Muscarinic Acetylcholine Receptors Modulate HCN Channel Properties in Vestibular Ganglion Neurons.

Efferent modulation of vestibular afferent excitability is linked to muscarinic signaling cascades that close low-voltage-gated potassium channels (i.e., KCNQ). Here, we show that muscarinic signaling cascades also depolarize the activation range of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. We compared the voltage activation range and kinetics of HCN channels and induced firing patterns before and after administering the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M (Oxo-M) in dissociated vestibular ganglion neurons (VGNs) from rats of either sex using perforated whole-cell patch-clamp methods. Oxo-M depolarized HCN channels' half-activation voltage (V 1/2) and sped up the rate of activation near resting potential twofold. HCN channels in large-diameter and/or transient firing VGN (putative cell bodies of irregular firing neuron from central epithelial zones) had relatively depolarized V 1/2 in control solution and were less sensitive to mAChR activation than those found in small-diameter VGN with sustained firing patterns (putatively belonging to regular firing afferents). The impact of mAChR on HCN channels is not a direct consequence of closing KCNQ channels since pretreating the cells with Linopirdine, a KCNQ channel blocker, did not prevent HCN channel depolarization by Oxo-M. Efferent signaling promoted ion channel configurations that were favorable to highly regular spiking in some VGN, but not others. This is consistent with previous observations that low-voltage gated potassium currents in VGN are conducted by mAChR agonist-sensitive and -insensitive channels. Connecting efferent signaling to HCN channels is significant because of the channel's impact on spike-timing regularity and nonchemical transmission between Type I hair cells and vestibular afferents.SIGNIFICANCE STATEMENT Vestibular afferents express a diverse complement of ion channels. In vitro studies identified low-voltage activated potassium channels and hyperpolarization-activated cyclic-nucleotide gated (HCN) channels as crucial for shaping the timing and sensitivity of afferent responses. Moreover, a network of acetylcholine-releasing efferent neurons controls afferent excitability by closing a subgroup of low-voltage activated potassium channels on the afferent neuron. This work shows that these efferent signaling cascades also enhance the activation of HCN channels by depolarizing their voltage activation range. The size of this effect varies depending on the endogenous properties of the HCN channel and on cell type (as determined by discharge patterns and cell size). Simultaneously controlling two ion-channel groups gives the vestibular efferent system exquisite control over afferent neuron activity.

Identification of Chemical and Pharmacological Chaperones for Correction of Trafficking-Deficient Mutant Cyclic Nucleotide-Gated A3 Channels.

Trafficking deficiency caused by missense mutations is a well known phenomenon that occurs for mutant, misfolded proteins. Typically, the misfolded protein is retained by the protein quality-control system and degraded by the endoplasmic reticulum-associated protein degradation pathway and thus does not reach its destination, although residual function of the protein may be preserved. Chemical and pharmacological chaperones can improve the targeting of trafficking-deficient proteins and thus may be promising candidates for therapeutic applications. Here, we report the application of a cellular bioassay based on the bioluminescent calcium reporter aequorin to quantify surface expression of mutant CNGA3 channels associated with the autosomal recessively inherited retinal disease achromatopsia. A screening of 77 compounds enabled the identification of effective chemical and pharmacological chaperones that result in a 1.5- to 4.8-fold increase of surface expression of mutant CNGA3. Using selected compounds, we confirmed that the rescue of the defective trafficking is not limited to a single mutation in CNGA3. Active compounds and our structure-activity correlated data for the dihydropyridine compound class may provide valuable information for developing a treatment of the trafficking defect in achromatopsia. SIGNIFICANCE STATEMENT: This study describes a novel luminescence-based assay to detect the surface expression of mutant trafficking-deficient CNGA3 channels based on the calcium-sensitive photoprotein aequorin. Using this assay for a compound screening, this study identifies novel chemical and pharmacological chaperones that restore the surface localization of mutant trafficking-deficient CNGA3 channels. The results from this work may serve as starting point for the development of potent compounds that rescue trafficking deficiencies in the autosomal recessively inherited retinal disease achromatopsia.

