Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (consisting of transmembrane segments 1–4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels.
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Electron Microscopy Data Bank
Protein Data Bank
3D cryo-EM density maps of TRPV1 complexes without low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-5776 (TRPV1–RTX/DkTx) and EMD-5777 (TRPV1–capsaicin). The coordinates of atomic models of TRPV1 in these two states have been deposited in the Protein Data Bank under the accession numbers 3J5Q and 3J5R.
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We thank X. Li for assistant in data acquisition using TF30 Polara and K2 Summit camera, and J.P Armache, C. Bohlen, J. Cordero-Morales and J. Osteen for discussion and reading of the manuscript. This work was supported by grants from the National Institutes of Health (R01GM098672 and S10RR026814 to Y.C. and R01NS065071 and R01NS047723 to D. J.), the National Science Foundation (DBI-0960271 to D. Agard and Y.C.) and the University of California, San Francisco Program for Breakthrough Biomedical Research (Y.C.). E.C. was a fellow of the Damon Runyon Cancer Research Foundation.
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Extended data figures and tables
a, b, Fourier transform (a) of a representative image (b). c, Two dimensional (2D) class averages of cryo-EM particles. d, Enlarged view of three representative 2D class averages. Arrows indicate DkTx densities near the channel pore. e, Gold-standard FSC curve (red) of the final 3D reconstruction, marked with the resolutions that correspond to FSC = 0.5 and 0.143. The FSC curve between the final map and that calculated from the atomic model is shown in blue. f, Euler angle distribution of all particles used for calculating the final 3D reconstruction. The sizes of balls represent the number of particles. The accuracy of rotation is 5.213°, as reported by RELION.
a–d, Four different views of the 3D reconstruction low-pass filtered at 6 Å and amplified by a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–RTX/DkTx complex (toxin is shown in magenta) built as described in Methods. e, f, Two views of the 3D reconstruction displayed at two different isosurface levels (high in yellow and low in grey). At the low isosurface level, the belt-shaped density of amphipols is visible with a thickness of ∼30 Å. DkTx-related densities are also clearly visible, including the linker peptide that connects the toxin’s two inhibitor cysteine knot (ICK) moieties, as noted.
a, b, Fourier transform (a) of a representative image (b). c, 2D class averages of cryo-EM particles. d, Enlarged view of three representative 2D class averages. e, Gold-standard FSC curve (red) of the final 3D reconstruction, marked with the resolutions that correspond to FSC = 0.5 and 0.143. The FSC curve between the final map and that calculated from the atomic model is shown in blue. f, Euler angle distribution of all particles used for calculating the final 3D reconstruction. The sizes of balls represent the number of particles. The accuracy of rotation is 4.989°, as reported by RELION.
Extended Data Figure 4 3D reconstruction of TRPV1–capsaicin complex low-pass filtered at 6 Å resolution.
a–d, Four different views of the 3D reconstruction low-pass filtered at 6 Å and amplified by a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–capsaicin complex built as described in Methods. e, f, Two views of the 3D reconstruction displayed at two different isosurface levels (high in yellow and low in grey). At the low isosurface level, the belt-shaped density of amphipols is visible with a thickness of ∼30 Å.
Extended Data Figure 5 3D reconstruction of TRPV1–RTX/DkTx complex low-pass filtered at 3.8 Å resolution.
a–d, Four different views of the 3D reconstruction low-pass filtered at 3.8 Å with a temperature factor of −100 Å2, fitted with de novo atomic model of TRPV1–RTX/DkTx complex (toxin is shown in magenta and indicated by arrows) built as described in Methods.
Extended Data Figure 6 3D reconstruction of TRPV1–capsaicin complex low-pass filtered at 4.2 Å resolution.
a–d, Four different views of the 3D reconstruction low-pass filtered to 4.2 Å with a temperature factor of −150 Å2, fitted with de novo atomic model of TRPV1–capsaicin complex built as described in Methods.
a, Non-protein associated densities in the region adjacent to S3–S4 transmembrane helices observed in 3D density maps of the apo TRPV1 structure (blue, 3.4 Å, −100 Å2) or TRPV1 in complex with RTX/DkTx (red, 3.8 Å, −150 Å2) or capsaicin (yellow, 3.9 Å, −150 Å2), as indicated. b, Density of bound capsaicin (blue) viewed from the side (left) and top-down (that is, from the extracellular face; right). Density is also observed in the apo-channel structure (purple), possibly representing an endogenous lipid or other small hydrophobic molecule. All maps were low-pass filtered to 4.5 Å with a temperature factor of −200 Å2, normalized and displayed at the same sigma level (8σ).
a–c, Density maps for pore region for two diagonally opposed monomers superimposed onto their atomic models (top). Distances between specific side-chain atoms along the pore are also indicated (bottom). d, Superimposed top-down view of apo and capsaicin-bound TRPV1 outer pore regions (orange and green, respectively). e, Density map of selectivity filter in the capsaicin-bound TRPV1 structure. The distance between carbonyl oxygens of diagonally opposed G643 residues (4.6 Å) does not differ from that of the apo structure (4.6 Å).
a, Superimposition of apo and RTX/DkTx-bound TRPV1 structures (orange and blue, respectively) from top-down view. S1–S4 domain (outlined in dashed box) shows near-complete overlap. b, Same comparison for apo and capsaicin-bound channel structures (orange and green, respectively). c, d, Superimposition of transmembrane core of apo versus RTX/DkTx- or capsaicin-bound TRPV1 structures (orange, blue and green, respectively). Dashed box denotes region highlighted in Fig. 6.
a, Pore helix and upper half of S5 helix are in close proximity and appear to be physically coupled, representing a potential mechanisms for allosteric coupling between upper and lower gates. Several residues on both helices are rendered as ball-and-stick to highlight close apposition. b, Downward tilt of pore helix away from the central pore is associated with movement of the S5 helix in RTX/DkTx structure (left). This structural arrangement is not observed in capsaicin-bound structure (right). c, Model depicting two gate mechanism of TRPV1 activation. Two main constriction points at the selectivity filter (1) and lower gate (2) block ion permeation in the apo, closed state (top left). Some pharmacological agents (for example, protons or spider toxins; gold hexagon) target the outer pore region of the channel to open or stabilize the conductive conformation of the selectivity filter (top right). Arrow denotes proposed coupling between the pore helix and S5. Small vanilloid ligands (for example, RTX and capsaicin; red ellipse) bind within a hydrophobic pocket formed by the S3–S4 helices, the S4–S5 linker and the pore module, eliciting conformational changes that expand the lower gate (bottom left). Arrows indicate proposed coupling between S4–S5 helix, S6 and TRP domain. Full expansion of the ion permeation pathway and ion conduction is achieved when both upper and lower gates are opened (bottom right). Pharmacological and mutagenesis data suggest that these gates are allosterically coupled.
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Cao, E., Liao, M., Cheng, Y. et al. TRPV1 structures in distinct conformations reveal activation mechanisms. Nature 504, 113–118 (2013). https://doi.org/10.1038/nature12823
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