Abstract
Transient receptor potential (TRP) channels are a large, eukaryotic ion channel superfamily that control diverse physiological functions, and therefore are attractive drug targets1,2,3,4,5. More than 210 structures from more than 20 different TRP channels have been determined, and all are tetramers4. Despite this wealth of structures, many aspects concerning TRPV channels remain poorly understood, including the pore-dilation phenomenon, whereby prolonged activation leads to increased conductance, permeability to large ions and loss of rectification6,7. Here, we used high-speed atomic force microscopy (HS-AFM) to analyse membrane-embedded TRPV3 at the single-molecule level and discovered a pentameric state. HS-AFM dynamic imaging revealed transience and reversibility of the pentamer in dynamic equilibrium with the canonical tetramer through membrane diffusive protomer exchange. The pentamer population increased upon diphenylboronic anhydride (DPBA) addition, an agonist that has been shown to induce TRPV3 pore dilation. On the basis of these findings, we designed a protein production and data analysis pipeline that resulted in a cryogenic-electron microscopy structure of the TRPV3 pentamer, showing an enlarged pore compared to the tetramer. The slow kinetics to enter and exit the pentameric state, the increased pentamer formation upon DPBA addition and the enlarged pore indicate that the pentamer represents the structural correlate of pore dilation. We thus show membrane diffusive protomer exchange as an additional mechanism for structural changes and conformational variability. Overall, we provide structural evidence for a non-canonical pentameric TRP-channel assembly, laying the foundation for new directions in TRP channel research.
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Data availability
All data and materials to draw the conclusions in this paper are presented in the main text, figures and the extended data figures and supplementary videos. The cryo-EM maps of the TRPV3 tetramer and pentamer have been deposited in the Electron Microscopy Data Bank with accession codes EMD-40181 and EMD-40183, respectively, and their structural models have been deposited in the PDB with accession codes 8GKA and 8GKG, respectively (Extended Data Table 1). Further data can be received from the corresponding author upon reasonable request.
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Acknowledgements
We thank A. Accardi and J. Dittman for important discussions. Negative-stain EM data were collected in the Electron Microscopy & Histology services of the Weill Cornell Medicine Microscopy & Image Analysis Core using a transmission electron microscope purchased with funds from an National Institutes of Health (NIH) Shared Instrumentation grant (no. S10RR027699) for Shared Resources. Cryo-EM data were collected at the Simons Electron Microscopy Center at the New York Structural Biology Center, with support from the Simons Foundation (grant no. SF349247). Work in the Scheuring laboratory is partly supported by grants from the NIH, National Center for Complementary and Integrative Health (NCCIH), grant no. DP1AT010874 (to S.S.) and National Institute of Neurological Disorders and Stroke (NINDS), grant no. R01NS110790 (to S.S.). Work in the Yuan laboratory is partly supported by grant no. NIH NINDS R01NS099341 (to P.Y.). Work in the Nimigean laboratory is partly supported by grant no. NIH NIMGS R01 GM088352 (to C.M.N.) and grant no. NIH NIMGS F32 GM145091 (to E.D.K.). S.L. is an awardee of the Weizmann Institute of Science Women’s Postdoctoral Career Development Award.
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S.L. and S.S. designed the study. J.Z. and P.Y. expressed and purified protein from P. pastoris cells. S.L., J.M.B. and E.D.K. expressed and purified protein from HEK GnTI− cells. S.L. and J.M.B. reconstituted protein. S.L. performed negative-stain EM imaging. S.L., E.D.K. and C.M.N. performed and analysed electrophysiology measurements. S.L. and J.M.B. performed HS-AFM imaging. S.L. and Y.J. performed HS-AFM data analysis. S.L. and J.M.B. performed nanoDSF experiments and analysis. S.L. and J.M.B. performed cryo-EM sample preparation and data collection. S.L., E.D.K. and N.P. analysed single-particle cryo-EM data. S.L. and S.S. performed channel structure analysis. Y.J. performed and analysed oligomer simulation. S.L. and S.S. wrote the paper. All authors edited the manuscript. S.S. supervised the project.
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Extended data figures and tables
Extended Data Fig. 1 TRP-channel structures.
Representative structures (out of >210 structures) of the 23 TRP-channels solved so far. All structures are tetramers, with the four subunits coloured in wheat, green, purple and yellow. Each structure is depicted in surface representation, shown from the intracellular (top) and side (bottom) views. The question mark in the crTRP1 panel signifies that the subfamily to which crTRP1 belongs is yet unknown.
Extended Data Fig. 2 Single-channel recordings of TRPV3.
