Hyperpolarization-activated Current Research Articles

Pharmacological modulation of the hyperpolarization-activated current ( If) in human atrial myocytes: focus on G protein-coupled receptors

Aims. – In human atrial myocytes (HuAM) two β-adrenergic receptors (β-AR) and four splicing-variants of the serotonin 5-HT 4 receptor are present. Multiple coupling with G stimulatory (G s) and G inhibitory (G i) proteins has been proposed for both β 2-AR and 5-HT (4b) subtypes, but no functional data exist in HuAM. Serotonin (5-HT) and catecholamines are able to trigger arrhythmias in human atrium, but the underlying cellular mechanisms are not completely understood. The pacemaker current ( I f) is an inward Na +/K + current, constitutively present in HuAM and directly modulated by cAMP; I f could play a role in triggering human atrial arrhythmias. This study evaluated the different G protein coupling of β 1-AR, β 2-AR and 5-HT 4 receptors by assessing the modulation of I f by selective stimuli. Methods. – HuAM were isolated from right atrial appendages and utilized for patch-clamp recording. The coupling of receptor subtypes with G i proteins was tested by incubating HuAM in pertussis toxin (PTX). Results. – β 1-AR stimulation (Isoprenaline [ISO] + ICI 118,551), and 5-HT caused a concentration-dependent significant shift of the half activation potential of I f activation curve (Δ V h), P < 0.01. β 2-AR stimulation (ISO 1 μM + CGP 20712A) also significantly shifted V h ( P < 0.0001), but with Δ V h[β 2-AR] significantly smaller than the effect caused by 1 μM β 1-AR stimulation ( P < 0.05). Pre-treatment of HuAM with PTX did not alter the effect of β 1-AR stimulation (both 0.1 and 1 μM) and 1 μM 5-HT on I f, but significantly increased the effect in response to β 2-AR stimulation and 0.1 μM 5-HT ( P < 0.05 for both), thus suggesting a G i protein coupling of these receptors. Conclusions. – Our results provide the first functional evidence of the different G protein coupling of β 1-AR, β 2-AR and 5-HT 4 receptors in HuAM. Further they support the view that I f current might play an important role in triggering catecholamines and serotonin-induced atrial arrhythmias.

The Mechanism of Intrinsic Amplification of Hyperpolarizations and Spontaneous Bursting in Striatal Cholinergic Interneurons

Striatal cholinergic interneurons pause their ongoing firing in response to sensory stimuli that have acquired meaning as a signal for learned behavior. In slices, these cells exhibit both spontaneous activity patterns and spontaneous pauses very similar to those seen in vivo. The mechanisms responsible for ongoing firing and spontaneous pauses were studied in striatal slices using perforated patch recordings. All hyperpolarizations, whether spontaneous or generated by current injection, were amplified and shaped by two hyperpolarization-activated currents. Hyperpolarization onsets were regeneratively amplified by a potassium current (KIR) whose activation promoted further hyperpolarization. The termination of hyperpolarizations was controlled by a time-dependent nonspecific cation current (HCN). The duration and even the sizes of spontaneous and driven hyperpolarizations and pauses in spontaneous activity in cholinergic interneurons are largely autonomous properties of the neuron, rather than reflections of characteristics of the input eliciting the response.

Investigation of PKC isoform‐specific translocation and targeting of the current of the late afterhyperpolarizing potential of myenteric AH neurons

AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (I(AHP)) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea-pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the I(AHP). Phorbol dibutyrate had no effect on the hyperpolarization-activated current (I(h)) or on the A current (I(A)). Stimulation of synaptic inputs to the neurons also reduced the I(AHP), and had no effect on I(h) or I(A). Phorbol dibutyrate also reduced a background outward current that was present after the I(AHP) current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCepsilon, in these neurons. Only phorbol dibutyrate caused translocation of PKCgamma, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the I(AHP) and another background K(+) current. The I(AHP) is targeted by a conventional PKC, suggested to be PKCgamma, as this is the only conventional PKC that is prominent in AH neurons.

