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- EMDB-16860: Ivabradine bound to HCN4 channel -

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Basic information

Entry
Database: EMDB / ID: EMD-16860
TitleIvabradine bound to HCN4 channel
Map datasharped map
Sample
  • Complex: HCN4
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
  • Ligand: Ivabradine
KeywordsHCN channels / ivabradine / ion transport / MEMBRANE PROTEIN / drug / hearth / hcn4
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSaponaro A / Chaves-Sanjuan A / Sharifzadeh AS / Clarke OB / Marabelli C / Bolognesi M / Thiel G / Moroni A
Funding support France, Italy, European Union, United States, 6 items
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, 2023-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_16860.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharped map
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.110308 - 3.1070626
Average (Standard dev.)0.004768307 (±0.0544208)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16860_msk_1.map
Projections & Slices
AxesZYX

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Additional map: unsharped map

Fileemd_16860_additional_1.map
Annotationunsharped map
Projections & Slices
AxesZYX

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Half map: half a

Fileemd_16860_half_map_1.map
Annotationhalf a
Projections & Slices
AxesZYX

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Half map: half b

Fileemd_16860_half_map_2.map
Annotationhalf b
Projections & Slices
AxesZYX

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Sample components

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Entire : HCN4

EntireName: HCN4
Components
  • Complex: HCN4
    • Protein or peptide: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
  • Ligand: Ivabradine

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Supramolecule #1: HCN4

SupramoleculeName: HCN4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: HCN4
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 400 KDa

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Macromolecule #1: Potassium/sodium hyperpolarization-activated cyclic nucleotide-ga...

MacromoleculeName: Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Molecular weightTheoretical: 98.522727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDKLPPSMRK RLYSLPQQVG AKAWIMDEEE DAEEEGAGGR QDPRRRSIRL RPLPSPSPSP SAAAAAAGGA ESRGAALGGA ADGEGPARG AAKSSTNGDC RRFRGSLASL GSRGGGGGGG STGGGSHGHL HDSAEERRLI AEGDASPGED RTPPGLAAEP E RPGAPAPP ...String:
MDKLPPSMRK RLYSLPQQVG AKAWIMDEEE DAEEEGAGGR QDPRRRSIRL RPLPSPSPSP SAAAAAAGGA ESRGAALGGA ADGEGPARG AAKSSTNGDC RRFRGSLASL GSRGGGGGGG STGGGSHGHL HDSAEERRLI AEGDASPGED RTPPGLAAEP E RPGAPAPP AASPPQVPSS CGEQRPADAA VKVEGGAAAG DQILPEAEAR LGQAGFMQRQ FGAMLQPGVN KFSLRMFGSQ KA VEREQER VKSAGFWIIH PYSDFRFYWD LTMLLLMVGN LIIIPVGITF FKDENTTPWI VFNVVSDTFF LIDLVLNFRT GIV VEDNTD IILDPRRIKM KYLKSWFVVD FVSSIPVDYI FLIVETRIDS EVYKTARALR IVRFTKILSL LRLLRLSRLI RYIH QWEEI FHMTYDLASA VVRIVNLIGM MLLLCHWDGC LQFLVPMLQD FPDDCWVSLN NMVNNSWGKQ YSYALFKAMS HMLCI GYGR QAPMGMSDVW LTMLSMIVGA TCYAMFIGHA TALIQSLDSS RRQYQEKYKQ VEQYMSFHKL PPDTRQRIHD YYEHRY QGK MFDEESILGE LSEPLREEII NFNCRKLVAS MPLFANADPN FVTSMLTKLR FEVFQPGDYI IREGTIGKKM YFIQHGV VS VLTKGNKETK LADGSYFGEI CLLTRGRRTA SVRADTYCRL YSLSVDNFNE VLEEYPMMRR AFETVALDRL DRIGKKNS I HKVQHDLSSG VSNYQENAIV QRIVQHDREM AHCARRAQAT TPVAPAIWTP LIQAPLQAAA QDLKLISASQ PALPQDGAQ TLRRASPHSS SGESVAALPP AGGPFPRAPG RPPGAGPGQH VTLTLPRKAS SGSLPPPLSL FGPRAAPRLT AAPQREPGAK SEPVRSKLP SNL

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Macromolecule #2: Ivabradine

MacromoleculeName: Ivabradine / type: ligand / ID: 2 / Number of copies: 1 / Formula: VNZ
Molecular weightTheoretical: 468.585 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7 / Component - Concentration: 200.0 mM / Component - Formula: NaCl / Component - Name: sodium chloride
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: 30mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 302 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 31670 / Average exposure time: 1.7 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 89136
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8ofi:
Ivabradine bound to HCN4 channel

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