Structure and gating mechanism of the transient receptor potential channel TRPV3

Abstract

Transient receptor potential vanilloid subfamily member 3 (TRPV3) channel plays a crucial role in skin physiology and pathophysiology. Mutations in TRPV3 are associated with various skin diseases, including Olmsted syndrome, atopic dermatitis, and rosacea. Here we present the cryo-electron microscopy structures of full-length mouse TRPV3 in the closed apo and agonist-bound open states. The agonist binds three allosteric sites distal to the pore. Channel opening is accompanied by conformational changes in both the outer pore and the intracellular gate. The gate is formed by the pore-lining S6 helices that undergo local α-to-π helical transitions, elongate, rotate, and splay apart in the open state. In the closed state, the shorter S6 segments are entirely α-helical, expose their nonpolar surfaces to the pore, and hydrophobically seal the ion permeation pathway. These findings further illuminate TRP channel activation and can aid in the design of drugs for the treatment of inflammatory skin conditions, itch, and pain.

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Fig. 1: 3D cryo-EM reconstruction and structure of TRPV3 in the apo (closed) state.
Fig. 2: Closed pore of TRPV3.
Fig. 3: Structure of 2-APB-bound TRPV3(Y564A).
Fig. 4: Open pore of TRPV3.
Fig. 5: Structural changes associated with TRPV3 channel opening.
Fig. 6: Comparison of gating rearrangements in different TRPV channels.
Fig. 7: TRPV3 gating mechanism.

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Acknowledgements

We thank H. Kao for computational support, R. Grassucci and F. Acosta-Reyes for assistance with microscope operation, and U. Baxa and T. Edwards for help with data collection. L.L.M. is supported by a National Institutes of Health grant (T32 GM008224). A.I.S. is supported by two NIH grants (R01 CA206573 and R01 NS083660), the Amgen Young Investigator Award, and the Irma T. Hirschl Career Scientist Award. Data were collected at the Frederick National Laboratory for Cancer Research National Cryo-EM Facility (NIH) and at the Simons Electron Microscopy Center and National Resource for Automated Molecular Microscopy (New York Structural Biology Center), supported by grants from the Simons Foundation (349247), NYSTAR, and the NIH (GM103310).

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A.K.S., L.L.M., and A.I.S. designed the project. A.K.S. and L.L.M. carried out protein expression, purification, cryo-EM sample preparation, and data collection. A.K.S., L.L.M., and A.I.S. processed and analyzed the cryo-EM data. A.K.S., L.L.M., and A.I.S. built the models and wrote the manuscript.

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Correspondence to Alexander I. Sobolevsky.

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Supplementary Figure 1 Functional characterization of wild-type and mutant TRPV3.

ac, Representative Fura-2 AM ratiometric fluorescence measurements of calcium uptake for HEK cells expressing wild-type TRPV3 (a), TRPV3(Y564A) (b) and TRPV3(H426A) (c). Changes in fluorescence were recorded in response to applications of 2-APB (ac) at different concentrations (indicated) or 5 mM camphor (c). Arrows indicate the time at which 2-APB or camphor was added. d, Dose–response curves for wild-type and mutant TRPV3 channel activation by 2-APB. For TRPV3(H426A) and TRPV3(R487A), the changes in F340/F380 elicited by the addition of 2-APB were normalized to the maximal change in F340/F380 in response to application of 5 mM camphor. For wild-type and other TRPV3 mutants, the changes in F340/F380 were normalized to their approximated maximal values at saturating concentrations of 2-APB. Curves through the points are fits with the logistic equation, with the mean ± s.e.m. values of the half-maximal effective concentration (EC50), 27.4 ± 4.5 μM (n = 4) for TRPV3, 1.35 ± 0.07 μM (n = 3) for TRPV3(Y564A), 33.1 ± 2.6 μM (n = 3) for TRPV3(H426A-Y564A), 97.8 ± 14.7 μM (n = 3) for TRPV3(Y540A), 147 ± 29 μM (n = 3) for TRPV3(Q483A) and 460 ± 20 μM (n = 3) for TRPV3(R487A). Each data point represents the average and s.e.m. of three independent measurements; for TRPV3(Y564A), the error bars were smaller than the symbol size

Supplementary Figure 2 Overview of single-particle cryo-EM for TRPV3.

