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- PDB-7np3: cAMP-free rabbit HCN4 stabilized in LMNG-CHS detergent mixture -

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Basic information

Entry
Database: PDB / ID: 7np3
TitlecAMP-free rabbit HCN4 stabilized in LMNG-CHS detergent mixture
ComponentsPotassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
KeywordsMEMBRANE PROTEIN / HCN channels / cAMP / ion transport
Function / homology
Function and homology information


intracellularly cAMP-activated cation channel activity / sodium channel activity / monoatomic ion channel complex / voltage-gated potassium channel activity / cAMP binding / cellular response to cAMP / potassium ion transmembrane transport / regulation of heart rate / plasma membrane
Similarity search - Function
Ion transport N-terminal / Ion transport protein N-terminal / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily ...Ion transport N-terminal / Ion transport protein N-terminal / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGiese, H.M. / Chaves-Sanjuan, A. / Saponaro, A. / Clarke, O. / Bolognesi, M. / Mancia, F. / Hendrickson, W.A. / Thiel, G. / Santoro, B. / Moroni, A.
Funding support United States, Italy, European Union, 6items
OrganizationGrant numberCountry
Leducq FoundationTNE19CVD03 United States
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: Mol Cell / Year: 2021
Title: Gating movements and ion permeation in HCN4 pacemaker channels.
Authors: Andrea Saponaro / Daniel Bauer / M Hunter Giese / Paolo Swuec / Alessandro Porro / Federica Gasparri / Atiyeh Sadat Sharifzadeh / Antonio Chaves-Sanjuan / Laura Alberio / Giacomo Parisi / ...Authors: Andrea Saponaro / Daniel Bauer / M Hunter Giese / Paolo Swuec / Alessandro Porro / Federica Gasparri / Atiyeh Sadat Sharifzadeh / Antonio Chaves-Sanjuan / Laura Alberio / Giacomo Parisi / Gabriele Cerutti / Oliver B Clarke / Kay Hamacher / Henry M Colecraft / Filippo Mancia / Wayne A Hendrickson / Steven A Siegelbaum / Dario DiFrancesco / Martino Bolognesi / Gerhard Thiel / Bina Santoro / Anna Moroni /
Abstract: The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy ...The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current I/I that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K/Na permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
History
DepositionFeb 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)394,0914
Polymers394,0914
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21200 Å2
ΔGint-124 kcal/mol
Surface area94400 Å2

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Components

#1: Protein
Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4,Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4 / Hyperpolarization-activated cation channel 4 / HAC-4


Mass: 98522.727 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Rabbit HCN4 with an internal deletion,Rabbit HCN4 with an internal deletion
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Gene: HCN4, HAC4 / Production host: Homo sapiens (human) / References: UniProt: Q9TV66

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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: all / Source: RECOMBINANT
Molecular weightExperimental 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: NaClSodium chloride
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
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 FIELDBright-field microscopy
Image recordingElectron dose: 71.85 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51758 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00816524
ELECTRON MICROSCOPYf_angle_d0.91822332
ELECTRON MICROSCOPYf_dihedral_angle_d23.7676124
ELECTRON MICROSCOPYf_chiral_restr0.0622448
ELECTRON MICROSCOPYf_plane_restr0.0062804

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