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Tallie Z Baram - One of the best experts on this subject based on the ideXlab platform.
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postnatal expression pattern of hcn channel isoforms in thalamic neurons relationship to maturation of thalamocortical oscillations
The Journal of Neuroscience, 2009Co-Authors: Tatyana Kanyshkova, Matthias Pawlowski, Patrick Meuth, Celine Dube, Arnd Baumann, Hanschristian Pape, Roland A Bender, Amy L Brewster, Tallie Z Baram, Thomas BuddeAbstract:Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels are the molecular substrate of the hyperpolarization-activated inward current ( I h). Because the developmental profile of HCN channels in the thalamus is not well understood, we combined electrophysiological, molecular, immunohistochemical, EEG recordings in vivo , and computer modeling techniques to examine HCN gene expression and I h properties in rat thalamocortical relay (TC) neurons in the dorsal part of the lateral geniculate nucleus and the functional consequence of this maturation. Recordings of TC neurons revealed an approximate sixfold increase in I h density between postnatal day 3 (P3) and P106, which was accompanied by significantly altered current kinetics, cAMP sensitivity, and steady-state activation properties. Quantification on tissue levels revealed a significant developmental decrease in cAMP. Consequently the block of basal adenylyl cyclase activity was accompanied by a hyperpolarizing shift of the I h activation curve in young but not adult rats. Quantitative analyses of HCN channel isoforms revealed a steady increase of mRNA and protein expression levels of HCN1, HCN2, and HCN4 with reduced relative abundance of HCN4. Computer modeling in a simplified thalamic network indicated that the occurrence of rhythmic delta activity, which was present in the EEG at P12, differentially depended on I h conductance and modulation by cAMP at different developmental states. These data indicate that the developmental increase in I h density results from increased expression of three HCN channel isoforms and that isoform composition and intracellular cAMP levels interact in determining I h properties to enable progressive maturation of rhythmic slow-wave sleep activity patterns.
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Formation of heteromeric hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in the hippocampus is regulated by developmental seizures.
Neurobiology of Disease, 2005Co-Authors: Amy L Brewster, Joie A. Bernard, Christine M. Gall, Tallie Z BaramAbstract:Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels mediate hyperpolarization-activated currents (Ih). In hippocampus, these currents contribute greatly to intrinsic cellular properties and synchronized neuronal activity. The kinetic and gating properties of HCN-mediated currents are largely determined by the type of subunits–for example, HCN1 and HCN2–that assemble to form homomeric channels. Recently, functional heteromeric HCN channels have been described in vitro, further enlarging the potential Ih repertoire of individual neurons. Because these heteromeric HCN channels may promote hippocampal hyperexcitability and the development of epilepsy, understanding the mechanisms governing their formation is of major clinical relevance. Here, we find that developmental seizures promote co-assembly of hippocampal HCN1/HCN2 heteromeric channels, in a duration-dependent manner. Long-lasting heteromerization was found selectively after seizures that provoked persistent hippocampal hyperexcitability. The mechanism for this enhanced heteromerization may involve increased relative abundance of HCN2-type subunits relative to the HCN1 isoform at both mRNA and protein levels. These data suggest that heteromeric HCN channels may provide molecular targets for intervention in the epileptogenic process.