Testing broad-spectrum and isoform-preferring HCN channel blockers for anticonvulsant properties in mice.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have been implicated in the pathogenesis of epilepsy and consequently as targets for anticonvulsant drugs. Consistent with this, broad-spectrum block of HCN-mediated current (Ih) reduces seizure susceptibility in a variety of epilepsy models. However, HCN channel isoforms have distinct biophysical characteristics and anatomical expression suggesting that they may play different roles in setting neuronal excitability. Here we confirm that the broad-spectrum blocker ivabradine is effective at reducing seizure susceptibility in the s.c.PTZ seizure assay and extend this, showing efficacy of this drug in a thermogenic assay that models febrile seizures. Ivabradine is also effective at reducing thermogenic seizures in the Scn1a mouse model of Dravet syndrome in which febrile seizures are a feature. HCN isoform-preferring drugs were tested in the s.c.PTZ seizure assay. We confirm that the HCN4-preferring drug, EC18, is efficacious in reducing seizure susceptibility. Conversely, the HCN2/1-preferring drug, MEL55A, increased seizure susceptibility in the s.c.PTZ seizure assay. MEL57A, an HCN1-preferring drug, had no effect on seizure susceptibility. Mouse pharmacokinetic studies (for MEL55A and MEL57A) and screening against additional ion channels have not been thoroughly investigated on the HCN isoform-preferring compounds. Our results need to be considered in this light. Nevertheless, these data suggest that HCN isoform-selective block can have a differential impact on seizure susceptibility. This motivates the need to develop more HCN isoform-selective compounds to better explore this idea.

Ca2+-activated Cl current predominates in threshold response of mouse olfactory receptor neurons.

In mammalian olfactory transduction, odorants activate a cAMP-mediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for ∼80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold.

ZD7288 and mibefradil inhibit the myogenic heartbeat in Daphnia magna indicating its dependency on HCN and T-type calcium ion channels.

Daphnia magna heartbeat is myogenic-originating within the animal's heart. However, the mechanism for this myogenic automaticity is unknown. The mechanism underlying the automaticity of vertebrate myogenic hearts involves cells (pacemaker cells), which have a distinct set of ion channels that include hyperpolarization activated cyclic nucleotide-gated (HCN) and T-type calcium ion channels. We hypothesized that these ion channels also underlie the automatic myogenic heartbeat of Daphnia magna. The drugs, ZD7288 and mibefradil dihydrochloride, block HCN and T-type calcium ion channels respectively. Application of these drugs, in separate experiments, show that they inhibit the heartbeat of Daphnia magna in a dose-dependent manner. Calculation of the percent difference between the heart rate of pretreatment (before drug application) and heart rate following drug application (post-treatment) allowed us to graph a dose-response curve for both ZD7288 and mibefradil, revealing that ZD7288 produces a greater effect on decreasing heart rate. This indicates the HCN ion channels play a foremost role in generating Daphnia magna heartbeat. Our results show conclusively that HCN and T-type calcium ion channels underlie the automatic myogenic heartbeat in Daphnia magna-and suggest a conserved mechanism for generating myogenic heartbeat within the animal kingdom. Thus, Daphnia magna represents a credible model system for further exploration of cardiac physiology.

Regulation of HCN Ion Channels by Non-canonical Cyclic Nucleotides.

The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) proteins are cAMP-regulated ion channels that play a key role in nerve impulse transmission and heart rate modulation in neuronal and cardiac cells, respectively. Although they are regulated primarily by cAMP, other cyclic nucleotides such as cGMP, cCMP, and cUMP serve as partial agonists for the HCN2 and HCN4 isoforms. By competing with cAMP for binding, these non-canonical ligands alter ion channel gating, and in turn, modulate the cAMP-dependent activation profiles. The partial activation of non-canonical cyclic nucleotides can be rationalized by either a partial reversal of a two-state inactive/active conformational equilibrium, or by sampling of a third conformational state with partial activity. Furthermore, different mechanisms and degrees of activation have been observed upon binding of non-canonical cyclic nucleotides to HCN2 versus HCN4, suggesting that these ligands control HCN ion channels in an isoform-specific manner. While more work remains to be done to achieve a complete understanding of ion channel modulation by non-canonical cyclic nucleotides, it is already clear that such knowledge will ultimately prove invaluable in achieving a more complete understanding of ion channel signaling in vivo, as well as in the development of therapeutics designed to selectively modulate ion channel gating.

