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A lower X-gate in TASK channels traps inhibitors within the vestibule

Abstract

TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K2P) channel family—are found in neurons1, cardiomyocytes2,3,4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8,9,10,11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12,13,14,15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.

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Fig. 1: TASK-1 has a unique crossed-helix X-gate.
Fig. 2: Mutation of X-gate residues results in an increase in ion conduction and loss of anaesthetic activation.
Fig. 3: The X-gate environment includes a hinge and a latch on either side of the vestibule, which are required for X-gate integrity.
Fig. 4: Two highly potent inhibitors bind TASK-1 in the vestibule, trapped by the X-gate.

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Data availability

X-ray crystallography structures and datasets generated during the current study are deposited in the Protein Data Bank (PDB) with the following accession codes: 6RV2 for the TASK-1 structure, 6RV3 for the structure of the TASK-1–BAY1000493 complex and 6RV4 for the structure of the TASK-1–BAY2341237 complex. Source data for the electrophysiology measurements generated during the current study are available in the Source Data files associated with Figs. 2, 3, 4 and Extended Data Figs. 1, 4, 5.

References

  1. Karschin, C. et al. Expression pattern in brain of TASK-1, TASK-3, and a tandem pore domain K+ channel subunit, TASK-5, associated with the central auditory nervous system. Mol. Cell. Neurosci. 18, 632–648 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Jones, S. A., Morton, M. J., Hunter, M. & Boyett, M. R. Expression of TASK-1, a pH-sensitive twin-pore domain K+ channel, in rat myocytes. Am. J. Physiol. Heart Circ. Physiol. 283, H181–H185 (2002).

    Article  CAS  PubMed  Google Scholar 

  3. Putzke, C. et al. The acid-sensitive potassium channel TASK-1 in rat cardiac muscle. Cardiovasc. Res. 75, 59–68 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Decher, N. et al. Knock-out of the potassium channel TASK-1 leads to a prolonged QT interval and a disturbed QRS complex. Cell. Physiol. Biochem. 28, 77–86 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Olschewski, A. et al. Impact of TASK-1 in human pulmonary artery smooth muscle cells. Circ. Res. 98, 1072–1080 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Donner, B. C. et al. Functional role of TASK-1 in the heart: studies in TASK-1-deficient mice show prolonged cardiac repolarization and reduced heart rate variability. Basic Res. Cardiol. 106, 75–87 (2011).

    Article  CAS  PubMed  Google Scholar 

  7. Gurney, A. M. et al. Two-pore domain K channel, TASK-1, in pulmonary artery smooth muscle cells. Circ. Res. 93, 957–964 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Steinberg, E. A., Wafford, K. A., Brickley, S. G., Franks, N. P. & Wisden, W. The role of K2P channels in anaesthesia and sleep. Pflügers Arch. 467, 907–916 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Putzke, C. et al. Differential effects of volatile and intravenous anesthetics on the activity of human TASK-1. Am. J. Physiol. Cell Physiol. 293, C1319–C1326 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Patel, A. J. et al. Inhalational anesthetics activate two-pore-domain background K+ channels. Nat. Neurosci. 2, 422–426 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Sirois, J. E., Lei, Q., Talley, E. M., Lynch, C., III & Bayliss, D. A. The TASK-1 two-pore domain K+ channel is a molecular substrate for neuronal effects of inhalation anesthetics. J. Neurosci. 20, 6347–6354 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Duprat, F. et al. TASK, a human background K+ channel to sense external pH variations near physiological pH. EMBO J. 16, 5464–5471 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Talley, E. M. & Bayliss, D. A. Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9) potassium channels: volatile anesthetics and neurotransmitters share a molecular site of action. J. Biol. Chem. 277, 17733–17742 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Lopes, C. M. et al. PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels. J. Physiol. 564, 117–129 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wilke, B. U. et al. Diacylglycerol mediates regulation of TASK potassium channels by Gq-coupled receptors. Nat. Commun. 5, 5540 (2014).

    Article  ADS  CAS  PubMed  Google Scholar 

  16. Kiper, A. K. et al. Kv1.5 blockers preferentially inhibit TASK-1 channels: TASK-1 as a target against atrial fibrillation and obstructive sleep apnea? Pflugers Arch. 467, 1081–1090 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Miller, A. N. & Long, S. B. Crystal structure of the human two-pore domain potassium channel K2P1. Science 335, 432–436 (2012).

