Hyperpolarization-activated Cation Channels Research Articles

Muscarinic receptor activation preferentially inhibits rebound in vulnerable dopaminergic neurons.

Dopaminergic subpopulations of the substantia nigra pars compacta (SNc) differentially degenerate in Parkinson's disease and are characterized by unique electrophysiological properties. The vulnerable population expresses a T-type calcium channel-mediated afterdepolarization (ADP) and shows rebound activity upon release from inhibition, whereas the resilient population does not have an ADP and is slower to fire after hyperpolarization. This rebound activity can trigger dopamine release in the striatum, an important component of basal ganglia function. Using whole-cell patch clamp electrophysiology on ex vivo slices from adult mice of both sexes, we find that muscarinic activation with the non-selective muscarinic agonist Oxotremorine inhibits rebound activity more strongly in vulnerable vs resilient SNc neurons. Here, we show that this effect depends on the direct activation of muscarinic receptors on the SNc dopaminergic neurons. Through a series of pharmacological and transgenic knock-out experiments, we tested whether the muscarinic inhibition of rebound was mediated through the canonical rebound-related ion channels: T-type calcium channels, hyperpolarization-activated cation channels (HCN), and A-type potassium channels. We find that muscarinic receptor activation inhibits HCN-mediated current (Ih) in vulnerable SNc neurons, but that Ih activity is not necessary for the muscarinic inhibition of rebound activity. Similarly, we find that Oxotremorine inhibits rebound activity independently of T-type calcium channels and A-type potassium channels. Together these findings reveal new principles governing acetylcholine and dopamine interactions, showing that muscarinic receptors directly affect SNc rebound activity in the midbrain at the somatodendritic level and differentially modify information processing in distinct SNc subpopulations.

Chronic but not acute nicotine treatment ameliorates acute inflammation-induced working memory impairment by increasing CRTC1 and HCN2 in adult male mice.

Systemic inflammation in which lipopolysaccharide (LPS) is released into circulation can cause cognitive dysfunction and we have previously shown that LPS impaired working memory (WM) which refers to the ability to guide incoming behavior by retrieving recently acquired information. However, the mechanism is not very clear, and currently, there is no approved strategy to improve inflammation-induced WM deficit. Notably, epidemiological studies have demonstrated a lower occurrence rate of inflammatory-related diseases in smoking patients, suggesting that inflammation-induced WM impairment may be improved by nicotine treatment. Here, our object is to investigate the effect and potential mechanisms of acute and chronic nicotine treatment on LPS-produced WM deficiency. Delayed alternation T-maze task (DAT) was applied for evaluating WM which includes both the short-term information storage and the ability to correct errors in adult male mice. Immunofluorescence staining and immunoblotting were used for assessing the levels and distribution of CREB-regulated transcription coactivator 1 (CRTC1) and hyperpolarization-activated cation channels 2 (HCN2) in the medial prefrontal cortex (mPFC) and hippocampus. Quantitative PCR and ELISA were employed for analyzing the mRNA and protein levels of TNF-α and IL-1β. Our results revealed that administration of LPS (i.p.) at a dose of 0.5 mg/kg significantly produced WM impairment in the DAT task accompanied by an increase in IL-1β and TNF-α expression in the mPFC. Moreover, intra-mPFC infusion of IL-1Ra, an IL-1 antagonist, markedly alleviated LPS-induced WM deficiency. More important, chronic (2 weeks) but not acute nicotine (0.2 mg/kg, subcutaneous) treatment significantly alleviated LPS-induced WM deficiency by upregulating CRTC1 and HCN2. Of note, intra-mPFC infusion of HCN blocker ZD7288 produced significant WM deficiency. In summary, in this study, we show that chronic nicotine treatment ameliorates acute inflammation-induced working memory deficiency by increasing CRTC1 and HCN2 in adult male mice.

Missing Puzzle Pieces in Dementia Research: HCN Channels and Theta Oscillations.

