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Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

Background: There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve., Methods: Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17--longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy., Results: In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells., Conclusions: Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.

Background: We compared cardiac electrophysiological indicators and regional expression levels of cardiac hyperpolarization-activated cyclic nucleotide-gated (HCN) channels between adult and aged dogs to identify possible mechanisms of age-related atrial fibrillation., Material/methods: Corrected sinus node recovery time (SNRTc) and effective refractory period (ERP) of the atrium and pulmonary veins were measured in 10 adult (3-6 years old) and 10 aged dogs (>9 years old). Expression levels of HCN2 and HCN4 channel mRNAs and proteins were measured in the sinoatrial node, atrium, and pulmonary veins by real-time PCR and Western blotting., Results: Aged dogs exhibited a higher induction rate of atrial fibrillation (AF) in response to electrical stimulation, longer AF duration after induction, longer SNRTc, longer right atrial effective refractory period (AERP), shorter left AERP, and increased AERP dispersion compared to adults. Expression levels of HCN2 and HCN4 channel mRNAs and proteins were lower in the sinoatrial node but higher in the atrium and pulmonary veins of aged dogs., Conclusions: Changes in atrial electrophysiological indicators in aged dogs revealed sinoatrial node dysfunction. There was a reversal in the local tissue distribution of HCN2 and HCN4 channel mRNA and protein, a decrease in sinoatrial node expression, and increase in atrial and pulmonary vein expression with age. Changes in atrial electrophysiological characteristics and regional HCN channel expression patterns were associated with the onset and maintenance of age-related atrial fibrillation.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels play prominent roles in shaping muscle and neuronal excitability. In the cardiovascular system, BK channels promote vascular relaxation and protect against ischemic injury. Recently, inhibition of BK channels has been shown to lower heart rate in intact rodents and isolated hearts, suggesting a novel role in heart function. However, the underlying mechanism is unclear. In the present study, we recorded ECGs from mice injected with paxilline (PAX), a membrane-permeable BK channel antagonist, and examined changes in cardiac conduction. ECGs revealed a 19 ± 4% PAX-induced reduction in heart rate in wild-type but not BK channel knockout (Kcnma1(-/-)) mice. The heart rate decrease was associated with slowed cardiac pacing due to elongation of the sinus interval. Action potential firing recorded from isolated sinoatrial node cells (SANCs) was reduced by 55 ± 15% and 28 ± 9% by application of PAX (3 μM) and iberiotoxin (230 nM), respectively. Furthermore, baseline firing rates from Kcnma1(-/-) SANCs were 33% lower than wild-type SANCs. The slowed firing upon BK current inhibition or genetic deletion was due to lengthening of the diastolic depolarization phase of the SANC action potential. Finally, BK channel immunoreactivity and PAX-sensitive currents were identified in SANCs with HCN4 expression and pacemaker current, respectively, and BK channels cloned from SANCs recapitulated similar activation as the PAX-sensitive current. Together, these data localize BK channels to SANCs and demonstrate that loss of BK current decreases SANC automaticity, consistent with slowed sinus pacing after PAX injection in vivo. Furthermore, these findings suggest BK channels are potential therapeutic targets for disorders of heart rate., (Copyright © 2014 the American Physiological Society.)

Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols., (© 2014. Published by The Company of Biologists Ltd.)

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.

Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn(2+) was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn(2+) change membrane potential (Em) and increase the concentration of intracellular Ca(2+) ([Ca(2+)]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn(2+) response in sperm of this species mainly involves an Em hyperpolarization caused by K(+) channel activation. The pharmacological profile of the Zn(2+)-induced hyperpolarization indicates that the cGMP-gated K(+) selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn(2+). Considering that Zn(2+) also induces [Ca(2+)]i fluctuations, our observations suggest that Zn(2+) activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn(2+) in male gamete function., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Fluorescent immunohistochemistry on the cardiac conduction system in whole mount mouse heart preparations demonstrates a particularly dense and complex network of nerve fibres and cardiomyocytes which are positive to the hyperpolarization activated cyclic nucleotide-gated potassium channel 4 (HCN4-positive cardiomyocytes) in the sinoatrial node region and adjacent areas around the root of right cranial vein. The present study was designed to investigate the morphologic and histochemical pattern of nerve fibres and HCN4-positive cardiomyocytes using fluorescent techniques and/or electron microscopy. Adrenergic and cholinergic nerve fibres together with HCN4-positive cardiomyocytes were identified using primary antibodies for tyrosine hydroxylase (TH), choline acetyltransferase (ChAT), and the HCN4 channel respectively. Amid HCN4-positive cardiomyocytes, fluorescence and electron microscopy data demonstrated a dense distribution of nerve fibres immunoreactive for ChAT and TH. In addition, novel electron microscopy data revealed that the mouse sinoatrial node contained exclusively unmyelinated nerve fibres, in which the majority of axons possess varicosities with clear mediatory vesicles that can be classified as cholinergic. Synapses occurred without any clear terminal connection with the effector cell, i.e. these synapes were of "en passant" type. In general, the morphologic pattern of innervation of mouse HCN4-positive cardiomyocytes identified using electron microscopy corresponds well to the dense network of nerve fibres demonstrated by fluorescent immunohistochemistry in mouse sinoatrial node and adjacent areas. The complex and extraordinarily dense innervation of HCN4-positive cardiomyocytes in mouse sinoatrial node underpins the importance of neural regulation for the cardiac conduction system. Based on the present observations, it is concluded that the occurrence of numerous nerve fibres nearby atrial cardiomyocytes serves as a novel reliable extracellular criterion for discrimination of SA nodal cardiomyocytes using electron microscopy., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABAB receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 μ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABAB receptors were pathologically altered and investigated neuroprotective effects of GABAB receptors activation and cross-talk networks between GABAB receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABAB R1, GABAB R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABAB receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABAB R1, GABAB R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 μ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABAB R1 and GABAB R2, and activation of GABAB receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 μ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.