Two olfactory receptors-OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation.

Olfactory receptors (ORs), which belong to the G-protein coupled receptor family, are expressed in various human tissues, including skin. Cells in non-olfactory tissues tend to express more than one individual OR gene, but function and interaction of two or more ORs in the same cell type has only been marginally analysed. Here, we revealed OR2A4/7 and OR51B5 as two new ORs in human skin cells and identified cyclohexyl salicylate and isononyl alcohol as agonists of these receptors. In cultured human keratinocytes, both odorants induce strong Ca2+ signals that are mediated by OR2A4/7 and OR51B5, as demonstrated by the receptor knockdown experiments. Activation of corresponding receptors induces a cAMP-dependent pathway. Localization studies and functional characterization of both receptors revealed several differences. OR2A4/7 is expressed in suprabasal keratinocytes and basal melanocytes of the epidermis and influences cytokinesis, cell proliferation, phosphorylation of AKT and Chk-2 and secretion of IL-1. In contrast, OR51B5 is exclusively expressed in suprabasal keratinocytes, supports cell migration and regeneration of keratinocyte monolayers, influences Hsp27, AMPK1 and p38MAPK phosphorylation and interestingly, IL-6 secretion. These findings underline that different ORs perform diverse functions in cutaneous cells, and thus offering an approach for the modulated treatment of skin diseases and wound repair.

Advances in the management of heart failure: the role of ivabradine.

A high resting heart rate (≥70-75 b.p.m.) is a risk factor for patients with heart failure (HF) with reduced ejection fraction (EF), probably in the sense of accelerated atherosclerosis, with an increased morbidity and mortality. Beta-blockers not only reduce heart rate but also have negative inotropic and blood pressure-lowering effects, and therefore, in many patients, they cannot be given in the recommended dose. Ivabradine specifically inhibits the pacemaker current (funny current, If) of the sinoatrial node cells, resulting in therapeutic heart rate lowering without any negative inotropic and blood pressure-lowering effect. According to the European Society of Cardiology guidelines, ivabradine should be considered to reduce the risk of HF hospitalization and cardiovascular death in symptomatic patients with a reduced left ventricular EF ≤35% and sinus rhythm ≥70 b.p.m. despite treatment with an evidence-based dose of beta-blocker or a dose below the recommended dose (recommendation class "IIa" = weight of evidence/opinion is in favor of usefulness/efficacy: "should be considered"; level of evidence "B" = data derived from a single randomized clinical trial or large nonrandomized studies). Using a heart rate cutoff of ≥ 75 b.p.m., as licensed by the European Medicines Agency, treatment with ivabradine 5-7.5 mg b.i.d. reduces cardiovascular mortality by 17%, HF mortality by 39% and HF hospitalization rate by 30%. A high resting heart rate is not only a risk factor in HF with reduced EF but also at least a risk marker in HF with preserved EF, in acute HF and also in special forms of HF. In this review, we discuss the proven role of ivabradine in the validated indication "HF with reduced EF" together with interesting preliminary findings, and the potential role of ivabradine in further, specific forms of HF.

Testosterone and atrial natriuretic peptide share the same pathway to induce vasorelaxation of human umbilical artery.

We recently observed in human umbilical artery smooth muscle cells that testosterone activates protein kinase G and stimulates large-conductance Ca²âº activated (BKCa) and voltage sensitive (KV) potassium channels. In the same work, we also show that atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase (pGC), stimulates the activity of BKCa and KV channels because of protein kinase G activation. The aim of this work was to prove that the relaxant effects of testosterone are also because of the increase of cGMP because of activation of the pGC. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (ICNG) in human umbilical artery smooth muscle cells expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Sodium nitroprusside (10 and 100 µM), ANP (0.1 and 1 µM), or testosterone (0.1, 1, and 10 µM) induced activation of ICNG. This activation induced by testosterone and ANP is bigger than that elicited by sodium nitroprusside. In summary, our study reveals that testosterone and ANP activate the pGC and induce vasorelaxation of human umbilical artery.

