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

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.

Download Full-text

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.

Download Full-text

Structural insights into the mechanisms of CNBD channel function

The Journal of General Physiology ◽

10.1085/jgp.201711898 ◽

2017 ◽

Vol 150 (2) ◽

pp. 225-244 ◽

Cited By ~ 32

Author(s):

Zachary M. James ◽

William N. Zagotta

Keyword(s):

Cyclic Nucleotide ◽

Ion Selectivity ◽

K Channel ◽

Hcn Channels ◽

Nucleotide Binding Domain ◽

Physiological Processes ◽

Structural Framework ◽

Voltage Dependent ◽

Compare And Contrast ◽

Structural Insights

Cyclic nucleotide-binding domain (CNBD) channels are a family of ion channels in the voltage-gated K+ channel superfamily that play crucial roles in many physiological processes. CNBD channels are structurally similar but functionally very diverse. This family includes three subfamilies: (1) the cyclic nucleotide-gated (CNG) channels, which are cation-nonselective, voltage-independent, and cyclic nucleotide-gated; (2) the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are weakly K+ selective, hyperpolarization-activated, and cyclic nucleotide-gated; and (3) the ether-à-go-go-type (KCNH) channels, which are strongly K+ selective, depolarization-activated, and cyclic nucleotide-independent. Recently, several high-resolution structures have been reported for intact CNBD channels, providing a structural framework to better understand their diverse function. In this review, we compare and contrast the recent structures and discuss how they inform our understanding of ion selectivity, voltage-dependent gating, and cyclic nucleotide–dependent gating within this channel family.

Download Full-text

S4 Movement in a Mammalian HCN Channel

The Journal of General Physiology ◽

10.1085/jgp.200308916 ◽

2003 ◽

Vol 123 (1) ◽

pp. 21-32 ◽

Cited By ~ 61

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.

Download Full-text

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.

Download Full-text

Voltage Sensor Movement and cAMP Binding Allosterically Regulate an Inherently Voltage-independent Closed−Open Transition in HCN Channels

The Journal of General Physiology ◽

10.1085/jgp.200609585 ◽

2007 ◽

Vol 129 (2) ◽

pp. 175-188 ◽

Cited By ~ 41

Author(s):

Shan Chen ◽

Jing Wang ◽

Lei Zhou ◽

Meena S. George ◽

Steven A. Siegelbaum

Keyword(s):

Cyclic Nucleotide ◽

Voltage Sensor ◽

Hcn Channels ◽

Nucleotide Binding Domain ◽

Voltage Range ◽

Slow Kinetics ◽

Rapid Kinetics ◽

Hcn1 Channels ◽

Rate Limiting ◽

Allosteric Model

The hyperpolarization-activated cyclic nucleotide-modulated cation (HCN) channels are regulated by both membrane voltage and the binding of cyclic nucleotides to a cytoplasmic, C-terminal cyclic nucleotide-binding domain (CNBD). Here we have addressed the mechanism of this dual regulation for HCN2 channels, which activate with slow kinetics that are strongly accelerated by cAMP, and HCN1 channels, which activate with rapid kinetics that are weakly enhanced by cAMP. Surprisingly, we find that the rate of opening of HCN2 approaches a maximal value with extreme hyperpolarization, indicating the presence of a voltage-independent kinetic step in the opening process that becomes rate limiting at very negative potentials. cAMP binding enhances the rate of this voltage-independent opening step. In contrast, the rate of opening of HCN1 is much greater than that of HCN2 and does not saturate with increasing hyperpolarization over the voltage range examined. Domain-swapping chimeras between HCN1 and HCN2 reveal that the S4–S6 transmembrane region largely determines the limiting rate in opening kinetics at negative voltages. Measurements of HCN2 tail current kinetics also reveal a voltage-independent closing step that becomes rate limiting at positive voltages; the rate of this closing step is decreased by cAMP. These results are consistent with a cyclic allosteric model in which a closed–open transition that is inherently voltage independent is subject to dual allosteric regulation by voltage sensor movement and cAMP binding. This mechanism accounts for several properties of HCN channel gating and has potentially important physiological implications.

