Hyperpolarization-activated Channels Research Articles

Structural mechanism of human HCN1 hyperpolarization-activated channel inhibition by ivabradine

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a crucial role in regulating neuronal excitability. Despite growing evidence supporting the therapeutic potential of HCN1 inhibition in treating neurological disorders, the structural basis of channel inhibition by inhibitor has remained elusive. Here, we present the cryo-electron microscopy structure of human HCN1 channel in complex with inhibitor ivabradine, the drug on the market that acts on HCN channels. Combining electrophysiology, mutagenesis, and molecular dynamics simulations, our findings reveal that ivabradine binds to a previously unidentified pocket formed between the S4, S1, and HCN domain. Furthermore, through structure-based virtual screening, we identify two FDA-approved drugs that can inhibit the HCN1 channel by interacting with the ivabradine-binding site. Our results not only provide insights into the structural intricacies of ivabradine-mediated inhibition, but also offer a potential pharmacological framework for developing novel drugs targeting the HCN1 channel. The elucidation of these molecular interactions serves as a foundational step in advancing therapeutic strategies for modulating HCN1 activity, contributing to the broader landscape of drug discovery and development in this area.

An LQT2-related mutation in the voltage-sensing domain is involved in switching the gating polarity of hERG

BackgroundCyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the “opposite” voltage-sensing properties of hERG and HCN remains unknown.ResultsWe found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y.ConclusionsOur data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Deficiency of Cullin 3, a Protein Encoded by a Schizophrenia and Autism Risk Gene, Impairs Behaviors by Enhancing the Excitability of Ventral Tegmental Area (VTA) DA Neurons.

The dopaminergic neuromodulator system is fundamental to brain functions. Abnormal dopamine (DA) pathway is implicated in psychiatric disorders, including schizophrenia (SZ) and autism spectrum disorder (ASD). Mutations in Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex, have been associated with SZ and ASD. However, little is known about the function and mechanism of CUL3 in the DA system. Here, we show that CUL3 is critical for the function of DA neurons and DA-relevant behaviors in male mice. CUL3-deficient mice exhibited hyperactive locomotion, deficits in working memory and sensorimotor gating, and increased sensitivity to psychostimulants. In addition, enhanced DA signaling and elevated excitability of the VTA DA neurons were observed in CUL3-deficient animals. Behavioral impairments were attenuated by dopamine D2 receptor antagonist haloperidol and chemogenetic inhibition of DA neurons. Furthermore, we identified HCN2, a hyperpolarization-activated and cyclic nucleotide-gated channel, as a potential target of CUL3 in DA neurons. Our study indicates that CUL3 controls DA neuronal activity by maintaining ion channel homeostasis and provides insight into the role of CUL3 in the pathogenesis of psychiatric disorders.SIGNIFICANCE STATEMENT This study provides evidence that Cullin 3 (CUL3), a core component of the Cullin-RING ubiquitin E3 ligase complex that has been associated with autism spectrum disorder and schizophrenia, controls the excitability of dopamine (DA) neurons in mice. Its DA-specific heterozygous deficiency increased spontaneous locomotion, impaired working memory and sensorimotor gating, and elevated response to psychostimulants. We showed that CUL3 deficiency increased the excitability of VTA DA neurons, and inhibiting D2 receptor or DA neuronal activity attenuated behavioral deficits of CUL3-deficient mice. We found HCN2, a hyperpolarization-activated channel, as a target of CUL3 in DA neurons. Our findings reveal CUL3's role in DA neurons and offer insights into the pathogenic mechanisms of autism spectrum disorder and schizophrenia.

