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Gating of human TRPV3 in a lipid bilayer

Nature Structural & Molecular Biology volume 27, pages 635–644 (2020)Cite this article

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Abstract

The transient receptor potential cation channel subfamily V member 3 (TRPV3) channel plays a critical role in skin physiology, and mutations in TRPV3 result in the development of a congenital skin disorder, Olmsted syndrome. Here we describe multiple cryo-electron microscopy structures of human TRPV3 reconstituted into lipid nanodiscs, representing distinct functional states during the gating cycle. The ligand-free, closed conformation reveals well-ordered lipids interacting with the channel and two physical constrictions along the ion-conduction pore involving both the extracellular selectivity filter and intracellular helix bundle crossing. Both the selectivity filter and bundle crossing expand upon activation, accompanied by substantial structural rearrangements at the cytoplasmic intersubunit interface. Transition to the inactivated state involves a secondary structure change of the pore-lining helix, which contains a π-helical segment in the closed and open conformations, but becomes entirely α-helical upon inactivation. Together with electrophysiological characterization, structures of TRPV3 in a lipid membrane environment provide unique insights into channel activation and inactivation mechanisms.

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Fig. 1: Cryo-EM structure of human TRPV3 in lipid nanodiscs.
Fig. 2: Two physical constrictions along the ion pore of TRPV3 in a lipid bilayer.
Fig. 3: TRPV3 opening in a lipid bilayer.
Fig. 4: Conformational changes upon channel opening.
Fig. 5: Structure of an inactivated conformation.
Fig. 6: 2-APB binding site.

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Data availability

The cryo-EM maps of the wild-type human TRPV3 and K169A have been deposited in the Electron Microscopy Data Bank with accession codes EMD-20917 (apo TRPV3), EMD-20918 (apo K169A), EMD-20919 (K169A with 3 min exposure to 2-APB) and EMD-20920 (K169A in the presence of 2-APB). Atomic coordinates have been deposited in the Protein Data Bank with accession codes 6UW4 (apo TRPV3), 6UW6 (apo K169A), 6UW8 (K169A with 3 min exposure to 2-APB) and 6UW9 (K169A in the presence of 2-APB).

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Acknowledgements

This work was supported by National Institutes of Health Grant R01NS099341 and the Mallinckrodt Foundation grant (to P.Y.), and by the McDonnell Center for Cellular and Molecular Neurobiology Postdoctoral Fellowship (to Z.D.). M.R. and J.A.J.F. are supported by the Washington University Center for Cellular Imaging, which is funded in part by Washington University School of Medicine through the Precision Medicine Initiative, the Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital (CDI-CORE-2015-505 and CDI-CORE-2019-813) and the Foundation for Barnes-Jewish Hospital (3770).

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  1. These authors contributed equally: Zengqin Deng, Grigory Maksaev.

Authors and Affiliations

  1. Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA

    Zengqin Deng, Grigory Maksaev, James A. J. Fitzpatrick & Peng Yuan

  2. Center for the Investigation of Membrane Excitability Diseases, Washington University School of Medicine, Saint Louis, MO, USA

    Zengqin Deng, Grigory Maksaev & Peng Yuan

  3. Washington University Center for Cellular Imaging, Washington University School of Medicine, Saint Louis, MO, USA

    Michael Rau & James A. J. Fitzpatrick

  4. Department of Anesthesiology, Washington University School of Medicine, Saint Louis, MO, USA

    Zili Xie & Hongzhen Hu

  5. Center for the Study of Itch, Washington University School of Medicine, Saint Louis, MO, USA

    Zili Xie & Hongzhen Hu

  6. Department of Neuroscience, Washington University School of Medicine, Saint Louis, MO, USA

    James A. J. Fitzpatrick

  7. Department of Biomedical Engineering, Washington University in Saint Louis, Saint Louis, MO, USA

    James A. J. Fitzpatrick

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Contributions

Z.D. performed biochemical preparations, cryo-EM experiments, structural determination and analysis. G.M. conducted electrophysiology experiments. M.R. and J.A.J.F. performed cryo-EM data acquisition in conjunction with Z.D. Z.X. and H.H. helped with functional studies. P.Y. designed and supervised the project. Z.D., G.M. and P.Y. analyzed the results and prepared the manuscript with input from all authors.

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Correspondence to Peng Yuan.