Download Full-text

Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels

AJP Cell Physiology ◽

10.1152/ajpcell.00154.2008 ◽

2008 ◽

Vol 295 (2) ◽

pp. C557-C565 ◽

Cited By ~ 15

Author(s):

Sriharsha Vemana ◽

Shilpi Pandey ◽

H. Peter Larsson

Keyword(s):

Binding Site ◽

Physiological Role ◽

Time Dependent ◽

Inward Rectification ◽

Hcn Channels ◽

Membrane Hyperpolarization ◽

Outward Currents ◽

Voltage Dependent ◽

Hcn1 Channels ◽

Pore Blocker

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents ( 5 ). In the present study, we show that intracellular Mg2+ functions as a voltage-dependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg2+ is in the physiological range, with Mg2+ binding with an IC50 of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at +50 mV. The effective electrical distance for the Mg2+ binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg2+, suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg2+ acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg2+ may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg2+ could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.

Download Full-text

Hysteresis in the Voltage Dependence of HCN Channels

The Journal of General Physiology ◽

10.1085/jgp.200409130 ◽

2005 ◽

Vol 125 (3) ◽

pp. 305-326 ◽

Cited By ~ 97

Author(s):

Roope Männikkö ◽

Shilpi Pandey ◽

H. Peter Larsson ◽

Fredrik Elinder

Keyword(s):

Voltage Dependence ◽

Rhythmic Activity ◽

Ionic Currents ◽

Hcn Channels ◽

Molecular Conformations ◽

Gating Charge ◽

Voltage Curve ◽

Current Change ◽

Voltage Hysteresis ◽

Hcn1 Channels

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (τ ≈ 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.

Download Full-text

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.

Download Full-text

Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805596115 ◽

2018 ◽

Vol 115 (34) ◽

pp. E8086-E8095 ◽

Cited By ~ 18

Author(s):

Galen E. Flynn ◽

William N. Zagotta

Keyword(s):

Ion Channels ◽

Cyclic Nucleotide ◽

Electromechanical Coupling ◽

Canonical Model ◽

Hcn Channels ◽

Voltage Gated ◽

Kv Channels ◽

Recovery From Inactivation ◽

Voltage Dependent ◽

Pore Domain

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are both voltage- and ligand-activated membrane proteins that contribute to electrical excitability and pace-making activity in cardiac and neuronal cells. These channels are members of the voltage-gated Kv channel superfamily and cyclic nucleotide-binding domain subfamily of ion channels. HCN channels have a unique feature that distinguishes them from other voltage-gated channels: the HCN channel pore opens in response to hyperpolarizing voltages instead of depolarizing voltages. In the canonical model of electromechanical coupling, based on Kv channels, a change in membrane voltage activates the voltage-sensing domains (VSD) and the activation energy passes to the pore domain (PD) through a covalent linker that connects the VSD to the PD. In this investigation, the covalent linkage between the VSD and PD, the S4-S5 linker, and nearby regions of spHCN channels were mutated to determine the functional role each plays in hyperpolarization-dependent activation. The results show that: (i) the S4-S5 linker is not required for hyperpolarization-dependent activation or ligand-dependent gating; (ii) the S4 C-terminal region (S4C-term) is not necessary for ligand-dependent gating but is required for hyperpolarization-dependent activation and acts like an autoinhibitory domain on the PD; (iii) the S5N-term region is involved in VSD–PD coupling and holding the pore closed; and (iv) spHCN channels have two voltage-dependent processes, a hyperpolarization-dependent activation and a depolarization-dependent recovery from inactivation. These results are inconsistent with the canonical model of VSD–PD coupling in Kv channels and elucidate the mechanism for hyperpolarization-dependent activation of HCN channels.

Download Full-text

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.

Download Full-text