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- PDB-8ofi: Ivabradine bound to HCN4 channel -

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Basic information

Entry
Database: PDB / ID: 8ofi
TitleIvabradine bound to HCN4 channel
ComponentsPotassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
KeywordsMEMBRANE PROTEIN / HCN channels / ivabradine / ion transport / drug / hearth / hcn4
Function / homology:
Function and homology information
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSaponaro, A. / Chaves-Sanjuan, A. / Sharifzadeh, A.S. / Clarke, O.B. / Marabelli, C. / Bolognesi, M. / Thiel, G. / Moroni, A.
Funding support France, Italy, European Union, United States, 6items
OrganizationGrant numberCountry
Leducq FoundationTNE19CVD03 France
Fondazione CARIPLO2018-0231 Italy
European Research Council (ERC)ERC Advanced grant noMAGIC 695078European Union
National Institutes of Health/National Center for Research Resources (NIH/NCRR)NIH grants R01-NS109366 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)P41-GM116799 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01-GM107462 via Type a message United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structural determinants of ivabradine block of the open pore of HCN4.
Authors: Andrea Saponaro / Jan H Krumbach / Antonio Chaves-Sanjuan / Atiyeh Sadat Sharifzadeh / Alessandro Porro / Roberta Castelli / Kay Hamacher / Martino Bolognesi / Dario DiFrancesco / Oliver B ...Authors: Andrea Saponaro / Jan H Krumbach / Antonio Chaves-Sanjuan / Atiyeh Sadat Sharifzadeh / Alessandro Porro / Roberta Castelli / Kay Hamacher / Martino Bolognesi / Dario DiFrancesco / Oliver B Clarke / Gerhard Thiel / Anna Moroni /
Abstract: HCN1-4 channels are the molecular determinants of the I/I current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), ...HCN1-4 channels are the molecular determinants of the I/I current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.
History
DepositionMar 15, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
B: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
C: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
D: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)394,5595
Polymers394,0914
Non-polymers4691
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4


Mass: 98522.727 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: HCN4, HAC4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Chemical ChemComp-VNZ / Ivabradine / 3-[3-[[(7S)-3,4-dimethoxy-7-bicyclo[4.2.0]octa-1,3,5-trienyl]methyl-methyl-amino]propyl]-7,8-dimethoxy-2,5-dihydro-1H-3-benzazepin-4-one / 3-[3-[[(7S)-3,4-dimethoxy-7-bicyclo[4.2.0]octa-1(6),2,4-trienyl]methyl-methyl-amino]propyl]-7,8-dimethoxy-2,5-dihydro-1H-3-benzazepin-4-one


Mass: 468.585 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H36N2O5 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HCN4 / Type: COMPLEX / Details: HCN4 / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.4 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
Buffer componentConc.: 200 mM / Name: sodium chloride / Formula: NaCl
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 302 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.7 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 31670
Image scansWidth: 6000 / Height: 4000

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Processing

EM software
IDNameVersionCategory
1RELION3.1particle selection
2Warpparticle selection
3EPUimage acquisition
5CTFFIND4.1.10CTF correction
8UCSF Chimeramodel fitting
10Cootmodel refinement
11PHENIXmodel refinement
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89136 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 7NMN

7nmn


Accession code: 7NMN / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00516557
ELECTRON MICROSCOPYf_angle_d0.82222388
ELECTRON MICROSCOPYf_dihedral_angle_d13.2786151
ELECTRON MICROSCOPYf_chiral_restr0.0512450
ELECTRON MICROSCOPYf_plane_restr0.0032826

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