Calcium's multifaceted functions: From nutrient to secondary messenger during stress

An essential macronutrient for plants, calcium (Ca2+) is needed for a variety of functions both under normal and stressful situations. It displays characteristics of a structural component and acts as a secondary messenger during several physiological processes. It behaves as a signaling molecule while under stress. It manifests a variety of symptoms, such as weakening the cell wall, at lower concentrations or in insufficient situations, demonstrating its significant significance at peak levels. Calcium is taken up by roots through a variety of channels or proteins that are positioned on the plasma membrane. These channels are divided into three categories based on their electrophysiological properties: voltage-independent channels (VICs), hyperpolarization-activated channels (HACCs), and depolarization-activated channels (DACCs). Protein channels allow it to enter the cytosol, where it is then exported towards the apoplast or xylem by Ca2+/H+ antiporters or Ca2+-ATPases. In addition to these, Ca2+ also functions as a secondary messenger throughout physiological, stress-related, and developmental events. The interaction of pumps and influx exporters/channels, also known as calcium signatures, is what causes the signal to be produced. The primary decoding proteins for reactions are calmodulins (CaM), calmodulin-like proteins (CMLs), calcineurin B-like proteins (CBLs), CBL-interacting protein kinases (CIPKs), and calcium-dependent protein kinases (CDPKs/CPKs). After the signals have been processed, transporters, transcription factors, and channels then transmit them into outputs, which lead to the shutting of stomata and gene expression. Ca2+ homeostasis is also preserved during signaling by several transporting components that control the outflow and import of Ca2+, respectively. These transporters include Ca2+-ATPases, Ca2+/H+ exchangers, Mechanosensitive channels (MSLs), Cyclic nucleotide-gated channels (CNGCs), Two Pore Channels (TPCs), Glutamate-like Receptor channels (GLRs), and Ca2+/cation exchangers (CCXs). Due to their sessile nature, plants have been subjected to a range of environmental challenges, including nutrient deficit, metal stress, temperature stress and drought. To investigate the many roles that calcium plays, this review has gone into detail about the various processes via which Ca2+ has been studied to play a part throughout these stresses.

Symmetry Reduction in a Hyperpolarization-Activated Homotetrameric Ion Channel.

Plants obtain nutrients from the soil via transmembrane transporters and channels in their root hairs, from which ions radially transport in toward the xylem for distribution across the plant body. We determined structures of the hyperpolarization-activated channel AKT1 from Arabidopsis thaliana, which mediates K+ uptake from the soil into plant roots. These structures of AtAKT1 embedded in lipid nanodiscs show that the channel undergoes a reduction of C4 to C2 symmetry, possibly to regulate its electrical activation.

HCN1 channels: A versatile tool for signal processing by primary sensory neurons

Most primary sensory neurons (PSNs) generate a slowly-activating inward current in response to membrane hyperpolarization (Ih) and express HCN1 along with additional isoforms coding for hyperpolarization-activated channels (HCN). Changes in HCN expression may affect the excitability and firing patterns of PSNs, but retinal and inner ear PSNs do not fire action potentials, suggesting HCN channel roles may extend beyond excitability and cell firing control. In patients taking Ih blockers, photopsia triggered in response to abrupt changes in luminance correlates with impaired visual signal processing via parallel rod and cone pathways. Furthermore, in a mouse model of inherited retinal degeneration, HCN blockers or Hcn1 genetic ablation may worsen photoreceptors' demise. PSN's use of HCN channels to adjust either their firing rate or process signals generated by sensory transduction in non-spiking PSNs indicates HCN1 channels as a versatile tool with a novel role in sensory processing beyond firing control.

A family of hyperpolarization-activated channels selective for protons

Proton (H+) channels are special: They select protons against other ions that are up to a millionfold more abundant. Only a few proton channels have been identified so far. Here, we identify a family of voltage-gated "pacemaker" channels, HCNL1, that are exquisitely selective for protons. HCNL1 activates during hyperpolarization and conducts protons into the cytosol. Surprisingly, protons permeate through the channel's voltage-sensing domain, whereas the pore domain is nonfunctional. Key to proton permeation is a methionine residue that interrupts the series of regularly spaced arginine residues in the S4 voltage sensor. HCNL1 forms a tetramer and thus contains four proton pores. Unlike classic HCN channels, HCNL1 is not gated by cyclic nucleotides. The channel is present in zebrafish sperm and carries a proton inward current that acidifies the cytosol. Our results suggest that protons rather than cyclic nucleotides serve as cellular messengers in zebrafish sperm. Through small modifications in two key functional domains, HCNL1 evolutionarily adapted to a low-Na+ freshwater environment to conserve sperm's ability to depolarize.