The apical dendrites of many neurons contain proximal and distal compartments that receive synaptic inputs from different brain regions. These compartments also contain distinct complements of ion channels that enable the differential processing of their respective synaptic inputs, making them functionally distinct. At present, the molecular mechanisms that specify dendritic compartments are not well understood. Here, we report that the extracellular matrix protein Reelin, acting through its downstream, intracellular Dab1 and Src family tyrosine kinase signaling cascade, is essential for establishing and maintaining the molecular identity of the distal dendritic compartment of cortical pyramidal neurons. We find that Reelin signaling is required for the striking enrichment of HCN1 and GIRK1 channels in the distal tuft dendrites of both hippocampal CA1 and neocortical layer 5 pyramidal neurons, where the channels actively filter inputs targeted to these dendritic domains., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present study investigated the electropharmacological effects of a traditional Chinese herbal drug, berberine, on the spontaneous activity of sinoatrial nodes (SANs) of the rabbit heart and on human hyperpolarization-activated cyclic nucleotide-gated 4 (hHCN4) channels, which are heterologously expressed in xenopus oocytes, and which contribute to pacemaker currents (Ifs). A standard microelectrode technique and standard two‑electrode voltage‑clamp recordings were employed to examine the properties of transmembrane potentials and cloned hHCN4 subunit currents, respectively, under control conditions and berberine administration. Berberine decreased the rate of pacemaker firing and the rate of diastolic depolarization, and modified the action potential parameters. In addition, berberine suppressed the hHCN4 channel currents in a concentration‑ (1‑300 µM) and use‑dependent manner, and simultaneously decreased the activation and deactivation kinetics of the hHCN4 channels. The ability of berberine to modulate the If of cardiac pacemaker cells may contribute to its antiarrhythmic action.

The pacemaker current If conducted by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels plays a critical role in the regulation of cardiac automaticity, with If density increased in hypertrophied ventricular myocytes. Amiodarone, a highly effective anti-arrhythmic agent, blocks human HCN currents and native If under normal conditions. To determine the effects of amiodarone under pathological conditions, we monitored If under after both acute (0.01, 0.1, 1, 10 and 100 μmol/L) and chronic (10 μmol/L) amiodarone treatment in ventricular myocytes from spontaneously hypertensive rats (SHR) with left ventricular hypertrophy using the whole-cell patch-clamp technique. The If current density was significantly greater in SHR ventricular myocytes than in cells from healthy normotensive control Wistar-Kyoto (WKY) rats. Acute application of amiodarone significantly decreased If density in myocytes from both SHR and WKY rats. The inhibition was concentration dependent with an IC50 of 4.9 ± 1.2 and 6.9 ± 1.3 μmol/L in myocytes from SHR and WKY rats, respectively. Amiodarone increased the activation and deactivation times of If in myocytes from SHR, although it did not alter the relationship of voltage-dependent activation and the reversal potential of If in myocytes from SHR. Chronic exposure of myocytes from SHR to amiodarone potently inhibited If and downregulated HCN2 and HCN4, the major channel subtypes underlying native If , at both the mRNA and protein level. These findings indicate that amiodarone inhibits If under hypertrophied conditions through dual mechanisms: (i) direct channel blockade of If currents; and (ii) indirect suppression via negative regulation of HCN channel gene expression. These unique properties of amiodarone may contribute to its anti-arrhythmic properties under pathological conditions., (© 2014 Wiley Publishing Asia Pty Ltd.)

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5'-O-(3-thio) triphosphate (ATPγS), 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5'-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca(2+) channel blockers GdCl3 and LaCl3. A hyperpolarization-activated Ca(2+) channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca(2+) channel significantly. Extracellular ATP-promoted Ca(2+) channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca(2+) channels., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca2+]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca2+]i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca2+] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na+ channels (HCN), [Ca2+]-dependent Cl- channels (CaCC) and [Ca2+]-dependent K+ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca2+]-dependent K+ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca2+] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.

