HCN Isoforms Research Articles

Molecular basis of funny current (I f) in normal and failing human heart

I f overexpression has been functionally demonstrated in ventricular myocytes from failing human hearts. Altered expression of I f-channels as a consequence of electrophysiological remodeling may represent an arrhythmogenic mechanism in heart failure; however, the molecular basis of I f overexpression in human cardiac disease is unknown. HCN1, 2 and 4 subtypes, which encode I f-channels, have been identified in the heart. The present study was designed to characterize HCN isoform expression in failing and non-failing hearts. Ventricular and atrial samples were obtained from normal or failing hearts explanted from patients with end-stage ischemic cardiomyopathy. I f was recorded in patch-clamped left ventricular myocytes. mRNA and protein expression of HCN subunits were measured in both atria and ventricles of control and diseased hearts. HCN2 and HCN4 were detected in human myocardium. Both mRNA and protein levels of HCN2/4 were significantly augmented in failing ventricles ( p < 0.01 for mRNA, p < 0.05 for protein). These results are consistent with the electrophysiological data showing that, in failing ventricular myocytes, I f is of larger amplitude and activates at less negative potential. Changes in mRNA and protein expression of both HCN2/4 isoforms in atrial specimens from patients with heart failure mirrored those observed in ventricles ( p < 0.001 for mRNA, p < 0.05 for protein). No disease-dependent alteration was detected for MiRP1, the putative β-subunit of the I f-channel. In conclusion, HCN4 is the predominant channel subtype in normal human heart, and its expression is further amplified by disease. HCN upregulation likely contributes to increased I f and may play a role in ventricular and atrial arrhythmogenesis in heart failure.

Inactivation of HCN channels slows rhythm differently in atria and ventricle and improves conduction in the anoxic‐reoxygenated embryonic chick heart

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels control heart rate and are expressed early during cardiogenesis. Distribution of the predominant HCN4 isoform and the involvement of HCN channels in myocardial dysfunction induced by anoxia‐reoxygenation were investigated in the embryonic heart.Expression of HCN4 channels was determined by western blotting in atria (A), ventricle (V) and outflow tract (OT) from hearts of 4‐day‐old chick embryos. Hearts or isolated ventricles were exposed to the selective inhibitor of HCN channels ivabradine. Effects of 0.3μM ivabradine on ECG, excitation‐contraction (EC) coupling and contractility were determined in hearts submitted to 30 min anoxia and 60 min reoxygenation.The distribution of HCN4 was inhomogeneous, i.e. A ≥ V ≫ OT. The intrinsic rate was 164±22 in A and 78±24 bpm in isolated V. Under normoxia, 0.3 μM ivabradine decreased A rate by 20% and V rate by 50% and increased A‐V conduction by 20%. These effects persisted throughout anoxia‐reoxygenation, whereas variations of QT duration, EC coupling, contractility and arrhythmias were not affected.These data indicate that HCN channels are inhomogeneously distributed and control finely atrial and ventricular pacemakers of the embryonic heart. Their inactivation during anoxia‐reoxygenation ameliorates atrial rhythmicity and conduction.Supported by Servier and SNSF n°3100A0‐105901

Hyperpolarization‐activated cyclic nucleotide‐modulated ‘HCN’ channels confer regular and faster rhythmicity to beating mouse embryonic stem cells

The hyperpolarization-activated cation current (I(f)), and the hyperpolarization-activated cyclic nucleotide-modulated 'HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. I(f) and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing I(f), and the density of I(f) in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The I(f) blocker ZD7288 (0.3 microm) reduced I(f) and the beating rate of embryoid bodies. Taken together, the activation kinetics of I(f) and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of I(f) to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.

Activity‐dependent heteromerization of the hyperpolarization‐activated, cyclic‐nucleotide gated (HCN) channels: role of N‐linked glycosylation

Formation of heteromeric complexes of ion channels via co-assembly of different subunit isoforms provides an important mechanism for enhanced channel diversity. We have previously demonstrated co-association of the hyperpolarization activated cyclic-nucleotide gated (HCN1/HCN2) channel isoforms that was regulated by network (seizure) activity in developing hippocampus. However, the mechanisms that underlie this augmented expression of heteromeric complexes have remained unknown. Glycosylation of the HCN channels has been implicated in the stabilization and membrane expression of heteromeric HCN1/HCN2 constructs in heterologous systems. Therefore, we used in vivo and in vitro systems to test the hypothesis that activity modifies HCN1/HCN2 heteromerization in neurons by modulating the glycosylation state of the channel molecules. Seizure-like activity (SA) increased HCN1/HCN2 heteromerization in hippocampus in vivo as well as in hippocampal organotypic slice cultures. This activity increased the abundance of glycosylated HCN1 but not HCN2-channel molecules. In addition, glycosylated HCN1 channels were preferentially co-immunoprecipitated with the HCN2 isoforms. Provoking SA in vitro in the presence of the N-linked glycosylation blocker tunicamycin abrogated the activity-dependent increase of HCN1/HCN2 heteromerization. Thus, hippocampal HCN1 molecules have a significantly higher probability of being glycosylated after SA, and this might promote a stable heteromerization with HCN2.

