining for the HCN channel isoform HCN1. Thus an age-dependent increase in apical dendritic HCN channel density ensures that L5 pyramidal neurons develop from compact temporal integrators to compartmentalized integrators of basal and apical dendritic synaptic input.

Effects of Antiarrhythmic Drugs on the Hyperpolarization-Activated Cyclic Nucleotide–Gated Channel Current

After the report of the Cardiac Arrhythmia Suppression Trial, a tabular framework of the Sicilian Gambit has been proposed to display actions of antiarrhythmic drugs on ion channels and receptors and to provide more rational pharmacotherapy of arrhythmias. However, because effects of antiarrhythmic drugs on If have not been thoroughly examined, we used patch clamp techniques to determine the effects of various antiarrhythmic drugs on the HCN (hyperpolarization-activated cyclic nucleotide-gated) channel currents. HCN4 channels, a dominant isoform of HCN channels in the heart, were expressed in HEK293 cells. Amiodarone and bepridil potently inhibited the HCN4 channel current with IC50 values of 4.5 and 4.9 microM, respectively, which were close to their therapeutic concentrations. The inhibitory effects of quinidine, disopyramide, cibenzoline, lidocaine, mexiletine, aprindine, propafenone, flecainide, propranolol, and verapamil on the HCN4 channel current were weak in their therapeutic concentrations, with IC50 values of 78.3, 249, 46.8, 276, 309, 43.7, 14.3, 1700, 50.5, and 44.9 microM, respectively, suggesting that the inhibitory effects on If would be clinically small. D,L-Sotalol hardly affected the HCN4 channel current. Information about the HCN4-channel effects of many antiarrhythmic drugs may be useful for determining the appropriate drug for treatment of various arrhythmias while minimizing adverse effects.

Dendritic HCN2 Channels Constrain Glutamate-Driven Excitability in Reticular Thalamic Neurons

Hyperpolarization activated cyclic nucleotide (HCN) gated channels conduct a current, I(h); how I(h) influences excitability and spike firing depends primarily on channel distribution in subcellular compartments. For example, dendritic expression of HCN1 normalizes somatic voltage responses and spike output in hippocampal and cortical neurons. We reported previously that HCN2 is predominantly expressed in dendritic spines in reticular thalamic nucleus (RTN) neurons, but the functional impact of such nonsomatic HCN2 expression remains unknown. We examined the role of HCN2 expression in regulating RTN excitability and GABAergic output from RTN to thalamocortical relay neurons using wild-type and HCN2 knock-out mice. Pharmacological blockade of I(h) significantly increased spike firing in RTN neurons and large spontaneous IPSC frequency in relay neurons; conversely, pharmacological enhancement of HCN channel function decreased spontaneous IPSC frequency. HCN2 deletion abolished I(h) in RTN neurons and significantly decreased sensitivity to 8-bromo-cAMP and lamotrigine. Recapitulating the effects of I(h) block, HCN2 deletion increased both temporal summation of EPSPs in RTN neurons as well as GABAergic output to postsynaptic relay neurons. The enhanced excitability of RTN neurons after I(h) block required activation of ionotropic glutamate receptors; consistent with this was the colocalization of HCN2 and glutamate receptor 4 subunit immunoreactivities in dendritic spines of RTN neurons. The results indicate that, in mouse RTN neurons, HCN2 is the primary functional isoform underlying I(h) and expression of HCN2 constrains excitatory synaptic integration.