Increasing evidence indicates a role of hyperpolarization activated cation (HCN) channels in controlling the resting membrane potential, pacemaker activity, memory formation, sleep, and arousal. Their disfunction may be associated with the development of epilepsy and age-related memory decline. Neuronal hyperexcitability involved in epileptogenesis and EEG desynchronization occur in the course of dementia in human Alzheimer's Disease (AD) and animal models, nevertheless the underlying ionic and cellular mechanisms of these effects are not well understood. Some suggest that theta rhythms involved in memory formation could be used as a marker of memory disturbances in the course of neurogenerative diseases, including AD. This review focusses on the interplay between hyperpolarization HCN channels, theta oscillations, memory formation and their role(s) in dementias, including AD. While individually, each of these factors have been linked to each other with strong supportive evidence, we hope here to expand this linkage to a more inclusive picture. Thus, HCN channels could provide a molecular target for developing new therapeutic agents for preventing and/or treating dementia.

Hyperpolarization-activated cation channels shape the spiking frequency preference of human cortical layer 5 pyramidal neurons.

Discerning the contribution of specific ionic currents to complex neuronal dynamics is a difficult, but important, task. This challenge is exacerbated in the human setting, although the widely-characterized uniqueness of the human brain as compared to preclinical models necessitates the direct study of human neurons. Neuronal spiking frequency preference is of particular interest given its role in rhythm generation and signal transmission in cortical circuits. Here, we combine the frequency-dependent gain (FDG), a measure of spiking frequency preference, and novel in silico analyses to dissect the contributions of individual ionic currents to the suprathreshold features of human L5 neurons captured by the FDG. We confirm that a contemporary model of such a neuron, primarily constrained to capture subthreshold activity driven by the hyperpolarization-activated cyclic nucleotide gated (h-) current, replicates key features of the in vitro FDG both with and without h-current activity. With the model confirmed as a viable approximation of the biophysical features of interest, we applied new analysis techniques to quantify the activity of each modeled ionic current in the moments prior to spiking, revealing unique dynamics of the h-current. These findings motivated patch-clamp recordings in analogous rodent neurons to characterize their FDG, which confirmed that a biophysically-detailed model of these neurons captures key inter-species differences in the FDG. These differences are correlated with distinct contributions of the h-current to neuronal activity. Together, this interdisciplinary and multi-species study provides new insights directly relating the dynamics of the h-current to suprathreshold spiking frequency preference in human L5 neurons.Significance StatementUnderstanding the contributions of individual ionic currents to neuronal activity is vital, considering the established role of ion channel modifications in neuropsychiatric conditions. We combine in vitro characterization of the spiking frequency preference of human L5 cortical pyramidal neurons via the frequency-dependent gain (FDG) with new analyses of a biophysically-detailed computational model of such a neuron to delineate the connection between the dynamics of the hyperpolarization-activated cyclic nucleotide gated (h-) current prior to spiking and key properties of the FDG. By further determining that both these FDG properties and h-current dynamics are distinct in analogous rodent neurons, we provide convincing evidence for the key role of the h-current in the suprathreshold frequency preference of human L5 cortical neurons.

An evolutionarily conserved pacemaker role for HCN ion channels in smooth muscle.