The Heart Rate‐Lowering Agent Ivabradine Inhibits the Pacemaker Current If in Human Atrial Myocytes

It has been speculated that pacemaker current (I(f)) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific I(f) inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on I(f) in atrial myocytes. Using patch-clamp technique, we studied effects of ivabradine on I(f) present in atrial myocytes isolated from human right appendages of patients undergoing cardiac surgery. The identification of HCN isoforms was obtained by means of multiplex single-cell RT-PCR. Ivabradine induced a marked concentration and use-dependent I(f) inhibition with an IC50 at steady state of 2.9 microM. Time constant of block development (Tau(on)) decreases with the increase in the ivabradine concentration. Use-dependent inhibition induced by ivabradine (3 microM) was not modified in the presence of cAMP (10 microM) in the pipette solution. Multiplex single-cell RT-PCR indicates that the major HCN gene subtype detected in atria was HCN2. HCN4 is detected weakly and HCN1 is not significantly detected. Ivabradine inhibits I(f) current in the nonpacemaker cell with characteristics similar to those described previously in rabbit sinus node cells, but revealed a lesser sensitivity for I(f) recorded in human atrial cell than hHCN4 subunits considered as the major contributors to native f-channels in human sinoatrial node. A potential protection of atrial arrhythmias by ivabradine is discussed.

HCN2 and HCN4 Isoforms Self-assemble and Co-assemble with Equal Preference to Form Functional Pacemaker Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.

Modulation of h-Channels in Hippocampal Pyramidal Neurons by p38 Mitogen-Activated Protein Kinase

Hyperpolarization-activated cyclic nucleotide-gated ion channels (h-channels; I(h); HCN) modulate intrinsic excitability in hippocampal and neocortical pyramidal neurons, among others. Whereas I(h) mediated by the HCN2 isoform is regulated by cAMP, there is little known about kinase modulation of I(h), especially for the HCN1 isoform predominant in pyramidal neurons. We used a computational method to identify a novel kinase modulator of h-channels, p38 mitogen-activated protein kinase (p38 MAPK). Inhibition of p38 MAPK in hippocampal pyramidal neurons caused a approximately 25 mV hyperpolarization of I(h) voltage-dependent activation. This downregulation of I(h) produced hyperpolarization of resting potential, along with increased input resistance and temporal summation of excitatory inputs. Activation of p38 MAPK caused a approximately 11 mV depolarizing shift in I(h) activation, along with depolarized resting potential, and decreased input resistance and temporal summation. Inhibition of related MAPKs, ERK1/2 (extracellular signal-related kinase 1/2) and JNK (c-Jun N-terminal kinase), produced no effect on I(h). These results show that p38 MAPK is a strong modulator of h-channel biophysical properties and may deserve additional exploration as a link between altered I(h) and pathological conditions such as epilepsy.

Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampus

The hyperpolarization-activated current (I(h)) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates I(h) conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of I(h) in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. I(h) amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of I(h) to cAMP, and the contribution of the slow component to the overall I(h), decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents.

I K1 and I f in ventricular myocytes isolated from control and hypertrophied rat hearts

Electrophysiological properties of inward rectifier potassium current (I (K1)) and hyperpolarization-activated inward current (I (f)) and the protein expression of the Kir2.1 subfamily and the hyperpolarization-activated cation channel 2 (HCN2) and HCN4 were studied in control and hypertrophied myocytes. Electrophysiological experiments were conducted by whole-cell patch-clamp technique, and protein levels of Kir2.1 subfamily and HCN2 and HCN4 isoforms were analysed by Western blot technique. The density of I (f) as well as the protein expression levels of the HCN2 isoform was found to be significantly higher in hypertrophied myocytes, whereas the protein expression level of HCN4 was not detected in any group. I (K1) density and Kir 2.1 protein expression were similar in control and hypertrophied myocytes, but the time-course of the currents was slower in hypertrophied myocytes. Analysis of I (f) and I (K1) in the same control and hypertrophied myocyte at -80 mV showed that cells in which I (f) was present had values of I (K1) density similar to those cells in which I (f) was not observed. In conclusion, although left ventricular hypertrophy involves an up-regulation of I (f) and its molecular correlate HCN2 in the rat ventricle, its contribution to diastolic depolarization would be limited by the low values of I (f) density at potentials close to the resting potential of the ventricular cells.