Electromechanical coupling in the hyperpolarization-activated K+ channel KAT1.

Voltage-gated potassium (Kv) channels orchestrate electrical signaling and control cell volume by gating in response to either membrane depolarization or hyperpolarization. Yet, while all voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges1–3, the general determinants of channel gating polarity remain poorly understood4. Here, we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped Kv channels based on the cryo-EM structure of KAT1, the hyperpolarization-activated Kv channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that a ~5–7 Å inward motion of the S4 sensor helix can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated (HCN) channels5, and may represent an unexpected link between depolarization and hyperpolarization-activated channels.

Gating mechanism of hyperpolarization-activated HCN pacemaker channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential for rhythmic activity in the heart and brain, and mutations in HCN channels are linked to heart arrhythmia and epilepsy. HCN channels belong to the family of voltage-gated K+ (Kv) channels. However, why HCN channels are activated by hyperpolarization whereas Kv channels are activated by depolarization is not clear. Here we reverse the voltage dependence of HCN channels by mutating only two residues located at the interface between the voltage sensor and the pore domain such that the channels now open upon depolarization instead of hyperpolarization. Our data indicate that what determines whether HCN channels open by hyperpolarizations or depolarizations are small differences in the energies of the closed and open states, due to different interactions between the voltage sensor and the pore in the different channels.

A Newly Identified Population of Gastrointestinal Neurons Expressing HCN4 Regulates Retrograde Peristalsis

Peristalsis is indispensable for physiological function of the gut. Enteric nervous system (ENS) plays an important role in regulating the peristalsis. While the neural network regulating the anterograde peristalsis, which migrates from the oral end to the anal end, is characterized to some extent, the retrograde peristalsis remains unresolved with regards to its neural regulation. Using forward genetics in zebrafish, we revealed that a population of neurons expressing a hyperpolarization-activated nucleotide-gated channel HCN4 specifically regulates retrograde peristalsis. HCN4(+) neurons are serotonergic, and are observed along the entire length of the gut. When blocked by a HCN channel inhibitor, retrograde peristalsis was specifically attenuated. Conversely, when HCN4(+) neurons expressing channelrhodopsin were activated by illumination, retrograde peristalsis was enhanced while anterograde peristalsis remained unchanged. Moreover, the effect of blocker or illumination was limited to the circular muscle, as opposed to the longitudinal muscle, resulting in the specific change of movement transverse to the gut axis. We propose that HCN4(+) neurons in the ENS forward activating signals toward the oral end and simultaneously stimulate local circuits regulating the circular muscle.

MP09-02 DIFFERENTIAL DISTRIBUTION OF HYPERPOLARIZATION-ACTIVATED CHANNELS IN PEPTIDERGIC AND CHOLINERGIC FIBERS OF THE HUMAN BLADDER

