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- EMDB-18595: Cryo-EM structure of the human inward-rectifier potassium 2.1 cha... -

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Basic information

Entry
Database: EMDB / ID: EMD-18595
TitleCryo-EM structure of the human inward-rectifier potassium 2.1 channel (Kir2.1) - R312H mutant
Map dataFull map
Sample
  • Organelle or cellular component: Human inward-rectifier potassium 2.1 channel (Kir2.1) containing the Andersen-syndrome related mutation R312H
    • Protein or peptide: Inward rectifier potassium channel 2
KeywordsIon channel / Potassium Channel / Kir channel / MEMBRANE PROTEIN
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 / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / Phase 4 - resting membrane potential / membrane repolarization during action potential / membrane repolarization during cardiac muscle cell action potential / regulation of membrane repolarization ...Sensory perception of sour taste / Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / relaxation of skeletal muscle / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / Phase 4 - resting membrane potential / membrane repolarization during action potential / membrane repolarization during cardiac muscle cell action potential / regulation of membrane repolarization / membrane depolarization during cardiac muscle cell action potential / regulation of resting membrane potential / regulation of monoatomic ion transmembrane transport / inward rectifier potassium channel activity / positive regulation of potassium ion transmembrane transport / cardiac muscle cell action potential involved in contraction / regulation of cardiac muscle cell contraction / relaxation of cardiac muscle / potassium ion import across plasma membrane / intracellular potassium ion homeostasis / regulation of heart rate by cardiac conduction / intercalated disc / phosphatidylinositol-4,5-bisphosphate binding / voltage-gated potassium channel complex / potassium ion transmembrane transport / T-tubule / cellular response to mechanical stimulus / potassium ion transport / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / protein homotetramerization / dendritic spine / postsynaptic membrane / neuronal cell body / glutamatergic synapse / identical protein binding / membrane / 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
Inward rectifier potassium channel 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.0 Å
AuthorsFernandes CAH / Zuniga D / Venien-Bryan C
Funding support France, European Union, 2 items
OrganizationGrant numberCountry
The French Muscular Dystrophy Telethon (AFM-Telethon)#23207 France
H2020 Marie Curie Actions of the European Commission101026386European Union
CitationJournal: FASEB J / Year: 2024
Title: Biochemical, biophysical, and structural investigations of two mutants (C154Y and R312H) of the human Kir2.1 channel involved in the Andersen-Tawil syndrome.
Authors: Dania Zuniga / Andreas Zoumpoulakis / Rafael F Veloso / Laurie Peverini / Sophie Shi / Alexandre Pozza / Valérie Kugler / Françoise Bonneté / Tahar Bouceba / Renaud Wagner / Pierre-Jean ...Authors: Dania Zuniga / Andreas Zoumpoulakis / Rafael F Veloso / Laurie Peverini / Sophie Shi / Alexandre Pozza / Valérie Kugler / Françoise Bonneté / Tahar Bouceba / Renaud Wagner / Pierre-Jean Corringer / Carlos A H Fernandes / Catherine Vénien-Bryan /
Abstract: Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to ...Inwardly rectifying potassium (Kir) channels play a pivotal role in physiology by establishing, maintaining, and regulating the resting membrane potential of the cells, particularly contributing to the cellular repolarization of many excitable cells. Dysfunction in Kir2.1 channels is implicated in several chronic and debilitating human diseases for which there are currently no effective treatments. Specifically, Kir2.1-R312H and Kir2.1-C154Y mutations are associated with Andersen-Tawil syndrome (ATS) in humans. We have investigated the impact of these two mutants in the trafficking of the channel to the cell membrane and function in Xenopus laevis oocytes. Despite both mutations being trafficked to the cell membrane at different extents and capable of binding PIP (phosphatidylinositol-4,5-bisphosphate), the main modulator for channel activity, they resulted in defective channels that do not display K current, albeit through different molecular mechanisms. Coexpression studies showed that R312H and C154Y are expressed and associated with the WT subunits. While WT subunits could rescue R312H dysfunction, the presence of a unique C154Y subunit disrupts the function of the entire complex, which is a typical feature of mutations with a dominant-negative effect. Molecular dynamics simulations showed that Kir2.1-C154Y mutation induces a loss in the structural plasticity of the selectivity filter, impairing the K flow. In addition, the cryo-EM structure of the Kir2.1-R312H mutant has been reconstructed. This study identified the molecular mechanisms by which two ATS-causing mutations impact Kir2.1 channel function and provide valuable insights that can guide potential strategies for the development of future therapeutic interventions for ATS.
History
DepositionOct 5, 2023-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18595.png