    Article  ADS  CAS  PubMed  Google Scholar 

  18. Brohawn, S. G., del Mármol, J. & MacKinnon, R. Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel. Science 335, 436–441 (2012).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  19. Brohawn, S. G., Campbell, E. B. & MacKinnon, R. Domain-swapped chain connectivity and gated membrane access in a Fab-mediated crystal of the human TRAAK K+ channel. Proc. Natl Acad. Sci. USA 110, 2129–2134 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dong, Y. Y. et al. K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac. Science 347, 1256–1259 (2015).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lolicato, M. et al. K2P2.1 (TREK-1)-activator complexes reveal a cryptic selectivity filter binding site. Nature 547, 364–368 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  22. Morton, M. J., O’Connell, A. D., Sivaprasadarao, A. & Hunter, M. Determinants of pH sensing in the two-pore domain K+channels TASK-1 and -2. Pflugers Arch. 445, 577–583 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Stansfeld, P. J. et al. Insight into the mechanism of inactivation and pH sensitivity in potassium channels from molecular dynamics simulations. Biochemistry 47, 7414–7422 (2008).

    Article  CAS  PubMed  Google Scholar 

  24. Goldstein, M. et al. Functional mutagenesis screens reveal the ‘cap structure’ formation in disulfide-bridge free TASK channels. Sci. Rep. 6, 19492 (2016).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  25. Klesse, G., Rao, S., Sansom, M. S. P. & Tucker, S. J. CHAP: a versatile tool for the structural and functional annotation of ion channel pores. J. Mol. Biol. 431, 3353–3365 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Streit, A. K. et al. A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore. J. Biol. Chem. 286, 13977–13984 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Piechotta, P. L. et al. The pore structure and gating mechanism of K2P channels. EMBO J. 30, 3607–3619 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Rinné, S. et al. TASK-1 and TASK-3 may form heterodimers in human atrial cardiomyocytes. J. Mol. Cell. Cardiol. 81, 71–80 (2015).

    Article  PubMed  CAS  Google Scholar 

  29. Renigunta, V. et al. Much more than a leak: structure and function of K2P-channels. Pflugers Arch. 467, 867–894 (2015).

    Article  CAS  PubMed  Google Scholar 

  30. Sterbulac, D. Molecular determinants of chemical modulation of two-pore domain potassium channels. Chem. Biol. Drug Design 94, 1596–1614 (2019).

    Article  CAS  Google Scholar 

  31. Delbeck, M. et al. 2-phenyl-3-(piperazinomethyl)imidazo[1,2-a]pyridine derivatives as blockers of TASK-1 and TASK-3 channels, for the treatment of sleep-related breathing disorders. WIPO patent WO2017097792A1 (2017).

  32. Chokshi, R. H., Larsen, A. T., Bhayana, B. & Cotten, J. F. Breathing stimulant compounds inhibit TASK-3 potassium channel function likely by binding at a common site in the channel pore. Mol. Pharmacol. 88, 926–934 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ma, L. et al. A novel channelopathy in pulmonary arterial hypertension. N. Engl. J. Med. 369, 351–361 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Barel, O. et al. Maternally inherited Birk Barel mental retardation dysmorphism syndrome caused by a mutation in the genomically imprinted potassium channel KCNK9. Am. J. Hum. Genet. 83, 193–199 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Sediva, M. et al. Novel variant in the KCNK9 gene in a girl with Birk Barel syndrome. Eur. J. Med. Genet. 63, 103619 (2020).

    Google Scholar 

  36. Armstrong, C. M. Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J. Gen. Physiol. 58, 413–437 (1971).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Bolshakov, K. V., Gmiro, V. E., Tikhonov, D. B. & Magazanik, L. G. Determinants of trapping block of N-methyl-d-aspartate receptor channels. J. Neurochem. 87, 56–65 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Linder, T., Saxena, P., Timin, E., Hering, S. & Stary-Weinzinger, A. Structural insights into trapping and dissociation of small molecules in K+ channels. J. Chem. Inf. Model. 54, 3218–3228 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Sitsel, O., Wang, K., Liu, X. & Gourdon, P. Crystallization of P-type ATPases by the high lipid-detergent (HiLiDe) method. Methods Mol. Biol. 1377, 413–420 (2016).

    Article  CAS  PubMed  Google Scholar 

  40. Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006).

    Article  PubMed  CAS  Google Scholar 

  42. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. STARANISO (Global Phasing, 2018).