HCN1 channels as targets for anesthetic and nonanesthetic propofol analogs in the amelioration of mechanical and thermal hyperalgesia in a mouse model of neuropathic pain.

Chronic pain after peripheral nerve injury is associated with afferent hyperexcitability and upregulation of hyperpolarization-activated, cyclic nucleotide-regulated (HCN)-mediated IH pacemaker currents in sensory neurons. HCN channels thus constitute an attractive target for treating chronic pain. HCN channels are ubiquitously expressed; analgesics targeting HCN1-rich cells in the peripheral nervous system must spare the cardiac pacemaker current (carried mostly by HCN2 and HCN4) and the central nervous system (where all four isoforms are expressed). The alkylphenol general anesthetic propofol (2,6-di-iso-propylphenol) selectively inhibits HCN1 channels versus HCN2-HCN4 and exhibits a modest pharmacokinetic preference for the periphery. Consequently, we hypothesized that propofol, and congeners, should be antihyperalgesic. Alkyl-substituted propofol analogs have different rank-order potencies with respect to HCN1 inhibition, GABA(A) receptor (GABA(A)-R) potentiation, and general anesthesia. Thus, 2,6- and 2,4-di-tertbutylphenol (2,6- and 2,4-DTBP, respectively) are more potent HCN1 antagonists than propofol, whereas 2,6- and 2,4-di-sec-butylphenol (2,6- and 2,4-DSBP, respectively) are less potent. In contrast, DSBPs, but not DTBPs, enhance GABA(A)-R function and are general anesthetics. 2,6-DTBP retained propofol's selectivity for HCN1 over HCN2-HCN4. In a peripheral nerve ligation model of neuropathic pain, 2,6-DTBP and subhypnotic propofol are antihyperalgesic. The findings are consistent with these alkylphenols exerting analgesia via non-GABA(A)-R targets and suggest that antagonism of central HCN1 channels may be of limited importance to general anesthesia. Alkylphenols are hydrophobic, and thus potential modifiers of lipid bilayers, but their effects on HCN channels are due to direct drug-channel interactions because they have little bilayer-modifying effect at therapeutic concentrations. The alkylphenol antihyperalgesic target may be HCN1 channels in the damaged peripheral nervous system.

Insulin modulates the electrical activity of subfornical organ neurons.

Insulin plays a crucial role in the regulation of energy balance. Within the central nervous system, hypothalamic nuclei such as the arcuate and ventromedial nuclei are targets of insulin; however, insulin may only access these nuclei after transport across the blood-brain barrier. Neurons of the subfornical organ are not protected by the blood-brain barrier and can rapidly detect and respond to circulating hormones such as leptin and ghrelin. Moreover, subfornical organ neurons form synaptic connections with hypothalamic control centers that regulate energy balance, including the arcuate and dorsomedial nuclei. However, it is unknown whether subfornical organ neurons respond to insulin. Using whole-cell current clamp, we examined the electrophysiological effects of insulin on rat subfornical organ neurons. Upon insulin application, 70% of neurons tested were responsive, with 33% of neurons tested (9/27) exhibiting hyperpolarization of membrane potential (-8.7 ± 1.7 mV) and 37% (10/27) exhibiting depolarization (10.5 ± 2.8 mV). Using pharmacological blockade, our data further indicate that the hyperpolarization was mediated by opening of KATP channels, whereas depolarization resulted from opening of Ih channels. These data are the first to show that insulin exerts a direct effect on the electrical activity of subfornical organ neurons and support the notion that the subfornical organ may act to communicate information on circulating satiety signals to homeostatic control centers.

HCN and KV7 (M-) channels as targets for epilepsy treatment.

