Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Nat Commun
2021 May 14;121:2802. doi: 10.1038/s41467-021-23062-7.
Show Gene links
Show Anatomy links
Electromechanical coupling mechanism for activation and inactivation of an HCN channel.
Dai G
,
Aman TK
,
DiMaio F
,
Zagotta WN
.
???displayArticle.abstract???
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 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.
Fig. 1. Structural features and properties of spHCN channels in the presence and absence of cyclic nucleotide.a Side view of a homology structure of spHCN channels based on the cryo-EM structure of human HCN1 (PDB 5U6P), highlighting the proximity of the S4 and S5 transmembrane helices relative to the C-linker domain of the adjacent subunit. Only two adjacent subunits are shown. b Top and bottom views of the same homology model of spHCN showing the arrangement of its quaternary structure. c Left: spHCN channel currents elicited by a hyperpolarizing voltage pulse from 0 to −100 mV, in the presence (red) or in the absence (black) of 1 mM cAMP. Middle: Channel inactivation without cyclic nucleotide, elicited by a series of hyperpolarizing voltages from 0 to −120 mV (with 10 mV steps), happened during the time frame indicated by the dashed box on the left panel. Note the blown up current and time scale. Right: Channel currents elicited by a series of hyperpolarizing voltages from 0 to −120 mV (with 10 mV steps), in the presence of 1 mM cAMP. d Left, cartoon showing the FRET between S346Anap in the S4 helix and Co2+ chelated by a di-histidine L182H and L186H in the HCN domain. e Fluorescence measurements for spHCN-S346Anap, L182H, and L186H channels with a −100 mV voltage pulse, before and after applying 1 mM Co2+. f Summary of the FRET efficiencies measured for spHCN-S346Anap, L182H, and L186H channels at 0 and −100 mV in the absence of cyclic nucleotide monophosphate (no cNMP; n = 4 independent patches), and the presence of cGMP (n=3 independent patches) or cAMP (n=3 independent patches). Data shown are mean ± s.e.m. Source data are provided in the Source Data file.
Fig. 2. tmFRET between the C-terminal end of the S4 helix and A′ or B′ helix of the C-linker.a Cartoon showing tmFRET between W355Anap in the S4 helix and Cu2+-TETAC attached to L481C in the A′ helix. b Cartoon showing tmFRET between W355Anap and Cu2+-TETAC attached to E506C in the B′ helix. c Summary of the fraction of Anap fluorescence quenched by Cu2+-TETAC for the spHCN-W355Anap channels in panels a and b, without (left) and with (middle, right) the introduced cysteines L481C or E506C in four conditions: the presence and absence of cAMP, and at 0 and −100 mV; n = 3–5 independent patches. Data shown are mean ± s.e.m.; Source data in the forms of FCys, and Fno Cys are provided in the Source Data file. d FRET efficiencies of spHCN-W355Anap, L481C channels measured in four conditions: the presence and absence of cAMP, and at 0 and −100 mV; n = 3 independent patches for cAMP conditions and n = 5 independent patches for no cNMP conditions. **p < 0.001 for the statistical comparisons. e FRET efficiencies of spHCN-W355Anap, E506C channels; n = 3 patches for cAMP and n = 4 patches for no cNMP conditions. **p < 0.001 for the statistical comparisons. Data shown are mean ± s.e.m.; source data are provided in the Source Data file.
Fig. 3. tmFRET between the N-terminal end of the S5 helix and A′ helix of the C-linker.a Cartoon showing tmFRET between C369Anap in the S5 helix and Cu2+-TETAC attached to Y477C in the A′ helix. b Cartoon showing tmFRET between W355Anap in the S4 helix and Cu2+-TETAC attached to Y477C in the A′ helix. c FRET efficiencies of spHCN-C369Anap, Y477C channels measured in four conditions: the presence and absence of cAMP, and at 0 and −100 mV; n = 7 independent patches for 0 mV conditions and n = 4 independent patches for −100 mV conditions. **p < 0.001; p = 0.1 between no cNMP, 0 mV and no cNMP, −100 mV conditions. d FRET efficiencies of spHCN-W355Anap, Y477C channels measured in four conditions: the presence and absence of cAMP, and at 0 and −100 mV; n = 3 independent patches for cAMP and n = 4 independent patches for no cNMP. **p < 0.001 and *p < 0.05. Data shown are mean ± s.e.m.; source data are provided in the Source Data file.