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enhanced expression of a specific hyperpolarization activated cyclic nucleotide gated cation channel hcn in surviving dentate gyrus granule cells of human and experimental epileptic hippocampus
The Journal of Neuroscience, 2003Co-Authors: Roland A Bender, Amy L Brewster, Sheila V Soleymani, Snow T Nguyen, Heinz Beck, Gary W Mathern, Tallie Z BaramAbstract:Changes in the expression of ion channels, contributing to altered neuronal excitability, are emerging as possible mechanisms in the development of certain human epilepsies. In previous immature rodent studies of experimental prolonged febrile seizures, isoform-specific changes in the expression of hyperpolarization-activated cyclic nucleotide-gated cation channels (HCNs) correlated with long-lasting hippocampal hyperexcitability and enhanced seizure susceptibility. Prolonged early-life seizures commonly precede human temporal lobe epilepsy (TLE), suggesting that transcriptional dysregulation of HCNs might contribute to the epileptogenic process. Therefore, we determined whether HCN isoform expression was modified in hippocampi of individuals with TLE. HCN1 and HCN2 expression were measured using in situ hybridization and immunocytochemistry in hippocampi from three groups: TLE with hippocampal sclerosis (HS; n = 17), epileptic hippocampi without HS, or non-HS (NHS; n = 10), and autopsy material (n = 10). The results obtained in chronic human epilepsy were validated by examining hippocampi from the pilocarpine model of chronic TLE. In autopsy and most NHS hippocampi, HCN1 mRNA expression was substantial in pyramidal cell layers and lower in dentate gyrus granule cells (GCs). In contrast, HCN1 mRNA expression over the GC layer and in individual GCs from epileptic hippocampus was markedly increased once GC neuronal density was reduced by >50%. HCN1 mRNA changes were accompanied by enhanced immunoreactivity in the GC dendritic fields and more modest changes in HCN2 mRNA expression. Furthermore, similar robust and isoform-selective augmentation of HCN1 mRNA expression was evident also in the pilocarpine animal model of TLE. These findings indicate that the expression of HCN isoforms is dynamically regulated in human as well as in experimental hippocampal epilepsy. After experimental febrile seizures (i.e., early in the epileptogenic process), the preserved and augmented inhibition onto principal cells may lead to reduced HCN1 expression. In contrast, in chronic epileptic HS hippocampus studied here, the profound loss of interneuronal and principal cell populations and consequent reduced inhibition, coupled with increased dendritic excitation of surviving GCs, might provoke a "compensatory" enhancement of HCN1 mRNA and protein expression.
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developmental febrile seizures modulate hippocampal gene expression of hyperpolarization activated channels in an isoform and cell specific manner
The Journal of Neuroscience, 2002Co-Authors: Amy L Brewster, Celine Dube, Roland A Bender, Yuncai Chen, Mariam Eghbalahmadi, Tallie Z BaramAbstract:Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work has demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization-activated depolarizing current (IH). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide-gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of the HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.
Martin Biel - One of the best experts on this subject based on the ideXlab platform.
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hcn3 ion channels roles in sensory neuronal excitability and pain
The Journal of Physiology, 2019Co-Authors: Sergio Lainez, Martin Biel, Christoforos Tsantoulas, Peter A McnaughtonAbstract:KEY POINTS: HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain. Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but the potential involvement of HCN3 in pain has not been investigated. HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih . HCN3 deletion increased the firing rate of medium but not small, sensory neurons. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick. We conclude that HCN3 plays little role in either acute or chronic pain sensation. ABSTRACT: HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, whereas HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, although the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, whereas the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 knockout mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to wild-type mice but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, although it has no major influence on acute or neuropathic pain processing.
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regulation of hyperpolarization activated cyclic nucleotide gated hcn channel activity by ccmp
Journal of Biological Chemistry, 2012Co-Authors: Xiangang Zong, Christian Gruner, Xiaochun Caoehlker, Stefanie Fenske, Christian Wahlschott, Jens Krüger, Chengchang Chen, S Krause, Martin BielAbstract:Abstract Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide, cyclic cytidine 3′, 5′-monophosphate (cCMP) as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide binding domain. We determined EC50 values of around 30 μM for cCMP as compared to 1 μM for cAMP. Notably, cCMP is a partial agonist of HCN channels displaying an efficacy of about 0.6. Cyclic CMP increased the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase was caused by a depolarizing shift of the activation curve of the native HCN current which in turn leads to an enhancement of the slope of the diastolic depolarization of SAN cells. In conclusion, our findings establish cCMP as gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel regulated processes.