Voltage-gated ion channels are important determinants of cellular excitability. The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) and KV7 (M-) channels are voltage-gated ion channels. Both channels are activated at sub-threshold potentials and have biophysical properties that mirror each other. KV7 channels inhibit neuronal excitability. Thus, mutations in KV7 channels that are associated with Benign Familial Neonatal Convulsions (BFNC) are likely to be epileptogenic. Mutations in HCN channels have also been associated with idiopathic epilepsies such as GEFS+. In addition, HCN channel expression and function are modulated during symptomatic epilepsies such as temporal lobe epilepsy. It is, though, unclear as to whether the changes in HCN channel expression and function associated with the various forms of epilepsy promote epileptogenesis or are adaptive. In this review, we discuss this as well as the potential for KV7 and HCN channels as drug targets for the treatment of epilepsy. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

[Endothelin-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes through a p38 MAPK-independent signaling pathway].

OBJECTIVE: To investigate the transcriptional regulation of pacemaker channel I(f) mediated by vasoactive peptide endothelin-1 (ET-1) in neonatal rat ventricular myocytes and its mechanism. METHODS: Neonatal rat ventricular myocytes were enzymatically isolated. I(f) current was recorded using the whole-cell patch-clamp technique. The expression of hyperpolarization-activated cyclic nucleotide-gated channel (HCN) isoforms HCN2 and HCN4 were measured by quantitative RT-PCR. RESULTS: ET-1 increased the expression of HCN2 and HCN4 mRNA in a dose- and time-dependent manner. These effects were blocked by specific ETA receptor antagonist BQ-123 but not the ETB receptor antagonist BQ-788. The effects of ET-1 on HCN2 and HCN4 mRNA expression were not affected by the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB-203580). CONCLUSION: These findings indicate that ET-1 stimulates the expression of pacemaker channel I(f) in cardiomyocytes via ETA receptor through a p38 MAPK-independent signaling pathway, which might be linked to the intrinsic arrhythmogenic potential of ET-1.

ZD7288 enhances long-term depression at early postnatal medial perforant path-granule cell synapses.

Hyperpolarization-activated, cyclic nucleotide-gated nonselective (HCN) channels modulate both membrane potential and resistance and play a significant role in synaptic plasticity. We compared the influence of HCN channels on long-term depression (LTD) at the medial perforant path-granule cell synapse in early postnatal (P9-15) and adult (P30-60) rats. LTD was elicited in P9-15 slices using low-frequency stimulation (LFS, 900 pulses, 1 Hz; 80 ± 4% of baseline). Application of the specific HCN channel blocker ZD7288 (10 µM) before LFS significantly enhanced LTD (62 ± 4%; P < 0.01), showing HCN channels restrain LTD induction. However, when ZD7288 was applied after LFS, LTD was similar to control values and significantly different from the values obtained with ZD7288 application before LFS (81 ± 5%; P < 0.01), indicating that HCN channels do not modulate LTD expression. LTD in slices from adult rats were only marginally lower compared to those in P9-15 slices (85 ± 6%), but bath application of ZD7288 prior to LFS resulted in the same amount of LTD (85 ± 5%). HCN channels in adult tissue hence lose their modulatory effect. In conclusion, we found that HCN channels at the medial perforant path-granule cell synapse compromise LFS-associated induction, but not expression of LTD in early postnatal, but not in adult, rats.

Electrophysiological characterization of Grueneberg ganglion olfactory neurons: spontaneous firing, sodium conductance, and hyperpolarization-activated currents.

Mammals rely on their acute olfactory sense for their survival. The most anterior olfactory subsystem in the nose, the Grueneberg ganglion (GG), plays a role in detecting alarm pheromone, cold, and urinary compounds. GG neurons respond homogeneously to these stimuli with increases in intracellular [Ca(2+)] or transcription of immediate-early genes. In this electrophysiological study, we used patch-clamp techniques to characterize the membrane properties of GG neurons. Our results offer evidence of functional heterogeneity in the GG. GG neurons fire spontaneously and independently in several stable patterns, including phasic and repetitive single-spike modes of discharge. Whole cell recordings demonstrated two distinct voltage-gated fast-inactivating Na(+) currents with different steady-state voltage dependencies and different sensitivities to tetrodotoxin. Hodgkin-Huxley simulations showed that these Na(+) currents confer dual mechanisms of action potential generation and contribute to different firing patterns. Additionally, GG neurons exhibited hyperpolarization-activated inward currents that modulated spontaneous firing in vitro. Thus, in GG neurons, the heterogeneity of firing patterns is linked to the unusual repertoire of ionic currents. The membrane properties described here will aid the interpretation of chemosensory function in the GG.