Although hyperpolarization-activated cation (HCN) ion channels are well established to underlie cardiac pacemaker activity, their role in smooth muscle organs remains controversial. HCN-expressing cells are localized to renal pelvic smooth muscle (RPSM) pacemaker tissues of the murine upper urinary tract and HCN channel conductance is required for peristalsis. To date, however, the Ih pacemaker current conducted by HCN channels has never been detected in these cells, raising questions on the identity of RPSM pacemakers. Indeed, the RPSM pacemaker mechanisms of the unique multicalyceal upper urinary tract exhibited by humans remains unknown. Here, we developed immunopanning purification protocols and demonstrate that 96% of isolated HCN+ cells exhibit Ih . Single-molecule STORM to whole-tissue imaging showed HCN+ cells express single HCN channels on their plasma membrane and integrate into the muscular syncytium. By contrast, PDGFR-α+ cells exhibiting the morphology of ICC gut pacemakers were shown to be vascular mural cells. Translational studies in the homologous human and porcine multicalyceal upper urinary tracts showed that contractions and pacemaker depolarizations originate in proximal calyceal RPSM. Critically, HCN+ cells were shown to integrate into calyceal RPSM pacemaker tissues, and HCN channel block abolished electrical pacemaker activity and peristalsis of the multicalyceal upper urinary tract. Cumulatively, these studies demonstrate that HCN ion channels play a broad, evolutionarily conserved pacemaker role in both cardiac and smooth muscle organs and have implications for channelopathies as putative aetiologies of smooth muscle disorders. KEY POINTS: Pacemakers trigger contractions of involuntary muscles. Hyperpolarization-activated cation (HCN) ion channels underpin cardiac pacemaker activity, but their role in smooth muscle organs remains controversial. Renal pelvic smooth muscle (RPSM) pacemakers trigger contractions that propel waste away from the kidney. HCN+ cells localize to murine RPSM pacemaker tissue and HCN channel conductance is required for peristalsis. The HCN (Ih ) current has never been detected in RPSM cells, raising doubt whether HCN+ cells are bona fide pacemakers. Moreover, the pacemaker mechanisms of the unique multicalyceal RPSM of higher order mammals remains unknown. In total, 97% of purified HCN+ RPSM cells exhibit Ih . HCN+ cells integrate into the RPSM musculature, and pacemaker tissue peristalsis is dependent on HCN channels. Translational studies in human and swine demonstrate HCN channels are conserved in the multicalyceal RPSM and that HCN channels underlie pacemaker activity that drives peristalsis. These studies provide insight into putative channelopathies that can underlie smooth muscle dysfunction.

A point mutation turns an inactivated potassium channel into a hyperpolarization activated cation channel.

In potassium channels, the slow inactivation (C-type) process decreases the ionic conduction by affecting the selectivity filter. The W434F mutation in Shaker potassium channel stabilizes it in the inactivated conformation. The recent determination of the structure of the Shaker W434F channel shows a dilated selectivity filter, compared to Shaker WT structure, reminiscent to the hyperpolarization activated cyclic nucleotide gated channel (HCN). This suggests a common link between inactivation and hyperpolarization gating, which is explored in this work. In Shaker the P475D (Sh-P475D) is a mutation in the bundle crossing region that stabilizes the activation gate in the open conformation over a wide voltage range (−140 mV to +100). Sh-P475D was expressed in Xenopus oocytes and inactivation was favored by removing external potassium. Under these conditions, the channel displays robust hyperpolarization activated currents reminiscent of HCN. (Similar currents are observed when the W434F mutation is added in Sh-P475D background.) The currents elicited by hyperpolarizing voltages in the Sh-P475D mutant are non-selective (PK/PNa∼1) and even N-methyl-D-glucamine and calcium are permeable in this mutant. We analyzed the role of residues known to affect pore inactivation, the contribution of the selectivity filter, and the influence of the voltage sensor movement on the kinetics and steady state characteristics of the P475D-induced hyperpolarization activated currents. Our results suggest a mechanism of hyperpolarization gating and inactivation at the selectivity filter that seems to be common among voltage gated cation channels. Supported by NIH GM03076.

Adenosine Downregulates the Activities of Glutamatergic Neurons in the Paraventricular Hypothalamic Nucleus Required for Sleep.

