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- PDB-7zdz: Cryo-EM structure of the human inward-rectifier potassium 2.1 cha... -

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Basic information

Entry
Database: PDB / ID: 7zdz
TitleCryo-EM structure of the human inward-rectifier potassium 2.1 channel (Kir2.1)
ComponentsInward rectifier potassium channel 2Inward-rectifier potassium channel
KeywordsMEMBRANE PROTEIN / Potassium channel / Inward-rectifier channel / inward rectification
Function / homology
Function and homology information


Sensory perception of sour taste / Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / membrane repolarization during action potential / membrane repolarization during cardiac muscle cell action potential / regulation of resting membrane potential ...Sensory perception of sour taste / Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / membrane repolarization during action potential / membrane repolarization during cardiac muscle cell action potential / regulation of resting membrane potential / regulation of membrane repolarization / intracellular potassium ion homeostasis / positive regulation of potassium ion transmembrane transport / membrane depolarization during cardiac muscle cell action potential / inward rectifier potassium channel activity / cardiac muscle cell action potential involved in contraction / regulation of cardiac muscle cell contraction / regulation of monoatomic ion transmembrane transport / relaxation of cardiac muscle / potassium ion import across plasma membrane / regulation of heart rate by cardiac conduction / smooth endoplasmic reticulum / intercalated disc / rough endoplasmic reticulum / voltage-gated potassium channel complex / potassium ion transmembrane transport / phosphatidylinositol-4,5-bisphosphate binding / T-tubule / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / potassium ion transport / cellular response to mechanical stimulus / postsynaptic membrane / protein homotetramerization / dendritic spine / neuronal cell body / glutamatergic synapse / Golgi apparatus / membrane / identical protein binding / plasma membrane
Similarity search - Function
Potassium channel, inwardly rectifying, Kir2.1 / Potassium channel, inwardly rectifying, Kir, N-terminal / Inward rectifier potassium channel N-terminal / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / Immunoglobulin E-set
Similarity search - Domain/homology
: / STRONTIUM ION / Inward rectifier potassium channel 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsFernandes, C.A.H. / Venien-Bryan, C. / Fagnen, C. / Zuniga, D.
Funding support France, 3items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-11-EQPX-0008 France
The French Muscular Dystrophy Telethon (AFM-Telethon)23207 France
The French Muscular Dystrophy Telethon (AFM-Telethon)23210 France
CitationJournal: Sci Adv / Year: 2022
Title: Cryo-electron microscopy unveils unique structural features of the human Kir2.1 channel.
Authors: Carlos A H Fernandes / Dania Zuniga / Charline Fagnen / Valérie Kugler / Rosa Scala / Gérard Péhau-Arnaudet / Renaud Wagner / David Perahia / Saïd Bendahhou / Catherine Vénien-Bryan /
Abstract: We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels ...We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen's syndrome. The structural analysis (cryo-electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP-binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP. Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.
History
DepositionMar 30, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Inward rectifier potassium channel 2
B: Inward rectifier potassium channel 2
C: Inward rectifier potassium channel 2
D: Inward rectifier potassium channel 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)193,5917
Polymers193,3774
Non-polymers2143
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24770 Å2
ΔGint-213 kcal/mol
Surface area67950 Å2

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Components

#1: Protein
Inward rectifier potassium channel 2 / Inward-rectifier potassium channel / Cardiac inward rectifier potassium channel / Inward rectifier K(+) channel Kir2.1 / IRK-1 / hIRK1 / ...Cardiac inward rectifier potassium channel / Inward rectifier K(+) channel Kir2.1 / IRK-1 / hIRK1 / Potassium channel / inwardly rectifying subfamily J member 2


Mass: 48344.141 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNJ2, IRK1 / Production host: Komagataella pastoris (fungus) / References: UniProt: P63252
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-SR / STRONTIUM ION / Strontium


Mass: 87.620 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Sr / 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: Human inward-rectifier potassium channel 2.1 (Kir2.1) / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 50 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMPotassium chlorideKCl1
220 mMTrizma hydrochlorideTris-HClTris1
30.03 %Dodecyl-B-D-maltosideDDM1
41 mMEthylenediamine tetraacetic acidEDTAEthylenediaminetetraacetic acid1
52 mMDichlorodiphenyltrichloroethaneDDT1
SpecimenConc.: 0.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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 / Nominal magnification: 105000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 61.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9895
Image scansSampling size: 10 µm / Width: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.6particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
7PHENIX1.2model fitting
9PHENIX1.2model refinement
10Coot0.9.7model refinement
11RELION3.0.7initial Euler assignment
12RELION3.0.7final Euler assignment
13RELION3.0.7classification
14RELION3.0.73D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1031472
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63584 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 404.73 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: For structural fitting, it was used dock-in-map (available at PHENIX) that uses both SSM and convolution-based shape searches to find a part of a map that is similar to a model. An initial ...Details: For structural fitting, it was used dock-in-map (available at PHENIX) that uses both SSM and convolution-based shape searches to find a part of a map that is similar to a model. An initial in silico homology model of human Kir2.1 was generated using I-TASSER using the crystal structure of chicken Kir2.2 channel (PDB ID 3JYC) as a template. For building and refinement of the atomic model, the transmembrane domain (TMD, 55-184 region) of this in silico model was placed into the final sharpened cryo-EM map using the Dock in Map tool available in PHENIX. For the cytoplasmic domain (CTD; 188-367 region), the crystal structure of the CTD from mice Kir2.1 channel (PDB ID 1U4F) was placed into the final cryo-EM map using the same approach. Once the models were placed in the electron density, the loops that connect the two domains (185-187 region) and a N-terminal loop (41-54 region) absent in the in silico model were manually built using Coot.
Atomic model buildingPDB-ID: 1U4F

1u4f


Pdb chain residue range: 188-367
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310595
ELECTRON MICROSCOPYf_angle_d0.82814342
ELECTRON MICROSCOPYf_dihedral_angle_d4.7661393
ELECTRON MICROSCOPYf_chiral_restr0.0471616
ELECTRON MICROSCOPYf_plane_restr0.0031807

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