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

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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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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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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NONCANONICAL ELECTROMECHANICAL COUPLING MECHANISM OF AN HCN CHANNEL

10.1101/2021.01.06.425635 ◽

2021 ◽

Author(s):

Gucan Dai ◽

Teresa K. Aman ◽

Frank DiMaio ◽

William N. Zagotta

Keyword(s):

Cyclic Nucleotide ◽

Metal Ion ◽

Electromechanical Coupling ◽

Transmembrane Domain ◽

Coupling Mechanism ◽

Hcn Channel ◽

Membrane Hyperpolarization ◽

Recovery From Inactivation ◽

Close Proximity ◽

Voltage Dependent

Pacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo an inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removal of cAMP produced a largely rigid-body rotation of the C-linker relative to transmembrane domain, bringing the A′ helix in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not in the presence of cAMP. These results suggest the A′ helix functions like a mechanical clutch to engage inactivation. cAMP binding disengages the clutch, permitting the hyperpolarization-dependent activation mediated by the S5 helix. Furthermore, rearrangement of A′ helix during recovery from inactivation of spHCN channels was similar to that for the activation of KCNH channels, suggesting a conserved mechanism for this noncanonical electromechanical coupling in the CNBD channel family.

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Electromechanical coupling mechanism for activation and inactivation of an HCN channel

Nature Communications ◽

10.1038/s41467-021-23062-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Gucan Dai ◽

Teresa K. Aman ◽

Frank DiMaio ◽

William N. Zagotta

Keyword(s):

Cyclic Nucleotide ◽

Metal Ion ◽

Electromechanical Coupling ◽

Transmembrane Domain ◽

Coupling Mechanism ◽

Hcn Channel ◽

Membrane Hyperpolarization ◽

Channel Inactivation ◽

Close Proximity ◽

Voltage Dependent

AbstractPacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET, patch-clamp fluorometry and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removing cAMP produced a largely rigid-body rotation of the C-linker relative to the transmembrane domain, bringing the A’ helix of the C-linker in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not the presence of cAMP. These results suggest that — in contrast to electromechanical coupling for channel activation — the A’ helix serves to couple the S4-helix movement for channel inactivation, which is likely a conserved mechanism for CNBD-family channels.

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Structural changes during HCN channel gating defined by high affinity metal bridges

The Journal of General Physiology ◽

10.1085/jgp.201210838 ◽

2012 ◽

Vol 140 (3) ◽

pp. 279-291 ◽

Cited By ~ 26

Author(s):

Daniel C.H. Kwan ◽

David L. Prole ◽

Gary Yellen

Keyword(s):

Structural Changes ◽

Structural Model ◽

Channel Gating ◽

Structural Basis ◽

Hcn Channels ◽

Hcn Channel ◽

Membrane Hyperpolarization ◽

Linker Region ◽

High Affinity ◽

Gating Mechanism

Hyperpolarization-activated cyclic nucleotide–sensitive nonselective cation (HCN) channels are activated by membrane hyperpolarization, in contrast to the vast majority of other voltage-gated channels that are activated by depolarization. The structural basis for this unique characteristic of HCN channels is unknown. Interactions between the S4–S5 linker and post-S6/C-linker region have been implicated previously in the gating mechanism of HCN channels. We therefore introduced pairs of cysteines into these regions within the sea urchin HCN channel and performed a Cd2+-bridging scan to resolve their spatial relationship. We show that high affinity metal bridges between the S4–S5 linker and post-S6/C-linker region can induce either a lock-open or lock-closed phenotype, depending on the position of the bridged cysteine pair. This suggests that interactions between these regions can occur in both the open and closed states, and that these regions move relative to each other during gating. Concatenated constructs reveal that interactions of the S4–S5 linker and post-S6/C-linker can occur between neighboring subunits. A structural model based on these interactions suggests a mechanism for HCN channel gating. We propose that during voltage-dependent activation the voltage sensors, together with the S4–S5 linkers, drive movement of the lower ends of the S5 helices around the central axis of the channel. This facilitates a movement of the pore-lining S6 helices, which results in opening of the channel. This mechanism may underlie the unique voltage dependence of HCN channel gating.

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

Biochemical Journal ◽

10.1042/bj20140692 ◽

2015 ◽

Vol 466 (1) ◽

pp. 77-84 ◽

Cited By ~ 5

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.

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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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Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression

AJP Cell Physiology ◽

10.1152/ajpcell.00389.2009 ◽

2010 ◽

Vol 298 (5) ◽

pp. C1066-C1076 ◽

Cited By ~ 20

Author(s):

Andrew P. Hegle ◽

Hamed Nazzari ◽

Andrew Roth ◽

Damiano Angoli ◽

Eric A. Accili

Keyword(s):

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Hcn Channels ◽

Hcn Channel ◽

Channel Surface ◽

Evolutionary Trajectories ◽

Evolutionary Emergence ◽

The Brain ◽

Beating Frequency

All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface.

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