Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Selective regulation of human TRAAK channels by biologically active phospholipids

Nature Chemical Biology volume 17, pages 89–95 (2021)Cite this article

Subjects

Abstract

TRAAK is an ion channel from the two-pore domain potassium (K2P) channel family with roles in maintaining the resting membrane potential and fast action potential conduction. Regulated by a wide range of physical and chemical stimuli, the affinity and selectivity of K2P4.1 toward lipids remains poorly understood. Here we show the two isoforms of K2P4.1 have distinct binding preferences for lipids dependent on acyl chain length and position on the glycerol backbone. The channel can also discriminate the fatty acid linkage at the SN1 position. Of the 33 lipids interrogated using native mass spectrometry, phosphatidic acid had the lowest equilibrium dissociation constants for both isoforms of K2P4.1. Liposome potassium flux assays with K2P4.1 reconstituted in defined lipid environments show that those containing phosphatidic acid activate the channel in a dose-dependent fashion. Our results begin to define the molecular requirements for the specific binding of lipids to K2P4.1.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Optimization of human K2P4.1 isoforms for high-resolution native MS studies.
Fig. 2: Determination of equilibrium dissociation constants (Kd) for individual lipid-binding events to K2P4.1a.
Fig. 3: Biophysical characterization of individual lipid-binding events to K2P4.1b.
Fig. 4: Liposome potassium flux assays of K2P4.1a reconstituted in defined lipid environments.

Similar content being viewed by others

Data availability

The native MS data are available on Zenodo (https://doi.org/10.5281/zenodo.3993214). Source data are provided with this paper.

Code availability

Python code is available at GitHub (https://github.com/LaganowskyLab/Laganowsky_Lab_Code.git).

References

  1. Heurteaux, C. et al. TREK-1, a K+ channel involved in neuroprotection and general anesthesia. EMBO J. 23, 2684–2695 (2004).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Alloui, A. et al. TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 25, 2368–2376 (2006).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Blondeau, N. et al. Polyunsaturated fatty acids are cerebral vasodilators via the TREK-1 potassium channel. Circ. Res. 101, 176–184 (2007).

    Article  CAS  PubMed  Google Scholar 

  4. Noel, J. et al. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception. EMBO J. 28, 1308–1318 (2009).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Enyedi, P. & Czirjak, G. Molecular background of leak K+ currents: two-pore domain potassium channels. Physiol. Rev. 90, 559–605 (2010).

    Article  CAS  PubMed  Google Scholar 

  6. Feliciangeli, S., Chatelain, F. C., Bichet, D. & Lesage, F. The family of K2P channels: salient structural and functional properties. J. Physiol. 593, 2587–2603 (2015).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Es-Salah-Lamoureux, Z., Steele, D. F. & Fedida, D. Research into the therapeutic roles of two-pore-domain potassium channels. Trends Pharmacol. Sci. 31, 587–595 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Noel, J., Sandoz, G. & Lesage, F. Molecular regulations governing TREK and TRAAK channel functions. Channels 5, 402–409 (2011).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Cabanos, C., Wang, M., Han, X. L. & Hansen, S. B. A soluble fluorescent binding assay reveals PIP2 antagonism of TREK-1 channels. Cell Rep. 20, 1287–1294 (2017).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Vivier, D., Bennis, K., Lesage, F. & Ducki, S. Perspectives on the two-pore domain potassium channel TREK-1 (TWIK-Related K(+) Channel 1). A novel therapeutic target? J. Med. Chem. 59, 5149–5157 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Chemin, J. et al. Up- and down-regulation of the mechano-gated K(2P) channel TREK-1 by PIP (2) and other membrane phospholipids. Pflug. Arch. 455, 97–103 (2007).

