Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I_f)/neuronal (I_h) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I_h channels in neurons. This raises the possibility of Ca²⁺ permeation in I_f, the I_h counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca²⁺ signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I_f in rat ventricular myocytes. We observed Ca²⁺ influx when HCN/I_f channels were activated. Ca²⁺ influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I_f channel blocker, inhibited I_f and Ca²⁺ influx at the same time. Quantitative analysis revealed that Ca²⁺ flux contributed to 0.5% of current produced by the HCN2 channel or I_f. The associated increase in Ca²⁺ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I_f current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca²⁺ chelator), preactivation of I_f channels significantly reduced the action potential duration, and the effect was blocked by another selective I_f channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I_f channels had no effects on action potential duration. Our data extend our previous discovery of Ca²⁺ influx in I_h channels in neurons to I_f channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels     

Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker

Background

Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (I _f) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K⁺ current (I _K1) flowing through inward rectifier potassium (K_ir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor I _K1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker in vitro.

Methods and Results

The rabbit Hcn4 gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the Kcnj2 gene encoding K_ir2.1, which is essential for generation of I _K1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the Hcn4 gene than did non-overexpressing mESC-CMs. Expression of the Cacna1h gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including Connexin40, Connexin43, and Connexin45 genes, which encode connexins forming gap junctions, and the Scn5a gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger I _f currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an I _f inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.

Conclusions

We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an I _f inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an in vitro co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.     

HCN1 and HCN2 in Rat DRG Neurons: Levels in Nociceptors and Non-Nociceptors,NT3-Dependence and Influence of CFA-Induced Skin Inflammation on HCN2 and NT3 Expression

I_h, which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie I_h were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via I_h, influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high I_h in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I_h. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I_h magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I_h in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.     

In situ investigation of allografted mouse HCN4 gene–transfected rat bone marrow mesenchymal stromal cells with the use of patch-clamp recording of ventricular slices

BackgroundRecently, proof-of-concept experiments have shown that genetically modified bone marrow mesenchymal stromal cells (MSCs) carrying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels were able to express the funny current (I_f) in vitro, which played a key role in the process of pacemaker generation for heart rate, and were capable of pacemaker function after transplantation into the host heart. Nevertheless, because of the lack of direct experimental access to the implanted cells in situ, the changes in electrophysiological characteristics and the mechanisms underlying the pacemaker function of engrafted HCN gene–transfected MSCs in vivo remain unclear.Methods and ResultsOn the basis of the improved preparation of ventricular slices, we successfully performed an in situ investigation of allografted mouse HCN4 gene (mHCN4)-transfected rat MSCs (rMSCs) with the use of patch-clamp recording in ventricular slices. We demonstrate that allografted mHCN4-transfected rMSCs survived in the host heart for >4 weeks; that they expressed I_f, which is generated by the mHCN4 channel, with a similar amplitude but greater negative activation compared with parallel cells cultured in vitro; that they did not express optical action potentials or depolarization-activated inward sodium or calcium currents; and that they exhibited a low incidence of gap-junctional coupling with host cardiomyocytes.ConclusionsThis study provides direct experimental access to investigate MSCs after transplantation into the host heart. We propose that mHCN4-transfected rMSCs survived in the host heart with altered electrophysiological characteristics of I_f and were accompanied by a low efficiency of connexin 43 expression at 4 weeks after transplantation, which may affect its pacemaker function in vivo.     

Functional contributions of HCN channels in the primary auditory neurons of the mouse inner ear

The hyperpolarization-activated current, I_h, is carried by members of the Hcn channel family and contributes to resting potential and firing properties in excitable cells of various systems, including the auditory system. I_h has been identified in spiral ganglion neurons (SGNs); however, its molecular correlates and their functional contributions have not been well characterized. To investigate the molecular composition of the channels that carry I_h in SGNs, we examined Hcn mRNA harvested from spiral ganglia of neonatal and adult mice using quantitative RT-PCR. The data indicate expression of Hcn1, Hcn2, and Hcn4 subunits in SGNs, with Hcn1 being the most highly expressed at both stages. To investigate the functional contributions of HCN subunits, we used the whole-cell, tight-seal technique to record from wild-type SGNs and those deficient in Hcn1, Hcn2, or both. We found that HCN1 is the most prominent subunit contributing to I_h in SGNs. Deletion of Hcn1 resulted in reduced conductance (G_h), slower activation kinetics (τ_fast), and hyperpolarized half-activation (V_1/2) potentials. We demonstrate that I_h contributes to SGN function with depolarized resting potentials, depolarized sag and rebound potentials, accelerated rebound spikes after hyperpolarization, and minimized jitter in spike latency for small depolarizing stimuli. Auditory brainstem responses of Hcn1-deficient mice showed longer latencies, suggesting that HCN1-mediated I_h is critical for synchronized spike timing in SGNs. Together, our data indicate that I_h contributes to SGN membrane properties and plays a role in temporal aspects of signal transmission between the cochlea and the brain, which are critical for normal auditory function.     