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Extended data

Extended Data Fig. 1 Reconstitution of human TRPV3 into lipid nanodiscs.

a, Size-exclusion chromatography of TRPV3 reconstituted into lipid nanodiscs made of soybean polar lipids and the scaffold protein MSP2N2 (left panel). Peaks indicating the void, the TRPV3-embedded nanodiscs, the empty nanodiscs, and GFP were labeled. The peak fraction corresponding to TRPV3 channels in nanodiscs was shown on SDS-PAGE (right panel). b, The collected TRPV3-nanodisc fraction ran as a monodisperse peak on size-exclusion chromatography. c, Representative micrograph for negative stain and reference-free 2D class averages indicating a tetrameric channel inserted into nanodiscs (right).

Extended Data Fig. 2 Cryo-EM reconstruction of the wild-type full-length human TRPV3 in lipid nanodiscs.

a, Flowchart of cryo-EM data processing. b, Fourier shell correlation before and after post-processing in RELION2. c, Fourier shell correlation between the refined model and the full map. d, Angular distribution plot of particles used for final reconstruction. e, Cryo-EM density map colored by local resolution. f, Representative cryo-EM density shown as blue mesh contoured at 5.0 σ.

Extended Data Fig. 3 Channel-lipid interactions in human TRPV3.

a, Lipid densities at sites 1, 2, and 3 located in the proximity of the outer pore region. Lipids are putatively modeled as phosphatidylethanolamine to illustrate interactions with the channel. Channel subunits are uniquely colored. Polar and charged residues potentially interacting with lipid head groups are highlighted in stick representation. b, Lipid density at site 4 in the intracellular cavity of the S1-S4 domain. c, Lipid density between the S4-S5 linkers of two neighboring subunits. The putative lipid densities are shown as orange mesh contoured at 3.5 σ.

Extended Data Fig. 4 Cryo-EM reconstruction of the human TRPV3 K169A variant in lipid nanodiscs.

a, Flowchart of cryo-EM data processing. Two major conformations, presumably representing the open (~69%) and inactivated states (31%), were refined to resolutions of 3.66 Å and 4.4 Å, respectively. b, Fourier shell correlation before and after post-processing in RELION3 for the open state. c, Fourier shell correlation between the refined model and the full map for the open state. d, Angular distribution plot of particles used for final reconstruction for the open state. e, Cryo-EM density map colored by local resolution for the open state. f, Representative cryo-EM density shown as blue mesh contoured at 5.0 σ.

Extended Data Fig. 5 Opening of TRPV1.

a, b, Orthogonal views of the closed (PDB 3J5P) and open (PDB 3J5Q) structures of TRPV1.

Extended Data Fig. 6 Lipid in the analogous vanilloid binding pocket in TRPV3.

a,b, Putative lipid density shown as orange mesh contoured at 4.5 σ in the analogous vanilloid binding pocket in the closed (a) and open (b) conformations.

Extended Data Fig. 7 Cryo-EM reconstruction of K169A accompanied by 2-APB in an inactivated state.

a, Flowchart of cryo-EM data processing. b, Fourier shell correlation before and after post-processing in RELION3. c, Fourier shell correlation between the refined model and the full map. d, Angular distribution plot of particles used for final reconstruction. e, Cryo-EM density map colored by local resolution. f, Representative cryo-EM density shown as blue mesh contoured at 5.0 σ.

Extended Data Fig. 8 Cryo-EM reconstruction of K169A briefly exposed to 2-APB for 3 minutes in an open conformation.

a, Flowchart of cryo-EM data processing. b, Fourier shell correlation before and after post-processing in RELION3. c, Fourier shell correlation between the refined model and the full map. d, Angular distribution plot of particles used for final reconstruction. e, Cryo-EM density map colored by local resolution. f, Representative cryo-EM density shown as blue mesh contoured at 5.0 σ.

Extended Data Fig. 9 TRPV3 pore structures in distinct functional states.

a–d, The pore structures in the apo closed wild-type TRPV3 channel (a), the apo open K169 mutant (b), the 2-APB-bound open K169A (c), and 2-APB-bound inactivated states (d). Also shown are cryo-EM densities of the normalized cryo-EM maps contoured at 6.0 σ.

Extended Data Fig. 10 Mapping the pore mutations enabling TRPV3-6M activation by RTX.

Two views of the closed TRPV3 pore, highlighting the pore mutations that render TRPV3-6M sensitive to RTX activation. These mutations, including V587L, A606V, F625L, F656I, and F666Y, are shown as stick representation and colored in green.

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Deng, Z., Maksaev, G., Rau, M. et al. Gating of human TRPV3 in a lipid bilayer. Nat Struct Mol Biol 27, 635–644 (2020). https://doi.org/10.1038/s41594-020-0428-2

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