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

2012 ◽  
Vol 140 (1) ◽  
pp. 29-39 ◽  
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.

scholarly journals Voltage-Controlled Gating at the Intracellular Entrance to a Hyperpolarization-Activated Cation Channel

2002 ◽  
Vol 119 (1) ◽  
pp. 83-91 ◽  
Author(s):  
Brad S. Rothberg ◽  
Ki Soon Shin ◽  
Prashant S. Phale ◽  
Gary Yellen
Keyword(s):  
Open Channel ◽  
Cardiac Cells ◽  
Hcn Channels ◽  
Hcn Channel ◽  
Channel One ◽  
Blocking Rate ◽  
Activation Gate ◽  
Flow Through ◽  
Controlled Structure ◽  
Pore Lining

Hyperpolarization-activated cation (HCN) channels regulate pacemaking activity in cardiac cells and neurons. Our previous work using the specific HCN channel blocker ZD7288 provided evidence for an intracellular activation gate for these channels because it appears that ZD7288, applied from the intracellular side, can enter and leave HCN channels only at voltages where the activation gate is opened (Shin, K.S., B.S. Rothberg, and G. Yellen. 2001. J. Gen. Physiol. 117:91–101). However, the ZD7288 molecule is larger than the Na+ or K+ ions that flow through the open channel. In the present study, we sought to resolve whether the voltage gate at the intracellular entrance to the pore for ZD7288 also can be a gate for permeant ions in HCN channels. Single residues in the putative pore-lining S6 region of an HCN channel (cloned from sea urchin; spHCN) were substituted with cysteines, and the mutants were probed with Cd2+ applied to the intracellular side of the channel. One mutant, T464C, displayed rapid irreversible block when Cd2+ was applied to opened channels, with an apparent blocking rate of ∼3 × 105 M−1s−1. The blocking rate was decreased for channels held at more depolarized voltages that close the channels, which is consistent with the Cd2+ access to this residue being gated from the intracellular side of the channel. 464C channels could be recovered from Cd2+ inhibition in the presence of a dithiol applied to the intracellular side. The rate of this recovery also was reduced when channels were held at depolarized voltages. Finally, Cd2+ could be trapped inside channels that were composed of WT/464C tandem-linked subunits, which could otherwise recover spontaneously from Cd2+ inhibition. Thus, Cd2+ escape is also gated at the intracellular side of the channel. Together, these results are consistent with a voltage-controlled structure at the intracellular side of the spHCN channel that can gate the flow of cations through the pore.

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

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.

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 ◽  
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

<b><i>Introduction:</i></b> Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents of <i>Ih</i> and absence epilepsy seizures are associated, but studies reveal differential results. <b><i>Objective:</i></b> 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. <b><i>Methods:</i></b> HCN isoform levels from isolated brains of both naïve nonepileptic Wistar and GAERS groups were evaluated by enzyme-linked immunosorbent assay. ZD7288, an <i>Ih</i> 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. <b><i>Results:</i></b> The HCN2 levels in the cortex and hippocampus of the GAERS group were lower compared to the naïve nonepileptic Wistar group (<i>p</i> &#x3c; 0.05). ZD7288 increased the number of SWDs at the 20th and 120th min with the highest administered dose of 7 μg (<i>p</i> &#x3c; 0.05). <b><i>Conclusion:</i></b> The <i>Ih</i> 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.

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

2015 ◽  
Vol 466 (1) ◽  
pp. 77-84 ◽  
Author(s):  
Mo Li ◽  
Lige Tonggu ◽  
Lan Tang ◽  
Liguo Wang
Keyword(s):  
Cell Membrane ◽  
Cyclic Nucleotide ◽  
Hcn Channels ◽  
Hcn Channel

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.

scholarly journals Allosteric Regulation of the Cyclic Nucleotide-Binding Domain in HCN Channels

2015 ◽  
Vol 108 (2) ◽  
pp. 192a-193a
Author(s):  
Hannah A. DeBerg ◽  
Shahidul M. Islam ◽  
Michael C. Puljung ◽  
Benoit Roux ◽  
William N. Zagotta ◽  
...  
Keyword(s):  
Cyclic Nucleotide ◽  
Allosteric Regulation ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Hcn Channels ◽  
Nucleotide Binding Domain ◽  
Cyclic Nucleotide Binding ◽  
Cyclic Nucleotide Binding Domain

scholarly journals S4 Movement in a Mammalian HCN Channel

2003 ◽  
Vol 123 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Sriharsha Vemana ◽  
Shilpi Pandey ◽  
H. Peter Larsson
Keyword(s):  
Voltage Sensor ◽  
K Channel ◽  
Hcn Channels ◽  
Dependent Manner ◽  
Hcn Channel ◽  
Voltage Range ◽  
Kv Channels ◽  
State Dependent ◽  
Voltage Dependent ◽  
Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

scholarly journals 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 ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3500-3511 ◽  
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.

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

2018 ◽  
Vol 150 (8) ◽  
pp. 1203-1213 ◽  
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.

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

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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