    Article  CAS  Google Scholar 

  12. Comoglio, Y. et al. Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid. Proc. Natl Acad. Sci. USA 111, 13547–13552 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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 Central  PubMed  Google Scholar 

  14. Chung, H. W. et al. A molecular target for an alcohol chain-length cutoff. J. Mol. Biol. 431, 196–209 (2019).

    Article  CAS  PubMed  Google Scholar 

  15. Fink, M. et al. A neuronal two P domain K+ channel stimulated by arachidonic acid and polyunsaturated fatty acids. EMBO J. 17, 3297–3308 (1998).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Chemin, J. et al. Lysophosphatidic acid-operated K+ channels. J. Biol. Chem. 280, 4415–4421 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Juarez-Contreras, R., Rosenbaum, T. & Morales-Lazaro, S. L. Lysophosphatidic acid and ion channels as molecular mediators of pain. Front Mol. Neurosci. 11, 462 (2018).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Maingret, F., Patel, A. J., Lesage, F., Lazdunski, M. & Honore, E. Lysophospholipids open the two-pore domain mechano-gated K(+) channels TREK-1 and TRAAK. J. Biol. Chem. 275, 10128–10133 (2000).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Brohawn, S. G., Su, Z. & MacKinnon, R. Mechanosensitivity is mediated directly by the lipid membrane in TRAAK and TREK1 K+ channels. Proc. Natl Acad. Sci. USA 111, 3614–3619 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 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  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Lolicato, M., Riegelhaupt, P. M., Arrigoni, C., Clark, K. A. & Minor, D. L. Jr. Transmembrane helix straightening and buckling underlies activation of mechanosensitive and thermosensitive K(2P) channels. Neuron 84, 1198–1212 (2014).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Brohawn, S. G., Campbell, E. B. & MacKinnon, R. Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel. Nature 516, 126–130 (2014).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. McClenaghan, C. et al. Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states. J. Gen. Physiol. 147, 497–505 (2016).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Bolla, J. R., Agasid, M. T., Mehmood, S. & Robinson, C. V. Membrane protein–lipid interactions probed using mass spectrometry. Annu. Rev. Biochem. 88, 85–111 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Laganowsky, A., Reading, E., Hopper, J. T. & Robinson, C. V. Mass spectrometry of intact membrane protein complexes. Nat. Protoc. 8, 639–651 (2013).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  28. Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  29. Hilton, G. R. & Benesch, J. L. Two decades of studying non-covalent biomolecular assemblies by means of electrospray ionization mass spectrometry. J. R. Soc. Interface 9, 801–816 (2012).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Liu, Y. et al. Selective binding of a toxin and phosphatidylinositides to a mammalian potassium channel. Nat. Commun. 10, 1352 (2019).

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  32. Cong, X. et al. Determining membrane protein-lipid binding thermodynamics using native mass spectrometry. J. Am. Chem. Soc. 138, 4346–4349 (2016).

    Article  CAS  PubMed  Google Scholar 

  33. Cong, X., Liu, Y., Liu, W., Liang, X. & Laganowsky, A. Allosteric modulation of protein-protein interactions by individual lipid binding events. Nat. Commun. 8, 2203 (2017).

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  34. Yen, H. Y. et al. PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling. Nature 559, 423–427 (2018).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  35. Patrick, J. W. et al. Allostery revealed within lipid binding events to membrane proteins. Proc. Natl Acad. Sci. USA 115, 2976–2981 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Marcoux, J. et al. Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump. Proc. Natl Acad. Sci. USA 110, 9704–9709 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Gault, J. et al. High-resolution mass spectrometry of small molecules bound to membrane proteins. Nat. Methods 13, 333–336 (2016).

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  38. 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  CAS  PubMed  PubMed Central  Google Scholar 

  39. Poltash, M. L, McCabe, J. W., Patrick, J. W., Laganowsky, A. & Russell, D. H. Development and evaluation of a reverse-entry ion source orbitrap mass spectrometer. J. Am. Soc. Mass Spec. 30, 192–198 (2018).

    Article  CAS  Google Scholar 

  40. Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970).

    Article  CAS  PubMed  Google Scholar 

  41. Lyu, J. et al. Discovery of potent charge-reducing molecules for native ion mobility mass spectrometry studies. Anal. Chem. 92, 11242–11249 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Ben-Nissan, G. & Sharon, M. The application of ion-mobility mass spectrometry for structure/function investigation of protein complexes. Curr. Opin. Chem. Biol. 42, 25–33 (2018).

    Article  CAS  PubMed  Google Scholar 

  43. Poltash, M. L. et al. Fourier transform-ion mobility-orbitrap mass spectrometer: a next-generation instrument for native mass spectrometry. Anal. Chem. 90, 10472–10478 (2018).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  44. Harayama, T. & Riezman, H. Understanding the diversity of membrane lipid composition. Nat. Rev. Mol. Cell Biol. 19, 281–296 (2018).