  44. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. BUSTER v.2.10.0 (Global Phasing, 2011).

  48. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).

    Article  CAS  Google Scholar 

  49. Zuzarte, M. et al. Intracellular traffic of the K+ channels TASK-1 and TASK-3: role of N- and C-terminal sorting signals and interaction with 14-3-3 proteins. J. Physiol.587, 929–952 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Rinné, S. et al. The molecular basis for an allosteric inhibition of K+-flux gating in K2P channels. eLife 8, e39476 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  51. The PyMOL Molecular Graphics System v.1.7.7.1 (Schrödinger, 2010).

  52. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson–Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Jurrus, E. et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci. 27, 112–128 (2018).

    Article  CAS  PubMed  Google Scholar 

  54. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Waterhouse, A. M., Procter, J. B., Martin, D. M., Clamp, M. & Barton, G. J. Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Bond, C. S. & Schüttelkopf, A. W. ALINE: a WYSIWYG protein-sequence alignment editor for publication-quality alignments. Acta Crystallogr. D 65, 510–512 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

K.E.J.R. and D.S. are supported by BBSRC grant BB/N009274/1 to E.P.C. and S.J.T. K.E.J.R., A.C.W.P., D.S., S.M.M.M., N.A.B.-B. and E.P.C. are members of the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome Canada, Janssen, Merck KGaA, Merck & Co., Novartis, the Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP and Takeda, as well as the Innovative Medicines Initiative Joint Undertaking ULTRA-DD grant 115766 and the Wellcome Trust (106169/Z/14/Z). L.J.C. is supported by a Wellcome Trust OXION PhD Studentship (109114/Z/15/Z). A.K.K. is supported by the von-Behring-Röntgen Stiftung grant 67-0015. N.D. is supported by Deutsche Forschungsgemeinschaft (DFG) grant DE1482-4/1. We thank the following: Diamond Light Source for access to the macromolecular crystallography beamlines, the Diamond Light Source staff for help with data collection; all members of the SGC Biotech team, including S. Venkaya, C. Strain-Damerell, K. Kupinska, D. Wang and K. Ellis; all members of the SGC IMP1 group, including Y. Y. Dong; D. Eberhardt for help with the electrophysiology experiments; R. Chalk, T. Moreira, G. Berridge and O. Borkowska for help with mass spectrometry; B. Marsden, D. Damerell, J. Bray, J. Crowe and C. Sluman for bioinformatics support; F. von Delft, T. Krojer and B. MacLean for assistance with crystallography infrastructure; O. Nowak for technical assistance; and T. Baukrowitz for critical reading of the manuscript.

Author information

Authors and Affiliations

Authors

Contributions

W.Z., S.R. and A.C.W.P. contributed equally to the project. The project was conceived and managed by E.P.C., N.D. and T.M. K.E.J.R. obtained crystals that diffracted to beyond 4 Å resolution, collected data and solved the structure of TASK-1 and the complexes with drug molecules, assisted by D.S. and supervised by A.C.W.P. and E.P.C. W.Z. was involved in optimization of the constructs, protein purification and production of initial crystals, supervised by A.Q., A.C.W.P. and E.P.C. A.K.K. performed voltage-clamp recordings, pharmacological experiments and single-channel recordings, S.R. performed voltage-clamp recordings, introduced all TASK-1 mutants and performed ELISA surface-expression assays, and M.G. performed all experiments with sevoflurane; all these experiments were supervised by N.D. Initial constructs for structural biology were screened for expression by L.S. and large-scale insect cell expressions were produced by S.M.M.M., supervised by N.A.B.-B. Tests of the relative activities of TASK-1 and the TASK-1Met1–Glu259 construct used in crystallisation were performed by L.J.C., supervised by S.J.T. The TASK-1 inhibitors were designed and produced by M.D., M.G.H., H.M. and T.M., supervised by T.M. M.P. was in charge of optimizing and performing TASK-1 and TASK-3 measurements in recombinant cell lines, including potency measurements of TASK antagonists with voltage-sensitive dyes and washout experiments in the same system. Data were analysed and the paper was written by K.E.J.R., A.C.W.P., A.K.K., S.R., L.J.C., S.J.T., T.M., N.D. and E.P.C.

Corresponding authors

Correspondence to Niels Decher or Elisabeth P. Carpenter.

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Competing interests

M.D., M.G.H., H.M. and T.M. are listed as inventors on patent number WO2017097792A1, priority date 10 December 2018, entitled ‘2-phenyl-3-(piperazinomethyl)imidazo[1,2-a]pyridine derivatives as blockers of TASK-1 and TASK-3 channels, for the treatment of sleep-related breathing disorders’. N.D. is listed as an inventor on patent number EP18189182.1, priority date 15 August 2018, entitled ‘TASK-1 inhibitors for treatment of atrial arrhythmias’. The other authors declare no competing interests.

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Peer review information Nature thanks Florian Lesage and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Functional properties of the crystallization construct TASK-1M1–E259 and pH-sensitivity of mutations near the pH sensor His98.

a, The C-terminally-truncated construct we used for crystallization (Met1–Glu259, denoted TASK-1M1–E259) has similar channel properties to the wild-type protein. The whole-cell wild-type TASK-1 and TASK-1M1–E259 currents recorded after expression in Xenopus oocytes show that the crystallization construct is functionally active (n = 7). b, The truncated crystallization construct TASK-1M1–E259 retains normal inhibition by the pore-blockers A1899 (wild-type TASK-1, n = 9; TASK-1M1–E259, n = 8) and ML365 (n = 5). c, The crystallization construct retains sensitivity to changes in extracellular pH (the number of biological repeats is shown on the graph). The small change in half-maximal activation between the two proteins (pH 7.2, wild-type TASK-1; pH 6.4, TASK-1M1–E259) suggests that the truncated cytoplasmic region may influence this process. However, overall the truncated construct exhibits a similar pharmacology and regulation to the wild-type channel. d, e, Representative two-electrode voltage-clamp recordings of wild-type TASK-1 (n = 6) (d) and the Q209E mutant (n = 6) (e) under different extracellular H+ concentrations. f, pH dose–response curves of wild-type TASK-1 and TASK-1 with mutations at Q77 and at Q209—residues positioned above the pH sensor H98. n = 6 for all proteins. Number of biological replicates (n) are illustrated in the legends or graphs. Data are presented as mean ± s.e.m. and shown as individual points.

Source Data

Extended Data Fig. 2 The structure of TASK-1 at 3.0 Å resolution indicates that the cap in TASK-1 forms the classic cap domain structure without an intramolecular disulfide bond.

a, Final 2FoFc electron density maps obtained using BUSTER, contoured at 1.0σ, showing the M1–M4 helices (first four maps) and the selectivity filter from the plane above the membrane (fifth map). b, The selectivity filter with a 2FoFc map contoured at 1.0σ shown from the plane of the membrane (left), the selectivity filter with a 2FoFc map contoured at 2.0σ (middle) and a simulated annealing mFoDFc K+ omit map contoured at 4.0σ (right). c, The pH-sensor residue, His98, the surrounding residues and the pore, with the K+ ions shown in green. d, Simulated annealing 2mFoDFc (blue) and mFoDFc (green) water omit maps shown for waters around H98 (top) and H98′ (bottom). Maps are contoured at 0.8σ and 3.0σ, respectively, and the 2mFoDFc map was sharpened in phenix.auto_sharpen with an applied B-factor sharpening of 40.23 Å2. e, Cartoon representation of the structure of TASK-1, with lipids, detergents and CHS shown as sticks, with expanded views of the binding sites. 2FoFc maps are contoured at 1.0σ. f, 2FoFc electron density maps of the cap, contoured at 1.0σ. g, View of the cap apex. h, Superposition of the TASK-1 cap (gold and purple) and the TREK-2 (PDB: 4XDJ) cap (yellow and blue), illustrating the lack of disulfide bond in TASK-1. i, View of the aligned cap apices. j, The M4 helix residues 233–257 represented as sticks, with the main-chain hydrogen-bonding pattern shown as dashed lines. k, Diagrams of the crystal packing showing overall packing interactions (top left), the crystal packing around the AB (gold and purple) and CD (blue and green) dimers (top middle and top right), and a close-up of the X-gate and distal M4 crystal contacts (bottom).

Extended Data Fig. 3 Alignment and phylogenetic trees for the TASK channels and the K2P channels reveal conservation of X-gate residues in TASK-1, TASK-3 and TASK-5, and a lack of an X-gate in other K2P channels.

a, Alignment of the human K2P proximal M1, cap and distal M4 regions. The cysteine residue in the cap apex is highlighted in yellow and the X-gate residues are highlighted in red. b, Sequence alignment of TASK channels from different species. c, Phylogenetic tree of the human K2P channels and the TASK channels in b. The TASK-1, TASK-3 and TASK-5 channels are highly conserved, whereas the K2P channels that were originally designated as TASK-2 and TASK-4 (TALK-2) have much lower homology and belong in the subfamily of TALK channels.