Adenosine is an endogenous substance that regulates sleep homeostasis. It plays an important role in sleep induction under physiological condition. So far, the neural mechanisms underlying sleep-promoting effects of adenosine are not completely clear. Recent studies have shown that glutamatergic neurons in the paraventricular hypothalamic nucleus (PVH) play an important role in wakefulness. Using whole-cell patch-clamp, we found that adenosine can inhibit glutamatergic neurons in PVH. This inhibition is mainly achieved by activating adenosine type 1 receptors, thereby reducing hyperpolarization-activated cyclic nucleotide-gated cation channels. By recording electroencephalogram (EEG) and electromyography (EMG), it was found that local administration of adenosine type 1 receptor blocker in PVH could significantly reduce the NREM sleep. On the contrary, if adenosine was given, it could increase the NREM sleep. These results suggest that adenosine can promote sleep by reducing the excitability of PVH neurons. This findings reveal a novel mechanism of adenosine regulating sleep homeostasis.

Spike-Rate Adaptation in a Computational Model of Human-Shaped Spiral Ganglion Neurons.

The purpose of this study is to develop a biophysical model of human spiral ganglion neurons (SGNs) that includes voltage-gated hyperpolarization-activated cation (HCN) channels and low-threshold potassium voltage-gated, delayed-rectifier low-threshold potassium (KLT) channels, providing for a more complete simulation of spike-rate adaptation, a feature of most spiking neurons in which spiking activity is reduced in response to sustained stimulation. Our model incorporates features of spike-rate adaptation reported from in vivo studies, whilst also displaying similar behaviour to existing models of human SGNs, including the dependence of electrically evoked thresholds on the polarity of electrical pulses. Hypothesizing that the mode of stimulation-intracellular or extracellular-predicts features of spike-rate adaptation similar to in vivo studies, including the influence of stimulus intensity and pulse-rate, we find that the mode of stimulation alters features of spike-rate adaptation. In particular, the reduction in spiking over time with sustained input was generally greater for extracellular, compared to intracellular, stimulation, when simulating a multi-compartment SGN with human morphological features. In contrast, time-constants of spike-rate adaption reported for in vivo data did not fit our predicted responses, highlighting the need for a more complete physiological understanding of the factors contributing to spike-rate adaptation in electrically stimulated human SGNs. Our model extends previous computational models of SGNs with human morphology with ionic channels accounting for features of spike-rate adaptation. The significance of this work resides in the ability to improve the modeling of cochlear implant (CI) stimulation and its effects on neural responses. This will help develop novel, and perhaps personalised, stimulation strategies to reduce variability in CI user outcomes.

Analgesic effect of ivabradine against inflammatory pain mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels expressed on primary afferent terminals in the spinal dorsal horn.

Ivabradine, a hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel blocker and clinically approved bradycardic agent, has analgesic effects against neuropathic pain. Although the expression of HCN channels in the spinal dorsal horn (SDH) is augmented under inflammatory pain, spinal responses to centrally and peripherally applied ivabradine remain poorly understood. We investigated the spinal action and cellular mechanisms underlying the drug's analgesic effects against inflammatory pain using inflammatory pain model rats. Intraperitoneal and intrathecal injections of ivabradine inhibited mechanical allodynia (6 rats/dose; P < 0.05), and immunohistochemical staining showed that ivabradine suppresses the phosphorylated extracellular signal-regulated kinase activation in the SDH (6 rats/group, P < 0.01). In vitro whole-cell patch-clamp and in vivo extracellular recordings showed that direct application of ivabradine to the spinal cord decreases the mean miniature excitatory postsynaptic currents' frequency (13 rats; P < 0.01), and direct and peripheral application of ivabradine suppresses the spinal response to mechanical stimulation-evoked firing (8 rats/group, P < 0.01). Moreover, ivabradine reduces the amplitudes of monosynaptic excitatory postsynaptic currents evoked by Aδ-fiber and C-fiber stimulation (6 rats; P < 0.01) and induces a stronger inhibition of those evoked by C-fiber stimulation. These phenomena were inhibited by forskolin, an activator of HCN channels. In conclusion, spinal responses mediated by HCN channels on primary afferent terminals are suppressed by central and peripheral administration of ivabradine; the drug also exhibits analgesic effects against inflammatory pain. In addition, ivabradine preferentially acts on C-fiber terminals of SDH neurons and induces a stronger inhibition of neuronal excitability in inflammatory pain.

