NONCANONICAL ELECTROMECHANICAL COUPLING MECHANISM OF AN HCN CHANNEL

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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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.

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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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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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The HCN Channel Voltage Sensor Undergoes A Large Downward Motion During Hyperpolarization

10.1101/522581 ◽

2019 ◽

Cited By ~ 1

Author(s):

Gucan Dai ◽

Teresa K. Aman ◽

Frank DiMaio ◽

William N. Zagotta

Keyword(s):

Ion Channels ◽

Metal Ion ◽

Voltage Sensor ◽

The Body ◽

Resting Membrane Potential ◽

Hcn Channels ◽

Hcn Channel ◽

Open Probability ◽

Membrane Hyperpolarization ◽

Transmembrane Voltage

Voltage-gated ion channels (VGICs) underlie almost all electrical signaling in the body1. They change their open probability in response to changes in transmembrane voltage, allowing permeant ions to flow across the cell membrane. Ion flow through VGICs underlies numerous physiological processes in excitable cells1. In particular, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which operate at the threshold of excitability, are essential for pacemaking activity, resting membrane potential, and synaptic integration2. VGICs contain a series of positively-charged residues that are displaced in response to changes in transmembrane voltage, resulting in a conformational change that opens the pore3–6. These voltage-sensing charges, which reside in the S4 transmembrane helix of the voltage-sensor domain (VSD)3 and within the membrane’s electric field, are thought to move towards the inside of the cell (downwards) during membrane hyperpolarization7. HCN channels are unique among VGICs because their open probability is increased by membrane hyperpolarization rather than depolarization8–10. The mechanism underlying this “reverse gating” is still unclear. Moreover, although many X-ray crystal and cryo-EM structures have been solved for the depolarized state of the VSD, including that of HCN channels11, no structures have been solved at hyperpolarized voltages. Here we measure the precise movement of the charged S4 helix of an HCN channel using transition metal ion fluorescence resonance energy transfer (tmFRET). We show that the S4 undergoes a significant (~10 Å) downward movement in response to membrane hyperpolarization. Furthermore, by applying constraints determined from tmFRET experiments to Rosetta modeling, we reveal that the carboxyl-terminal part of the S4 helix exhibits an unexpected tilting motion during hyperpolarization activation. These data provide a long-sought glimpse of the hyperpolarized state of a functioning VSD and also a framework for understanding the dynamics of reverse gating in HCN channels. Our methods can be broadly applied to probe short-distance rearrangements in other ion channels and membrane proteins.

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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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Rapid recovery from K current inactivation on membrane hyperpolarization in molluscan neurons.

The Journal of General Physiology ◽

10.1085/jgp.84.6.861 ◽

1984 ◽

Vol 84 (6) ◽

pp. 861-875 ◽

Cited By ~ 5

Author(s):

P Ruben ◽

S Thompson

Keyword(s):

Time Course ◽

Outward Current ◽

Rapid Recovery ◽

Resting Potential ◽

Molluscan Neurons ◽

Membrane Hyperpolarization ◽

Time Constants ◽

Recovery From Inactivation ◽

Voltage Dependent ◽

K Current

Recovery from K current inactivation was studied in molluscan neurons using two-microelectrode and internal perfusion voltage clamps. Experiments were designed to study the voltage-dependent delayed outward current (IK) without contamination from other K currents. The amount of recovery from inactivation and the rate of recovery increase dramatically when the membrane potential is made more negative. The time course of recovery at the resting potential, -40 mV, is well fit by a single exponential with a time constant of 24.5 s (n = 7). At more negative voltages, the time course is best fit by the sum of two exponentials with time constants at -90 mV of 1.7 and 9.8 s (n = 7). In unclamped cells, a short hyperpolarization can cause rapid recovery from inactivation that results in a shortening of the action potential duration. We conclude that there are two inactivated states of the channel and that the time constants for recovery from both states are voltage dependent. The results are discussed in terms of the multistate model for K channel gating that was developed by R. N. Aldrich (1981, Biophys. J., 36:519-532).

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Acute effects of repetitive depolarization on sodium current in chick myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1991.260.6.h1810 ◽

1991 ◽

Vol 260 (6) ◽

pp. H1810-H1818

Author(s):

M. R. Gold ◽

G. R. Strichartz

Keyword(s):

Time Course ◽

Sodium Current ◽

Complete Recovery ◽

Acute Effects ◽

Na Current ◽

Patch Clamp Technique ◽

Membrane Hyperpolarization ◽

Atrial Cells ◽

Whole Cell Patch Clamp ◽

Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

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