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- PDB-9mel: Structure of the human TWIK-2 potassium channel in complex with p... -

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Basic information

Entry
Database: PDB / ID: 9mel
TitleStructure of the human TWIK-2 potassium channel in complex with pimozide
ComponentsPotassium channel subfamily K member 6
KeywordsMEMBRANE PROTEIN / Two-pore potassium channel K2P6 TWIK2 K2P channel pimozide drug NLRP3 inflammasome
Function / homology
Function and homology information


regulation of lysosome size / Tandem of pore domain in a weak inwardly rectifying K+ channels (TWIK) / Phase 4 - resting membrane potential / potassium ion leak channel activity / regulation of resting membrane potential / inward rectifier potassium channel activity / outward rectifier potassium channel activity / negative regulation of systemic arterial blood pressure / positive regulation of NLRP3 inflammasome complex assembly / potassium channel activity ...regulation of lysosome size / Tandem of pore domain in a weak inwardly rectifying K+ channels (TWIK) / Phase 4 - resting membrane potential / potassium ion leak channel activity / regulation of resting membrane potential / inward rectifier potassium channel activity / outward rectifier potassium channel activity / negative regulation of systemic arterial blood pressure / positive regulation of NLRP3 inflammasome complex assembly / potassium channel activity / voltage-gated potassium channel complex / potassium ion transmembrane transport / potassium ion transport / late endosome membrane / lysosomal membrane / metal ion binding / plasma membrane
Similarity search - Function
Two pore domain potassium channel, TWIK-2 / Two pore domain potassium channel, TWIK family / Two pore domain potassium channel, TASK family / Two pore domain potassium channel / Potassium channel domain / Ion channel
Similarity search - Domain/homology
Chem-1II / : / Potassium channel subfamily K member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsKhanra, N.K. / Long, S.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structure of the human TWIK-2 potassium channel and its inhibition by pimozide.
Authors: Nandish K Khanra / Chongyuan Wang / Bryce D Delgado / Stephen B Long /
Abstract: The potassium channel TWIK-2 is crucial for ATP-induced activation of the NLRP3 inflammasome in macrophages. The channel is a member of the two-pore domain potassium (K2P) channel superfamily and an ...The potassium channel TWIK-2 is crucial for ATP-induced activation of the NLRP3 inflammasome in macrophages. The channel is a member of the two-pore domain potassium (K2P) channel superfamily and an emerging therapeutic target to mitigate severe inflammatory injury involving NLRP3 activation. We report the cryo-EM structure of human TWIK-2. In comparison to other K2P channels, the structure reveals an unusual "up" conformation of Tyr111 in the selectivity filter and a resulting SF1-P1 pocket behind the filter. Density for acyl chains is present in fenestrations within the transmembrane region that connects the central cavity of the pore to the lipid membrane. Despite its importance as a drug target, limited pharmacological tools are available for TWIK-2. A previous study suggested that the FDA-approved small molecule pimozide might inhibit TWIK-2. Using a reconstituted system, we show that pimozide directly inhibits the channel and we determine a cryo-EM structure of a complex with the drug. Pimozide displaces the acyl chains within the fenestrations and binds below the selectivity filter where it would impede ion permeation. The drug may access its binding site by lateral diffusion in the membrane, suggesting that other hydrophobic small molecules could have utility for inhibiting TWIK-2. The work defines the structure of TWIK-2 and provides a structural foundation for development of more specific inhibitors with potential utility as anti-inflammatory drugs.
History
DepositionDec 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 23, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Potassium channel subfamily K member 6
B: Potassium channel subfamily K member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,1296
Polymers67,5502
Non-polymers5794
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 6 / Inward rectifying potassium channel protein TWIK-2 / TWIK-originated similarity sequence


Mass: 33775.113 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KCNK6, TOSS, TWIK2 / Production host: Homo sapiens (human) / References: UniProt: Q9Y257
#2: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-1II / 3-[1-[4,4-bis(4-fluorophenyl)butyl]piperidin-4-yl]-1~{H}-benzimidazol-2-one


Mass: 461.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H29F2N3O / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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 TWIK-2 potassium channel in complex with pimozide
Type: COMPLEX
Details: human TWIK-2 potassium channel in complex with pimozide
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.03374728 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Details: 20 mM HEPES, 150 mM KCl, pH 7.5, 0.01 % LMNG, 0.001 % CHS, 0.0033 % GDN, 0.5 mM pimozide
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acidHEPES1
2150 mMPotassium ChlorideKCl1
30.01 %2,2-didecylpropane-1,3-bis-beta-D-maltopyranosideLMNG1
40.001 %Cholesteryl HemisuccinateCHS1
50.0033 %glyco-diosgeninGDN1
60.5 mM1-[1-[4,4-Bis(4-fluorophenyl)butyl]-4-piperidinyl]-1,3-dihydro-2H-benzimidazol-2-onePimozide1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse recombinantly purified TWIK2 bound to pimozide
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K / Details: 4.0 uL sample, blot time = 3.0 sec, blot force = 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 51.38 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
2Leginonimage acquisition
4RELION3.1CTF correctionCTFFIND 4.1
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6263529
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 156956 / Symmetry type: POINT
Atomic model buildingB value: 124.2
RefinementHighest resolution: 3.17 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0043676
ELECTRON MICROSCOPYf_angle_d0.6435046
ELECTRON MICROSCOPYf_dihedral_angle_d11.541532
ELECTRON MICROSCOPYf_chiral_restr0.037636
ELECTRON MICROSCOPYf_plane_restr0.003600

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