Hypoxia with inflammation and reperfusion alters membrane resistance by dynamically regulating voltage-gated potassium channels in hippocampal CA1 neurons

Hypoxia typically accompanies acute inflammatory responses in patients and animal models. However, a limited number of studies have examined the effect of hypoxia in combination with inflammation (Hypo-Inf) on neural function. We previously reported that neuronal excitability in hippocampal CA1 neurons decreased during hypoxia and greatly rebounded upon reoxygenation. We attributed this altered excitability mainly to the dynamic regulation of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels and input resistance. However, the molecular mechanisms underlying input resistance changes by Hypo-Inf and reperfusion remained unclear. In the present study, we found that a change in the density of the delayed rectifier potassium current (IDR) can explain the input resistance variability. Furthermore, voltage-dependent inactivation of A-type potassium (IA) channels shifted in the depolarizing direction during Hypo-Inf and reverted to normal upon reperfusion without a significant alteration in the maximum current density. Our results indicate that changes in the input resistance, and consequently excitability, caused by Hypo-Inf and reperfusion are at least partially regulated by the availability and voltage dependence of KV channels. Moreover, these results suggest that selective KV channel modulators can be used as potential neuroprotective drugs to minimize hypoxia- and reperfusion-induced neuronal damage.

Uterine Excitability and Ion Channels and Their Changes with Gestation and Hormonal Environment.

We address advances in the understanding of myometrial physiology, focusing on excitation and the effects of gestation on ion channels and their relevance to labor. This review moves through pioneering studies to exciting new findings. We begin with the myometrium and its myocytes and describe how excitation might initiate and spread in this myogenic smooth muscle. We then review each of the ion channels in the myometrium: L- and T-type Ca2+ channels, KATP (Kir6) channels, voltage-dependent K channels (Kv4, Kv7, and Kv11), twin-pore domain K channels (TASK, TREK), inward rectifier Kir7.1, Ca2+-activated K+ channels with large (KCNMA1, Slo1), small (KCNN1-3), and intermediate (KCNN4) conductance, Na-activated K channels (Slo2), voltage-gated (SCN) Na+ and Na+ leak channels, nonselective (NALCN) channels, the Na K-ATPase, and hyperpolarization-activated cation channels. We finish by assessing how three key hormones- oxytocin, estrogen, and progesterone-modulate and integrate excitability throughout gestation.

Modeling Reveals Human-Rodent Differences in H-Current Kinetics Influencing Resonance in Cortical Layer 5 Neurons.

While our understanding of human neurons is often inferred from rodent data, inter-species differences between neurons can be captured by building cellular models specifically from human data. This includes understanding differences at the level of ion channels and their implications for human brain function. Thus, we here present a full spiking, biophysically detailed multi-compartment model of a human layer 5 (L5) cortical pyramidal cell. Model development was primarily based on morphological and electrophysiological data from the same human L5 neuron, avoiding confounds of experimental variability. Focus was placed on describing the behavior of the hyperpolarization-activated cation (h-) channel, given increasing interest in this channel due to its role in pacemaking and differentiating cell types. We ensured that the model exhibited post-inhibitory rebound spiking considering its relationship with the h-current, along with other general spiking characteristics. The model was validated against data not used in its development, which highlighted distinctly slower kinetics of the human h-current relative to the rodent setting. We linked the lack of subthreshold resonance observed in human L5 neurons to these human-specific h-current kinetics. This work shows that it is possible and necessary to build human-specific biophysical neuron models in order to understand human brain dynamics.

