Effects of N-glycosylation on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

The results suggest that N-glycosylation is not required for the opening of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and some but not all of the four subunits of the HCN channel need to be glycosylated for trafficking to cell membrane.

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Inner activation gate in S6 contributes to the state-dependent binding of cAMP in full-length HCN2 channel

The Journal of General Physiology ◽

10.1085/jgp.201110749 ◽

2012 ◽

Vol 140 (1) ◽

pp. 29-39 ◽

Cited By ~ 17

Author(s):

Shengjun Wu ◽

Weihua Gao ◽

Changan Xie ◽

Xinping Xu ◽

Christina Vorvis ◽

...

Keyword(s):

Ligand Binding ◽

Cyclic Nucleotide ◽

Binding Domain ◽

Hcn Channels ◽

Channel Blockers ◽

Structural Elements ◽

Hcn Channel ◽

Channel Interaction ◽

State Dependent ◽

Activation Gate

Recently, applications of the patch-clamp fluorometry (PCF) technique in studies of cyclic nucleotide–gated (CNG) and hyperpolarization-activated, cyclic nucleotide–regulated (HCN) channels have provided direct evidence for the long-held notion that ligands preferably bind to and stabilize these channels in an open state. This state-dependent ligand–channel interaction involves contributions from not only the ligand-binding domain but also other discrete structural elements within the channel protein. This insight led us to investigate whether the pore of the HCN channel plays a role in the ligand–whole channel interaction. We used three well-characterized HCN channel blockers to probe the ion-conducting passage. The PCF technique was used to simultaneously monitor channel activity and cAMP binding. Two ionic blockers, Cs+ and Mg2+, effectively block channel conductance but have no obvious effect on cAMP binding. Surprisingly, ZD7288, an open channel blocker specific for HCN channels, significantly reduces the activity-dependent increase in cAMP binding. Independent biochemical assays exclude any nonspecific interaction between ZD7288 and isolated cAMP-binding domain. Because ZD7228 interacts with the inner pore region, where the activation gate is presumably located, we did an alanine scanning of the intracellular end of S6, from T426 to A435. Mutations of three residues, T426, M430, and H434, which are located at regular intervals on the S6 α-helix, enhance cAMP binding. In contrast, mutations of two residues in close proximity, F431A and I432A, dampen the response. Our results demonstrate that movements of the structural elements near the activation gate directly affect ligand binding affinity, which is a simple mechanistic explanation that could be applied to the interpretation of ligand gating in general.

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HCN domain is required for HCN channel expression and couples voltage- and cAMP-dependent gating mechanisms

10.1101/2020.03.02.973560 ◽

2020 ◽

Author(s):

Ze-Jun Wang ◽

Ismary Blanco ◽

Sebastien Hayoz ◽

Tinatin I. Brelidze

Keyword(s):

Cyclic Nucleotide ◽

Transmembrane Domain ◽

Rhythmic Activity ◽

Surface Expression ◽

Hcn Channels ◽

Hcn Channel ◽

Structural Element ◽

Functional Link ◽

Membrane Hyperpolarization ◽

Membrane Spanning

ABSTRACTHyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity, and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD) and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane domain via the C-linker. Previous functional analysis of HCN channels suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of the coupling were unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed HCN domain (HCND), forms a direct structural link between the VSD and C-linker/CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified R237 and G239 residues on the S2 of the VSD that form direct interactions with I135 on the HCND. Disrupting these interactions abolished HCN2 currents. We then identified three residues on the C-linker/CNBD (E478, Q382 and H559) that form direct interactions with residues R154 and S158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the surface expression of HCN channels, and provides a functional link between the voltage- and cAMP-dependent mechanisms of HCN channel gating.

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Decreased Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 2 Activity in a Rat Model of Absence Epilepsy and the Effect of ZD7288, an Ih Inhibitor, on the Spike-and-Wave Discharges

Pharmacology ◽

10.1159/000520059 ◽

2022 ◽

pp. 1-8

Author(s):

Melis Yavuz ◽

Banu Aydın ◽

Nihan Çarçak ◽

Filiz Onat

Keyword(s):

Rat Model ◽

Cyclic Nucleotide ◽

Total Duration ◽

Absence Epilepsy ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Hcn Channels ◽