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2018MP09-02 DIFFERENTIAL DISTRIBUTION OF HYPERPOLARIZATION-ACTIVATED CHANNELS IN PEPTIDERGIC AND CHOLINERGIC FIBERS OF THE HUMAN BLADDER Pradeep Tyagi, Mahendra Kashyap, and Naoki Yoshimura Pradeep TyagiPradeep Tyagi More articles by this author , Mahendra KashyapMahendra Kashyap More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.328AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Hyperpolarization-activated cyclic nucleotide-gated channels (HCN) have been previously identified by us in the cell bodies of rat bladder afferent neurons, where they carry currents to perform diverse functions, including the hyperpolarization activated Ih current. Recent studies indicate expression of HCN channels in human bladder and suggest their involvement in the modulation of bladder contractility. HCN channels in neurons are responsible for setting the resting membrane potential, regulate discharge patterns and network activity. Present study was conducted to identify and characterize HCN1 and HCN4 immunoreactive (IR) nerve fibers in urothelium, suburothelium and detrusor regions of human bladder by immunocytochemistry. METHODS Human bladders were obtained from 3 deceased organ donors (either male or female between 16 to 82 years old) with full ethical approval. Bladders were surgically separated into urothelium (containing suburothelium) and detrusor section. Tissue regions from trigone region were cryopreserved and sectioned into 10μm thick cryosections. Sections were fixed in chilled acetone and processed for immunohistochemistry using antibodies for either HCN1 and HCN4 primary antibody. Double immunostaining with neuronal markers of calcitonin gene related peptide (CGRP) and choline acetyltransferase (ChAT) was performed. RESULTS The expression of HCN1 was relatively stronger in urothelium, sub urothelium and detrusor, whereas HCN4 expression was strongest in detrusor and suburothelium, while expression in urothelium was minimal. The co-localization of HCN1 and CGRP+ve peptidergic neuron fibers was higher than the co-localization of HCN1 with ChAT +ve cholinergic fibers in suburothelium and detrusor regions. The HCN4 immunoreactivity co-localized with the red immunoreactivity of ChAT much strongly in detrusor than in sub-urothelium. CONCLUSIONS Based on these findings, HCN ion channel mechanisms are postulated to modulate the release of neurotransmitters and initiate action potentials in response to transmitter-evoked depolarization forafferent (HCN1) and efferent ( HCN4) arm of micturition reflex. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e105-e106 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Pradeep Tyagi More articles by this author Mahendra Kashyap More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Association between hyperpolarization-activated channel in interstitial cells of Cajal and gastrointestinal dysmotility induced by malignant ascites.

Advanced malignant ascites is accompanied by gastrointestinal dysmotility, and patients often feel abdominal pain, abdominal distention, nausea and constipation. Gastrointestinal dysmotility is not only painful for the patients, but it reduces the absorption of nutrients and affects the physical recovery of patients with malignant ascites. It is reported that changes in interstitial cells of Cajal (ICCs) are responsible for the gastrointestinal dysmotility induced by malignant ascites, but the mechanism is not completely understood. The present study observed a significantly decreased expression of ion channels, including hyperpolarization-activated cyclic nucleotide-gated potassium channel 2 (HCN2) and cyclic adenosine monophosphate, in the condition of malignant ascites. Using electrophysiology, it was identified that malignant ascites led to lower amplitude and slower frequency signals in cells of the small intestine. In addition, when ICCs were cultured with malignant ascites in vitro, the expression of HCN2 of ICCs was significantly reduced, and the data of flow cytometry revealed that the Ca2+ concentration of ICCs was also decreased. The results of electron microscopy analysis demonstrated the nuclei of ICCs were pyknotic, and the processes of ICCs were reduced in malignant ascites. The present study suggests the small intestinal dysmotility caused by malignant ascites may be associated with changes in HCN2 of ICCs, which offers a potential therapeutic target for gastrointestinal dysmotility in advanced malignant ascites.

Structural Changes and Lack of HCN1 Channels in the Binaural Auditory Brainstem of the Naked Mole-Rat (Heterocephalus glaber).