Integration of Within-Cell Experimental Data With Multi-Compartmental Modeling Predicts H-Channel Densities and Distributions in Hippocampal OLM Cells.

Determining biophysical details of spatially extended neurons is a challenge that needs to be overcome if we are to understand the dynamics of brain function from cellular perspectives. Moreover, we now know that we should not average across recordings from many cells of a given cell type to obtain quantitative measures such as conductance since measures can vary multiple-fold for a given cell type. In this work we examine whether a tight combination of experimental and computational work can address this challenge. The oriens-lacunosum/moleculare (OLM) interneuron operates as a “gate” that controls incoming sensory and ongoing contextual information in the CA1 of the hippocampus, making it essential to understand how its biophysical properties contribute to memory function. OLM cells fire phase-locked to the prominent hippocampal theta rhythms, and we previously used computational models to show that OLM cells exhibit high or low theta spiking resonance frequencies that depend respectively on whether their dendrites have hyperpolarization-activated cation channels (h-channels) or not. However, whether OLM cells actually possess dendritic h-channels is unknown at present. We performed a set of whole-cell recordings of OLM cells from mouse hippocampus and constructed three multi-compartment models using morphological and electrophysiological parameters extracted from the same OLM cell, including per-cell pharmacologically isolated h-channel currents. We found that the models best matched experiments when h-channels were present in the dendrites of each of the three model cells created. This strongly suggests that h-channels must be present in OLM cell dendrites and are not localized to their somata. Importantly, this work shows that a tight integration of model and experiment can help tackle the challenge of characterizing biophysical details and distributions in spatially extended neurons. Full spiking models were built for two of the OLM cells, matching their current clamp cell-specific electrophysiological recordings. Overall, our work presents a technical advancement in modeling OLM cells. Our models are available to the community to use to gain insight into cellular dynamics underlying hippocampal function.

A single residue exerts two contrasting effects on autoinhibition of HCN channels

Hyperpolarization‐activated cation (HCN) channels regulate electrical activity in the brain and heart in a cAMP‐dependent manner. The voltage‐gating of these channels is mediated by a transmembrane (TM) region but is additionally regulated by direct binding of cAMP to a cyclic nucleotide‐binding (CNB) fold in the cytoplasmic C‐terminal region. In the unliganded form, the CNB fold mediates an autoinhibitory mechanism that kinetically and thermodynamically stabilizes the closed state, and cAMP binding relieves this inhibition. This autoinhibition is an important regulatory mechanism, relief of which provides a molecular mechanism for how cAMP potentiation occurs. The magnitude of this autoinhibition can be calculated by comparing thermodynamic or kinetic parameters of a channel to the parameters from a channel that is autoinhibition‐free due to truncation of the CNB fold. Recently, I have found that the magnitude of autoinhibition is regulated by interdomain interactions between the C‐terminal region and TM region structures. Specifically, the magnitude of both kinetic and thermodynamic autoinhibition was augmented by replacing the TM region of the HCN2 channel with the TM region of HCN4. I hypothesized that interdomain interactions between the HCN4 TM region and charged residues in the C‐linker, which connects the CNB fold to the TM region, can modify the magnitude of autoinhibition. I used site directed mutagenesis to identify that the E457 residue formed an electrostatic interaction with the HCN4 TM region to stabilize the open state. This residue also has an important role in both the kinetic and thermodynamic components of the autoinhibition mechanism. A charge reversal mutation (E457R) limited the thermodynamic effect of autoinhibition while augmenting the kinetic effect of autoinhibition. Overall, this work suggests that the molecular mechanisms of autoinhibition must include participation of TM region structures and provides functional evidence for interdomain interactions that facilitate HCN channel regulation by CNB fold‐mediated mechanisms.Support or Funding InformationNSERC, Simon Fraser University

Inhibitory Effective Perturbations of Cilobradine (DK-AH269), A Blocker of HCN Channels, on the Amplitude and Gating of Both Hyperpolarization-Activated Cation and Delayed-Rectifier Potassium Currents.