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Exploring HCN channels as novel drug targets
Nature Reviews Drug Discovery, 2011Co-Authors: Otilia Postea, Martin BielAbstract:Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of heart rate and neuronal excitability. Ivabradine is the first compound acting on HCN channels to be clinically approved for the treatment of angina pectoris. HCN channels may offer excellent opportunities for the development of novel anticonvulsant, anaesthetic and analgesic drugs. In support of this idea, some well-established drugs that act on the central nervous system — including lamotrigine, gabapentin and propofol — have been found to modulate HCN channel function. This Review gives an up-to-date summary of compounds acting on HCN channels, and discusses strategies to further explore the potential of these channels for therapeutic intervention. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known for their role in controlling the rhythmic activity of cardiac pacemaker cells and spontaneously firing neurons. They are now emerging as interesting targets not only for the development of drugs to lower heart rate but also for the treatment of diseases related to impaired neuronal activity, such as epilepsy and neuropathic pain. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are members of the voltage-gated ion channel superfamily that are dually gated by membrane hyperpolarization and cyclic nucleotides. The current produced by HCN channels is called I_h (hyperpolarization-activated current) or I_f (funny current). HCN channels have a key role in controlling cardiac pacemaker activity and are important regulators of neuronal excitability. Dysfunction of HCN channels has been implicated in arrhythmogenic diseases of the heart and several diseases of the nervous system, including pain disorders, epilepsy and ataxia. Ivabradine is the first HCN channel blocker that has been clinically approved. Ivabradine efficiently lowers heart rate by blocking the sinoatrial HCN4 channel, and is used for the treatment of stable angina pectoris. HCN1 and HCN2 channels are involved in the pathologies of inflammatory and neuropathic pain disorders. Compounds acting on these two channels are promising candidates for treating peripheral pain modalities. Compounds acting on HCN1 and HCN2 channels might be also valuable in other indications such as anaesthesia and the treatment of various types of epilepsies. The design of agents that selectively target each of the four HCN channel isoforms is an important goal for future drug development. To avoid side effects on cardiac rhythmicity, compounds targeting the central nervous system should have a low affinity for the sinoatrial HCN4 channel. A first series of subtype-selective HCN channel compounds has been developed based on the ivabradine backbone. Other compounds acting on HCN channels (for example, clonidine or nicotine) may serve as alternative lead structures for the development of next-generation drugs targeting HCN channels.
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Electrophysiological Evaluation of Novel Blockers of If Current
Biophysical Journal, 2009Co-Authors: Martina Del Lungo, Maria Novella Romanelli, Michele Melchiorre, Laura Sartiani, Martin Biel, András Varró, Elisabetta CerbaiAbstract:In the sino-atrial node (SAN) a major role in rhythm generation is played by f-channels. They mediate a sodium-potassium inward current (If) activated upon hyperpolarization and encoded by HCN genes (HCN1-4). If is overexpressed in cardiac diseases at ventricular level, where it may contribute to the increased propensity for arrhythmias. Selective f-channel blockers have a potential therapeutic use as bradycardic and antiarrhythmic agents. Zatebradine and ivabradine act as f-channel blockers but they lack cardiac selectivity, blocking the neuronal HCN isoforms. As a consequence an unmet need exists to develop new blockers selective for the mammalian SAN channel isoform, HCN4. Zatebradine analogues (C1-C5) were synthesized and the effect on If was measured on patch-clamped HEK293 cells expressing mHCN1, mHCN2 and hHCN4 and native guinea-pig and rabbit SAN cells. At 10 μM concentration all compounds reduced maximal If amplitude; however, potencies (defined by EC50) differed considerably. Ivabradine, taken as reference compound, showed no isoform selectivity; C1 and C4 were more potent on HCN1, the ratio being 4.3 (HCN2/HCN1) and 7.5 (HCN4/HCN1). C2 was more potent on HCN4, the ratio being 6 and 17 vs. HCN1 and HCN2, respectively. C3 was equipotent on HCN1 and HCN4 and C5 had low activity on all isoforms. Blockade was concentration-dependent, did not reverse upon drug removal and did not change current activation properties. Finally, data obtained in SAN cells show that effects on native If resemble those obtained on HCN4 isoform, in accordance with the hypothesis that HCN4 has a major contribution in SAN cells. Present results indicate that drug interaction with different HCN isoforms has diverse structural requirements. Current investigations are aimed to characterize the pharmacological profile of the new f-channel blockers and improve their isoform selectivity (Supported by EU - LSH M/CT/2006/018676, Normacor).
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Molecular basis for the different activation kinetics of the pacemaker channels HCN2 and HCN4.