    Article  CAS  PubMed  Google Scholar 

  45. Brohawn, S. G. et al. The mechanosensitive ion channel TRAAK is localized to the mammalian node of Ranvier. eLife 8, https://doi.org/10.7554/eLife.50403 (2019).

  46. Farooqui, A. A., Farooqui, T. & Horrocks, L. A. (eds) In Metabolism and Functions of Bioactive Ether Lipids in the Brain 85–106 (Springer, 2008).

  47. Hargreaves, K. & Clandinin, M. T. Dietary lipids in relation to postnatal development of the brain. Ups. J. Med Sci. Suppl. 48, 79–95 (1990).

    CAS  PubMed  Google Scholar 

  48. Qiao, P., Liu, Y., Zhang, T., Benavides, A. & Laganowsky, A. Insight into the selectivity of Kir3.2 toward phosphatidylinositides. Biochemistry 59, https://doi.org/10.1021/acs.biochem.0c00163 (2020).

  49. Robinson, C. V., Rohacs, T. & Hansen, S. B. Tools for understanding nanoscale lipid regulation of ion channels. Trends Biochem. Sci. 44, 795–806 (2019).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Levitz, J. et al. Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels. Proc. Natl Acad. Sci. USA 113, 4194–4199 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).

    Article  CAS  PubMed  Google Scholar 

  52. Weis, R. High-throughput screening and selection of Pichia pastoris strains. Methods Mol. Biol. 1923, 169–185 (2019).

    Article  CAS  PubMed  Google Scholar 

  53. Chen, P. S., Toribara, T. Y. & Warner, H. Microdetermination of phosphorus. Anal. Chem. 28, 1756–1758 (1956).

    Article  CAS  Google Scholar 

  54. Allison, T. M. et al. Quantifying the stabilizing effects of protein-ligand interactions in the gas phase. Nat. Commun. 6, 8551 (2015); https://doi.org/10.1038/ncomms9551

  55. Marklund, E. G., Degiacomi, M. T., Robinson, C. V., Baldwin, A. J. & Benesch, J. L. Collision cross sections for structural proteomics. Structure 23, 791–799 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Virtanen, P. et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat. Methods 17, 261–272 (2020).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  57. Alexander, W. M., Ficarro, S. B., Adelmant, G. & Marto, J. A. multiplierz v2.0: a Python-based ecosystem for shared access and analysis of native mass spectrometry data. Proteomics 17, 1700091 (2017).

    Article  CAS  Google Scholar 

  58. Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  59. Hunter, J. D. Matplotlib: a 2D graphics environment. Comput. Sci. Eng. 9, 90–95 (2007).

    Article  Google Scholar 

  60. Su, Z. W., Brown, E. C., Wang, W. W. & MacKinnon, R. Novel cell-free high-throughput screening method for pharmacological tools targeting K+ channels. PNAS 113, 5748–5753 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) awarded to A.L. (grant no. DP2GM123486) and development of custom instrumentation supported by NIH (grant no. P41GM128577-01, D.R.).

Author information

Author notes

  1. These authors contributed equally: Samantha Schrecke, Yun Zhu.

Authors and Affiliations

  1. Department of Chemistry, Texas A&M University, College Station, TX, USA

    Samantha Schrecke, Yun Zhu, Jacob W. McCabe, Mariah Bartz, Charles Packianathan, David Russell & Arthur Laganowsky

  2. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Minglei Zhao

  3. Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA

    Ming Zhou

Authors
  1. Samantha Schrecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yun Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jacob W. McCabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mariah Bartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Charles Packianathan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Minglei Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ming Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. David Russell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Arthur Laganowsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.S., Y.Z. and A.L. designed the research. S.S., Y.Z. and C.P. expressed and purified the protein. S.S. and M.B. carried out molecular biology experiments. S.S. and Y.Z. performed the native MS experiments and Y.Z. conducted functional assays. S.S., Y.Z. and A.L. analyzed the data. S.S., Y.Z. and A.L. wrote the manuscript with input from the other authors.