Downloads & links

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Map

FileDownload / File: emd_18595.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
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Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 280 pix.
= 204.4 Å		0.73 Å/pix.
x 280 pix.
= 204.4 Å		0.73 Å/pix.
x 280 pix.
= 204.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0297
Minimum - Maximum-0.015066323 - 0.09245007
Average (Standard dev.)0.0025583091 (±0.008528329)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 204.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened map

Fileemd_18595_additional_1.map
AnnotationSharpened map
Projections & Slices
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Half map: Half-map

Fileemd_18595_half_map_1.map
AnnotationHalf-map
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Half map: Half-map

Fileemd_18595_half_map_2.map
AnnotationHalf-map
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Sample components

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Entire : Human inward-rectifier potassium 2.1 channel (Kir2.1) containing ...

EntireName: Human inward-rectifier potassium 2.1 channel (Kir2.1) containing the Andersen-syndrome related mutation R312H
Components
  • Organelle or cellular component: Human inward-rectifier potassium 2.1 channel (Kir2.1) containing the Andersen-syndrome related mutation R312H
    • Protein or peptide: Inward rectifier potassium channel 2

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Supramolecule #1: Human inward-rectifier potassium 2.1 channel (Kir2.1) containing ...

SupramoleculeName: Human inward-rectifier potassium 2.1 channel (Kir2.1) containing the Andersen-syndrome related mutation R312H
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Inward rectifier potassium channel 2

MacromoleculeName: Inward rectifier potassium channel 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 36.148816 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: RFVKKDGHCN VQFINVGEKG QRYLADIFTT CVDIRWRWML VIFCLAFVLS WLFFGCVFWL IALLHGDLDA SKEGKACVSE VNSFTAAFL FSIETQTTIG YGFRCVTDEC PIAVFMVVFQ SIVGCIIDAF IIGAVMAKMA KPKKRNETLV FSHNAVIAMR D GKLCLMWR ...String:
RFVKKDGHCN VQFINVGEKG QRYLADIFTT CVDIRWRWML VIFCLAFVLS WLFFGCVFWL IALLHGDLDA SKEGKACVSE VNSFTAAFL FSIETQTTIG YGFRCVTDEC PIAVFMVVFQ SIVGCIIDAF IIGAVMAKMA KPKKRNETLV FSHNAVIAMR D GKLCLMWR VGNLRKSHLV EAHVRAQLLK SRITSEGEYI PLDQIDINVG FDSGIDRIFL VSPITIVHEI DEDSPLYDLS KQ DIDNADF EIVVILEGMV EATAMTTQCH SSYLANEILW GHRYEPVLFE EKHYYKVDYS RFHKTYEVPN TPLCSARDLA

UniProtKB: Inward rectifier potassium channel 2

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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

Concentration0.6 mg/mL
BufferpH: 7.4 / Component - Concentration: 20.0 mM / Component - Formula: tris-hcl
Component - Name: Tris(hydroxymethyl)aminomethane hydrochloride
Details: 20 mM Tris-HCl pH 7.4, 150 mM KCl, 1 mM EDTA, 0.59 mM DDM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting force 0, blotting time 3s.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X
DetailsPreliminary grid screening was performed manually at a Glacios 2 microscope.
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 10762 / Average exposure time: 2.35 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: 2.4 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 165000
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

Particle selectionNumber selected: 2787753
Details: A blob picking followed by 2D classification was performed to generate templates for automated template-picking. Initially, 2,787,753 particles were selected after template-picking, which ...Details: A blob picking followed by 2D classification was performed to generate templates for automated template-picking. Initially, 2,787,753 particles were selected after template-picking, which were submitted to three rounds do 2D classification to remove false picks, ice contamination and classes with unclear features.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7zdz

Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 146539
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7zdz


Chain - Residue range: 195-367 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8qql:
Cryo-EM structure of the human inward-rectifier potassium 2.1 channel (Kir2.1) - R312H mutant

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