Hcn Channel ◽

Gated Channel ◽

Channel Currents

Introduction: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents of Ih and absence epilepsy seizures are associated, but studies reveal differential results. Objective: In our study, we aimed to investigate the role of the HCN channels on the expression of spike-and-wave discharges (SWDs) using the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) model. Methods: HCN isoform levels from isolated brains of both naïve nonepileptic Wistar and GAERS groups were evaluated by enzyme-linked immunosorbent assay. ZD7288, an Ih inhibitor as well as an HCN channel antagonist, was administered intracerebroventricularly to the adult GAERS groups, and to evaluate their SWD activities, electroencephalography was recorded. The effect of ZD7288 on the cumulative total duration and number of SWDs and the mean duration of each SWD complex was evaluated. Results: The HCN2 levels in the cortex and hippocampus of the GAERS group were lower compared to the naïve nonepileptic Wistar group (p < 0.05). ZD7288 increased the number of SWDs at the 20th and 120th min with the highest administered dose of 7 μg (p < 0.05). Conclusion: The Ih inhibitor ZD7288 increased the number of SWDs in a genetic absence epilepsy rat model, although this increase may not be significant due to the inconsistent time-dependent effects. In GAERS, the cortical and hippocampal HCN2 channel levels were significantly lower compared to the control group. Further studies are needed with higher doses of ZD7288 to determine if the effects will increase drastically.

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Gonadotropin-Releasing Hormone-1 Neuronal Activity Is Independent of Hyperpolarization-Activated Cyclic Nucleotide-Modulated Channels but Is Sensitive to Protein Kinase A-Dependent Phosphorylation

Endocrinology ◽

10.1210/en.2007-1508 ◽

2008 ◽

Vol 149 (7) ◽

pp. 3500-3511 ◽

Cited By ~ 18

Author(s):

Stephanie Constantin ◽

Susan Wray

Keyword(s):

Protein Kinase ◽

Neuronal Activity ◽

Cyclic Nucleotide ◽

Excitatory Input ◽

Membrane Receptors ◽

Induced Response ◽

Hcn Channels ◽

Hcn Channel ◽

Stimulation Experiments

Pulsatile release of GnRH-1 stimulates the anterior pituitary and induces secretion of gonadotropin hormones. GnRH-1 release is modulated by many neurotransmitters that act via G protein-coupled membrane receptors. cAMP is the most ubiquitous effector for these receptors. GnRH-1 neurons express hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel protein in vivo. HCN channels are involved in neuronal pacemaking and can integrate cAMP signals. cAMP-dependent protein kinase (PKA) is also activated by cAMP signals, and PKA-dependent phosphorylation modulates voltage-activated channels. In this report, these two pathways were examined in GnRH-1 neurons as integrators of forskolin (FSK)-induced stimulation. The HCN3 isoform was detected in GnRH-1 neurons obtained from mouse nasal explants. ZD7288, a HCN channel blocker, significantly reduced the efficiency of FSK to stimulate GnRH-1 neurons, whereas blockade of PKA with Rp-adenosine-3′,5′-cyclic monophosphorothioate triethylammonium did not attenuate the FSK-induced stimulation. To ensure that disruption of HCN channels on GnRH-1 neurons was responsible for reduction of FSK stimulation, experiments were performed removing γ-aminobutyric acid (GABA), the major excitatory input to GnRH-1 neurons in nasal explants. Under these conditions, Rp-adenosine-3′,5′-cyclic monophosphorothioate triethylammonium, but not ZD7288, altered the FSK-induced response of GnRH-1 neurons. These studies indicate that PKA-dependent phosphorylation is involved in the FSK-induced stimulation of GnRH-1 neurons rather than HCN channels, and HCN channels integrate the FSK-induced stimulation on GABAergic neurons. In addition, blockade of HCN channels did not modify basal GnRH-1 neuronal activity when GABAergic input was intact or removed, negating a role for these channels in basal GABAergic or GnRH-1 neuronal activity.

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Minimal molecular determinants of isoform-specific differences in efficacy in the HCN channel family

The Journal of General Physiology ◽

10.1085/jgp.201812031 ◽

2018 ◽

Vol 150 (8) ◽

pp. 1203-1213 ◽

Cited By ~ 4

Author(s):

Claudia P. Alvarez-Baron ◽

Vadim A. Klenchin ◽

Baron Chanda

Keyword(s):