The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca(2+)]i release and Ca(2+) handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels control neuronal and cardiac electrical rhythmicity. There are four homologous isoforms (HCN1-4) sharing a common multidomain architecture that includes an N-terminal transmembrane tetrameric ion channel followed by a cytoplasmic "C-linker," which connects a more distal cAMP-binding domain (CBD) to the inner pore. Channel opening is primarily stimulated by transmembrane elements that sense membrane hyperpolarization, although cAMP reduces the voltage required for HCN activation by promoting tetramerization of the intracellular C-linker, which in turn relieves auto-inhibition of the inner pore gate. Although binding of cAMP has been proposed to relieve auto-inhibition by affecting the structure of the C-linker and CBD, the nature and extent of these cAMP-dependent changes remain limitedly explored. Here, we used NMR to probe the changes caused by the binding of cAMP and of cCMP, a partial agonist, to the apo-CBD of HCN4. Our data indicate that the CBD exists in a dynamic two-state equilibrium, whose position as gauged by NMR chemical shifts correlates with the V½ voltage measured through electrophysiology. In the absence of cAMP, the most populated CBD state leads to steric clashes with the activated or "tetrameric" C-linker, which becomes energetically unfavored. The steric clashes of the apo tetramer are eliminated either by cAMP binding, which selects for a CBD state devoid of steric clashes with the tetrameric C-linker and facilitates channel opening, or by a transition of apo-HCN to monomers or dimer of dimers, in which the C-linker becomes less structured, and channel opening is not facilitated., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neuronal nitric oxide (NO) plays an important role in gastric motor activity and modulates the mechanosensitivity of gastro-oesophageal vagal afferents. Effects of NO on food intake are dependent on feeding status. We sought to determine the effect of NO on gastro-oesophageal vagal afferent activity in the normally fed and food-restricted states and the second messenger pathways mediating these effects. Eight week old female C56BL/6 mice were fed ad libitum or food restricted for 14 h. An in vitro preparation was used to determine the functional effects of NO and the second messenger pathways involved. Expression of NO signal transduction molecules in vagal afferents was determined by reverse-transcription polymerase chain reaction (RT-PCR). Endogenous NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited vagal mucosal afferent responses to tactile stimuli in mice fed ad libitum. After a 14 h fast endogenous NO and SNAP potentiated tension and mucosal afferent responses to mechanical stimulation. The excitatory effect of NO was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. After a 14 h fast expression of NADPH oxidase 2 (NOX2) mRNA in whole nodose ganglia was significantly reduced and the excitatory effect of NO on gastro-oesophageal vagal afferents was lost. Under fasting conditions the inhibitory effect of NO was blocked with the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channel blocker ivabradine and mRNA expression of HCN3 in the nodose ganglia was elevated. In conclusion, the role of NO in the peripheral modulation of gastro-oesophageal vagal afferents is dynamic and dependent on feeding status., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated potassium ion channels. They are, however, activated by hyperpolarizing potentials and are permeable to cations. Four HCN subunits have been cloned, of which HCN1 and HCN2 subunits are predominantly expressed in the cortex. These subunits are principally located in pyramidal cell dendrites, although they are also found at lower concentrations in the somata of pyramidal neurons as well as other neuron subtypes. HCN channels are actively trafficked to dendrites by binding to the chaperone protein TRIP8b. Somato-dendritic HCN channels in pyramidal neurons modulate spike firing and synaptic potential integration by influencing the membrane resistance and resting membrane potential. Intriguingly, HCN channels are present in certain cortical axons and synaptic terminals too. Here, they regulate synaptic transmission but the underlying mechanisms appear to vary considerably amongst different synaptic terminals. In conclusion, HCN channels are expressed in multiple neuronal subcellular compartments in the cortex, where they have a diverse and complex effect on neuronal excitability., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model) but couple more loosely (as envisioned in a modular model of protein activation). Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile "slow" channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.

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., (© The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The subiculum, considered to be the output structure of the hippocampus, modulates information flow from the hippocampus to various cortical and sub-cortical areas such as the nucleus accumbens, lateral septal region, thalamus, nucleus gelatinosus, medial nucleus and mammillary nuclei. Tonic inhibitory current plays an important role in neuronal physiology and pathophysiology by modulating the electrophysiological properties of neurons. While the alterations of various electrical properties due to tonic inhibition have been studied in neurons from different regions, its influence on intrinsic subthreshold resonance in pyramidal excitatory neurons expressing hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is not known. Using pharmacological agents, we show the involvement of α5βγ GABAA receptors in the picrotoxin-sensitive tonic current in subicular pyramidal neurons. We further investigated the contribution of tonic conductance in regulating subthreshold electrophysiological properties using current clamp and dynamic clamp experiments. We demonstrate that tonic GABAergic inhibition can actively modulate subthreshold properties, including resonance due to HCN channels, which can potentially alter the response dynamics of subicular pyramidal neurons in an oscillating neuronal network., (© 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

The purpose of this research is to explore the distribution and expression of hyperpolarization-activated cyclic nucleotide-gated channels subtype 2 (HCN2) in cerebrospinal fluid (CSF)-contacting nucleus in neuropathic pain, and provide experimental evidence to reveal the biological function and regulation mechanisms of CSF-contacting nucleus in neuropathic pain. Neuropathic pain model was produced by chronic constriction injury (CCI) in Sprague-Dawley (SD) rats. Intracerebroventricular injection of cholera toxin subunit B (CTb) labeled with horseradish peroxidase (CB-HRP) was used to specifically mark distal CSF-contacting nucleus. The thermal withdrawal latency and mechanical withdrawal threshold of rats were recorded to detect the change of pain threshold. The expressions HCN2 channel and c-Fos proteins in CSF-contacting nucleus were detected by immunofluorescence and Western blot. The results showed that, compared with the control group, CTb-treated rats did not show any differences in the expressions of HCN2 channel and c-Fos proteins in CSF-contacting nucleus, as well as pain threshold. At 7, 14 d after CCI operation, the model rats showed not only significantly increased expressions of HCN2 channel and c-Fos in CSF-contacting nucleus, but also decreased pain threshold. ZD7288, a HCN2 channel blocker, could reverse the above changes in neuropathic pain model rats. These results suggest that the CSF-contacting nucleus may be involved in the process of neuropathic pain via the HCN2 channel.