Interaction of the Pacemaker Channel HCN1 with Filamin A

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltage-gated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin+), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin-) indicating that interaction with filamin A affects membrane localization. Also, in filamin- cells the gating kinetics of HCN1 were strongly accelerated relative to filamin+ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.

Structural Elements of Instantaneous and Slow Gating in Hyperpolarization-activated Cyclic Nucleotide-gated Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (If) and an instantaneous voltage-independent current (Iinst) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs+ like If and unlike Iinst. Together with a concomitant reduction in If, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "If-like," and not an "Iinst-like," state. The "If-like" instantaneous current represented approximately 70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K+/Na+ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing Iinst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for Iinst. The data are consistent with Iinst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.

The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart.

Hyperpolarization-activated, cyclic nucleotide-gated cation currents, termed If or Ih, are generated by four members of the hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channel family. These currents have been proposed to contribute to several functions including pacemaker activity in heart and brain, control of resting potential, and neuronal plasticity. Transcripts of the HCN4 isoform have been found in cardiomyocytes and neurons, but the physiological role of this channel is unknown. Here we show that HCN4 is essential for the proper function of the developing cardiac conduction system. In wild-type embryos, HCN4 is highly expressed in the cardiac region where the early sinoatrial node develops. Mice lacking HCN4 channels globally, as well as mice with a selective deletion of HCN4 in cardiomyocytes, died between embryonic days 9.5 and 11.5. On average, If in cardiomyocytes from mutant embryos is reduced by 85%. Hearts from HCN4-deficient embryos contracted significantly slower compared with wild type and could not be stimulated by cAMP. In both wild-type and HCN4-/- mice, cardiac cells with "primitive" pacemaker action potentials could be found. However, cardiac cells with "mature" pacemaker potentials, observed in wild-type embryos starting at day 9.0, were not detected in HCN4-deficient embryos. Thus, HCN4 channels are essential for the proper generation of pacemaker potentials in the emerging sinoatrial node.

Hyperpolarization-activated, cyclic AMP-gated, hcn1-like cation channel: the primary, full-length hcn isoform expressed in a saccular hair-cell layer

The expression of transcript for hyperpolarization-activated, cyclic nucleotide-sensitive cation channel (HCN) isoforms underlying hyperpolarization-activated, inward current ( I h) has been determined for a model hair-cell preparation from the saccule of the rainbow trout, Oncorhynchus mykiss. Based upon identification from homology to known vertebrate HCN cDNA sequence, cloning of PCR products amplified with degenerate primers indicated an expression frequency of 7:2:1 (HCN1:HCN2:HCN4) for the hair-cell sheet compared with 1:1:7 for brain. Full-length sequence has been obtained for the HCN1-like isoform representing the primary HCN transcript expressed in the hair-cell preparation. The channel protein is 938 amino acids in length with 93% amino acid identity for the region extending from the S1–S6 membrane spanning domains through the voltage-pore and cyclic nucleotide-binding domains, compared with HCN1 for rabbit, rat, mouse and human. The N- and C-terminal regions are less homologous, with 39–51% and 43–44% amino acid identities, respectively. Compared with other vertebrate HCN1, the hair-cell HCN1 contains additional consensus phosphorylation sites associated with unique repeats in the carboxy terminus. The HCN1-like transcript has been localized to hair cells of the saccular sensory epithelia by in situ hybridization. Previous electrophysiological studies have identified I h as the sole inwardly rectifying ion channel in a specific population of hair cells of the saccule of frogs [J Neurophysiol (1995) 73:1484] and fish [J Physiol (1996) 495:665]. I h is an important determinant of the resting membrane potential, and for this population of hair cells, is predicted to maintain the membrane potential within a voltage range allowing the voltage-gated calcium channels to open, permitting “spontaneous” release of transmitter. The molecular properties of the HCN1-like isoform underlying I h expressed in the saccular hair cells of the teleost, trout, may consequently impact spontaneous release of transmitter from hair cells of the saccule.