Fig. 4. Distance determination using measured FRET efficiencies, guided by FCG and homologous structures.a Eight FRET pairs used to obtain the Förster Convolved with Gaussian (FCG) relation to determine the distance dependence of the FRET efficiency. The Cβ–Cβ distances were measured from the homology models based on human HCN1 (PDB:5U6P and PDB: 6UQF) assuming they correspond to the structures at 0 and −100 mV respectively (in the presence of cAMP). b The measured FRET efficiencies are plotted versus the distances measure in panel a (in R0 units) as 4 black (0 mV) and 4 purple (−100 mV) circles. Predicted distance dependencies of the Förster equation (black) and the FCG relation (green) are shown. The FCG relation used a Gaussian distribution with a standard deviation, σ, of 5 Å (inset).
Fig. 5. Comparison of the measured distances from tmFRET and the corresponding distances from Rosetta models.a–d Comparison of the distances of FRET pairs measured from tmFRET (circle, mean ± s.e.m, n = 3–7) using the FCG relation in Fig. 4b and mean Cβ–Cβ distances of these FRET pairs in the 17 top scoring Rosetta models (square) based on the experimentally-determined distance constraints in four states: resting-cAMP (a), activated (b), resting-apo (c), and inactivated (d). The standard deviation of the Cβ–Cβ distances from the top 17 scoring Rosetta models for each state was less than 0.3 Å.
Fig. 6. Rosetta models for activation and inactivation of spHCN channels and their regulation by cAMP.a–d Modeled structures (side view and bottom view) highlighting the rearrangement of the C-linker region of one subunit relative to the S4 and S5 helices of the adjacent subunit for four states: resting-cAMP (a), activated (b), resting-apo (c), and inactivated (d). The S4 helix moves downward and bends into two segments after hyperpolarization in both activated and inactivated states. The bottom view highlights the rigid-body rotational movements of the C-linker around the central axis of the pore in the absence of cyclic nucleotide. See also Supplementary Movies 1–4.
Aldrich,
Fifty years of inactivation.
2001, Pubmed
Aldrich,
Fifty years of inactivation.
2001,
Pubmed
Aman,
Regulation of CNGA1 Channel Gating by Interactions with the Membrane.
2016,
Pubmed
Baker,
Functional Characterization of Cnidarian HCN Channels Points to an Early Evolution of Ih.
2015,
Pubmed
Catterall,
The chemical basis for electrical signaling.
2017,
Pubmed
Chatterjee,
A genetically encoded fluorescent probe in mammalian cells.
2013,
Pubmed
Clark,
Electromechanical coupling in the hyperpolarization-activated K+ channel KAT1.
2020,
Pubmed
Cowgill,
Bipolar switching by HCN voltage sensor underlies hyperpolarization activation.
2019,
Pubmed
Craven,
CNG and HCN channels: two peas, one pod.
2006,
Pubmed
Craven,
Salt bridges and gating in the COOH-terminal region of HCN2 and CNGA1 channels.
2004,
Pubmed
Dai,
The HCN channel voltage sensor undergoes a large downward motion during hyperpolarization.
2019,
Pubmed
,
Echinobase
Dai,
Molecular mechanism of voltage-dependent potentiation of KCNH potassium channels.
2017,
Pubmed
DeBerg,
Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides.
2016,
Pubmed
Decher,
Voltage-dependent gating of hyperpolarization-activated, cyclic nucleotide-gated pacemaker channels: molecular coupling between the S4-S5 and C-linkers.
2004,
Pubmed
DiFrancesco,
Characterization of single pacemaker channels in cardiac sino-atrial node cells.
NULL,
Pubmed
Evans,
Allosteric conformational change of a cyclic nucleotide-gated ion channel revealed by DEER spectroscopy.
2020,
Pubmed
Flynn,
Insights into the molecular mechanism for hyperpolarization-dependent activation of HCN channels.
2018,
Pubmed
,
Echinobase
Flynn,
Molecular mechanism underlying phosphatidylinositol 4,5-bisphosphate-induced inhibition of SpIH channels.
2011,
Pubmed
,
Echinobase
Gauss,
Molecular identification of a hyperpolarization-activated channel in sea urchin sperm.