Characterization and functional role of voltage gated cation conductances in the glucagon‐like peptide‐1 secreting GLUTag cell line

Glucagon-like peptide-1 (GLP-1) is released from intestinal L-cells in response to nutrient ingestion. It is currently under therapeutic evaluation because it enhances insulin secretion in type 2 diabetes. Previous studies using the GLP-1 secreting cell line GLUTag have shown that the cells are electrically active, and that the action potential frequency is regulated by nutrients. In this study we characterize voltage gated currents underlying this electrical activity and correlate the electrophysiological findings with gene expression determined by microarrays. Whole cell voltage clamp experiments designed to separate different ionic components revealed rapidly inactivating sodium currents sensitive to tetrodotoxin, calcium currents sensitive to nifedipine and omega-conotoxin GVIA, and sustained as well as rapidly inactivating potassium currents, which were sensitive to TEA and 4-AP, respectively. In perforated patch experiments we also observed hyperpolarization-activated currents which were inhibited by ZD7288. The amplitude of the sodium current was approximately 10 times that of the other depolarizing currents and tetrodotoxin abolished action potential firing. In secretion experiments, however, nifedipine, but not tetrodotoxin, omega-conotoxin GVIA or ZD7288, inhibited glucose-induced GLP-1 release. Consistent with this finding, the intracellular Ca2+ response to glucose was impaired by nifedipine but not by tetrodotoxin. Thus, in GLUTag cells, GLP-1 release is not dependent on the firing of Na+-carrying action potentials but requires membrane depolarization and Ca2+ entry through L-type Ca2+ channels. Understanding the characteristics of the currents and the molecular identification of the underlying channels in GLP-1 secreting cells might facilitate the development of agents to enhance GLP-1 secretion in vivo.

Oscillatory activity within rat substantia gelatinosa in vitro: a role for chemical and electrical neurotransmission

Although rhythmic behaviour of mammalian spinal ventral horn networks has been extensively studied little is known about oscillogenesis in the spinal dorsal horn. The aims of this in vitro study were to record and determine the underlying mechanisms of potassium-evoked network field oscillations in the substantia gelatinosa of the neonatal rat dorsal horn, a lamina involved in nociceptive processing. Transient pressure ejection of a potassium solution evoked reproducible rhythmic activity in discrete areas of the substantia gelatinosa which lasted for 5-15 s with a single prominent peak in the 4-12 Hz frequency band (7.7 +/- 0.1 Hz, n = 60). Oscillations of similar frequency and amplitude were also observed in isolated dorsal horn quadrants. Application of CNQX (10 microm) reduced peak power amplitude and integrated power area (from 4 to 12 Hz) of the power spectrum, whereas D-AP5 (50 microm) had no effect on the potassium-evoked rhythm. Bicuculline (30 microm) or strychnine (10 microm) reduced the power amplitude and area. On combination of bicuculline (30 microm) and strychnine (10 microm) the reductions in power amplitude and area were not significantly different (P > 0.05) when compared with application of either drug alone. The gap junction blockers carbenoxolone (100 microm) or octanol (1 mM) significantly reduced power amplitude and area. Although TTX (1 microm) or a calcium-free perfusate both caused reductions in the power amplitude and area, potassium-evoked rhythmic activity persisted. However, this persistent rhythm was further reduced on combination of calcium-free perfusate with octanol (1 mM) and was abolished using a cocktail of drugs. Blockade of the potassium delayed rectifier current by tetraethylammonium (5 mM) or the hyperpolarization-activated current (I(h)) by ZD7288 (10 microm) disrupted the synchronization of the potassium-induced oscillation. The frequency of potassium-induced rhythms was unaffected by any of the drugs tested. These novel findings demonstrate that transient pressure ejection of potassium evokes oscillatory activity in the substantia gelatinosa in vitro. This rhythm is partly dependent upon various receptors (AMPA/kainate, GABA(A) and glycine), ion channels (potassium delayed rectifier and I(h)) and gap junctions. Oscillatory behaviour in the substantia gelatinosa could potentially play a role in the processing of nociceptive signals.

Spontaneous low-frequency voltage oscillations in frog saccular hair cells.