Cyclic Nucleotide ◽

Rhythmic Activity ◽

Structural Similarity ◽

Hcn Channels ◽

Hcn Channel ◽

Nucleotide Binding Domain ◽

Functional Differences ◽

Molecular Determinants ◽

Voltage Dependent ◽

Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated (HCN) channels generate rhythmic activity in the heart and brain. Isoform-specific functional differences reflect the specializations required for the various roles that they play. Despite a high sequence and structural similarity, HCN isoforms differ greatly in their response to cyclic nucleotides. Cyclic AMP (cAMP) enhances the activity of HCN2 and HCN4 isoforms by shifting the voltage dependence of activation to more depolarized potentials, whereas HCN1 and HCN3 isoforms are practically insensitive to this ligand. Here, to determine the molecular basis for increased cAMP efficacy in HCN2 channels, we progressively mutate residues in the C-linker and cyclic nucleotide–binding domain (CNBD) of the mouse HCN2 to their equivalents in HCN1. We identify two clusters of mutations that determine the differences in voltage-dependent activation between these two isoforms. One maps to the C-linker region, whereas the other is in proximity to the cAMP-binding site in the CNBD. A mutant channel containing just five mutations (M485I, G497D, S514T, V562A, and S563G) switches cAMP sensitivity of full-length HCN2 to that of HCN1 channels. These findings, combined with a detailed analysis of various allosteric models for voltage- and ligand-dependent gating, indicate that these residues alter the ability of the C-linker to transduce signals from the CNBD to the pore gates of the HCN channel.

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Simulation and calculation of the contribution of hyperpolarization-activated cyclic nucleotide-gated channels to action potentials

Archives of Biological Sciences ◽

10.2298/abs150416144l ◽

2016 ◽

Vol 68 (1) ◽

pp. 217-224

Author(s):

Liping Liao ◽

Xianguang Lin ◽

Jielin Hu ◽

Xin Wu ◽

Xiaofei Yang ◽

...

Keyword(s):

Membrane Potential ◽

Action Potential ◽

Cyclic Nucleotide ◽

Ganglion Cells ◽

Recovery Phase ◽

Hcn Channels ◽

Action Potential Generation ◽

Hcn Channel ◽

Potential Generation ◽

Cyclic Nucleotide Gated Channels

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, which mediates the influx of cations, has an important role in action potential generation. In this article, we describe the contribution of the HCN channel to action potential generation. We simulated several common ion channels in neuron membranes based on data from rat dorsal root ganglion cells and modeled the action potential. The ion channel models employed in this paper were based on the Markov model. After modifying and calibrating these models, we compared the simulated action potential curves under the presence and absence of an HCN channel and calculated that the proportional contribution of the HCN channel in the potential recovery phase was 33.39%. This result indicates that the HCN channel is critical in assisting membrane potential recovery from a hyperpolarized state to a resting state. Furthermore, we showed how the HCN channel modifies the firing of the action potential using mathematic modeling. Our results indicated that although the loss of the HCN channel made recovery of the membrane potential more difficult from the most negative point to resting in comparison with the control, the firing rate of the action potential increased in certain circumstances. We present a novel explanation for the HCN channels? mechanism in neuron action potential generation using mathematical models.

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Upper and lower limb motor axons demonstrate differential excitability and accommodation to strong hyperpolarizing currents during induced hyperthermia

Journal of Neurophysiology ◽

10.1152/jn.00464.2018 ◽

2019 ◽

Vol 121 (6) ◽

pp. 2061-2070

Author(s):

Oliver R. Marmoy ◽

Paul L. Furlong ◽

Christopher E. G. Moore

Keyword(s):

Peripheral Neuropathy ◽

Differential Expression ◽

Cyclic Nucleotide ◽

Lower Limbs ◽

Sensory Loss ◽

Hcn Channels ◽

Pathophysiological Mechanism ◽

Hcn Channel ◽

Disease States ◽

Insidious Onset

Length-dependent peripheral neuropathy typically involves the insidious onset of sensory loss in the lower limbs before later progressing proximally. Recent evidence proposes hyperpolarization-activated cyclic nucleotide-gated (HCN) channels as dysfunctional in rodent models of peripheral neuropathy, and therefore differential expression of HCN channels in the lower limbs was hypothesized as a pathophysiological mechanism accounting for the pattern of symptomatology within this study. We studied six healthy participants, using motor axon excitability including strong and long [−70% and −100% hyperpolarizing threshold electrotonus (TEh)] hyperpolarizing currents to preferably study HCN channel function from the median and tibial nerves from high (40%) and low (20%) threshold. This was recorded at normothermia (~32°C) and then repeated during hyperthermia (~40°C) as an artificial hyperpolarizing axon stress. Significant differences between recovery cycle, superexcitability, accommodation to small depolarizing currents, and alterations in late stages of the inward-rectifying currents of strongest (−70% and −100% TEh) currents were observed in the lower limbs during hyperthermia. We demonstrate differences in late IH current flow, which implies higher expression of HCN channel isoforms. The findings also indicate their potential inference in the symptomatology of length-dependent peripheral neuropathies and may be a unique target for minimizing symptomatology and pathogenesis in acquired disease. NEW & NOTEWORTHY This study demonstrates nerve excitability differences between the upper and lower limbs during hyperthermia, an experimentally induced axonal stress. The findings indicate that there is differential expression of slow hyperpolarization-activated cyclic nucleotide-gated (HCN) channel isoforms between the upper and lower limbs, which was demonstrated through strong, long hyperpolarizing currents during hyperthermia. Such mechanisms may underlie postural control but render the lower limbs susceptible to dysfunction in disease states.