Ethanol enhances human hyperpolarization-activated cyclic nucleotide-gated currents.

BACKGROUND: There is a clear association between excessive ethanol ( EtOH ) consumption and the risk of sudden cardiac death. The hyperpolarization-activated cyclic nucleotide-gated (HCN) current, I (f) , is known to contribute to spontaneous pacemaker activity of sinoatrial (SA) node cells. However, the exact mechanisms of EtOH on arrhythmia induction are not well understood. METHODS: The preparations of SA node were excised from rabbit heart, transmembrane potentials were recorded by standard glass microelectrode technique, and a whole-cell patch clamp technique was used to record I (f) in enzymatically isolated rabbit SA node pacemaker cells. Human HCN2 (hHCN2) and HCN4 channels were heterologously expressed in Xenopus oocytes and studied using 2-electrode voltage clamp technique. RESULTS: Superfusion of EtOH increased the spontaneous firing frequency of SA node cells in a reversible fashion. Treatment with ivabradine irreversibly depressed basal firing frequency and markedly attenuated the enhancement effect of EtOH on firing. The stimulatory effects of EtOH on I (f) were concentration-dependent in the range of 1 to 100 mM, with an average EC (50) value of 20.81 ± 6.71 mM and Hill coefficient of 1.19 ± 0.10. Furthermore, EtOH reversibly enhanced the HCN currents in a concentration-dependent fashion with an EC (50) value of 18.41 ± 2.75 mM for the HCN2 channel and 21.98 ± 3.54 mM for the HCN4 channel, which was accompanied by the acceleration of activation and deactivation kinetics. In addition, EtOH , at both moderate and high doses, caused a shift in the voltage dependence of hHCN4 channel activation to more depolarizing potentials. However, superfusion of high, not moderate, concentration of EtOH caused a shift in the voltage dependence of hHCN2 channel activation to more hyperpolarizing potentials. CONCLUSIONS: This study provides insight into the molecular interaction of EtOH and the hHCN channels, which may shed light on elucidating the potentially proarrhythmic mechanism of EtOH .

Dopamine modulation of Ih improves temporal fidelity of spike propagation in an unmyelinated axon.

We studied how conduction delays of action potentials in an unmyelinated axon depended on the history of activity and how this dependence was changed by the neuromodulator dopamine (DA). The pyloric dilator axons of the stomatogastric nervous system in the lobster, Homarus americanus, exhibited substantial activity-dependent hyperpolarization and changes in spike shape during repetitive activation. The conduction delays varied by several milliseconds per centimeter, and, during activation with realistic burst patterns or Poisson-like patterns, changes in delay occurred over multiple timescales. The mean delay increased, whereas the resting membrane potential hyperpolarized with a time constant of several minutes. Concomitantly with the mean delay, the variability of delay also increased. The variability of delay was not a linear or monotonic function of instantaneous spike frequency or spike shape parameters, and the relationship between these parameters changed with the increase in mean delay. Hyperpolarization was counteracted by a hyperpolarization-activated inward current (I(h)), and the magnitude of I(h) critically determined the temporal fidelity of spike propagation. Pharmacological block of I(h) increased the change in delay and the variability of delay, and increasing I(h) by application of DA diminished both. Consequently, the temporal fidelity of pattern propagation was substantially improved in DA. Standard measurements of changes in excitability or delay with paired stimuli or tonic stimulation failed to capture the dynamics of spike conduction. These results indicate that spike conduction can be extremely sensitive to the history of axonal activity and to the presence of neuromodulators, with potentially important consequences for temporal coding.

HCN channels and heart rate.