Spontaneous membrane voltage oscillations were found in 27 of 130 isolated frog saccular hair cells. Voltage oscillations had a mean peak-to-peak amplitude of 23 mV and a mean oscillatory frequency of 4.6 Hz. When compared with non-oscillatory cells, oscillatory cells had significantly greater hyperpolarization-activated and lower depolarization-activated current densities. Two components, the hyperpolarization-activated cation current, I(h), and the K(+)-selective inward-rectifier current, I(K1), contributed to the hyperpolarization-activated current, as assessed by the use of the I(K1)-selective inhibitor Ba(2+) and the I(h)-selective inhibitor ZD-7288. Five depolarization-activated currents were present in these cells (transient I(BK), sustained I(BK), I(DRK), I(A), and I(Ca)), and all were found to have significantly lower densities in oscillatory cells than in non-oscillatory cells (revealed by using TEA to block I(BK), 4-AP to block I(DRK), and prepulses at different voltages to isolate I(A)). Bath application of either Ba(2+) or ZD-7288 suppressed spontaneous voltage oscillations, indicating that I(h) and I(K1) are required for generating this activity. On the contrary, TEA or Cd(2+) did not inhibit this activity, suggesting that I(BK) and I(Ca) do not contribute. A mathematical model has been developed to test the interpretation derived from the pharmacological and biophysical data. This model indicates that spontaneous voltage oscillations can be generated when the electrophysiological features of oscillatory cells are used. The oscillatory behaviour is principally driven by the activity of I(K1) and I(h), with I(A) playing a modulatory role. In addition, the model indicates that the high densities of depolarization-activated currents expressed by non-oscillatory cells help to stabilize the resting membrane potential, thus preventing the spontaneous oscillations.

Enhanced Learning in Channel Knockout

Apparently, a single ion channel can have very different roles in learning and memory depending on the cell type in which it is expressed and the pattern of connectivity of those cells. Nolan et al . derive this conclusion from their studies of mice lacking the HCN1 protein, a subunit of a hyperpolarization-activated, cyclic nucleotide-gated, cation nonselective channel that accounts for hyperpolarization-activated inward currents ( I h ). HCN1 knockout mice have defective motor learning that appears to result from loss of function in Purkinje cells. However, the authors analyzed mice specifically lacking HCN1 in forebrain neurons and found that the animals actually performed better than wild-type animals in learning to accomplish a spatial memory task in which they have to use spatial cues to find a platform that is hidden under water. Loss of the channel also enhanced long-term memory of how to perform the task. In the CA1 region of the hippocampus, a brain area known to be important in spatial learning and memory, enhanced low-frequency oscillations in neuronal network activity were detected in the knockout animals. Single pyramidal cells of this region also showed greater response to low-frequency oscillatory currents when HCN1 was lacking. The pyramidal cells integrate inputs that come directly from the cerebral cortex (the perforant pathway) with hippocampal inputs from the Schaffer collateral pathway. HCN1 channels are more abundant in the distal dendrites where perforant pathway inputs are localized, and loss of HCN1 preferentially enhanced postsynaptic responses to a single input from the perforant pathway. Similarly, long-term potentiation was enhanced at these perforant path synapses. The authors propose that learning may be suppressed by HCN1 channels because they inhibit postsynaptic changes at distal dendrites that could otherwise result in synaptic plasticity. The loss of HCN1 changes the way in which pyramidal cells integrate incoming signals, enhancing response to low-frequency waveforms and also favoring response to the distal rather than proximal dendrites. This may be particularly important for spatial learning and memory because CA1 pyramidal neurons are thought to compare sensory input from the perforant pathway with stored information from the CA3 region. M. F. Nolan, G. Malleret, J. T. Dudman, D. L. Buhl, B. Santoro, E. Gibbs, S. Vronskaya, G. Buzsáki, S. A. Siegelbaum, E. R. Kandel, A. Morozov, A behavioral role for dendritic integration: HCN1 channels constrain spatial memory and plasticity at inputs to distal dendrites of CA1 pyramidal neurons. Cell 119 , 719-732 (2004). [Online Journal]

Design, synthesis and preliminary biological evaluation of zatebradine analogues as potential blockers of the hyperpolarization-activated current

A series of zatebradine analogues, differing in the basic moiety and in the methylene spacer, have been synthesized; their negative chronotropic activity has been determined in guinea pig atria. The most active compounds have been studied for their blocking properties on the hyperpolarization-activated current I f (which is one of the main currents underlying automatic activity in the sinus node) measured on ventricular myocytes of old spontaneously-hypertensive rats (SHR) by means of the patch-clamp technique. The majority of the substances were able to block I f, with one of them ( 15) being slightly more potent than zatebradine. Surprisingly one analogue ( 6), while showing good negative chronotropic activity, was found to inhibit I f only at high concentration and to markedly reduce outward currents, suggesting for this substance a different mechanism of action responsible for the negative chronotropic effect.