Journal of Biological Chemistry, 2003Co-Authors: Juliane Stieber, Anna Thomer, Barbara Much, Martin Biel, Angela Schneider, Franz HofmannAbstract:Abstract The pacemaker channels HCN2 and HCN4 have been identified in cardiac sino-atrial node cells. These channels differ considerably in several kinetic properties including the activation time constant (τact), which is fast for HCN2 (144 ms at –140 mV) and slow for HCN4 (461 ms at –140 mV). Here, by analyzing HCN2/4 chimeras and mutants we identified single amino acid residues in transmembrane segments 1 and 2 and the connecting loop between S1 and S2 that are major determinants of this difference. Replacement of leucine 272 in S1 of HCN4 by the corresponding phenylalanine present in HCN2 decreased τact of HCN4 to 149 ms. Conversely, activation of the fast channel HCN2 was decreased 3-fold upon the corresponding mutation of F221L in the S1 segment. Mutation of N291T and T293A in the linker between S1 and S2 of HCN4 shifted τact to 275 ms. While residues 272, 291, and 293 of HCN4 affected the activation speed at basal conditions they had no obvious influence on the cAMP-dependent acceleration of activation kinetics. In contrast, mutation of I308M in S2 of HCN4 abolished the cAMP-dependent decrease in τact. Surprisingly, this mutation also prevented the acceleration of channel activation observed after deletion of the C-terminal cAMP binding site. Taken together our results indicate that the speed of activation of the HCN4 channel is determined by structural elements present in the S1, S1-S2 linker, and the S2 segment.
Andreas Ludwig - One of the best experts on this subject based on the ideXlab platform.
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Pathophysiology of HCN channels
Pflügers Archiv - European Journal of Physiology, 2007Co-Authors: Stefan Herrmann, Juliane Stieber, Andreas LudwigAbstract:Hyperpolarization-activated cation currents termed I _f/h are observed in many neurons and cardiac cells. Four genes (HCN1-4) encode the channels underlying these currents. New insights into the pathophysiological significance of HCN channels have been gained recently from analyses of mice engineered to be deficient in HCN genes. Lack of individual subunits results in markedly different phenotypes. Disruption of HCN1 impairs motor learning but enhances spatial learning and memory. Deletion of HCN2 results in absence epilepsy, ataxia, and sinus node dysfunction. Mice lacking HCN4 die during embryonic development and develop no sinoatrial node-like action potentials. In the present review, we summarize the physiology and pathophysiology of HCN channel family members based primarily on information from the transgenic mouse models and on data from human patients carrying defects in HCN4 channels.
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Bradycardic and proarrhythmic properties of sinus node inhibitors.
Molecular Pharmacology, 2005Co-Authors: Juliane Stieber, Karen Wieland, Georg Stöckl, Andreas Ludwig, Franz HofmannAbstract:Sinus node inhibitors reduce the heart rate presumably by blocking the pacemaker current If in the cardiac conduction system. This pacemaker current is carried by four hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels. We tested the potential subtype-specificity of the sinus node inhibitors cilobradine, ivabradine, and zatebradine using cloned HCN channels. All three substances blocked the slow inward current through human HCN1, HCN2, HCN3, and HCN4 channels. There was no subtype-specificity for the steady-state block, with mean IC50 values of 0.99, 2.25, and 1.96 microM for cilobradine, ivabradine, and zatebradine, respectively. Native If, recorded from mouse sinoatrial node cells, was slightly more efficiently blocked by cilobradine (IC50 value of 0.62 microM) than were the HCN currents. The block of I(f) in sinoatrial node cells resulted in slower and dysrhythmic spontaneous action potentials. The in vivo action of these blockers was analyzed using telemetric ECG recordings in mice. Each compound reduced the heart rate dose-dependently from 600 to 200 bpm with ED50 values of 1.2, 4.7, and 1.8 mg/kg for cilobradine, ivabradine, and zatebradine, respectively. beta-Adrenergic stimulation or forced physical activity only partly reversed this bradycardia. In addition to bradycardia, all three drugs induced increasing arrhythmia at concentrations greater than 5 mg/kg for cilobradine, greater than 10 mg/kg for zatebradine, or greater than 15 mg/kg for ivabradine. This dysrhythmic heart rate is characterized by periodic fluctuations of the duration between the T and P wave, resembling a form of sick sinus syndrome in humans. Hence, all available sinus node inhibitors possess an as-yet-unrecognized proarrhythmic potential.
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pacemaker channels and sinus node arrhythmia
Trends in Cardiovascular Medicine, 2004Co-Authors: Juliane Stiebe, Franz Hofma, Andreas LudwigAbstract:Abstract Cardiac pacemaker activity is regulated by at least five different classes of ion channels and by the opposing influence of sympathetic and parasympathetic stimulation. Inactivation of several genes, including a subunit coding for the potassium channel activated by the muscarinic receptor, I KACh ; the calcium channel, I Ca, ; and the hyperpolarization-activated channel, I f , results in sinus node arrhythmia. Inactivation of the gene for the hyperpolarization-activated, cyclic nucleotide-gated channel isoform HCN2 or HCN4 and the use of pacemaker channel blockers show that (a) HCN2 prevents the diastolic membrane potential from becoming too negative, (b) HCN4 is the major channel mediating sympathetic stimulation of the pacemaker activity, and (3) complete blockage of the I f current is compatible with slow sinus node rhythm.