HCN Channels Are Not Required for Mechanotransduction in Sensory Hair Cells of the Mouse Inner Ear

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were orginally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as I_f, I_Q or I_h. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50–70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.     

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.     

PKA-independent activation of If by cAMP in mouse sinoatrial myocytes

Hyperpolarization-activated, cyclic nucleotide-sensitive (HCN4) channels produce the “funny current,” I_f, which contributes to spontaneous pacemaking in sinoatrial myocytes (SAMs). The C-terminus of HCN channels inhibits voltage-dependent gating, and cAMP binding relieves this “autoinhibition.” We previously showed 1) that autoinhibition in HCN4 can be relieved in the absence of cAMP in some cellular contexts and 2) that PKA is required for β adrenergic receptor (βAR) signaling to HCN4 in SAMs. Together, these results raise the possibility that native HCN channels in SAMs may be insensitive to direct activation by cAMP. Here, we examined PKA-independent activation of I_f by cAMP in SAMs. We observed similar robust activation of I_f by exogenous cAMP and Rp-cAMP (an analog than cannot activate PKA). Thus PKA-dependent βAR-to-HCN signaling does not result from cAMP insensitivity of sinoatrial HCN channels and might instead arise via PKA-dependent limitation of cAMP production and/or cAMP access to HCN channels in SAMs.     

HCN1 subunits contribute to the kinetics of Ih in neonatal cortical plate neurons

The distribution of ion channels in neurons regulates neuronal activity and proper formation of neuronal networks during neuronal development. One of the channels is the hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channel constituting the molecular substrate of hyperpolarization‐activated current (I_h). Our previous study implied a role for the fastest activating subunit HCN1 in the generation of I_h in rat neonatal cortical plate neurons. To better understand the impact of HCN1 in early neocortical development, we here performed biochemical analysis and whole‐cell recordings in neonatal cortical plate and juvenile layer 5 somatosensory neurons of HCN1^?/? and control HCN1^+/+ mice. Western Blot analysis revealed that HCN1 protein expression in neonatal cortical plate tissue of HCN^+/+ mice amounted to only 3% of the HCN1 in young adult cortex and suggested that in HCN1^?/? mice other isoforms (particularly HCN4) might be compensatory up‐regulated. At the first day after birth, functional ablation of the HCN1 subunit did not affect the proportion of I_h expressing pyramidal cortical plate neurons. Although the contribution of individual subunit proteins remains open, the lack of HCN1 markedly slowed the current activation and deactivation in individual I_h expressing neurons. However, it did not impair maximal amplitude/density, voltage dependence of activation, and cAMP sensitivity. In conclusion, our data imply that, although expression is relatively low, HCN1 contributes substantially to I_h properties in individual cortical plate neurons. These properties are significantly changed in HCN1^?/?, either due to the lack of HCN1 itself or due to compensatory mechanisms. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 785–797, 2013     

State-dependent and site-directed photodynamic transformation of HCN2 channel by singlet oxygen

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

Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.     

Flavonoid Regulation of HCN2 Channels

The hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are pacemaker channels whose currents contribute to rhythmic activity in the heart and brain. HCN channels open in response to hyperpolarizing voltages, and the binding of cAMP to their cyclic nucleotide-binding domain (CNBD) facilitates channel opening. Here, we report that, like cAMP, the flavonoid fisetin potentiates HCN2 channel gating. Fisetin sped HCN2 activation and shifted the conductance-voltage relationship to more depolarizing potentials with a half-maximal effective concentration (EC₅₀) of 1.8 μm. When applied together, fisetin and cAMP regulated HCN2 gating in a nonadditive fashion. Fisetin did not potentiate HCN2 channels lacking their CNBD, and two independent fluorescence-based binding assays reported that fisetin bound to the purified CNBD. These data suggest that the CNBD mediates the fisetin potentiation of HCN2 channels. Moreover, binding assays suggest that fisetin and cAMP partially compete for binding to the CNBD. NMR experiments demonstrated that fisetin binds within the cAMP-binding pocket, interacting with some of the same residues as cAMP. Together, these data indicate that fisetin is a partial agonist for HCN2 channels.     

