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- PDB-9e94: Cryo-EM structure of human TWIK-2 at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 9.0E+94
TitleCryo-EM structure of human TWIK-2 at pH 7.5
ComponentsPotassium channel subfamily K member 6
KeywordsTRANSPORT PROTEIN / Potassium ion channel K2P
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 / negative regulation of systemic arterial blood pressure / outward rectifier potassium channel activity / 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 / negative regulation of systemic arterial blood pressure / outward rectifier potassium channel activity / 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
DECANE / : / Potassium channel subfamily K member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsMa, Q. / Kumar, A. / Navratna, V. / Mosalaganti, S.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Commun / Year: 2026
Title: Insights into the structure and modulation of human TWIK-2.
Authors: Qianqian Ma / Ciria C Hernandez / Vikas Navratna / Arvind Kumar / Jaimin K Rana / Jiameng Zong / Abraham Lee / Shyamal Mosalaganti /
Abstract: The Tandem of pore domain in a Weak Inward Rectifying K channel 2 (TWIK-2; KCNK6) is a member of the Two-Pore Domain K (K) channel family, which is associated with pulmonary hypertension, lung ...The Tandem of pore domain in a Weak Inward Rectifying K channel 2 (TWIK-2; KCNK6) is a member of the Two-Pore Domain K (K) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. Despite its physiological relevance, the structure, regulatory mechanisms, and selective modulators of TWIK-2 remain largely unknown. Here, we present a 3.7 Å single particle cryo-electron microscopy structure of human TWIK-2 and highlight its conserved and distinctive features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing site-directed mutagenesis and provide insights into the possible lipid-mediated mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising- anti-inflammatory therapeutics.
History
DepositionNov 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 11, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Feb 11, 2026Data 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 6
B: Potassium channel subfamily K member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)68,66312
Polymers67,5502
Non-polymers1,11310
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 / Plasmid: pEG BacMam N term Strep / Details (production host): Addgene #160683 / Cell line (production host): HEK293S GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9Y257
#2: Chemical
ChemComp-D10 / DECANE


Mass: 142.282 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C10H22
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 3 / 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: Two-pore domain Weak Inwardly rectifying K+ channel 2 (TWIK2)
Type: COMPLEX
Details: Full length human TWIK2 expressed as recombinant protein in mamalian cells.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.063 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 GnTI-
Buffer solutionpH: 7.5 / Details: 25 mM Tris-HCl, 150 mM KCl, 0.1% DMNG
Buffer component
IDConc.NameFormulaBuffer-ID
125 mM4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acidTris1
2150 mMPotassium ChlorideKCl1
30.1 %Decyl maltose neopentyl glycolDMNG1
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: TWIK2 in DMNG micelle, purified by Strep-Tactin affinity chromotography.
Specimen supportDetails: 15 mA current / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 2
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM9.03image acquisition
9cryoSPARC4.5initial Euler assignment
12cryoSPARC4.53D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104997 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementHighest resolution: 3.67 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.013445
ELECTRON MICROSCOPYf_angle_d1.6854680
ELECTRON MICROSCOPYf_dihedral_angle_d8.491521
ELECTRON MICROSCOPYf_chiral_restr0.089576
ELECTRON MICROSCOPYf_plane_restr0.025566

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