(a) and (b) Representative single-channel recordings of TRPV3 in the absence (a) and presence (b) of 100 μM DPBA, at −50 and 50 mV. (c) Since-channel current-voltage (IV) curves of TRPV3 obtained from −100 to 100 mV, in the absence and presence of 100 μM DPBA. (d) Single-channel open probabilities determined from recordings obtained at −50 mV, in the absence and presence of 100 μM DPBA. Open probability values (0.27 ± 0.01 and 0.78 ± 0.05 respectively) were derived as the mean values +/− s.e.m. from n ≥ 3 independent experiments (circles). Statistical significance was assessed with the one-tailed Welch’s T-test, yielding a significant (p-value = 0.0007) increase in open channel probability following DPBA addition. All recordings were performed on TRPV3 channels from one purification following the same protein expression and purification protocol as for the cryo-EM analysis, and reconstituted following the same protocol as for the HS-AFM analysis though at higher lipid-to-protein ratio (LPR between 5 and 20 for electrophysiology recordings vs. LPR between 0.5 and 2.5 for HS-AFM experiments). *** p-value < 0.005.
Extended Data Fig. 3 The TRPV3 tetramer and pentamer are reversible.
(a) to (f) Tetramer to pentamer transitions. (g) to (k) Pentamer to tetramer transitions. (l) Tetramer-pentamer-tetramer transition. (m) Pentamer-tetramer-pentamer-tetramer-pentamer-tetramer transition. White arrowheads indicate the occasionally observed monomers ‘attacking’ and inserting into tetramers to yield pentamers, and the observed monomers dissociating from pentamers to yield tetramers.
Extended Data Fig. 4 TRPV3 tetramers can breakup into fragments and reform.
(a) TRPV3 tetramers and pentamers coexist alongside TRPV3 ≤ 3 protomer fragments. Grey arrowheads indicate monomer (1), dimer (2), and trimer (3) fragments. (b) and (c) TRPV3 tetramer breakups into trimer and dimer. (d) TRPV3 fragments form a stable tetramer, which then breaks apart.
Extended Data Fig. 5 Workflow for cryo-EM reconstruction of the TRPV3 tetramer and pentamer.
Flowchart for the cryo-EM data processing, particle picking, classification, and reconstruction, enabling map reconstruction of the tetramer at 2.55Å resolution and for the pentamer at 4.38Å resolution. Unless otherwise stated, all processing steps were conducted in cryoSPARC version 3.3.2. Dashed lines indicate the inputs used for the iterative cycles of heterogenous refinement.
Extended Data Fig. 6 Cryo-EM density maps of the TRPV3 tetramer and pentamer.
(a) and (b) Cryo-EM reconstructed maps of the TRPV3 tetramer (a) and pentamer (b), colored according to local resolution using a rainbow colour scale. (c) and (d) Representative cryo-EM densities of the tetramer (contour level at 5.5 RMSD) (c) and pentamer (contour level at 4.16 RMSD) (d), at 2.55Å and 4.38Å resolution, respectively (the TMDs in the pentamer map are of ~5.0-5.5 Å resolution, see local resolution color scheme in (b)).
Extended Data Fig. 7 Structural comparison of the TRPV3 tetramer and pentamer cryo-EM structures.
(a) and (b) TRPV3 tetramer (a) and pentamer (b) structures, colored according to domains: ARD in purple, VSLD in yellow, PD in pink, SF in green, TRP helix in wheat, coupling domain in light blue. (c) to (e) Superposition of a pentamer subunit (purple) onto the tetramer subunit (green), aligned with respect to the PD, indicating a hinge-motion in the pentamer monomer by 18º, as manifested by rotation of the ARD (c), VSLD, and TRP helix (d). This hinge-motion enables preservation of the inter-subunit interactions of S5 with S1 and S4, and the SF-SF and S6-S6 interactions (e). Neighboring subunits in gray. Open book graphics of the tetramer (f, green) and pentamer (g, purple) inter-subunit contact areas. Contact areas are colored in pink (in the tetramer) and yellow (in the pentamer).
Extended Data Fig. 8 Model of the reversible transition between the tetrameric and pentameric TRPV3 states.
TRPV3 tetramers may dissociate. Dissociation is favored by activation, due to destabilization of the interprotomer interaction, notably the VSLD-PD domain-swap interface, by helix-intercalating molecules (e.g., capsaicin or LPA in TRPV1, DPBA in TRPV3, temperature). Monomers may ‘attack’ and insert into tetramers to yield pentamers with an estimated ~2.4-fold enlarged pore diameter at the SF. Pentamers, lifetime of ~3 min, are less stable than tetramers due to more fragile VSLD-PD interfaces, and shed subunits to regain the tetrameric state (figure created with BioRender.com).
Extended Data Fig. 9 Simulation of oligomeric state transitions.