Extended Data Fig. 4 Functional studies of TASK X-gate and latch mutants and role of cholesterol in channel function.

a, Relative surface expression of wild-type TASK-1 and TASK-1 with gain-of-function mutations at the bend or at the X-gate (two-sided Mood’s median test, PA237V = 3.2 × 10−9, PL241A = 3.2 × 10−9, PR245A = 1.4 × 10−9; two-sided Mann–Whitney U-test PL244A = 3.0 × 10−5). b, Relative current amplitudes of wild-type TASK-3 (n = 15) and the Leu244Ala mutant (n = 15) (two-sided Welch’s t-test, P = 1.8 × 10−4). c, Analysis activation after the application of 1 mM sevoflurane to wild-type TASK-1 and the indicated TASK-1 mutants (two-sided Student’s t-test, PV243A = 0.15, PR245A = 5.3 × 10−4, PM247A = 3.8 × 10−7; two-sided Mann–Whitney U-test PL244A = 2.4 × 10−6; two-sided Welch’s t-test PF246A = 1.6 × 10−6, PT248A = 0.0043). d, Representative two-electrode voltage-clamp recordings of wild-type TASK-1 (n = 32) and the latch-destabilizing mutants Arg7Asp (n = 48) and Arg131Asp (n = 15). e, Relative surface expression of wild-type TASK-1 and TASK-1 with mutations in the latch region (two-sided Mood’s median test, PN5C = 2.9 × 10−12, PR7D = 1.3 × 10−5, PR131C = 2.3 × 10−13, PR131D = 1.4 × 10−9, PN133C = 1.2 × 10−11, PN250C = 1.8 × 10−7). f, Normal extracellular pH gating of wild-type TASK-1 (n = 6) and of mutant channels in which the inner gate is destabilized by latch mutations R7D (n = 7) and R131D (n = 5). g, Representative inside-out macropatch recordings of wild-type TASK-1 channels after consecutive application of CHS, control, tetraethylammonium (TEA) and control solution again (n = 6). h, Analysis of current after CHS was applied to inside-out patch-clamp recordings using a rapid perfusion system (two-sided paired Student’s t-test, Pcontrol-CHS = 0.12, PCHS-washout = 0.076). i, Representative two-electrode voltage-clamp recordings before and after the application of β-cyclodextrin for 30 min (n = 10). j, Analysis of relative inhibition by 2.5 mM β-cyclodextrin. All data were recorded in X. laevis oocytes. Number of biological replicates (n) are illustrated in the respective panels. ***P < 0.001. Data in ac, e, f, j are presented as mean ± s.e.m. For box plots in h, the centre line represents the median, the box limits are the 25th and 75th percentiles, and the whiskers are s.e.m. For full statistical details see Methods and Source Data.

Source Data

Extended Data Fig. 5 Effects of BAY1000493 and BAY2341237 on TASK-1 and TASK-3, and inhibitor washout data for BAY1000493 and A1899.

a, Representative two-electrode voltage-clamp recordings of wild-type TASK-1 (n = 5), and the Leu122Ala (n = 5) and Leu244Ala (n = 4) mutants before and after the application of BAY1000493 (100 nM). b, Representative recordings of wild-type TASK-1 (n = 5) and TASK-3 (n = 7) channels before and after the application of different concentrations of BAY1000493. c, Dose–response curves for BAY1000493 with TASK-1 (n = 5) and TASK-3 (n = 7). d, Dose response curves for BAY2341237 with TASK-1 (n = 5) and TASK-3 (n = 6; except for 1 nM, n = 5). e, Representative recordings of wild-type TASK-1 before (n = 5) and after (n = 5) application of BAY1000493 (100 nM) under high extracellular K+ concentration (symmetrical conditions), using an ND96 solution in which 96 mM NaCl was replaced by KCl. A step protocol with an increment of +10 mV was applied every 12 s. f, g, Analyses of the voltage-dependence of TASK-1 inhibition by BAY1000493 (f) and BAY2341237 (g). h, i, The EC50 values were determined in an ND96 solution containing 2 mM extracellular K+ or in ND96 solution with 96 mM NaCl replaced by KCl. j, Washout of BAY1000493 from wild-type TASK-1 (n = 5) and the TASK-1 mutants Arg131Cys (n = 7), Thr134Cys (n = 5) and Leu244Ala (n = 5). k, Washout of BAY1000493 (n = 5) compared to A1899 (n = 4). l, Relative washout rates for BAY1000493 (n = 5) and A1899 (n = 4). m, Analysis of the washout rates for the compounds shown in l (using two-sided Student’s t-test, PA1899 = 2.0 × 10−3). Number of biological replicates (n) are illustrated in the respective panels. **P < 0.01. Data are presented as mean ± s.e.m. For full statistical details see Methods and Source Data.