Background: Atrial fibrillation (AF) is the most common arrhythmia, and a recent genome-wide association study identified the hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) as a novel AF susceptibility locus. HCN4 encodes for the cardiac pacemaker channel, and HCN4 mutations are associated with familial sinus bradycardia and AF., Objective: The purpose of this study was to determine whether novel variants in the coding region of HCN4 contribute to the susceptibility for AF., Methods: We sequenced the coding region of HCN4 for novel variants from 527 cases with early-onset AF from the Massachusetts General Hospital AF Study and 443 referents from the Framingham Heart Study. We used site-directed mutagenesis, cellular electrophysiology, immunocytochemistry, and confocal microscopy to functionally characterize novel variants., Results: We found the frequency of novel coding HCN4 variants was 2-fold greater for individuals with AF (7 variants) compared to the referents (3 variants). We determined that of the 7 novel HCN4 variants in our AF cases, 1 (p.Pro257Ser, located in the amino-terminus adjacent to the first transmembrane spanning domain) did not traffic to cell membrane, whereas the remaining 6 were not functionally different from wild type. In addition, the 3 novel variants in our referents did not alter function compared to wild-type. Coexpression studies showed that the p.Pro257Ser mutant channel failed to colocalize with the wild-type HCN4 channel on the cell membrane., Conclusion: Our findings are consistent with HCN4 haploinsufficiency as the likely mechanism for early-onset AF in the p.Pro257Ser carrier., (Copyright © 2014 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system.

Background: Hyperpolarization-activated cyclic nucleotide (HCN) channels are pacemaker channels that regulate heart rate and neuronal rhythm in spontaneously active cardiac and neuronal cells. Interstitial cells of Cajal (ICCs) are also spontaneously active pacemaker cells in the gastrointestinal tract. Here, we investigated the existence of HCN channel and its role on pacemaker activity in colonic ICCs., Methods: We performed whole-cell patch clamp, RT-PCR, and Ca(2+)-imaging in cultured ICCs from mouse mid colon., Results: SQ-22536 and dideoxyadenosine (adenylate cyclase inhibitors) decreased the frequency of pacemaker potentials, whereas both rolipram (cAMP-specific phosphodiesterase inhibitor) and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. CsCl, ZD7288, zatebradine, clonidine (HCN channel blockers), and genistein (a tyrosine kinase inhibitor) suppressed the pacemaker activity. RT-PCR revealed expression of HCN1 and HCN3 channels in c-kit and Ano1 positive colonic ICCs. In recordings of spontaneous intracellular Ca(2+) [Ca(2+)]i oscillations, rolipram and 8-bromo-cAMP increased [Ca(2+)]i oscillations, whereas SQ-22536, CsCl, ZD7288, and genistein decreased [Ca(2+)]i oscillations., Conclusions: HCN channels in colonic ICCs are tonically activated by basal cAMP production and participate in regulation of pacemaking activity.

Deciphering the molecular basis of HCN channel regulation by cGMP leads to the serendipitous discovery of cyclic dinucleotides as potent inhibitors of I(f) current in the heart.

Endurance athletes exhibit sinus bradycardia, that is a slow resting heart rate, associated with a higher incidence of sinus node (pacemaker) disease and electronic pacemaker implantation. Here we show that training-induced bradycardia is not a consequence of changes in the activity of the autonomic nervous system but is caused by intrinsic electrophysiological changes in the sinus node. We demonstrate that training-induced bradycardia persists after blockade of the autonomous nervous system in vivo in mice and in vitro in the denervated sinus node. We also show that a widespread remodelling of pacemaker ion channels, notably a downregulation of HCN4 and the corresponding ionic current, If. Block of If abolishes the difference in heart rate between trained and sedentary animals in vivo and in vitro. We further observe training-induced downregulation of Tbx3 and upregulation of NRSF and miR-1 (transcriptional regulators) that explains the downregulation of HCN4. Our findings provide a molecular explanation for the potentially pathological heart rate adaptation to exercise training.

HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

Singlet oxygen ((1)O2), which is generated through metabolic reactions and oxidizes numerous biological molecules, has been a useful tool in basic research and clinical practice. However, its role as a signaling factor, as well as a mechanistic understanding of the oxidation process, remains poorly understood. Here, we show that hyperpolarization-activated, cAMP-gated (HCN) channels--which conduct the hyperpolarization-activated current (Ih) and the voltage-insensitive instantaneous current (Iinst), and contribute to diverse physiological functions including learning and memory, cardiac pacemaking, and the sensation of pain--are subject to modification by (1)O2. To increase the site specificity of (1)O2 generation, we used fluorescein-conjugated cAMP, which specifically binds to HCN channels, or a chimeric channel in which an in-frame (1)O2 generator (SOG) protein was fused to the HCN C terminus. Millisecond laser pulses reduced Ih current amplitude, slowed channel deactivation, and enhanced Iinst current. The modification of HCN channel function is a photodynamic process that involves (1)O2, as supported by the dependence on dissolved oxygen in solutions, the inhibitory effect by a (1)O2 scavenger, and the results with the HCN2-SOG fusion protein. Intriguingly, (1)O2 modification of the HCN2 channel is state dependent: laser pulses applied to open channels mainly slow down deactivation and increase Iinst, whereas for the closed channels, (1)O2 modification mainly reduced Ih amplitude. We identified a histidine residue (H434 in S6) near the activation gate in the pore critical for (1)O2 modulation of HCN function. Alanine replacement of H434 abolished the delay in channel deactivation and the generation of Iinst induced by photodynamic modification. Our study provides new insights into the instantaneous current conducted by HCN channels, showing that modifications to the region close to the intracellular gate underlie the expression of Iinst, and establishes a well-defined model for studying (1)O2 modifications at the molecular level.

