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Gating mechanism of the two-pore-domain potassium channel THIK1

Nature Structural & Molecular Biology volume 32, pages 1175–1182 (2025)Cite this article

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Abstract

TWIK-related halothane-inhibited potassium channel (THIK1) maintains the resting membrane potential and regulates potassium efflux in microglia. It is a potential therapeutic target for neurodegenerative disorders, neuropathic pain and inflammation. However, the mechanism underlying its function remains unclear. Here we used cryo-electron microscopy to solve the structures of full-length human THIK1, revealing two inner gates and a C-type selectivity filter gate, distinct from other two-pore-domain potassium channels. One inner gate, formed by a short helix in the distal C terminus, introduces a unique gating mechanism involving the distal cytoplasmic domain. The other, beneath the selectivity filter, is constricted by Y273 in the M4 helix, dividing the cavity. In addition, the selectivity filter gate is modulated by polyunsaturated fatty acids. These structural insights into THIK1 gating, through the distal C-terminal helices, hydrophilic residues and selectivity filter, advance our understanding of THIK1’s role in microglial homeostasis and neuropathologies.

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Fig. 1: Overall structure of THIK1.
Fig. 2: The C-terminal helix as an inner gate.
Fig. 3: A gate beneath the SF.
Fig. 4: The lipid in the modulator pocket stabilizes the SF conformation.
Fig. 5: Structural model for THIK1 gating mechanism.

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Data availability

THIK1 protein sequence is available from UniProt under accession number Q9HB14. Structure coordinates and cryo-EM density maps were deposited to the PDB and EM Data Bank under accession numbers 9JGZ and EMD-61467 for THIK1WT and 9JH1 and EMD-61469 for THIK1S136P, respectively. Data and materials can be obtained from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank Q. Chen for critical reading and discussing this paper. We thank the staff members of the Center of Cryo-EM of Fudan University for technical support and assistance and members of the B.L. laboratory for feedback on the paper. This work was supported in part by grants from The National Science and Technology Innovation 2030 Major Projects of China (STI2030-Major Projects-2022ZD0207800 to B.L. and J.W.), the National Natural Science Foundation of China (32371261 to B.L. and 32301011 to R.Z.) and the Fundamental Research Funds for the Central Universities (2632024ZD10 to J.W.).

Author information

Author notes

  1. These authors contributed equally: Xiangyun Fang, Haichao Jin.

Authors and Affiliations

  1. Department of Anesthesiology, Fudan University, Shanghai, China

    Xiangyun Fang, Ran Zhang & Baobin Li

  2. Institute for Translational Brain Research, Fudan University, Shanghai, China

    Xiangyun Fang, Ran Zhang & Baobin Li

  3. State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, China

    Xiangyun Fang, Ran Zhang & Baobin Li

  4. MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China

    Xiangyun Fang, Ran Zhang & Baobin Li

  5. School of Science, China Pharmaceutical University, Nanjing, China

    Haichao Jin & Jin Wang

  6. School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, China

    Haichao Jin

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  1. Xiangyun Fang
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Contributions

B.L., R.Z. and J.W. conceptualized and supervised the project. X.F. prepared the samples. X.F., R.Z. and B.L. performed data acquisition, image processing and structure determination. J.W. and H.J. performed electrophysiology and analyzed the data. B.L. wrote the paper with the input from R.Z., J.W., X.F. and H.J.

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Correspondence to Jin Wang, Ran Zhang or Baobin Li.

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Nature Structural & Molecular Biology thanks Youxing Jiang, Marcos Matamoros and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Cryo-EM analysis of THIK1WT.

a, Cryo-EM data processing steps in Relion 4.0 and cryoSPARC4.4.1. See Methods for details. b, A representative cryo-EM micrograph of THIK1WT. Scale bar, 200 Å. c, 2D class average images of THIK1WT. d, Local resolution distribution for the density map of THIK1WT. e, Euler angle distribution of the set of particles that contributed to the final density map of THIK1WT. f, The GSFSC curve for the reconstruction of THIK1WT. g, Cryo-EM densities for various TMs in the THIK1WT.

Extended Data Fig. 2 Sequence alignment of human K2P channels.

Alignment of human K2P channels colored by conservation. THIK1 secondary structure is drawn above the sequences, and selectivity filters as orange lines.

Extended Data Fig. 3 The full-length THIK1 structure predicted by AlphaFold 3.

viewed from the membrane plane (a) and the intracellular side (b).

Extended Data Fig. 4 Molecular dynamics simulation of C-terminus helix gate.

a, MD simulation started from the cryo-EM structure. The plots of the root mean square deviations (RMSD) for all Cα atoms (green) and selected Cα atoms of residues 393-404 (black) are shown. b, MD simulation of the cryo-EM structure with missing loop added. The conformation of the missing loop is derived from AlphaFold3. The RMSD for Cα atoms of residues in the cryo-EM structure (green) and residues 393-404 (black) are shown.

Source data

Extended Data Fig. 5 Cryo-EM analysis of THIK1 S136P.

a, cryo-EM data processing steps in Relion 4.0 and cryoSPARC4.4.1. See Methods for details. b, A representative cryo-EM micrograph of THIK1S136P. Scale bar, 200 Å. c, 2D class average images of THIK1S136P. d, Local resolution distribution for the density map of THIK1S136P. e, Euler angle distribution of the set of particles that contributed to the final density map of THIK1S136P. f, The GSFSC curve for the reconstruction of THIK1S136P. g, Cryo-EM densities for various TMs in the THIK1S136P.

Extended Data Fig. 6 Conformational changes of TM2 and TM4 induced by the S136P mutation.

a-c, View from the membrane plane showing one TM2 from THIK1WT, THIK1S136P, and both structures overlaid. The dashed line in (c) indicates the central axis of the helix, and the rotation degree is labeled. d, Overlaid structures of THIK1WT and THIK1S136P. The dashed arrows indicate the motion directions of TM2 and TM4.

Extended Data Fig. 7 Lipid binding in the modulator pocket.

a, Hydrophobic surface representation of the modulator pocket in THIK1WT (blue represents the most hydrophilic regions, transitioning to white, and then to orange-red for the most hydrophobic regions). b, Ligplot of the lipid in the modulator pocket. Key residues are labeled.

Extended Data Fig. 8

Densities for the lipid and surrounding residues in THIK1WT (a) and THIK1S136P (b).

Extended Data Fig. 9 Conformational changes of the selectivity filter.

a, d, Ion occupancy in the selectivity filter for THIK1WT and THIK1S136P. b-c, e-f, Inter-carbonyl distances at K+-binding sites S3 and S4 for THIK1WT and THIK1S136P.

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Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Extended Data Fig. 4

Statistical source data.

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Fang, X., Jin, H., Wang, J. et al. Gating mechanism of the two-pore-domain potassium channel THIK1. Nat Struct Mol Biol 32, 1175–1182 (2025). https://doi.org/10.1038/s41594-025-01542-4

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