Luisa Garciacuenllas - One of the best experts on this subject based on the ideXlab platform.
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a rare HCN4 variant with combined sinus bradycardia left atrial dilatation and hypertrabeculation left ventricular noncompaction phenotype
Revista Espanola De Cardiologia, 2020Co-Authors: Marta Alonsofernandezgatta, Ricardo Caballero, Eduardo Villacorta, Maria Gallegodelgado, Elena Diazpelaez, Belen Garciaberrocal, Teresa Crespogarcia, Beatriz Plataizquierdo, Elena Marcosvadillo, Luisa GarciacuenllasAbstract:INTRODUCTION AND OBJECTIVES HCN4 variants have been reported to cause combined sick sinus syndrome (SSS) and left ventricular noncompaction (LVNC) cardiomyopathy. This relationship has been proven in few cases and no previous patients have associated left atrial dilatation (LAD). Our objective was to study a familial disorder characterized by SSS, LAD, and hypertrabeculation/LVNC and to identify the underlying genetic and electrophysiological characteristics. METHODS A family with SSS and LVNC underwent a clinical, genetic, and electrophysiological assessment. They were studied via electrocardiography, Holter recording, echocardiography, and exercise stress tests; cardiac magnetic resonance imaging was additionally performed in affected individuals. Genetic testing was undertaken with targeted next-generation sequencing, as well as a functional study of the candidate variant in Chinese hamster ovary cells. RESULTS Twelve members of the family had sinus bradycardia, associated with complete criteria of LVNC in 4 members and hypertrabeculation in 6 others, as well as LAD in 9 members. A HCN4 c.1123C>T;(p.R375C) variant was present in heterozygosis in all affected patients and absent in unaffected individuals. Electrophysiological analyses showed that the amplitude and densities of the HCN4 currents (IHCN4) generated by mutant p.R375C HCN4 channels were significantly lower than those generated by wild-type channels. CONCLUSIONS The combined phenotype of SSS, LAD, and LVNC is associated with the heritable HCN4 c.1123C>T;(p.R375C) variant. HCN4 variants should be included in the genetic diagnosis of LVNC cardiomyopathy and of patients with familial forms of SSS, as well as of individuals with sinus bradycardia and LAD.
Ira S Cohen - One of the best experts on this subject based on the ideXlab platform.
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HCN212-channel biological pacemakers manifesting ventricular tachyarrhythmias are responsive to treatment with If blockade
Heart Rhythm, 2007Co-Authors: Alexei N. Plotnikov, Annalisa Bucchi, Richard B. Robinson, Ira S Cohen, Iryna N. Shlapakova, Peter Danilo, Peter R. Brink, Michael R RosenAbstract:Background A potential concern about biological pacemakers is their possible malfunction, which might create ventricular tachycardias (VTs). Objective The purpose of this study was to test our hypothesis that should VTs complicate implantation of HCN-channel-based biological pacemakers, they would be suppressed by inhibitors of the pacemaker current, I f . Methods We created a chimeric channel (HCN212) containing the N- and C-termini of mouse HCN2 and the transmembrane region of mouse HCN1 and implanted it in HEK293 cells. Forty-eight hours later, in whole-cell patch clamp recordings, mean steady state block induced by 3 μM ivabradine (IVB) showed HCN1=HCN212 > HCN2 currents. The HCN212 adenoviral construct was then implanted into the canine left bundle branch in 11 dogs. Complete AV block was created via radiofrequency ablation, and a ventricular demand electronic pacemaker was implanted (VVI 45 bpm). Electrocardiogram, 24-hour Holter monitoring, and pacemaker log record check were performed for 11 days. Results All dogs developed rapid VT (>120 bpm, maximum rate=285 ± 37 bpm) at 0.9 ± 0.3 days after implantation that persisted through 5 ± 1 days. IVB, 1 mg/kg over 5 minutes, was administered during rapid VT, and three dogs received a second dose 24 hours later. While VT terminated with IBV in all instances within 3.4 ± 0.6 minutes, no effect of IVB on sinus rate was noted. Conclusion We conclude that (1) I f -associated tachyarrhythmias—if they occur with HCN-based biological pacemakers—can be controlled with I f -inhibiting drugs such as IVB; (2) in vitro , IVB appears to have a greater steady state inhibiting effect on HCN1 and HCN212 isoforms than on HCN4; and (3) VT originating from the HCN212 injection site is suppressed more readily than sinus rhythm. This suggests a selectivity of IVB at the concentration attained for ectopic over HCN4-based pacemaker function. This might confer a therapeutic benefit.
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Constitutively active Src tyrosine kinase changes gating of HCN4 channels through direct binding to the channel proteins.
Journal of Cardiovascular Pharmacology, 2006Co-Authors: Suzanne S. Arinsburg, Ira S CohenAbstract:Cardiac pacemaker current, if, is generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Our previous studies demonstrated that altered tyrosine phosphorylation can modulate the properties of both if and HCN channels. To assess a hypothesis that the intracellular tyrosine kinase Src may play a role in modulation by tyrosine phosphorylation of if, we cotransfected HEK293 cells with HCN4 and Src proteins. When HCN4 was cotransfected with a constitutively activated Src protein (Src529), the resultant voltage-dependent HCN4 activation was positively shifted (HCN4: V1/2 = −93 mV; Src529: V1/2 = −80 mV). The activation kinetics were accelerated at some potentials but not over the entire voltage range tested (eg, at −95 mV, τ_act(HCN4) = 3243 ms; τ_act(Src529) = 1113 ms). When HCN4 was cotransfected with a dominant negative Src protein (Src296), the HCN4 activation was shifted more negative to a smaller degree (HCN4: V1/2 = −93 mV; Src296: V1/2 = −98 mV; statistically insignificant) and the activation kinetics were slowed at most test potentials (eg, at −95 mV, τ_act(Src296) = 7396 ms). Neither Src529 nor Src296 significantly altered HCN4 current density. Coimmunoprecipitation experiments revealed that Src forms a complex with HCN4 in HEK293 cells and in rat ventricular myocytes. Our data provide a novel mechanism of if regulation by Src tyrosine phosphorylation.
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Tyrosine kinase inhibition differentially regulates heterologously expressed HCN channels
Pflügers Archiv, 2004Co-Authors: Hangang Yu, Zhongju Lu, Ira S CohenAbstract:The HCN ion channel subunit gene family encodes hyperpolarization-activated cation channels that are permeable to Na^+ and K^+. There are four members of this channel family, three of which, HCN1, HCN2, and HCN4, are expressed in the heart. Current evidence suggests that the HCN ion channel subunit family is the molecular correlate of the alpha subunit of the cardiac pacemaker current i _f. Our previous work has shown that HCN4 is the dominant isoform expressed in the rabbit sinoatrial (SA) node and that changes in tyrosine phosphorylation, either by kinase inhibition or growth factor activation, lead to changes in rabbit SA node i _f conductance with no change in voltage dependence. In the present study we investigate the actions of genistein, a tyrosine kinase inhibitor, on heterologously expressed HCN currents in Xenopus oocytes. Genistein had no effect on HCN1-induced currents, but reduced whole-cell currents induced by HCN2 or HCN4 and slowed activation kinetics at voltages near the midpoint of activation. In the case of HCN2 there was also a negative shift in the voltage dependence of activation that accompanies the current reduction. We have shown previously that HCN2 is the dominant isoform expressed in rat ventricular myocytes. The above results predict that genistein should reduce i _f in the rat ventricle and cause a negative shift of voltage dependence and kinetics of activation. We tested this hypothesis by studying the effects of genistein on isolated rat ventricular myocytes. Genistein significantly reduced i _f current density (pA/pF) (control: 12.2±1.8; genistein: 3.5±0.5; washout: 7.7±0.8; n =10), and caused a negative shift of the midpoint of activation by 14 mV (−133±1 mV for genistein and −119±1 mV for washout, n =7) with no change in slope factor. Our results thus suggest that i _f in the heart and i _f-like currents in other tissues can be regulated differentially by tyrosine phosphorylation based on isoform expression patterns.
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distribution and prevalence of hyperpolarization activated cation channel hcn mrna expression in cardiac tissues
Circulation Research, 1999Co-Authors: Richard B. Robinson, Randy S Wymore, Hangang Yu, Jiying Wu, Jane E Dixon, David Mckinnon, Ira S CohenAbstract:Abstract—HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be ≈−70 mV in neonatal r...