Hyperpolarization and Cyclic Nucleotide (HCN) -gated channels represent the molecular correlates of the "funny" pacemaker current (I(f)), a current activated by hyperpolarization and considered able to influence the sinus node function in generating cardiac impulses. HCN channels are a family of six transmembrane domain, single pore-loop, hyperpolarization activated, non-selective cation channels. This channel family comprises four members: HCN1-4, but there is a general agreement to consider HCN4 as the main isoform able to control heart rate. This review aims to summarize advanced insights into the structure, function and cellular regulation of HCN channels in order to better understand the role of such channels in regulating heart rate and heart function in normal and pathological conditions. Therefore, we evaluated the possible therapeutic application of the selective HCN channels blockers in heart rate control.

Role of calcium in activation of hyperpolarization-activated cyclic nucleotide-gated channels caused by cholecystokinin octapeptide in interstitial cells of cajal.

BACKGROUND: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate pacemaker activity in some cardiac cells and neurons. Little is known about the effects of cholecystokinin octapeptide (CCK-8) on HCN channels and excitability of murine interstitial cells of Cajal (ICCs). METHODS: In the present study, the effects and mechanisms of CCK-8 on HCN channels were investigated by measuring mechanical contraction of smooth muscle strips and ionic channels of ICCs in murine gastric antrum. RESULTS: Sulfated CCK-8 (CCK-8S) was used, and we found that CCK-8S increased the contraction of smooth muscle strips in the gastric antrum, which could be suppressed by specific HCN channel blockers CsCl and ZD7288. Extracellular calcium could also intensify the contraction. Under the same conditions, when antral strips were exposed to calcium ion (Ca²âº)-free solution, no significant changes could be recorded with CCK-8S or ZD7288. Isolated ICCs from the murine gastric antrum identified by specific c-Kit antibody primers were chosen for electrophysiological recordings. HCN current (I(h)) of cultured ICCs was studied by whole-cell patch clamp techniques. A spontaneous transient inward current was recorded in ICCs, which could be inhibited by addition of CsCl and ZD7288; the current proved to be I(h). CCK-8S-facilitated I(h) in cultured ICCs could be inhibited by CsCl and ZD7288. When cultured ICCs were exposed to Ca²âº-free solution, no significant changes could be recorded by application of CCK-8S on I(h), which proved extracellular calcium might have an excitatory effect on HCN channels. CONCLUSION: We demonstrate that HCN channels are present in ICCs in the murine gastric antrum; they might be an important regulator of ICC excitability and pacemaker activity and are strongly affected by CCK-8S. Extracellular calcium might be a trigger in the activation of HCN channels caused by CCK-8S in cultured ICCs.

SK- and h-current contribute to the generation of theta-like resonance of rat substantia nigra pars compacta dopaminergic neurons at hyperpolarized membrane potentials.

Oscillation activities are the feature of neural network and correlated to different physiological states. The theta (θ) oscillation (2-7 Hz) has been reported in the basal ganglia, and the intrinsic resonance properties of individual neurons have provided a basis for this network oscillation. The basal ganglia neurons receive comprehensive modulation arising from dopaminergic (DA) neurons located in the substantia nigra pars compacta (SNc), but how the oscillation is regulated in SNc DA neurons remains poorly understood. In this paper, whole-cell patch-clamp recordings were performed on SNc DA neurons in rat brain slices to reveal the resonance properties and underlying mechanisms. After swept-sine-wave (ZAP protocol) current was injected into SNc DA neurons, θ resonance was induced, whose peak impedance went up with the rising of temperature, demonstrating the dependency of resonance on temperature. Voltage dependency of resonance was also observed at hyperpolarized membrane potentials. Further investigation demonstrated two individual components: (1) SK-current generated resonance at around -65 mV, which could be blocked by apamin (300 nM), a specific antagonist of the small-conductance calcium-dependent potassium channel; (2) h-current (I (h)) generated resonance at around -75 mV, which could be abolished by ZD7288 (10 µM), a selective blocker of HCN channels. We concluded that in SNc DA neurons, θ resonance was mediated by two distinct ionic channels at hyperpolarized potentials. Our results imply that θ frequency resonance of individual SNc DA neurons may participate in coordinating rhythmic firing activity and contribute to the physiological or pathophysiological behaviors of Parkinson's disease.