Cilobradine (CIL, DK-AH269), an inhibitor of hyperpolarization-activated cation current (Ih), has been observed to possess pro-arrhythmic properties. Whether and how CIL is capable of perturbing different types of membrane ionic currents existing in electrically excitable cells, however, is incompletely understood. In this study, we intended to examine possible modifications by it or other structurally similar compounds of ionic currents in pituitary tumor (GH3) cells and in heart-derived H9c2 cells. The standard whole-cell voltage-clamp technique was performed to examine the effect of CIL on ionic currents. GH3-cell exposure to CIL suppressed the density of hyperpolarization-evoked Ih in a concentration-dependent manner with an effective IC50 of 3.38 μM. Apart from its increase in the activation time constant of Ih during long-lasting hyperpolarization, the presence of CIL (3 μM) distinctly shifted the steady-state activation curve of Ih triggered by a 2-s conditioning pulse to a hyperpolarizing direction by 10 mV. As the impedance-frequency relation of Ih was studied, its presence raised the impedance magnitude at the resonance frequency induced by chirp voltage. CIL also suppressed delayed-rectifier K+ current (IK(DR)) followed by the accelerated inactivation time course of this current, with effective IC50 (measured at late IK(DR)) or KD value of 3.54 or 3.77 μM, respectively. As the CIL concentration increased 1 to 3 μM, the inactivation curve of IK(DR) elicited by 1- or 10-s conditioning pulses was shifted to a hyperpolarizing potential by approximately 10 mV, and the recovery of IK(DR) inactivation during its presence was prolonged. The peak Na+ current (INa) during brief depolarization was resistant to being sensitive to the presence of CIL, yet to be either decreased by subsequent addition of A-803467 or enhanced by that of tefluthrin. In cardiac H9c2 cells, unlike the CIL effect, the addition of either ivabradine or zatebradine mildly led to a lowering in IK(DR) amplitude with no conceivable change in the inactivation time course of the current. Taken together, the compound like CIL, which was tailored to block hyperpolarization-activated cation (HCN) channels effectively, was also capable of altering the amplitude and gating of IK(DR), thereby influencing the functional activities of electrically excitable cells, such as GH3 cells.

Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels.

Hyperpolarization-activated cationic HCN channels comprise four members (HCN1–4) that control dendritic integration, synaptic transmission and action potential firing. In the kidney, HCN1, HCN2 and HCN3 are differentially expressed and contribute to the transport of sodium, potassium (K+) and ammonium into the nephrons. HCN3 is regulated by K+ diets in the kidney. In this work we performed a proteomic analysis of HCN3 expressed in human embryonic kidney cells (HEK293 cells). More than 50% of the interacting proteins belonged to mitochondria. Therefore, we explored the presence of HCN channels in kidney mitochondria. By immunoblotting and immunogold electron microscopy HCN3 protein expression was found in rat kidney mitochondria; it was also confirmed in human kidney. Patch-clamp recordings of renal mitochondria and mitochondria from HEK293 cells overexpressing HCN1, HCN2 and HCN3 channels, stained with MitoTracker Green FM, indicated that only HCN3 could produce inwardly K+ currents that were inhibited by ZD7288, a specific blocker of HCN channels. Furthermore, ZD7288 caused inhibition of the oxygen consumption coupled to ATP synthesis and hyperpolarization of the inner mitochondrial membrane. In conclusion, we show for the first time that pacemaker HCN channels contribute to K+ transport in mitochondria facilitating the activity of the respiratory chain and ATP synthesis by controlling the inner mitochondrial membrane potential.