Naked mole-rats (Heterocephalus glaber) live in large eu-social, underground colonies in narrow burrows and are exposed to a large repertoire of communication signals but negligible binaural sound localization cues, such as interaural time and intensity differences. We therefore asked whether monaural and binaural auditory brainstem nuclei in the naked mole-rat are differentially adjusted to this acoustic environment. Using antibody stainings against excitatory and inhibitory presynaptic structures, namely the vesicular glutamate transporter VGluT1 and the glycine transporter GlyT2 we identified all major auditory brainstem nuclei except the superior paraolivary nucleus in these animals. Naked mole-rats possess a well structured medial superior olive, with a similar synaptic arrangement to interaural-time-difference encoding animals. The neighboring lateral superior olive, which analyzes interaural intensity differences, is large and elongated, whereas the medial nucleus of the trapezoid body, which provides the contralateral inhibitory input to these binaural nuclei, is reduced in size. In contrast, the cochlear nucleus, the nuclei of the lateral lemniscus and the inferior colliculus are not considerably different when compared to other rodent species. Most interestingly, binaural auditory brainstem nuclei lack the membrane-bound hyperpolarization-activated channel HCN1, a voltage-gated ion channel that greatly contributes to the fast integration times in binaural nuclei of the superior olivary complex in other species. This suggests substantially lengthened membrane time constants and thus prolonged temporal integration of inputs in binaural auditory brainstem neurons and might be linked to the severely degenerated sound localization abilities in these animals.

Alterations in CA1 pyramidal neuronal intrinsic excitability mediated by Ih channel currents in a rat model of amyloid beta pathology

Amyloid beta (Aβ) accumulation plays an important role in the pathogenesis of Alzheimer’s disease (AD) by changing the neuronal excitability. However, the cellular mechanisms by which accumulation of Aβ affects intrinsic neuronal properties are not well understood. The effect of bilateral intra-frontal cortex Aβ (1–42) peptide injection on the intrinsic excitability of hippocampal CA1 pyramidal neurons with particular focus on the contribution of hyperpolarization-activated (Ih) channel currents was examined using whole-cell patch-clamp recording. Passive avoidance memory impairment and morphological changes in rats receiving intra-frontal Aβ treatment were observed, which was associated with significant changes both in passive and active intrinsic electrical membrane properties of CA1 pyramidal neurons. Electrophysiological recording showed a significant decrease in neuronal excitability associated with an augmentation in the first spike after-hyperpolarization (AHP) amplitude. In addition, the depolarizing sag voltage was altered in neurons recorded from Aβ-treated group. In voltage-clamp condition, a hyperpolarizing activated inward current sensitive to ZD7288 and capsaicin was significantly increased in neurons from Aβ-treated rats. The Ih current density was increased and the activation curve was shifted toward less negative potential in the Aβ-treated group as compared to control group. The enhancing effect of Aβ treatment on Ih current was confirmed by showing upregulation of the mRNA of HCN1 channel in the CA1 pyramidal layer of hippocampi.These findings suggest the contribution of Ih and possibly TRPV1 channel currents to the changes induced by Aβ treatment in the intrinsic membrane properties, which, in turn, may provide therapeutic targets for treatment of AD.

Increased excitability of medium-sized dorsal root ganglion neurons by prolonged interleukin-1β exposure is K(+) channel dependent and reversible.

