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:

An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel

Nature Chemical Biology volume 14, pages 396–404 (2018)Cite this article

This article has been updated

Abstract

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation–deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical ‘lipid clamp’ identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: Optical control of TRPC3 conductances expressed in HEK293 cells by DAG photoswitches.
Fig. 2: Localization of critical residues within a ‘lipid-gating fenestration’ in TRPC3.
Fig. 3: G652 plays a critical role in TRPC3 gating and activation by DAGs.
Fig. 4: G652A mutation alters discrimination between DAGs by TRPC3.
Fig. 5: Photopharmacological determination of the DAG sensitivity of TRPC3-WT and G652A channels.
Fig. 6: Optical cycling of TRPC3-WT and G652A mutant channels in the presence of OptoDArG (30 μM) occurs with divergent kinetics.

Similar content being viewed by others

Change history

  • 08 August 2018

    The version of this article originally published included an extended version of the Life Sciences Reporting Summary. The error has been corrected in the HTML and PDF versions of the paper.

References

  1. Hofmann, T. et al. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Bandyopadhyay, B. C. et al. Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells. Role in apical Ca2+ influx. J. Biol. Chem. 280, 12908–12916 (2005).

    Article  CAS  PubMed  Google Scholar 

  3. Dietrich, A. et al. Increased vascular smooth muscle contractility in TRPC6−/− mice. Mol. Cell. Biol. 25, 6980–6989 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Neuner, S. M. et al. TRPC3 channels critically regulate hippocampal excitability and contextual fear memory. Behav. Brain Res. 281, 69–77 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Quick, K. et al. TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells. Open Biol. 2, 120068 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Poteser, M. et al. PKC-dependent coupling of calcium permeation through transient receptor potential canonical 3 (TRPC3) to calcineurin signaling in HL-1 myocytes. Proc. Natl Acad. Sci. USA 108, 10556–10561 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Fuchs, B. et al. Diacylglycerol regulates acute hypoxic pulmonary vasoconstriction via TRPC6. Respir. Res. 12, 20 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kim, M. S. et al. Genetic and pharmacologic inhibition of the Ca2+ influx channel TRPC3 protects secretory epithelia from Ca2+-dependent toxicity. Gastroenterology 140, 2107–2115.e4 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Numaga-Tomita, T. et al. TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis. Sci. Rep. 6, 39383 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Phelan, K. D., Shwe, U. T., Abramowitz, J., Birnbaumer, L. & Zheng, F. Critical role of canonical transient receptor potential channel 7 in initiation of seizures. Proc. Natl Acad. Sci. USA 111, 11533–11538 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Smedlund, K. B., Birnbaumer, L. & Vazquez, G. Increased size and cellularity of advanced atherosclerotic lesions in mice with endothelial overexpression of the human TRPC3 channel. Proc. Natl Acad. Sci. USA 112, E2201–E2206 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Spassova, M. A., Hewavitharana, T., Xu, W., Soboloff, J. & Gill, D. L. A common mechanism underlies stretch activation and receptor activation of TRPC6 channels. Proc. Natl Acad. Sci. USA 103, 16586–16591 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. van Rossum, D. B. et al. TRP_2, a lipid/trafficking domain that mediates diacylglycerol-induced vesicle fusion. J. Biol. Chem. 283, 34384–34392 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhang, L. & Saffen, D. Muscarinic acetylcholine receptor regulation of TRP6 Ca2+ channel isoforms. Molecular structures and functional characterization. J. Biol. Chem. 276, 13331–13339 (2001).

    Article  CAS  PubMed  Google Scholar 

  15. Cao, E., Liao, M., Cheng, Y. & Julius, D. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gao, Y., Cao, E., Julius, D. & Cheng, Y. TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action. Nature 534, 347–351 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Frank, J. A. et al. Photoswitchable fatty acids enable optical control of TRPV1. Nat. Commun. 6, 7118 (2015).

    Article  PubMed  Google Scholar 

  18. Frank, J. A. et al. Photoswitchable diacylglycerols enable optical control of protein kinase C. Nat. Chem. Biol. 12, 755–762 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Doleschal, B. et al. TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1. Cardiovasc. Res. 106, 163–173 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hofmann, T., Schaefer, M., Schultz, G. & Gudermann, T. Subunit composition of mammalian transient receptor potential channels in living cells. Proc. Natl Acad. Sci. USA 99, 7461–7466 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lichtenegger, M. et al. A novel homology model of TRPC3 reveals allosteric coupling between gate and selectivity filter. Cell Calcium 54, 175–185 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Zagotta, W. N., Hoshi, T., Dittman, J. & Aldrich, R. W. Shaker potassium channel gating. II: transitions in the activation pathway. J. Gen. Physiol. 103, 279–319 (1994).

    CAS  PubMed  Google Scholar 

  23. Mauco, G., Dangelmaier, C. A. & Smith, J. B. Inositol lipids, phosphatidate and diacylglycerol share stearoylarachidonoylglycerol as a common backbone in thrombin-stimulated human platelets. Biochem. J. 224, 933–940 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hindenes, J.-O. et al. Physical properties of the transmembrane signal molecule, sn-1-stearoyl 2-arachidonoylglycerol. Acyl chain segregation and its biochemical implications. J. Biol. Chem. 275, 6857–6867 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. 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  PubMed Central  Google Scholar 

  26. Aryal, P., Abd-Wahab, F., Bucci, G., Sansom, M. S. & Tucker, S. J. Influence of lipids on the hydrophobic barrier within the pore of the TWIK-1 K2P channel. Channels 9, 44–49 (2015).

    Article  PubMed  Google Scholar 

  27. Storch, U. et al. Dynamic NHERF interaction with TRPC4/5 proteins is required for channel gating by diacylglycerol. Proc. Natl Acad. Sci. USA 114, E37–E46 (2017).

    Article  CAS  PubMed  Google Scholar 

  28. Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Barad, B. A. et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).

    Article  CAS  PubMed  Google Scholar 

  31. Shen, M.-Y. & Sali, A. Statistical potential for assessment and prediction of protein structures. Protein Sci. 15, 2507–2524 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

M. Lichtenegger was a fellow of the BioTechMed Graz program (project: Deciphering the lipid sensing machinery of TRPC channels); O. Tiapko, B. Svobodova and N. Shrestha are members of the PhD program (DK) “Metabolic and Cardovascular Disease” (W1226-B18). The authors wish to thank H. Janovjak for helpful discussions, M. Janschitz for assistance with graphics and T. Schmidt for critically reading the manuscript. The work was supported by FWF (P28701 and P26067 to R. Schindl., P27263 to C.R., P28243 to T.G., W1226-B18 to K.G. and SFB35 subproject F3524 to T.S.) as well as BMWFW HSRSM (PromOpt2.0 to K.G. & C.R.).

Author information

Author notes

  1. These authors contributed equally: Michaela Lichtenegger and Oleksandra Tiapko.

Authors and Affiliations

  1. Gottfried Schatz Research Center, Biophysics, Medical University of Graz, Graz, Austria

    Michaela Lichtenegger, Oleksandra Tiapko, Barbora Svobodova, Wolfgang Schreibmayer, Dieter Platzer, Sarah Krenn, Niroj Shrestha, Rainer Schindl & Klaus Groschner

  2. Institute of Pharmacology, Medical University of Vienna, Vienna, Austria

    Thomas Stockner

  3. Institute of Chemistry, University of Graz, Graz, Austria

    Toma N. Glasnov & Gema Guedes de la Cruz

  4. Institute of Biophysics, University of Linz, Linz, Austria

    Romana Schober & Christoph Romanin

Authors
  1. Michaela Lichtenegger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oleksandra Tiapko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Barbora Svobodova
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Thomas Stockner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Toma N. Glasnov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wolfgang Schreibmayer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Dieter Platzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gema Guedes de la Cruz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sarah Krenn
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Romana Schober
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Niroj Shrestha
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Rainer Schindl
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Christoph Romanin
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Klaus Groschner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.L. and O.T. performed experiments, analyzed data and wrote parts of the manuscript; T.S. performed homology modeling; B.S., D.P., W.S., R. Schober, N.S., S.K. and R. Schindl analyzed data; T.N.G. and G.G.d.l.C. synthesized compounds; C.R. contributed with experimental planning and data interpretation; K.G. designed the project and wrote the manuscript.

Corresponding author

Correspondence to Klaus Groschner.

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lichtenegger, M., Tiapko, O., Svobodova, B. et al. An optically controlled probe identifies lipid-gating fenestrations within the TRPC3 channel. Nat Chem Biol 14, 396–404 (2018). https://doi.org/10.1038/s41589-018-0015-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0015-6

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