Dopamine Deficiency Reduces Striatal Cholinergic Interneuron Function in Models of Parkinson’s Disease

Motor and cognitive functions depend on the coordinated interactions between dopamine (DA) and acetylcholine (ACh) at striatal synapses. Increased ACh availability was assumed to accompany DA deficiency based on the outcome of pharmacological treatments and measurements in animals that were critically depleted of DA. Using Slc6a3DTR/+ diphtheria-toxin-sensitive mice, we demonstrate that a progressive and L-dopa-responsive DA deficiency reduces ACh availability and the transcription of hyperpolarization-activated cation (HCN) channels that encode the spike timing of ACh-releasing tonically active striatal interneurons (ChIs). Although the production and release of ACh and DA are reduced, the preponderance of ACh over DA contributes to the motor deficit. The increase in striatal ACh relative to DA is heightened via D1-type DA receptors that activate ChIs in response to DA release from residual axons. These results suggest that stabilizing the expression of HCN channels may improve ACh-DA reciprocity and motor function in Parkinson's disease (PD). VIDEO ABSTRACT.

The Hyperpolarization-Activated HCN4 Channel is Important for Proper Maintenance of Oscillatory Activity in the Thalamocortical System.

Hyperpolarization-activated cation channels are involved, among other functions, in learning and memory, control of synaptic transmission and epileptogenesis. The importance of the HCN1 and HCN2 isoforms for brain function has been demonstrated, while the role of HCN4, the third major neuronal HCN subunit, is not known. Here we show that HCN4 is essential for oscillatory activity in the thalamocortical (TC) network. HCN4 is selectively expressed in various thalamic nuclei, excluding the thalamic reticular nucleus. HCN4-deficient TC neurons revealed a massive reduction of Ih and strongly reduced intrinsic burst firing, whereas the current was normal in cortical pyramidal neurons. In addition, evoked bursting in a thalamic slice preparation was strongly reduced in the mutant mice probes. HCN4-deficiency also significantly slowed down thalamic and cortical oscillations during active wakefulness. Taken together, these results establish that thalamic HCN4 channels are essential for the production of rhythmic intrathalamic oscillations and determine regular TC oscillatory activity during alert states.

Inhibitory effects of class I antiarrhythmic agents on Na+ and Ca2+ currents of human iPS cell-derived cardiomyocytes.

IntroductionHuman induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called “iCell cardiomyocytes” under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents.MethodsiCell cardiomyocytes were cultured for 8–14 days (38–44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp.ResultsiCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were −62.7 ± 13.7, −8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 μM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 μM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 μM, respectively.ConclusionsIn iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.

Development of a Rat Model of Sick Sinus Syndrome Using Pinpoint Press Permeation.

Objective Sick sinus syndrome (SSS) is one of the most common causes of cardiac impairment necessitating pacemaker implantation. However, studies of SSS pathogenesis are neither comprehensive nor conclusive due to limited success in achieving a stable rat SSS model. Here, we modified pinpoint press permeation to establish a stable rat SSS model. Methods We randomly assigned 138 male Sprague-Dawley rats into three groups: normal control (n = 8), sham (n = 10), and SSS (n = 120). Postoperatively, the SSS group was further divided into SSSA (n = 40), SSSB (n = 40), and SSSC (n = 40), based on reduction in heart rates by 20–30%, 31–40%, and 41–50%, respectively. We also assessed histomorphological characteristics and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) expression in the sinoatrial node (SAN) at 1, 2, 3, and 4 weeks after surgery. ResultsMortality was statistically higher in SSSC compared to SSSA and SSSB (7.5% versus 90.0% and 87.5%; P < 0.05). Heart rate in SSSA was gradually restored to preoperative levels by week 4 after surgery. In contrast, heart rate in SSSB was stable at 2–3 weeks after surgery. However, we observed that the tissues and cells in SAN were severely injured and also found a time-dependent increase in collagen content and atrium myocardium in SSSB. HCN4 expression was significantly reduced at all 4 time points in SSSB, with statistically significant differences among the groups (P < 0.01). ConclusionWe successfully developed a rat SSS model that was sustainable for up to 4 weeks.