Excitability of auditory brainstem neurons, in vivo, is increased by cyclic-AMP

Physiological control of auditory neural responses is critical for accurate representation of acoustic information, such as sound source localization and speech perception. Central auditory neural responses are almost certainly regulated by a range of mechanisms, including second messenger systems, such as the cAMP pathway. An increase in spontaneous neural discharge is known to accompany cochlear insults. Here we report that an increase in spontaneous as well as tone-evoked discharge can also be induced by pressure application of forskolin, a pharmacological agent that elevates intracellular cAMP level by activating adenyl cyclase. The forskolin induced increase in superior olivary complex (SOC) brainstem neurons is specific, dose-dependent, and reversible, whereas application of artificial cerebrospinal fluid (aCSF, the vehicle) does not alter activity. Forskolin-application also has a relatively greater effect on spontaneous activity compared to tone evoked responses. Blockade of the hyperpolarization-activated current, I h, by ZD7288, consistently reversed the effects of forskolin. Based on these findings, we propose that the second messenger, cAMP, can significantly modulate neural excitability and spontaneous discharge in SOC neurons, principally by shifting the activation of I h channels.

Atrial natriuretic peptide modulates the hyperpolarization-activated current (If) in human atrial myocytes.

The relationship between atrial stretching and changes in cell excitability is well documented. Once stretched, human atrial myocytes (HuAM) release atrial natriuretic peptide (hANP). Receptors for hANP (NPR) are coupled to a guanylyl cyclase (GC) activity, and are present on HuAM, but the electrophysiological effects of hANP are largely unknown. We investigated the effect of hANP on If, the hyperpolarization-activated current present in HuAM, and the underlying intracellular pathway. HuAM were isolated from atrial appendages and utilized for patch-clamp recording. hANP caused a significant and concentration dependent shift of the midpoint activation potential (DeltaVh) toward less negative potentials of 6.9 +/- 1.0 mV at 0.1 nM; 13.0 +/- 2.6 mV at 1 nM and 15.3 +/- 2.2 mV at 10 nM (p < 0.001 for all); a parallel increase of If rate of activation occurred. The effect of hANP was completely blocked by isatin, a potent antagonist of NPR (p < 0.01 vs. hANP). In the presence of the inhibitors of guanylyl cyclase (ODQ and LY83583), hANP caused a significantly smaller DeltaVh (p < 0.01 vs. hANP for both). 8Br-cGMP mimicked the effect of hANP, both in the presence and absence of KT5823, a selective inhibitor of Protein kinase G. Pretreatment with pertussis toxin (PTX) did not change the effect of hANP, thus excluding a major role for the coupling of NPR with the Gi-Proteins system. Pretreating cells with cyclopentyladenosine (CPA), an A1-adenosine receptor agonist, completely blocked hANP effect. Adding hANP to maximal serotonin concentration produced an additive response. Our data demonstrate for the first time that ANP is able to increase If, likely through a modulation of intracellular cGMP and cAMP levels. This effect could have implications in the relationship between stretch and arrhythmogenesis in the human atrium.

Functional interaction between extracellular sodium, potassium and inactivation gating in HERG channels.

We have studied the interaction between extracellular K(+) (K(+)(o)) and extracellular Na(+) (Na(+)(o)) in human ether-à-go-go related gene (HERG)-encoded K(+) channels expressed in Chinese hamster ovary (CHO-K1) cells, using the whole-cell voltage clamp technique. Prior studies indicate that Na(+)(o) potently inhibits HERG current (IC(50) 3 mm) by binding to an outer pore site, and also speeds recovery from inactivation. In this study, we sought to explore the relationship between the Na(+)(o) effect on recovery and Na(+)(o) inhibition of HERG current, and to determine whether inactivation gating plays a critical role in Na(+)(o) inhibition of HERG current. Na(+)(o) concentration-response relationships for current inhibition and speeding of recovery were different, with Na(+)(o) less potent at speeding recovery. Na(+)(o) inhibition of HERG current was relieved by physiological [K(+)](o), while Na(+)(o) speeded recovery from inactivation similarly in the absence or presence of physiological [K(+)](o). To examine the link between Na(+)(o) block and inactivation using an independent approach, we studied hyperpolarization-activated currents uncoupled from inactivation in the S4-S5 linker mutant D540K. Depolarization-activated D540K currents were inhibited by Na(+)(o), while hyperpolarization-activated currents were augmented by Na(+)(o). This result reveals a direct link between Na(+)(o) inhibition and a depolarization-induced conformational change, most likely inactivation. We attempted to simulate the disparate concentration-response relationships for the two effects of Na(+)(o) using a kinetic model that included Na(+)(o) site(s) affected by permeation and gating. While a model with only a single dynamic Na(+)(o) site was inadequate, a model with two distinct Na(+)(o) sites was sufficient to reproduce the data.

Architectural and functional asymmetry of the His-Purkinje system of the murine heart.

The aim of this work was to target a vital reporter gene in the mouse cardiac conduction system (CS) to distinguish this tissue from the surrounding myocardium in the adult heart. A transgenic mouse line has been created in which EGFP is expressed under the control of the Cx40 gene. Correlative investigations associating EGFP imaging and electrophysiological techniques were carried out on the adult heart and isolated cardiomyocytes. In the heart of the Cx40(EGFP/+) mice, EGFP signal was seen in the coronary arteries, the atria, the atrioventricular (AV) node and the His-Purkinje system. The latter was found to be structurally and functionally asymmetrical. The anatomical asymmetry was apparent in both the number of strands or fasciculi making up the His bundle branches (BBs) (1 strand on the right, 20 or so on the left), and the density (low on the right, high on the left) of the network of Purkinje fibers (PFs) that extends over the ventricular wall surfaces. The profiles of the electrical activation patterns recorded on the right and left flanks of the septum were also asymmetrical, mirroring the architecture of the branches. EGFP made it easy to identify the Purkinje cells in populations of dissociated cardiomyocytes and they were investigated using the patch-clamp technique. The hyperpolarization-activated current (If) was recorded in all spontaneously active Purkinje cells. This investigation provides positive evidence of the asymmetry of the His-Purkinje system of the adult mouse, and the first patch-clamp recording data on murine cardiac Purkinje cells. This mouse model opens up new perspectives for investigating the contribution of specific genes to the morphology and function of the His-Purkinje system.

Regulation of orexin neurons by the monoaminergic and cholinergic systems

Orexins are a pair of neuropeptides implicated in energy homeostasis and arousal. Here we characterize the electrophysiological properties of orexin neurons using slice preparations from transgenic mice in which orexin neurons specifically express green fluorescent protein. Orexin neurons showed high frequency firing with little adaptation by injecting a positive current. The hyperpolarization-activated current was observed in orexin neurons by a negative current injection. The neurotransmitters, which were implicated in sleep/wake regulation, affected the activity of orexin neurons; noradrenaline and serotonin hyperpolarized, while carbachol depolarized orexin neurons in either the presence or absence of tetrodotoxin. It has been reported that orexins directly or indirectly activate the nuclei that are the origin of the neurons containing these neurotransmitters. Our data suggest that orexin neurons have reciprocal neural circuitries between these nuclei for either a positive or negative feedback loop and orchestrate the activity of these neurons to regulate the vigilance states.

Differential distribution and function of hyperpolarization-activated channels in sensory neurons and mechanosensitive fibers.

Sensory neurons express hyperpolarization-activated currents (I(H)) that differ in magnitude and kinetics within the populations. We investigated the structural basis for these differences and explored the functional role of the I(H) channels in sensory neurons isolated from rat nodose ganglia. Immunohistochemical studies demonstrated a differential distribution of hyperpolarization-activated cyclic nucleotide-gated (HCN) protein (HCN1, HCN2, HCN4) in sensory neurons and peripheral terminals. HCN2 and HCN4 immunoreactivity was present in all nodose neurons. In contrast, only 20% of the total population expressed HCN1 immunoreactivity. HCN1 did not colocalize with IB4 (a marker for C-type neurons), and only 15% of HCN1-positive neurons colocalized with immunoreactivity for the vanilloid receptor VR1, another protein associated primarily with C-type neurons. Therefore, most HCN1-containing neurons were A-type neurons. In further support, HCN1 was present in the mechanosensitive terminals of myelinated but not unmyelinated sensory fibers, whereas HCN2 and HCN4 were present in receptor terminals of both myelinated and unmyelinated fibers. In voltage-clamp studies, cell permeant cAMP analogs shifted the activation curve for I(H) to depolarized potentials in C-type neurons but not A-type neurons. In current-clamp recording, CsCl, which inhibits only I(H) in nodose neurons, hyperpolarized the resting membrane potential from -63 +/- 1 to -73 +/- 2 mV and nearly doubled the input resistance from 1.3 to 2.2 GOmega. In addition, action potentials were initiated at lower depolarizing current injections in the presence of CsCl. At the sensory receptor terminal, CsCl decreased the threshold pressure for initiation of mechanoreceptor discharge. Therefore, elimination of the I(H) increases excitability of both the soma and the peripheral sensory terminals.

Characterisation of hyperpolarization-activated currents ( Ih) in the medial septum/diagonal band complex in the mouse

Hyperpolarization-activated cyclic nucleotide gated (HCN) channel subunits are distributed widely, but selectively, in the central nervous system, and underlie hyperpolarization-activated currents ( I h) that contribute to rhythmicity in a variety of neurons. This study investigates, using current and voltage-clamp techniques in brain slices from young mice, the properties of I h currents in medial septum/diagonal band (MS/DB) neurons. Subsets of neurons in this complex, including GABAergic and cholinergic neurons, innervate the hippocampal formation, and play a role in modulating hippocampal theta rhythm. In support of a potential role for I h in regulating MS/DB firing properties and consequently hippocampal neuron rhythmicity, I h currents were present in around 60% of midline MS/DB complex neurons. The I h currents were sensitive to the selective blocker ZD7288 (10 μM). The I h current had a time constant of activation of around 220 ms (at −130 mV), and tail current analysis revealed a half-activation voltage of −98 mV. Notably, the amplitude and kinetics of I h currents in MS/DB neurons were insensitive to the cAMP membrane permeable analogue 8-bromo-cAMP (1 mM), and application of muscarine (100 μM). Immunofluoresence using antibodies against HCN1, 2 and 4 channel subunits revealed that all three HCN subunits are expressed in neurons in the MS/DB, including neurons that express the calcium binding protein parvalbumin (marker of fast spiking GABAergic septo-hippocampal projection neurons). The results demonstrate, for the first time, that specific HCN channel subunits are likely to be coexpressed in subsets of MS/DB neurons, and that the resultant I h currents show both similarities, and differences, to previously described I h currents in other CNS neurons.

Functional basis of sinus bradycardia in congenital heart block.

Congenital heart block (CHB) is a conduction abnormality characterized by complete atrioventricular (AV) block. CHB affects fetuses and/or newborn of mothers with autoantibodies reactive with ribonucleoproteins 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro. We recently established animal models of CHB and reported, for the first time, significant sinus bradycardia preceding AV block. This unexpected observation implies that the spectrum of conduction abnormalities extends beyond the AV node to also affect the SA node. To test this hypothesis, we investigated the functional basis of this sinus bradycardia by characterizing the effects of antibodies from mothers with CHB children (positive IgG) on ionic currents that are known to significantly contribute to spontaneous pacing in SA node cells. We recorded L- (I(Ca.L)) and T- (I(Ca.T)) type Ca2+, delayed rectifier K+ (I(K)), hyperpolarization-activated (I(f)) currents, and action potentials (APs) from young rabbit SA node cells. We demonstrated that positive IgG significantly inhibited both I(Ca.T) and I(Ca.L) and induced sinus bradycardia but did not affect I(f) and I(K). Normal IgG from mothers with healthy children did not affect all the currents studied and APs. These results establish that IgG from mothers with CHB children causes substantial inhibition of I(Ca.T) and I(Ca.L), two important pacemaker currents in rabbit SA node cells and point to both I(Ca.T) and I(Ca.L) as major players in the ionic mechanism by which maternal antibodies induce sinus bradycardia in CHB. These novel findings have important clinical significance and suggest that sinus bradycardia may be a potential marker in the detection and prevention of CHB. The full text of this article is available online at http://circres.ahajournals.org

ZD7288 inhibits postsynaptic glutamate receptor‐mediated responses at hippocampal perforant path–granule cell synapses

Hyperpolarization-activated channels (Ih) are widely expressed in the nervous system and believed to play an important role in the regulation of membrane excitability and rhythmic activity. Recent evidence suggests that Ih may be involved in long-term potentiation (LTP) in the hippocampus; however, the results are controversial. To explore the possible causes of these differing results, the effects of Ih blockers on synaptic activity were evaluated in mouse hippocampal slices. ZD7288 (20 micro m), a selective Ih blocker, apparently prevented the induction of LTP, while Cs+ (1 mm), a commonly used Ih blocker, had no effect on LTP at hippocampal perforant path-dentate granule cell synapses. In addition, ZD7288 but not Cs+ abolished basal synaptic transmission. Results from voltage-clamp experiments showed that ZD7288 produced a very little inhibition on hyperpolarization-activated currents, indicating a weak expression of the Ih in granule neurons. Outside-out patch recordings revealed that ZD7288 inhibited glutamate receptor-mediated responses, while Cs+ had no effect on them. Meanwhile, ZD7288 reduced both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents. The results suggest that ZD7288-induced reduction of synaptic transmission may result from its inhibition of the postsynaptic glutamate receptors on dentate granule neurons.

Effects of C-type natriuretic peptide on ionic currents in mouse sinoatrial node: a role for the NPR-C receptor.

The effects of C-type natriuretic peptide (CNP) on heart rate and ionic currents were demonstrated by recording the ECG from adult mice and performing voltage-clamp experiments on single sinoatrial (SA) node cells isolated from mouse heart. The selective natriuretic peptide type C receptor (NPR-C) agonist cANF (10(-7) M) significantly decreased heart rate in the presence of isoproterenol (5 x 10(-9) M), as indicated by an increase in the R-R interval of ECGs obtained from Langendorff-perfused hearts. Voltage-clamp measurements in enzymatically isolated single pacemaker myocytes revealed that CNP (10(-8) M) and cANF (10(-8) M) significantly inhibited L-type Ca2+ current [ICa(L)]. These findings suggest that the CNP effect on this current is mediated by NPR-C. Further support for an NPR-C-mediated inhibition of ICa(L) in SA node myocytes was obtained by altering the functional coupling between the G protein Gi and NPR-C. In these experiments, a "Gi-activator peptide," which consists of a 17-amino acid segment of NPR-C containing a specific Gi protein-activator sequence, was dialyzed into SA node myocytes. This peptide decreased ICa(L) significantly, suggesting that NPR-C activation can result in a reduction in ICa(L) when CNP is bound and the Gi protein pathway is activated. This effect of CNP appears to be selective for ICa(L), because the hyperpolarization-activated current was unaffected by CNP or cANF. These results provide the first demonstration that CNP has a negative chronotropic effect on heart rate and suggest that this effect is mediated by selectively activating NPR-C and reducing ICa(L) through coupling to Gi protein.

Dendritic IhSelectively Blocks Temporal Summation of Unsynchronized Distal Inputs in CA1 Pyramidal Neurons

The active dendritic conductances shape the input-output properties of many principal neurons in different brain regions, and the various ways in which they regulate neuronal excitability need to be investigated to better understand their functional consequences. Using a realistic model of a hippocampal CA1 pyramidal neuron, we show a major role for the hyperpolarization-activated current, Ih, in regulating the spike probability of a neuron when independent synaptic inputs are activated with different degrees of synchronization and at different distances from the soma. The results allowed us to make the experimentally testable prediction that the Ih in these neurons is needed to reduce neuronal excitability selectively for distal unsynchronized, but not for synchronized, inputs.

Characterization of recombinant and native Ih-channels from Apis mellifera

Recently, a novel class of genes coding for I h-channels has been identified in several vertebrates and invertebrates. We isolated a cDNA (AMIH) encoding a putative member of these ion channels from Apis mellifera heads by means of polymerase chain reaction and homology screening. High similarity (88% identical amino acids) to the putative Drosophila melanogaster I h-channel suggests that the Apis cDNA codes for a hyperpolarization-activated and cyclic nucleotide-gated channel. Functional expression of recombinant AMIH in HEK293 cells gave unitary currents that were preferentially selective for potassium over sodium ions and were activated by hyperpolarizing voltage steps. Cyclic nucleotides shifted the voltage activation curve to more positive membrane potentials. The current kinetics, activation by hyperpolarizing voltage steps and modulatory influence of cyclic nucleotides properties closely resemble those of mammalian I h-channels. RT-PCR analysis showed pronounced mRNA expression in the antennae, head and body of Apis mellifera. Investigation of hyperpolarization-activated currents in olfactory receptor neurons (ORNs) in a primary cell culture of Apis mellifera antennal cells revealed activation properties similar to the heterologously expressed I h-channel. By in-situ hybridization and immunohistochemistry, expression of AMIH was seen in olfactory receptor neurons of the bee antennae. We conclude that AMIH is the ion channel responsible for the hyperpolarization-activated currents in olfactory receptor neurons of bee.