Filamin A Promotes Dynamin-dependent Internalization of Hyperpolarization-activated Cyclic Nucleotide-gated Type 1 (HCN1) Channels and Restricts Ih in Hippocampal Neurons

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

Activity-dependent regulation of HCN1 protein in cortical neurons

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I_h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons.     

Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits

Background

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I_h, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.

Methodology/Principal Findings

We investigated the effects of Kcne2 gene deletion on I_h properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2 ^+/+ and Kcne2 ^−/− mice. Kcne2 deletion shifted the voltage-dependence of I_h activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I_h density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2 ^−/− neurons.

Conclusions/Significance

Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.     

Processing of retinal signals in normal and HCN deficient mice

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1^−/− mice or during a pharmacological blockade of I_h show that, contrary to previous reports, I_kx alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I_h and I_kx to the rods'' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.     

HCN-Encoded Pacemaker Channels: From Physiology and Biophysics to Bioengineering

The depolarizing membrane ionic current I _h (also known as I _f, “f” for funny), encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN1-4) channel gene family, was first discovered in the heart over 25 years ago. Later, I _h was also found in neurons, retina, and taste buds. HCN channels structurally resemble voltage-gated K⁺ (Kv) channels but the molecular features underlying their opposite gating behaviors (activation by hyperpolarization rather than depolarization) and non-selective permeation profiles (≥25 times less selective for K⁺ than Kv channels) remain largely unknown. Although I _h has been functionally linked to biological processes from the autonomous beating of the heart to pain transmission, the underlying mechanistic actions remain largely inferential and, indeed, somewhat controversial due to the slow kinetics and negative operating voltage range relative to those of the bioelectrical events involved (e.g., cardiac pacing). This article reviews the current state of our knowledge in the structure-function properties of HCN channels in the context of their physiological functions and potential HCN-based therapies via bioengineering.     

Cellular context and multiple channel domains determine cAMP sensitivity of HCN4 channels: Ligand-independent relief of autoinhibition in HCN4

Hyperpolarization-activated, cyclic nucleotide–sensitive (HCN) channels produce the I_f and I_h currents, which are critical for cardiac pacemaking and neuronal excitability, respectively. HCN channels are modulated by cyclic AMP (cAMP), which binds to a conserved cyclic nucleotide–binding domain (CNBD) in the C terminus. The unliganded CNBD has been shown to inhibit voltage-dependent gating of HCNs, and cAMP binding relieves this “autoinhibition,” causing a depolarizing shift in the voltage dependence of activation. Here we report that relief of autoinhibition can occur in the absence of cAMP in a cellular context- and isoform-dependent manner: when the HCN4 isoform was expressed in Chinese hamster ovary (CHO) cells, the basal voltage dependence was already shifted to more depolarized potentials and cAMP had no further effect on channel activation. This “pre-relief” of autoinhibition was specific both to HCN4 and to CHO cells; cAMP shifted the voltage dependence of HCN2 in CHO cells and of HCN4 in human embryonic kidney (HEK) cells. The pre-relief phenotype did not result from different concentrations of soluble intracellular factors in CHO and HEK cells, as it persisted in excised cell-free patches. Likewise, it did not arise from a failure of cAMP to bind to the CNBD of HCN4 in CHOs, as indicated by cAMP-dependent slowing of deactivation. Instead, a unique ∼300–amino acid region of the distal C terminus of HCN4 (residues 719–1012, downstream of the CNBD) was found to be necessary, but not sufficient, for the depolarized basal voltage dependence and cAMP insensitivity of HCN4 in CHO cells. Collectively, these data suggest a model in which multiple HCN4 channel domains conspire with membrane-associated intracellular factors in CHO cells to relieve autoinhibition in HCN4 channels in the absence of cAMP. These findings raise the possibility that such ligand-independent regulation could tune the activity of HCN channels and other CNBD-containing proteins in many physiological systems.