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- PDB-9ft7: Structure of the human two pore domain potassium ion channel THIK... -

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Basic information

Entry
Database: PDB / ID: 9ft7
TitleStructure of the human two pore domain potassium ion channel THIK-1 (K2P13.1) in a closed conformation
ComponentsPotassium channel subfamily K member 13
KeywordsMEMBRANE PROTEIN / Potassium channel
Function / homology
Function and homology information


regulation of excitatory synapse pruning / Tandem pore domain halothane-inhibited K+ channel (THIK) / regulation of NLRP3 inflammasome complex assembly / Phase 4 - resting membrane potential / potassium ion leak channel activity / regulation of resting membrane potential / outward rectifier potassium channel activity / monoatomic ion channel complex / potassium channel activity / potassium ion transmembrane transport ...regulation of excitatory synapse pruning / Tandem pore domain halothane-inhibited K+ channel (THIK) / regulation of NLRP3 inflammasome complex assembly / Phase 4 - resting membrane potential / potassium ion leak channel activity / regulation of resting membrane potential / outward rectifier potassium channel activity / monoatomic ion channel complex / potassium channel activity / potassium ion transmembrane transport / protein heterodimerization activity / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Two pore domain potassium channel, THIK / Two pore domain potassium channel / Potassium channel domain / Ion channel
Similarity search - Domain/homology
LINOLEIC ACID / : / Potassium channel subfamily K member 13
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsRodstrom, K.E.J. / Tucker, S.J.
Funding support United Kingdom, 5items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T002018/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S008608/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/W017741/1 United Kingdom
Wellcome TrustWT084655MA United Kingdom
Wellcome Trust102161/B/13/Z United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Cryo-EM structure of the human THIK-1 K2P K channel reveals a lower Y gate regulated by lipids and anesthetics.
Authors: Karin E J Rödström / Bisher Eymsh / Peter Proks / Mehtab S Hayre / Sönke Cordeiro / Edward Mendez-Otalvaro / Christian Madry / Anna Rowland / Wojciech Kopec / Simon Newstead / Thomas ...Authors: Karin E J Rödström / Bisher Eymsh / Peter Proks / Mehtab S Hayre / Sönke Cordeiro / Edward Mendez-Otalvaro / Christian Madry / Anna Rowland / Wojciech Kopec / Simon Newstead / Thomas Baukrowitz / Marcus Schewe / Stephen J Tucker /
Abstract: THIK-1 (KCNK13) is a halothane-inhibited and anionic-lipid-activated two-pore domain (K2P) K channel implicated in microglial activation and neuroinflammation, and a current target for the treatment ...THIK-1 (KCNK13) is a halothane-inhibited and anionic-lipid-activated two-pore domain (K2P) K channel implicated in microglial activation and neuroinflammation, and a current target for the treatment of neurodegenerative disorders, for example Alzheimer's disease and amyothropic lateral sclerosis (ALS). However, compared to other K2P channels, little is known about the structural and functional properties of THIK-1. Here we present a 3.16-Å-resolution cryo-EM structure of human THIK-1 that reveals several distinct features, in particular, a tyrosine in M4 that contributes to a lower 'Y gate' that opens upon activation by physiologically relevant G-protein-coupled receptor and lipid signaling pathways. We demonstrate that linoleic acid bound within a modulatory pocket adjacent to the filter influences channel activity, and that halothane inhibition involves a binding site within the inner cavity, both resulting in conformational changes to the Y gate. Finally, the extracellular cap domain contains positively charged residues that line the ion exit pathway and contribute to the distinct biophysical properties of this channel. Overall, our results provide structural insights into THIK-1 function and identify distinct regulatory sites that expand its potential as a drug target for the modulation of microglial function.
History
DepositionJun 24, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Mask / Part number: 2 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Mar 5, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Potassium channel subfamily K member 13
B: Potassium channel subfamily K member 13
hetero molecules


Theoretical massNumber of molelcules
Total (without water)67,9156
Polymers67,2762
Non-polymers6394
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Potassium channel subfamily K member 13 / Tandem pore domain halothane-inhibited potassium channel 1 / THIK-1


Mass: 33637.980 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: K2P13.1 residues G9-G297 / Source: (gene. exp.) Homo sapiens (human) / Gene: KCNK13 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9HB14
#2: Chemical ChemComp-EIC / LINOLEIC ACID / 9,12-LINOLEIC ACID


Mass: 280.445 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H32O2
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K
Has ligand of interestN
Has protein modificationN

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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: K2P13.1 homodimer / Type: COMPLEX
Details: Protein generated by removal of the 10xHis and FLAG purification tags with HRV 3C protease cleavage
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.67 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 200 mM KCl, 0.12% w/v OGNG, 0.012% w/v CHS, pH was adjusted using potassium hydroxide
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acidC8H18N2O4S1
2200 mMpotassium chlorideKCl1
30.12 % w/voctyl glucose neopentyl glycolC27H52O121
40.012 % w/vcholesteryl hemisuccinate tris saltC31H50O4C4H11NO31
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Au-flat 1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Grid blotted for 4 seconds

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42.54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 21171
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2EPUimage acquisition
4cryoSPARC4.2.1CTF correction
9PHENIX1.20.1-4487model refinement
10cryoSPARC4.4.0initial Euler assignment
11cryoSPARC4.4.0final Euler assignment
13cryoSPARC4.4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 302189 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: An initial model was built manually in COOT
RefinementCross valid method: NONE

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