1998,
Pubmed
,
Echinobase
Gordon,
Visualizing conformational dynamics of proteins in solution and at the cell membrane.
2018,
Pubmed
Guo,
Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana.
2016,
Pubmed
Horrocks,
Energy transfer between terbium (III) and cobalt (II) in thermolysin: a new class of metal--metal distance probes.
1975,
Pubmed
Idikuda,
cAMP binds to closed, inactivated, and open sea urchin HCN channels in a state-dependent manner.
2019,
Pubmed
,
Echinobase
James,
Structural insights into the mechanisms of CNBD channel function.
2018,
Pubmed
James,
CryoEM structure of a prokaryotic cyclic nucleotide-gated ion channel.
2017,
Pubmed
Kasimova,
Helix breaking transition in the S4 of HCN channel is critical for hyperpolarization-dependent gating.
2019,
Pubmed
Lee,
Structures of the Human HCN1 Hyperpolarization-Activated Channel.
2017,
Pubmed
Lee,
Voltage Sensor Movements during Hyperpolarization in the HCN Channel.
2019,
Pubmed
Li,
Structure of a eukaryotic cyclic-nucleotide-gated channel.
2017,
Pubmed
Long,
Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment.
2007,
Pubmed
Long,
Voltage sensor of Kv1.2: structural basis of electromechanical coupling.
2005,
Pubmed
Lu,
Coupling between voltage sensors and activation gate in voltage-gated K+ channels.
2002,
Pubmed
Lörinczi,
Voltage-dependent gating of KCNH potassium channels lacking a covalent link between voltage-sensing and pore domains.
2015,
Pubmed
Marchesi,
An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel.
2018,
Pubmed
Männikkö,
Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages.
2002,
Pubmed
,
Echinobase
Prole,
Reversal of HCN channel voltage dependence via bridging of the S4-S5 linker and Post-S6.
2006,
Pubmed
,
Echinobase
Ramentol,
Gating mechanism of hyperpolarization-activated HCN pacemaker channels.
2020,
Pubmed
Robertson,
hERG Function in Light of Structure.
2020,
Pubmed
Robinson,
Hyperpolarization-activated cation currents: from molecules to physiological function.
2003,
Pubmed
Rohl,
Protein structure prediction using Rosetta.
2004,
Pubmed
Schmidpeter,
Prolyl isomerization controls activation kinetics of a cyclic nucleotide-gated ion channel.
2020,
Pubmed
Schneider,
NIH Image to ImageJ: 25 years of image analysis.
2012,
Pubmed
Shin,
Inactivation in HCN channels results from reclosure of the activation gate: desensitization to voltage.
2004,
Pubmed
Song,
High-resolution comparative modeling with RosettaCM.
2013,
Pubmed
Taraska,
Fluorescence applications in molecular neurobiology.
2010,
Pubmed
Taraska,
Mapping the structure and conformational movements of proteins with transition metal ion FRET.
2009,
Pubmed
Tomczak,
A new mechanism of voltage-dependent gating exposed by KV10.1 channels interrupted between voltage sensor and pore.
2017,
Pubmed
Vemana,
S4 movement in a mammalian HCN channel.
2004,
Pubmed
,
Echinobase
Wainger,
Molecular mechanism of cAMP modulation of HCN pacemaker channels.
2001,
Pubmed
Wang,
Cryo-EM Structure of the Open Human Ether-à-go-go-Related K+ Channel hERG.
2017,
Pubmed
Whicher,
Structure of the voltage-gated K⁺ channel Eag1 reveals an alternative voltage sensing mechanism.
2016,
Pubmed
Whicher,
Regulation of Eag1 gating by its intracellular domains.
2019,
Pubmed
Wisedchaisri,
Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel.
2019,
Pubmed
Xue,
Structural mechanisms of gating and selectivity of human rod CNGA1 channel.
2021,
Pubmed
Zagotta,
Structural basis for modulation and agonist specificity of HCN pacemaker channels.
2003,
Pubmed
Zheng,
Patch-clamp fluorometry recording of conformational rearrangements of ion channels.
2003,
Pubmed
Zheng,
Mechanism of ligand activation of a eukaryotic cyclic nucleotide-gated channel.
2020,
Pubmed
Zhou,
A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization.
2004,
Pubmed