Neuropathic pain resulting from peripheral nerve injury is initiated and maintained by persistent ectopic activity in primary afferent neurons. Sciatic nerve injury increases the excitability of medium-sized dorsal root ganglion (DRG) neurons. Levels of the inflammatory cytokine interleukin 1β (IL-1β) increase and peak after 7days. Five to six days of exposure of medium sized DRG neurons to 100pm IL-1β promotes persistent increases in excitability which abate within 3-4days of cytokine removal. This is associated with a profound attenuation of K(+) channel currents but only modest increases in function of cyclic nucleotide-sensitive hyperpolarization-activated channels (HCNs) and of voltage-gated Na(+) and Ca(2+) channel currents. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons is capable of initiating an enduring phenotypic shift in their electrophysiological properties that follows sciatic nerve injury. The findings also underline the importance of K(+) channel modulation in the actions of inflammatory mediators on peripheral neurons. Chronic constriction injury of rat sciatic nerve promotes signs of neuropathic pain. This is associated with an increase in the level of interleukin 1β (IL-1β) in primary afferents that peaks at 7days. This initial cytokine exposure has been proposed to trigger an enduring alteration in neuronal phenotype that underlies chronic hyper-excitability in sensory nerves, which initiates and maintains chronic neuropathic pain. We have shown previously that 5-6days of exposure of rat dorsal root ganglia (DRGs) to 100pm IL-1β increases the excitability of medium-sized neurons. We have now found using whole-cell recording that this increased excitability reverts to control levels within 3-4days of cytokine removal. The effects of IL-1β were dominated by changes in K(+) currents. Thus, the amplitudes of A-current, delayed rectifier and Ca(2+) -sensitive K(+) currents were reduced by ∼68%, ∼64% and ∼36%, respectively. Effects of IL-1β on other cation currents were modest by comparison. There was thus a slight decrease in availability of high voltage-activated Ca(2+) channel current, a small increase in rates of activation of hyperpolarization-activated cyclic nucleotide-gated channel current (IH ), and a shift in the voltage dependence of activation of tetrodotoxin-sensitive sodium current (TTX-S INa ) to more negative potentials. It is unlikely, therefore, that direct interaction of IL-1β with DRG neurons initiates an enduring phenotypic shift in their electrophysiological properties following sciatic nerve injury. Persistent increases in primary afferent excitability following nerve injury may instead depend on altered K(+) channel function and on the continued presence of slightly elevated levels IL-1β and other cytokines.

Dendritic distributions of I h channels in experimentally-derived multi-compartment models of oriens-lacunosum/moleculare (O-LM) hippocampal interneurons.

The O-LM cell type mediates feedback inhibition onto hippocampal pyramidal cells and gates information flow in the CA1. Its functions depend on the presence of voltage-gated channels (VGCs), which affect its integrative properties and response to synaptic input. Given the challenges associated with determining densities and distributions of VGCs on interneuron dendrites, we take advantage of computational modeling to consider different possibilities. In this work, we focus on hyperpolarization-activated channels (h-channels) in O-LM cells. While h-channels are known to be present in O-LM cells, it is unknown whether they are present on their dendrites. In previous work, we used ensemble modeling techniques with experimental data to obtain insights into potentially important conductance balances. We found that the best O-LM models that included uniformly distributed h-channels in the dendrites could not fully capture the “sag” response. This led us to examine activation kinetics and non-uniform distributions of h-channels in the present work. In tuning our models, we found that different kinetics and non-uniform distributions could better reproduce experimental O-LM cell responses. In contrast to CA1 pyramidal cells where higher conductance densities of h-channels occur in more distal dendrites, decreasing conductance densities of h-channels away from the soma were observed in O-LM models. Via an illustrative scenario, we showed that having dendritic h-channels clearly speeds up back-propagating action potentials in O-LM cells, unlike when h-channels are present only in the soma. Although the present results were morphology-dependent, our work shows that it should be possible to determine the distributions and characteristics of O-LM cells with recordings and morphologies from the same cell. We hypothesize that h-channels are distributed in O-LM cell dendrites and endow them with particular synaptic integration properties that shape information flow in hippocampus.

Integrated Omic Analysis of a Guinea Pig Model of Heart Failure and Sudden Cardiac Death