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The HCN domain is required for HCN channel cell-surface expression and couples voltage- and cAMP-dependent gating mechanisms

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013281 ◽

2020 ◽

Vol 295 (24) ◽

pp. 8164-8173

Author(s):

Ze-Jun Wang ◽

Ismary Blanco ◽

Sebastien Hayoz ◽

Tinatin I. Brelidze

Keyword(s):

Cell Surface ◽

Cyclic Nucleotide ◽

Rhythmic Activity ◽

Surface Expression ◽

Cell Surface Expression ◽

Hcn Channels ◽

Hcn Channel ◽

Structural Element ◽

Functional Link ◽

Membrane Hyperpolarization

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are major regulators of synaptic plasticity and rhythmic activity in the heart and brain. Opening of HCN channels requires membrane hyperpolarization and is further facilitated by intracellular cyclic nucleotides (cNMPs). In HCN channels, membrane hyperpolarization is sensed by the membrane-spanning voltage sensor domain (VSD), and the cNMP-dependent gating is mediated by the intracellular cyclic nucleotide-binding domain (CNBD) connected to the pore-forming S6 transmembrane segment via the C-linker. Previous functional analysis of HCN channels has suggested a direct or allosteric coupling between the voltage- and cNMP-dependent activation mechanisms. However, the specifics of this coupling remain unclear. The first cryo-EM structure of an HCN1 channel revealed that a novel structural element, dubbed the HCN domain (HCND), forms a direct structural link between the VSD and C-linker–CNBD. In this study, we investigated the functional significance of the HCND. Deletion of the HCND prevented surface expression of HCN2 channels. Based on the HCN1 structure analysis, we identified Arg237 and Gly239 residues on the S2 of the VSD that form direct interactions with Ile135 on the HCND. Disrupting these interactions abolished HCN2 currents. We also identified three residues on the C-linker–CNBD (Glu478, Gln482, and His559) that form direct interactions with residues Arg154 and Ser158 on the HCND. Disrupting these interactions affected both voltage- and cAMP-dependent gating of HCN2 channels. These findings indicate that the HCND is necessary for the cell-surface expression of HCN channels and provides a functional link between voltage- and cAMP-dependent mechanisms of HCN channel gating.

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The Expression and Role of Hyperpolarization-Activated and Cyclic Nucleotide-Gated Channels in Endocrine Anterior Pituitary Cells

Molecular Endocrinology ◽

10.1210/me.2011-1207 ◽

2012 ◽

Vol 26 (1) ◽

pp. 153-164 ◽

Cited By ~ 18

Author(s):

Karla Kretschmannova ◽

Marek Kucka ◽

Arturo E. Gonzalez-Iglesias ◽

Stanko S. Stojilkovic

Keyword(s):

Cell Membrane ◽

Electrical Activity ◽

Adenylyl Cyclase ◽

Phospholipase C ◽

Cyclic Nucleotide ◽

Anterior Pituitary ◽

Hcn Channels ◽

Pituitary Cells ◽

Stimulation Of

Abstract Pituitary cells fire action potentials independently of external stimuli, and such spontaneous electrical activity is modulated by a large variety of hypothalamic and intrapituitary agonists. Here, we focused on the potential role of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in electrical activity of cultured rat anterior pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from normal and GH3 immortalized pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3, and HCN4. Electrophysiological experiments showed the presence of a slowly developing hyperpolarization-activated inward current, which was blocked by Cs+ and ZD7288, in gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. Inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity. These experiments indicate that cAMP and cGMP have opposite effects on the excitability of endocrine pituitary cells, that basal cAMP production in cultured cells is sufficient to integrate the majority of HCN channels in electrical activity, and that depletion of phosphatidylinositol 4,5-bisphosphate caused by activation of phospholipase C silences them.

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