Article | Published:

TRPV1 structures in nanodiscs reveal mechanisms of ligand and lipid action

Nature volume 534, pages 347351 (16 June 2016) | Download Citation

Abstract

When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.

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Primary accessions

Electron Microscopy Data Bank

Data deposits

The three-dimensional cryo-EM density maps of the TRPV1–nanodisc complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-8118 (TRPV1–nanodisc), EMD-8117 (TRPV1–RTX/DkTx–nanodisc), EMD-8119 (TRPV1–capsazepine–nanodisc) and EMD-8120 (TRPV1–capsazepine in amphipol). Particle image stacks after motion correction related to TRPV1–nanodisc and TRPV1–RTX/DkTx–nanodisc have been deposited in the Electron Microscopy Pilot Image Archive (http://www.ebi.ac.uk/pdbe/emdb/empiar/) under accession number EMPIAR-10059. Atomic coordinates for the atomic model of TRPV1 in nanodisc, TRPV1–RTX/DkTx in nanodisc and TRPV1–capsazepine in nanodisc have been deposited in the Protein Data Bank under accession numbers 5IRZ, 5IRX and 5IS0.

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Acknowledgements

We thank our laboratory colleagues, past and present, for many helpful discussions and manuscript critiques, C. Paulsen and E. Green for helping with initial screening for nanodisc reconstitution, and E. Palovcak for providing scripts for focused classification. This work was supported by grants from the National Institutes of Health (R01NS047723, R37NS065071 and R01NS055299 to D.J., S10OD020054, R01GM098672, P01GM111126 and P50GM082250 to Y.C.). Y.C. is an Investigator with the Howard Hughes Medical Institute.

Author information

Author notes

    • Erhu Cao

    Present address: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650, USA.

Affiliations

  1. Department of Physiology, University of California, San Francisco, California 94143, USA

    • Yuan Gao
    • , Erhu Cao
    •  & David Julius
  2. Keck Advanced Microscopy Laboratory and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143, USA

    • Yuan Gao
    •  & Yifan Cheng
  3. Howard Hughes Medical Institute, University of California, San Francisco, California 94143, USA

    • Yifan Cheng

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Contributions

Y.G. carried out protein purification, nanodisc reconstitution, and detailed cryo-EM experiments, including data acquisition, image processing, atomic model building and refinement of TRPV1–nanodisc complexes. E.C. carried out cryo-EM experiments of the TRPV1–capsazepine complex solubilized in amphipol. All authors contributed to experimental design, data analysis, and manuscript preparation.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to David Julius or Yifan Cheng.

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https://doi.org/10.1038/nature17964

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