Mechanistic understanding of heart failure (HF) and sudden cardiac death (SCD) has been hampered by the lack of suitable experimental models with features of human disease. We have developed a guinea pig model of cardiac hypertrophy (HYP) and HF, characterized by predisposition to SCD (Liu et al. Circ. Res. 2014). Our objective was to refine guinea pig models of HF progression by integrating protein, metabolite and transcript levels. Relative protein abundances from sham-operated, HYP and HF hearts were assessed using isobaric tags for relative and absolute quantification, prior to liquid chromatography and tandem mass spectrometry (LC-MS/MS). Metabolites were quantified by LC-MS/MS or gas chromatography coupled to MS. Transcriptome profiles were obtained from Affymetrix microarrays. Theguinea pig HF proteome exhibited classic biosignatures of cardiac HYP, left ventricular dysfunction, fibrosis, cellular degeneration, inflammation and extravasation. Fatty acid metabolism, mitochondrial transcription/translation factors, and other mitochondrial processes, were downregulated. Processes/proteins upregulated in HF include fibrillogenesis, extracellular matrix remodeling, cytoskeletal proteins, the unfolded-protein response, and acute phase inflammation markers. Among metabolites, downregulation of acyl-carnitines was observed in HYP, while fatty acids accumulated in HF. Levels of the TCA cycle metabolite, citrate, and the potent inhibitor, 2-methylcitrate, increased upon transition from HYP to HF. Among transcripts, downregulation of repolarizing K+ channels, SERCA2a, phospholamban and ryanodine receptor was noted, and upregulation of mRNAs for cyclic-nucleotide gated hyperpolarization-activated channels and TRPC6. The biosignature of decompensation in the guinea pig HF/SCD model parallels prior rodent models but spotlights metabolic bottlenecks. Fatty acid use is likely abrogated by impaired mitochondrial import and oxidation, while compensation by glycolytic flux is predicted to be impaired, along with TCA cycle dysfunction and accumulation of inhibitors (e.g. citrate). Pathway analysis suggests avenues for metabolic therapy beyond PPAR activation.

Contribution of hyperpolarization-activated channels to heat hypersensitivity and ongoing activity in the neuritis model

Neuritis can cause pain hypersensitivities in the absence of axonal degeneration. Such hypersensitivities are reputed to be maintained by ongoing activity into the spinal cord, which, in the neuritis model, is mainly generated from intact C-fiber neurons. The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of ion channels has been implicated in nerve injury-induced pain hypersensitivities. The present study has examined the role of these channels in the development of heat and mechanical hypersensitivities in the neuritis model. The systemic administration of the HCN-specific blocker ZD7288 produced a reversal of heat but not mechanical hypersensitivity within one hour post-administration. Recordings from C-fiber neurons were performed to determine whether ZD7288 acts by inhibiting ongoing activity. ZD7288 (0.5mM) caused a 44.1% decrease in the ongoing activity rate following its application to the neuritis site. Immunohistochemical examination of the HCN2 channel subtype within the L5 dorsal root ganglia revealed an increase in expression in neuronal cell bodies of all sizes post-neuritis. In conclusion, HCN channels contribute to the development of neuritis-induced heat hypersensitivity and ongoing activity. Drugs that target HCN channels may be beneficial in the treatment of neuropathic pain in patients with nerve inflammation.

CAMP control of HCN2 channel Mg2+ block reveals loose coupling between the cyclic nucleotide-gating ring and the pore.

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

Biochemical and Structural Analysis of the Hyperpolarization-Activated K+ Channel MVP

In contrast to the majority of voltage-gated ion channels, hyperpolarization-activated channels remain closed at depolarizing potentials and are activated at hyperpolarizing potentials. The basis for this reverse polarity is thought to be a result of differences in the way the voltage-sensing domain (VSD) couples to the pore domain. In the absence of structural data, the molecular mechanism of this reverse polarity coupling remains poorly characterized. Here we report the characterization of the structure and local dynamics of the closed activation gate (lower S6 region) of MVP, a hyperpolarization-activated potassium channel from Methanococcus jannaschii, by electron paramagnetic resonance (EPR) spectroscopy. We show that a codon-optimized version of MVP has high expression levels in Escherichia coli, is purified as a stable tetramer, and exhibits expected voltage-dependent activity when reconstituted in liposomes. EPR analysis of the mid to lower S6 region revealed positions exhibiting strong spin–spin coupling, indicating that the activation gate of MVP is closed at 0 mV. A comparison of local environmental parameters along the activation gate for MVP and KcsA indicates that MVP adopts a different closed conformation. These structural details set the stage for future evaluations of reverse electromechanical coupling in MVP.