Corresponding author

Correspondence to Arthur Laganowsky.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Optimization of expression and purification of human K2P4.1 for native ion mobility mass spectrometry studies.

a, Shown are representative mass spectra of the K2P4.1b fusion protein acquired on a Synapt G1 HDMS instrument. Denoted in the inset is the detergent abbreviation: dodecyl maltoside (DDM); decyl maltoside (DM); lauryldimethylamine-N-oxide (LDAO); pentaethylene glycol monodecyl ether (C10E5); and octaethylene glycol monododecyl ether (C12E8). The polyethylene glycol detergents, C10E5 and C12E8, yielded well resolved mass spectra compared to the other detergents screened. b, Membrane proteins were expressed with affinity tags fused to both termini. Shown is the native mass spectrum for K2P4.1b with tags removed by TEV protease in C10E5 acquired on an Orbitrap. c, Native ion mobility mass spectra of the K2P4.1a fusion protein solubilized in (left) 2x CMC C10E5 and with the addition of (right) 50 mM spermidine acquired on Synapt G1. In the absence of charge-reducing molecule, the arrival time distributions are broad for high charge states indicating collisional activation (or partial unfolding) of the protein complex. d, Collision induced unfolding (CIU) plot of arrival time distribution for the 9+ charge state of K2P4.1a with tags removed in C10E5 and spermidine acquired under different collision voltages (10 V step size) on a Synapt G1 instrument. The arrival time distribution for the 9+ and other charge states of K2P4.1a remain constant showing that charge reduction aids retention of native-like structure, even at the highest collision voltage (240 V). e, Ion mobility mass spectrum for K2P4.1a in C10E5 and spermidine recorded on an Orbitrap UHMR equipped with a REIS and 1.5 m drift tube operating in the FT-mode. f, Collision cross section profiles for different charge states of K2P4.1a. The centroid CCS for the 10+, 9+, and 8+ are 4559, 4528, 4547 Å2, respectively. The calculated CCS for PDB 4WFE is 4656 Å2.

Extended Data Fig. 2 Native mass spectra for weakly (or no) binding lipids to K2P4.1a.

Shown are native mass spectra for K2P4.1a in the presence of 20 µM of lipid. Lipid abbreviations are provided in Supplementary Table 1.

Extended Data Fig. 3 Determination of equilibrium dissociation constants (KD) for lipids binding K2P4.1a.

Shown are representative native mass for K2P4.1a in the presence of 20 µM of lipid. Plots of mole fraction (right panel) for apo K2P4.1a and bound to different number of lipids determined from a titration series (dots with error bars to represent the three measurements) and resulting fit from a sequential lipid-binding model (solid lines). Lipid abbreviations are provided in Supplementary Table 1.

Extended Data Fig. 4 Determination of KD for lysolipids and PIPs binding K2P4.1a.

Shown as described in Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 5 Determination of KD for lipids binding K2P4.1b.

Shown as described for Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 6 Determination of KD for lysolipids, PIPs lipids binding K2P4.1b.

Shown as described for Extended Data Fig. 3. * and ** denote lipid concentrations of 15 μM and 10 μM, respectively.

Extended Data Fig. 7 Liposome potassium flux assays of K2P4.1a reconstituted in POPC.

a-d, Flux assay traces for K2P4.1a reconstituted in POPC and POPC doped with POPG or POPA. Empty liposomes of the same lipid composition are shown in gray. The mole fraction (%) of doped lipid is shown in the inset. e, Photograph of a silver stained SDS-PAGE of different proteoliposome mixtures used in the liposome potassium flux assays. Bands corresponding to monomer and dimer K2P4.1a are denoted by a single and double asterisk, respectively. The Precision Plus Protein Dual Color Standard (Bio-Rad) protein ladder was loaded in the first lane and molecular weight in kDa is shown to the left. The white scale bar (lower right corner) represents 1 cm. f, Densitometry analysis of K2P4.1a monomeric bands. No statistical differences were found between the different samples. Shown are the mean and standard deviation (n = 3) for three independent experiments (white dots).

Source data

Supplementary information

Source data

Source Data Fig. 2

Kd values for each replicate

Source Data Fig. 3

Kd values for each replicate

Source Data Fig. 4

Liposome flux for each experiment

Source Data Extended Data Fig. 7

Unprocessed stained gel, densitometry measurements

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schrecke, S., Zhu, Y., McCabe, J.W. et al. Selective regulation of human TRAAK channels by biologically active phospholipids. Nat Chem Biol 17, 89–95 (2021). https://doi.org/10.1038/s41589-020-00659-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-00659-5

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing