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

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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.

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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.

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  • 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.

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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.).

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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.

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Correspondence to Klaus Groschner.

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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

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