Source Data

Extended Data Fig. 6 Structures, electron density maps and bromine anomalous data for TASK-1 in complex with BAY1000493 or BAY2341237.

a, The structure of the TASK-1–BAY1000493 complex, viewed from the selectivity filter towards the vestibule, with the 2FoFc map (blue) and the anomalous difference map at 3.3 Å (contoured at 3.5σ) collected at the Br edge (magenta). The two 50%-occupancy BAY1000493 molecules shown with carbons coloured teal or light blue, oxygens in red, nitrogens in dark blue, fluorines in cyan and bromines in maroon. b, The structure of the TASK-1–BAY2341237 complex, as for a, with the 2FoFc map in blue. The BAY1000493 molecule (100% occupancy) is shown with carbons in green, chlorines in dark green and other atoms coloured as in a. c, The structure of TASK-1, with a 2FoFc map shown in blue. There is some residual density below the selectivity filter, as is seen in many K2P structures. df represent the same structures as in ac, with the positive FoFc difference density from omit maps (green) calculated with the inhibitors excluded. 2FoFc and FoFc maps are contoured at 1.0σ and 2.5σ, respectively. g, Superposition of the two complexes viewed from the membrane plane, with BAY1000493 in teal and BAY2341237 in green. The BAY1000493 complex is shown as a cartoon with side chains as sticks (gold and purple), and side chains from the BAY2341237 complex are shown in in red and blue. h, Schematic showing how the 50:50 distribution of BAY1000493 orientations occurs in crystals. i, Schematic showing how asymmetry could lead to 100% occupancy of one orientation in the case of BAY2341237. j, k, Interactions between TASK-1 and BAY1000493 (j) or BAY2341237 (k). Close contacts (less than 4.0 Å) between the protein and inhibitor atoms are shown as blue lines. For clarity, only the three closest contacts are shown if there were more than three contacts.

Extended Data Fig. 7 Model of the conformation that TASK-1 could adopt with a straight, continuous M4 helix, and a gating scheme for TASK-1.

a, b, Model of TASK-1 with M4 adopting a straight α-helical conformation (in red and blue) aligned with the TASK-1 structure shown from the side (a) and below the membrane (b). c, Open and closed models of TASK-1 viewed from below the membrane. d, A model of TASK-1 activation, with the closed X-gate state shown in orange, and two schematics for possible open states, based on the down and up states seen in TRAAK and TREK-2. The version based on the down state, with an open X-gate, is shown in light blue, and the version with the helices in the up state, potentially giving a more active state, is shown in dark blue. Asterisks indicate the TASK-1 conformations for which structures have been obtained. At acidic pHs, we predict that His98 would move, causing the selectivity filter to become less conductive—a change that could, in theory, occur in any of the conformations shown above.

Extended Data Fig. 8 The location of TASK-1 mutations associated with PPH4 in the structure.

a, b, Location of mutations in TASK-1 and TASK-3 mapped onto the TASK-1 structure shown from the membrane plane (a) and from the extracellular side (b). cg, Expanded views of Thr8Lys in the latch region on M1 (c), Tyr192Cys (d), Gly97Arg and Gly203Asp in the pore loops (e), Glu182Lys (f) and Val221Leu (g). h, Left, the TASK-3 mutations Gly236Arg and Ala237Asp in M4 (highlighted in yellow), which are associated with Birk-Barel syndrome, shown from the membrane plane. Right, a 90° rotation, with the vestibule shown in blue.

Extended Data Table 1 X-ray crystallography data, refinement and model statistics
Extended Data Table 2 EC50 values of a series of TASK-1 compounds and their chemical structures

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Rödström, K.E.J., Kiper, A.K., Zhang, W. et al. A lower X-gate in TASK channels traps inhibitors within the vestibule. Nature 582, 443–447 (2020). https://doi.org/10.1038/s41586-020-2250-8

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