A Conserved Domain in the NH2 Terminus Important for Assembly and Functional Expression of Pacemaker Channels

Pacemaker channels are formed by co-assembly of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits. Previously, we suggested that the NH(2) termini of the mouse HCN2 isoform were important for subunit co-assembly and functional channel expression. Using an alignment strategy together with yeast two-hybrid assays, patch clamp electrophysiology, and confocal imaging, we have now identified a domain within the NH(2) terminus of the HCN2 subunit that is responsible for interactions between NH(2) termini and promoting the trafficking of functional channels to the plasma membrane. This domain is composed of 52 amino acids, is located adjacent to the putative first transmembrane segment, and is highly conserved among the mammalian HCN isoforms. This conserved domain, but not the remaining unconserved NH(2)-terminal regions of HCN2, specifically interacted with itself in yeast two-hybrid assays. Moreover, the conserved domain was important for expression of currents. Whereas relatively normal whole cell HCN2 currents were produced by channels containing only the conserved domain, further deletion of this region, leaving only a more polar and putative coiled-coil segment, eliminated HCN2 currents and resulted in proteins that localized predominantly in perinuclear compartments. Thus, we suggest that this conserved domain is the critical NH(2)-terminal determinant of subunit co-assembly and trafficking of pacemaker channels.

Keeping Pace with Pacemaker Channels.

Developmental Febrile Seizures Modulate Hippocampal Gene Expression of Hyperpolarization-activated Channels in an Isoform and Cell-specific Manner Brewter A, Bender RA, Chen Y, Dube C, Eghbal-Ahmadi M, and Baram TZ J Neurosci 2002;22:4591–4599 Febrile seizures, in addition to being the most common seizure type of the developing human, may contribute to the generation of subsequent limbic epilepsy. Our previous work demonstrated that prolonged experimental febrile seizures in the immature rat model increased hippocampal excitability in the long term, enhancing susceptibility to future seizures. The mechanisms for these profound proepileptogenic changes did not require cell death and were associated with long-term slowed kinetics of the hyperpolarization activated depolarizing current (Ih). Here we show that these seizures modulate the expression of genes encoding this current, the hyperpolarization-activated, cyclic nucleotide–gated channels (HCNs): In CA1 neurons expressing multiple HCN isoforms, the seizures induced a coordinated reduction of HCN1 mRNA and enhancement of HCN2 expression, thus altering the neuronal HCN phenotype. The seizure-induced augmentation of HCN2 expression involved CA3 in addition to CA1, whereas for HCN4, mRNA expression was not changed by the seizures in either hippocampal region. This isoform- and region-specific transcriptional regulation of HCNs required neuronal activity rather than hyperthermia alone, correlated with seizure duration, and favored the formation of slow-kinetics HCN2-encoded channels. In summary, these data demonstrate a novel, activity-dependent transcriptional regulation of HCN molecules by developmental seizures. These changes result in long-lasting alteration of the HCN phenotype of specific hippocampal neuronal populations, with profound consequences on the excitability of the hippocampal network.

Different Roles for the Cyclic Nucleotide Binding Domain and Amino Terminus in Assembly and Expression of Hyperpolarization-activated, Cyclic Nucleotide-gated Channels

In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms. To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy. We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells. We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels. This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein. However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel. Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays. Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels.

Dominant-negative suppression of HCN1- and HCN2-encoded pacemaker currents by an engineered HCN1 construct: insights into structure-function relationships and multimerization.

I(f), a diastolic depolarizing current activated by hyperpolarization, is a key player in cardiac pacing. Despite the fact that I(f) has been known for over 20 years, the encoding genes, namely HCN1 to 4, have only recently been identified. Functional data imply that different HCN isoforms may coassemble to form heteromeric channel complexes, but little direct evidence is available. Subunit stoichiometry is also unknown. Although the pore region of HCN channels contains the glycine-tyrosine-glycine (GYG) signature motif found in K+-selective channels, they permeate both Na+ and K+. In the present study, we probed the functional importance of the GYG selectivity motif in pacemaker channels by replacing this triplet in HCN1 with alanines (GYG(349-351)AAA or HCN1-AAA). HCN1-AAA did not yield functional currents; coexpression of HCN1-AAA with wild-type (WT) HCN1 suppressed normal channel activity in a dominant-negative manner (55.2+/-3.2%, 68.3+/-4.3%, 78.7+/-1.6%, 91.7+/-0.8%, and 97.9+/-0.2% current reduction at -140 mV for WT:AAA cRNA ratios of 4:1, 3:1, 2:1, 1:1, and 1:2, respectively) without affecting gating (steady-state activation, activation and deactivation kinetics) or permeation (reversal potential) properties. HCN1-AAA coexpression, however, did not alter the expressed current amplitudes of Kv1.4 and Kv2.1 channels, indicating that its suppressive effect was channel-specific. Statistical analysis reveals that a single HCN channel is composed of 4 monomeric subunits. Interestingly, HCN1-AAA also inhibited HCN2 in a dominant-negative manner with the same efficacy. We conclude that the GYG motif is a critical determinant of ion permeation for HCN channels, and that HCN1 and HCN2 readily coassemble to form heterotetrameric complexes.

Differential and age-dependent expression of hyperpolarization-activated, cyclic nucleotide-gated cation channel isoforms 1–4 suggests evolving roles in the developing rat hippocampus

Hyperpolarization-activated cation currents ( I h) are found in several brain regions including thalamus and hippocampus. Important functions of these currents in promoting synchronized network activity and in determining neuronal membrane properties have been progressively recognized, but the molecular underpinnings of these currents are only emerging. I h currents are generated by hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCNs). These channel proteins are encoded by at least four HCN genes, that govern the kinetic and functional properties of the resulting channels. Because of the potential impact of I h-mediated coordinated neuronal activity on the maturation of the functional hippocampal network, this study focused on determining the expression of the four members of the HCN gene family throughout postnatal hippocampal development at both the regional and single cell level. The results of these experiments demonstrated that HCNs 1, 2 and 4 are differentially expressed in interneuronal and principal cell populations of the rat hippocampal formation. Expression profiles of each HCN isoform evolve during postnatal development, and patterns observed during early postnatal ages differ significantly from those in mature hippocampus. The onset of HCN expression in interneurons of the hippocampus proper precedes that in the dentate gyrus, suggesting that HCN-mediated pacing activity may be generated in hippocampal interneurons prior to those in the hilus. Taken together, these findings indicate an age-dependent spatiotemporal evolution of specific HCN expression in distinct hippocampal cell populations, and suggest that these channels serve differing and evolving functions in the maturation of coordinated hippocampal activity.

Molecular mechanism of cAMP modulation of HCN pacemaker channels.

Hyperpolarization-activated cation channels of the HCN gene family contribute to spontaneous rhythmic activity in both heart and brain. All four family members contain both a core transmembrane segment domain, homologous to the S1-S6 regions of voltage-gated K+ channels, and a carboxy-terminal 120 amino-acid cyclic nucleotide-binding domain (CNBD) motif. Homologous CNBDs are responsible for the direct activation of cyclic nucleotide-gated channels and for modulation of the HERG voltage-gated K+ channel--important for visual and olfactory signalling and for cardiac repolarization, respectively. The direct binding of cyclic AMP to the cytoplasmic site on HCN channels permits the channels to open more rapidly and completely after repolarization of the action potential, thereby accelerating rhythmogenesis. However, the mechanism by which cAMP binding modulates HCN channel gating and the basis for functional differences between HCN isoforms remain unknown. Here we demonstrate by constructing truncation mutants that the CNBD inhibits activation of the core transmembrane domain. cAMP binding relieves this inhibition. Differences in activation gating and extent of cAMP modulation between the HCN1 and HCN2 isoforms result largely from differences in the efficacy of CNBD inhibition.

Integrated allosteric model of voltage gating of HCN channels.

Hyperpolarization-activated (pacemaker) channels are dually gated by negative voltage and intracellular cAMP. Kinetics of native cardiac f-channels are not compatible with HH gating, and require closed/open multistate models. We verified that members of the HCN channel family (mHCN1, hHCN2, hHCN4) also have properties not complying with HH gating, such as sigmoidal activation and deactivation, activation deviating from fixed power of an exponential, removal of activation "delay" by preconditioning hyperpolarization. Previous work on native channels has indicated that the shifting action of cAMP on the open probability (Po) curve can be accounted for by an allosteric model, whereby cAMP binds more favorably to open than closed channels. We therefore asked whether not only cAMP-dependent, but also voltage-dependent gating of hyperpolarization-activated channels could be explained by an allosteric model. We hypothesized that HCN channels are tetramers and that each subunit comprises a voltage sensor moving between "reluctant" and "willing" states, whereas voltage sensors are independently gated by voltage, channel closed/open transitions occur allosterically. These hypotheses led to a multistate scheme comprising five open and five closed channel states. We estimated model rate constants by fitting first activation delay curves and single exponential time constant curves, and then individual activation/deactivation traces. By simply using different sets of rate constants, the model accounts for qualitative and quantitative aspects of voltage gating of all three HCN isoforms investigated, and allows an interpretation of the different kinetic properties of different isoforms. For example, faster kinetics of HCN1 relative to HCN2/HCN4 are attributable to higher HCN1 voltage sensors' rates and looser voltage-independent interactions between subunits in closed/open transitions. It also accounts for experimental evidence that reduction of sensors' positive charge leads to negative voltage shifts of Po curve, with little change of curve slope. HCN voltage gating thus involves two processes: voltage sensor gating and allosteric opening/closing.