(a) Visualization of simulated traces. Each column represents an independent space that can be either empty (0) or occupied by a molecule with a specific oligomeric state (1, 2, 3, 4 or 5). Total space: 5250. Total time step: 2000. Initial setup (time step = 1): 5000 out of 5250 spaces had a value of 4 (tetramers) and 250 out of the 5250 spaces had a value of 0 (empty). (b) and (c) Close-up views of the simulated traces in (a) as indicated by the dashed boxes. 1: Empty → monomer → dimer → trimer → tetramer → pentamer transition. 2: Transitions between tetramer and pentamer states (with short pentamer dwell-times). 3: A long pentamer state event. 4: pentamer → tetramer → trimer → dimer → monomer → empty transition. (d) Time-evolution of oligomer counts. Top panel: Tetramers. Middle panel: Pentamers and lower oligomers (trimer, dimer, and monomer) aggregated. Bottom panel: Trimers, dimers and monomers. (e) Oligomer state dwell-times. Left to right: Lower oligomers (n = 34984), tetramer (n = 278141), and pentamer (n = 331579).
Supplementary information
Supplementary Information
This file contains Supplementary Figs. 1–6, Tables 1–3 and Code.
Supplementary Video 1
Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.80 nm per pixel.
Supplementary Video 2
Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 2 s per frame. Pixel sampling, 0.80 nm per pixel.
Supplementary Video 3
Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.27 nm per pixel.
Supplementary Video 4
Overview HS-AFM video of TRPV3 reconstitution. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.
Supplementary Video 5
Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1.5 s per frame. Pixel sampling, 0.33 nm per pixel.
Supplementary Video 6
Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.50 nm per pixel.
Supplementary Video 7
Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.
Supplementary Video 8
Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 0.5 s per frame. Pixel sampling, 0.48 nm per pixel.
Supplementary Video 9
Overview HS-AFM video of TRPV3 reconstitution revealing several channels with pentameric oligomeric states. Frame rate, 2 s per frame. Pixel sampling, 0.40 nm per pixel.
Supplementary Video 10
High-resolution HS-AFM videos of tetrameric TRPV3 channels. Frame rate, 1 s per frame. Pixel sampling, 0.20 nm per pixel.
Supplementary Video 11
High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 0.3 s per frame. Pixel sampling, 0.12 nm per pixel.
Supplementary Video 12
High-resolution HS-AFM videos of pentameric TRPV3 channels. Frame rate, 1.5 s per frame. Pixel sampling, 0.25 nm per pixel.
Supplementary Video 13
High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 1 s per frame. Pixel sampling, 0.35 nm per pixel.
Supplementary Video 14
High-resolution HS-AFM videos of a tetrameric and pentameric TRPV3 channel. Frame rate, 1 s per frame. Pixel sampling, 0.20 nm per pixel.
Supplementary Video 15
HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 2 s per frame. Pixel sampling, 0.28 nm per pixel.
Supplementary Video 16
HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 2 s per frame. Pixel sampling, 0.67 nm per pixel.
Supplementary Video 17
HS-AFM video of TRPV3 reconstitution revealing a tetramer–pentamer transition. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule tetramer–pentamer transition. Bottom right panel shows the average (t,t + 2) of single-molecule tetramer–pentamer transition. Frame rate, 1 s per frame. Pixel sampling, 0.32 nm per pixel.
Supplementary Video 18
HS-AFM video of TRPV3 reconstitution revealing two pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows single-molecule pentamer–tetramer transitions. Bottom right panel shows the verage (t,t + 2) of single-molecule pentamer–tetramer transitions. Frame rate, 1 s per frame. Pixel sampling, 0.25 nm per pixel.
Supplementary Video 19
HS-AFM video of TRPV3 reconstitution revealing a pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule pentamer–tetramer transition. Frame rate, 1 s per frame. Pixel sampling, 0.35 nm per pixel.
Supplementary Video 20
HS-AFM video of TRPV3 reconstitution revealing a pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule pentamer–tetramer transition. Frame rate, 1 s per frame. Pixel sampling, 0.32 nm per pixel.
Supplementary Video 21
HS-AFM video of TRPV3 reconstitution revealing a complete tetramer–pentamer–tetramer transitions. Left panel shows an overview of TRPV3 reconstitution. Top right panel shows the single-molecule complete tetramer–pentamer–tetramer transition. Bottom right panel shows the average (t,t + 2) of single-molecule complete tetramer–pentamer–tetramer transition. Frame rate, 1.5 s per frame. Pixel sampling, 0.33 nm per pixel.
Supplementary Video 22
Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.
Supplementary Video 23
Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 2 s per frame. Pixel sampling, 0.67 nm per pixel.
Supplementary Video 24
Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.33 nm per pixel.
Supplementary Video 25
Overview HS-AFM video of TRPV3 reconstitution in the presence of 320 μM DPBA, revealing several channels with pentameric oligomeric states. Frame rate, 1 s per frame. Pixel sampling, 0.40 nm per pixel.
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Lansky, S., Betancourt, J.M., Zhang, J. et al. A pentameric TRPV3 channel with a dilated pore. Nature 621, 206–214 (2023). https://doi.org/10.1038/s41586-023-06470-1
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DOI: https://doi.org/10.1038/s41586-023-06470-1
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