The GABAAγ2(R43Q) mouse is an established model of absence epilepsy displaying spontaneous spike-and-wave discharges (SWD) and associated behavioral arrest. Absence epilepsy typically results from cortico-thalamic networks. Nevertheless, there is increasing evidence for changes in hippocampal metabolism and electrical behavior, consistent with a link between absence seizures and hippocampus-related co-morbidities. Hyperpolarization-activated-cyclic-nucleotide-gated (HCN) channels are known to be transcriptionally regulated in a number of seizure models. Here we investigate the expression and function of these channels in the hippocampus of the genetic epilepsy model. A reduction in HCN1, but not HCN2 transcript, was observed in GABAAγ2(R43Q) mice relative to their littermate controls. In contrast, no change in HCN1 transcript was noted at an age prior to seizure expression or in a SWD-free model in which the R43Q mutation has been crossed into a seizure-resistant genetic background. Whole-cell recordings from CA1 pyramidal neurons confirm a reduction in Ih in the GABAAγ2(R43Q) mouse. Further, a left-shift in half-activation of the Ih conductance-voltage relationship is consistent with a reduction in HCN1 with no change in HCN2 channel expression. Behavioral analysis using the Morris water maze indicates that GABAAγ2(R43Q) mice are unable to learn as effectively as their wildtype littermates suggesting a deficit in hippocampal-based learning. SWD-free mice harboring the R43Q mutation had no learning deficit. We conclude that SWDs reduce hippocampal HCN1 expression and function, and that the reduction associates with a spatial learning deficit., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Eukaryotic CBDs (cAMP-binding domains) control multiple cellular functions (e.g. phosphorylation, guanine exchange and ion channel gating). Hence the manipulation of cAMP-dependent signalling pathways has a high translational potential. However, the ubiquity of eukaryotic CBDs also poses a challenge in terms of selectivity. Before the full translational potential of cAMP signalling can be tapped, it is critical to understand the structural basis for selective cAMP agonism and antagonism. Recent NMR investigations have shown that structurally homologous CBDs respond differently to several CBD ligands and that these unexpected differences arise at the level of either binding (i.e. affinity) or allostery (i.e. modulation of the autoinhibitory equilibria). In the present article, we specifically address how the highly conserved CBD fold binds cAMP with markedly different affinities in PKA (protein kinase A) relative to other eukaryotic cAMP receptors, such as Epac (exchange protein directly activated by cAMP) and HCN (hyperpolarization-activated cyclic-nucleotide-modulated channel). A major emerging determinant of cAMP affinity is hypothesized to be the position of the autoinhibitory equilibrium of the apo-CBD, which appears to vary significantly across different CBDs. These analyses may assist the development of selective CBD effectors that serve as potential drug leads for the treatment of cardiovascular diseases.

Coordinated ureteric peristalsis propels urine from the kidney to the bladder. Cells in the renal pelvis and ureter spontaneously generate and propagate electrical activity to control this process. Recently, c-kit tyrosine kinase and hyperpolarization-activated cyclic nucleotide-gated channel 3 (HCN3) were identified in the upper urinary tract. Both of these proteins are required for coordinated proximal to distal contractions in the ureter. Alterations in pacemaker cell expression are present in multiple congenital kidney diseases, suggesting a functional contribution by these cells to pathologic states. In contrast to gut and heart pacemaker cells, the developmental biology of ureteric pacemaker cells, including cell lineage and signaling mechanisms, is undefined. Here, we review pacemaker cell identify and function in the urinary pelvis and ureter and the control of pacemaker function by Hedgehog-GLI signaling. Next, we highlight current knowledge of gut and heart pacemaker cells that is likely to provide insight into developmental mechanisms that could control urinary pacemaker cells.

Depressed heart rate variability in severe inflammatory diseases can be partially explained by the lipopolysaccharide (LPS)-dependent modulation of cardiac pacemaker channels. Recently, we showed that LPS inhibits pacemaker current in sinoatrial node cells and in HEK293 cells expressing cloned pacemaker channels, respectively. The present study was designed to verify whether this inhibition involves LPS-dependent intracellular signalling and to identify structures of LPS responsible for pacemaker current modulation. We examined the effect of LPS on the activity of human hyperpolarization-activated cyclic nucleotide-gated channel 2 (hHCN2) stably expressed in HEK293 cells. In whole-cell recordings, bath application of LPS decreased pacemaker current (IhHCN2) amplitude. The same protocol had no effect on channel activity in cell-attached patch recordings, in which channels are protected from the LPS-containing bath solution. This demonstrates that LPS must interact directly with or close to the channel protein. After cleavage of LPS into lipid A and the polysaccharide chain, neither of them alone impaired IhHCN2, which suggests that modulation of channel activity critically depends on the integrity of the entire LPS molecule. We furthermore showed that β-cyclodextrin interfered with LPS-dependent channel modulation predominantly via scavenging of lipid A, thereby abrogating the capability of LPS to intercalate into target cell membranes. We conclude that LPS impairs IhHCN2 by a local mechanism that is restricted to the vicinity of the channels. Furthermore, intercalation of lipid A into target cell membranes is a prerequisite for the inhibition that is suggested to depend on the direct interaction of the LPS polysaccharide chain with cardiac pacemaker channels.

Disruptions of endoplasmic reticulum (ER) Ca(2+) homeostasis are heavily linked to neuronal pathology. Depletion of ER Ca(2+) stores can result in cellular dysfunction and potentially cell death, although adaptive processes exist to aid in survival. We examined the age and region dependence of one postulated, adaptive response to ER store-depletion (SD), hyperpolarization-activated cation-nonspecific (h)-channel plasticity in neurons of the dorsal and ventral hippocampus (DHC and VHC, respectively) from adolescent and adult rats. With the use of whole-cell patch-clamp recordings from the soma and dendrites of CA1 pyramidal neurons, we observed a change in h-sensitive measurements in response to SD, induced by treatment with cyclopiazonic acid, a sarcoplasmic reticulum/ER Ca(2+)-ATPase blocker. We found that whereas DHC and VHC neurons in adolescent animals respond to SD with a perisomatic expression of SD h plasticity, adult animals express SD h plasticity with a dendritic and somatodendritic locus of plasticity in DHC and VHC neurons, respectively. Furthermore, SD h plasticity in adults was dependent on membrane potential and on the activation of L-type voltage-gated Ca(2+) channels. These results suggest that cellular responses to the impairment of ER function, or ER stress, are dependent on brain region and age and that the differential expression of SD h plasticity could provide a neural basis for region- and age-dependent disease vulnerabilities.

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties. Whereas regulation of HCN1 channels by FLNa has been observed in melanoma cell lines, its physiological relevance to neuronal function and the underlying cellular pathways that govern this regulation remain unknown. Using a combination of mutational, pharmacological, and imaging approaches, we find here that FLNa facilitates a selective and reversible dynamin-dependent internalization of HCN1 channels in HEK293 cells. This internalization is accompanied by a redistribution of HCN1 channels on the cell surface, by accumulation of the channels in endosomal compartments, and by reduced Ih density. In hippocampal neurons, expression of a truncated dominant-negative FLNa enhances the expression of native HCN1. Furthermore, acute abrogation of HCN1-FLNa interaction in neurons, with the use of decoy peptides that mimic the FLNa-binding domain of HCN1, abolishes the punctate distribution of HCN1 channels in neuronal cell bodies, augments endogenous Ih, and enhances the rebound-response ("voltage-sag") of the neuronal membrane to transient hyperpolarizing events. Together, these results support a major function of FLNa in modulating ion channel abundance and membrane trafficking in neurons, thereby shaping their biophysical properties and function.

Renal pacemakers set the origin and frequency of the smooth muscle contractions that propel wastes from the kidney to the bladder. Although congenital defects impairing this peristalsis are a leading cause of pediatric renal failure, the mechanisms underlying renal pacemaker activity remain unknown. Using ratiometric optical mapping and video microscopy, we discovered that hyperpolarization-activated cation (HCN) channel block with the specific anatagonist ZD7288 (30 μm; IC50) abolished the pacemaker depolarizations that initiate murine upper urinary tract peristalsis. Optical mapping and immunohistochemistry indicate that pacemaker potentials are generated by cells expressing HCN isoform-3, and that HCN3(+) cells are coupled to definitive smooth muscle via gap junctions. Furthermore, we demonstrate that HCN3(+) cells coexpress T-type Ca(2+) (TTC) channels and that TTC channel inhibition with R(-)efonidipine or NNC55-0396 decreased contractile frequency in a dose-dependent manner. Collectively, these data demonstrate that HCN3(+)/TTC(+) cells are the pacemakers that set the origin and rate of upper urinary tract peristalsis. These results reveal a conserved mechanism controlling autorhythmicity in 2 distinct muscle types, as HCN and TTC channels also mediate cardiac pacemaker activity. Moreover, these findings have translational applications, including the development of novel diagnostics to detect fetal urinary tract motility defects prior to renal damage.-Hurtado, R., Bub, G., Herzlinger, D. A molecular signature of tissues with pacemaker activity in the heart and upper urinary tract involves coexpressed hyperpolarization-activated cation and T-type Ca(2+) channels.

The spiral ganglion conveys afferent auditory information predominantly through a single class of type I neurons that receive signals from inner hair cell sensory receptors. These auditory primary afferents, like in other systems (Puopolo and Belluzzi, 1998; Gascon and Moqrich, 2010; Leao et al., 2012) possess a marked diversity in their electrophysiological features (Taberner and Liberman, 2005). Consistent with these observations, when the auditory primary afferents were assessed in neuronal explants separated from their peripheral and central targets it was found that individual neurons were markedly heterogeneous in their endogenous electrophysiological features. One aspect of this heterogeneity, obvious throughout the ganglion, was their wide range of excitability as assessed by voltage threshold measurements (Liu and Davis, 2007). Thus, while neurons in the base differed significantly from apical and middle neurons in their voltage thresholds, each region showed distinctly wide ranges of values. To determine whether the resting membrane potentials (RMPs) of these neurons correlate with the threshold distribution and to identify the ion channel regulatory elements underlying heterogeneous neuronal excitability in the ganglion, patch-clamp recordings were made from postnatal day (P5-8) murine spiral ganglion neurons in vitro. We found that RMP mirrored the tonotopic threshold distribution, and contributed an additional level of heterogeneity in each cochlear location. Pharmacological experiments further indicated that threshold and RMP was coupled through the Kv1 current, which had a dual impact on both electrophysiological parameters. Whereas, hyperpolarization-activated cationic channels decoupled these two processes by primarily affecting RMP without altering threshold level. Thus, beyond mechanical and synaptic specializations, ion channel regulation of intrinsic membrane properties imbues spiral ganglion neurons with different excitability levels, a feature that contributes to primary auditory afferent diversity., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are cation-selective channels present in retina, brain and heart. The activity of HCN channels contributes to signal integration, cell excitability and pacemaker activity. HCN1 channels expressed in photoreceptors participate in keeping light responses transient and are required for normal mesopic vision. The subcellular localization of HCN1 varies among cell types. In photoreceptors HCN1 is concentrated in the inner segments while in other retinal neurons, HCN1 is evenly distributed though the cell. This is in contrast to hippocampal neurons where HCN1 is concentrated in a subset of dendrites. A key regulator of HCN1 trafficking and activity is tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b). Multiple splice isoforms of TRIP8b are expressed throughout the brain and can differentially regulate the surface expression and activity of HCN1. The purpose of the present study was to determine which isoforms of TRIP8b are expressed in the retina and to test if loss of TRIP8b alters HCN1 expression or trafficking. We found that TRIP8b colocalizes with HCN1 in multiple retina neurons and all major splice isoforms of TRIP8b are expressed in the retina. Photoreceptors express three different isoforms. In TRIP8b knockout mice, the ability of HCN1 to traffic to the surface of retinal neurons is unaffected. However, there is a large decrease in the total amount of HCN1. We conclude that TRIP8b in the retina is needed to achieve maximal expression of HCN1.

Background:  Loss-of-function mutations in the HCN4 gene have been shown to be associated with sinus dysfunction, but there are no reports on HCN4-mediated atrioventricular (AV) block. A novel missense HCN4 mutation G1097W was identified in a 69 year-old Japanese male with AV block, and we characterized the functional consequences of If-like channels reconstituted with the heterozygous HCN4 mutation., Methods and Results:  Wild-type (WT) HCN4 or/and HCN4-G1097W were expressed in a heterologous cell expression system. A functional assay using a whole-cell patch-clamp demonstrated that the mutant If-like currents were activated at more negative voltages compared to WT currents, while they retained the sensitivity to changes in intracellular cyclic adenosine monophosphate (cAMP) levels. Co-expression of G1097W with WT channels showed dominant-negative effects, including a reduction in peak currents and a negative voltage shifting on reconstituted currents., Conclusions:  The HCN4-G1097W mutant channels displayed a loss-of-function type modulation on cardiac If channels and thus could predispose them to AV nodal dysfunction. These data provide a novel insight into the genetic basis for the AV block.

Among the different transport systems present in plant cells, Shaker channels constitute the major pathway for K(+) in the plasma membrane. Plant Shaker channels are members of the 6 transmembrane-1 pore (6TM-1P) cation channel superfamily as the animal Shaker (Kv) and HCN channels. All these channels are voltage-gated K(+) channels: Kv channels are outward-rectifiers, opened at depolarized voltages and HCN channels are inward-rectifiers, opened by membrane hyperpolarization. Among plant Shaker channels, we can find outward-rectifiers, inward-rectifiers and also weak-rectifiers, with weak voltage dependence. Despite the absence of crystal structures of plant Shaker channels, functional analyses coupled to homology modeling, mostly based on Kv and HCN crystals, have permitted the identification of several regions contributing to plant Shaker channel gating. In the present mini-review, we make an update on the voltage-gating mechanism of plant Shaker channels which seem to be comparable to that proposed for HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in a broad range of cell types, but the expression of the various HCN isoforms is still poorly understood. In the present study we have compared the expression of HCN isoforms in rat excitable and non-excitable tissues at both the mRNA and protein levels. Real-time PCR and Western blot analysis revealed distinct expression patterns of the four HCN isoforms in brain, heart, pituitary and kidney, with inconsistent mRNA-protein expression correlation. The HCN2 was the most abundant mRNA transcript (95.6, 78.0 and 59.0 % in kidney heart and pituitary, respectively) except in the brain (42.0 %) whereas HCN4 was the most abundant protein isoform. Our results suggest that HCN channels are mostly produced by the HCN4 isoform in heart, which contrasts with the sharp differences in the isoform stoichiometry in pituitary (15 HCN4:2 HCN2:1 HCN1:1 HCN3), kidney (24 HCN4:2 HCN3:1 HCN2:1 HCN1) and brain (3 HCN4:2 HCN2:1 HCN1:1 HCN3). Moreover, deviations of the electrophoretic molecular weight (MW) of the HCN isoforms relative to the theoretical MW were observed, suggesting that N-glycosylation and enzymatic proteolysis influences HCN channel surface expression. We hypothesize that selective cleavage of HCN channels by membrane bound metalloendopeptidases could account for the multiplicity of properties of native HCN channels in different tissues.

Background/aims: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis., Methods: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology., Results: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and If currents as well as functional hormonal regulation and sensitivity to If channel blockers., Conclusion: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes., (© 2014 S. Karger AG, Basel.)

Background: Sinus node dysfunction (SND) is a major clinically relevant disease that is associated with sudden cardiac death and requires surgical implantation of electric pacemaker devices. Frequently, SND occurs in heart failure and hypertension, conditions that lead to electric instability of the heart. Although the pathologies of acquired SND have been studied extensively, little is known about the molecular and cellular mechanisms that cause congenital SND., Methods and Results: Here, we show that the HCN1 protein is highly expressed in the sinoatrial node and is colocalized with HCN4, the main sinoatrial pacemaker channel isoform. To characterize the cardiac phenotype of HCN1-deficient mice, a detailed functional characterization of pacemaker mechanisms in single isolated sinoatrial node cells, explanted beating sinoatrial node preparation, telemetric in vivo electrocardiography, echocardiography, and in vivo electrophysiology was performed. On the basis of these experiments we demonstrate that mice lacking the pacemaker channel HCN1 display congenital SND characterized by bradycardia, sinus dysrhythmia, prolonged sinoatrial node recovery time, increased sinoatrial conduction time, and recurrent sinus pauses. As a consequence of SND, HCN1-deficient mice display a severely reduced cardiac output., Conclusions: We propose that HCN1 stabilizes the leading pacemaker region within the sinoatrial node and hence is crucial for stable heart rate and regular beat-to-beat variation. Furthermore, we suggest that HCN1-deficient mice may be a valuable genetic disease model for human SND.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel-mediated currents, known as I h, are involved in the control of rhythmic activity in neuronal circuits and in determining neuronal properties including the resting membrane potential. Recent studies have shown that HCN channels play a role in seizure susceptibility and in absence and limbic epilepsy including temporal lobe epilepsy following long febrile seizures (FS). This study focused on the potential contributions of abnormalities in the HCN2 isoform and their role in FS. A novel heterozygous missense mutation in HCN2 exon 1 leading to p.S126L was identified in two unrelated patients with FS. The mutation was inherited from the mother who had suffered from FS in a pedigree. To determine the effect of this substitution we conducted whole-cell patch clamp electrophysiology. We found that mutant channels had elevated sensitivity to temperature. More specifically, they displayed faster kinetics at higher temperature. Kinetic shift by change of temperature sensitivity rather than the shift of voltage dependence led to increased availability of I h in conditions promoting FS. Responses to cyclic AMP did not differ between wildtype and mutant channels. Thus, mutant HCN2 channels cause significant cAMP-independent enhanced availability of I h during high temperatures, which may contribute to hyperthermia-induced neuronal hyperexcitability in some individuals with FS.

Objective: To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells., Methods: Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group., Results: After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 +/- 0.41) was significantly higher than that of the control group (23.39 +/- 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current., Conclusion: The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.

Introduction: Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF., Methods and Results: Right atrial samples were obtained from sinus rhythm (SR, n = 49) or cAF (duration >1 year, n = 31) patients undergoing corrective cardiac surgery. Among f-channel isoforms expressed in the human atrium (HCN1, 2 and 4), HCN4 mRNA levels measured by RT-PCR were significantly reduced. However, protein expression was preserved in cAF compared to SR (+85% for HCN4); concurrently, miR-1 expression was significantly reduced. In patch-clamped atrial myocytes, current-specific conductance (gf) was significantly increased in cAF at voltages around the threshold for If activation (-60 to -80 mV); accordingly, a 10-mV rightward shift of the activation curve occurred (P < 0.01). β-Adrenergic and 5-HT4 receptor stimulation exerted similar effects on If in cAF and SR cells, while the ANP-mediated effect was significantly reduced (P < 0.02), suggesting downregulation of natriuretic peptide signaling., Conclusions: In human cAF modifications in transcriptional and posttranscriptional mechanisms of HCN channels occur, associated with a slight yet significant gain-of-function of If , which may contribute to enhanced atrial ectopy., (© 2013 Wiley Periodicals, Inc.)

The aneural heart of the Pacific hagfish, Eptatretus stoutii, varies heart rate fourfold during recovery from anoxia. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which play an important role in establishing the pacemaker rate of vertebrate hearts, were postulated to be present in this ancestral vertebrate heart, and it was also theorized that changes in hagfish heart rate with oxygen availability involved altered HCN expression. Partial gene cloning revealed six HCN isoforms in the hagfish heart. Hagfish representatives of HCN2, HCN3 and HCN4 were discovered, with HCN2 and HCN3 existing as isoforms designated as HCN2a, HCN2b, HCN3a, two paralogs of HCN3b, and HCN3c. Phylogenetic analysis revealed HCN3b and HCN3c to be ancestral, followed by HCN3a, HCN4 and HCN2. Moreover, HCN3a expression was dominant in both the atrial and ventricular chambers, suggesting that the HCN4 dominance in adult mammalian hearts appeared after hagfish divergence. HCN expression was higher in the atrium than in the ventricle, as might be expected given that atrial beating rate is known to be faster than the ventricular rate. In addition, mRNA expression under normoxic conditions was compared with that following 24 h of anoxia, and either a 2-h or 36-h recovery in normoxic water. In the ventricle, anoxia decreased HCN3a but not HCN4 expression. In contrast, atrial HCN3a expression significantly increased following 2 h of recovery, before returning to control levels following 36 h of recovery, possibly contributing to heart rate changes previously observed under these conditions.