a, Example cryo-EM micrograph for TRPV3 with example particles circled in red. b, Reference-free 2D class averages illustrating different particle orientations. c, Local resolution mapped on TRPV3 density at a 0.010 threshold level (UCSF Chimera) calculated using ResMap (Bioinformatics 21, 3327–3328, 2005) and two unfiltered half maps, with the highest resolution observed for the channel core. d, Orientation distribution of particles that contributed to the final 4.3-Å reconstruction; longer red rods represent orientations that comprise more particles. e, FSC curve calculated between half maps. e, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map 1 was used for refinement with PHENIX (Acta Crystallogr. D Biol. Crystallogr. 68, 352–367, 2012)) and the unfiltered summed map

Supplementary Figure 3 Cryo-EM density of TRPV3.

a, Cryo-EM density (blue mesh) at 4σ for a single TRPV3 subunit; the structure, shown as a ribbon, is colored according to domain. bh, Fragments of the TRPV3 transmembrane and C-terminal domains with the corresponding cryo-EM density shown as a blue mesh at 4σ

Supplementary Figure 4 Structural comparison of TRPV channels.

aj, Comparison of the structures of mouse TRPV3 in the closed state (yellow) and rat TRPV1 in the closed state (red; PDB 3J5P) (a,f), rabbit TRPV2 in a putative desensitized state (teal; PDB 5AN8) (b,g), Xenopus tropicalis TRPV4 in the closed state (purple; PDB 6BBJ) (c,h), rabbit TRPV5 in the closed state (green; PDB 6B5V) (d,i) and human TRPV6 in the open state (pink; PDB 6BO8) (e,j) viewed intracellularly (ae) or parallel to the membrane (fj). In fj, only the TMD and the TRP helix from one subunit are shown. The structures are aligned based on their pore domains

Supplementary Figure 5 Sequence alignment of mouse TRPV channels.

Above the sequences, α-helices and β-strands are depicted as cylinders and arrows, respectively. The *, ¥ and Ω symbols indicate residues contributing to sites 2, 3 and 4, respectively. The location of the selectivity filter (S.F.) is indicated by a red box

Supplementary Figure 6 Comparison of the pore region density in different cryo-EM reconstructions.

ac, Fragments of cryo-EM density for the S6 and TRP helices in TRPV3 (a), TRPV3(Y564A)2-APB (b) and TRPV32-APB (c) viewed intracellularly. The density for M677, which forms the gate in the closed state, is indicated by arrows. df, Fragments of cryo-EM density for the S5, S6 and TRP helices in TRPV3 (d), TRPV3(Y564A)2-APB (e) and TRPV3(Y564A) (f), all filtered to the same (6.5–Å) resolution

Supplementary Figure 7 Overview of single-particle cryo-EM for TRPV3(Y564A)2-APB.

a, Example cryo-EM micrograph for TRPV3(Y564A)2-APB with example particles circled in red. b, Reference-free 2D class averages illustrating different particle orientations. c, Local resolution mapped on TRPV3(Y564A)2-APB density at a 0.012 threshold level (UCSF Chimera) calculated using ResMap (Bioinformatics 21, 3327–3328, 2005) and two unfiltered half maps, with the highest resolution observed for the channel core. d, Orientation distribution of particles that contributed to the final 4.1-Å reconstruction; longer red rods represent orientations that comprise more particles. e, FSC curve calculated between half maps. e, Cross-validation FSC curves for the refined model versus unfiltered half maps (only half map 1 was used for refinement with PHENIX (Acta Crystallogr. D Biol. Crystallogr. 68, 352–367, 2012)) and the unfiltered summed map

Supplementary Figure 8 Comparison of sites 2–4 in TRPV3 and TRPV3(Y564A)2-APB.

a,b, Extracellular part of the S1–S4 domain in TRPV3 (a) and TRPV3(Y564A)2-APB (b) viewed parallel to the membrane with cryo-EM density shown as blue mesh at 4σ. Note that the S1–S2 loop occupies site 4 in TRPV3 (a). In TRPV3(Y564A)2-APB (b), this loop is displaced upward and site 4 is occupied by a nonprotein density into which a 2-APB molecule (green sticks) can be easily fit. c, Superposition of the region in one subunit encompassing sites 2 and 3 of TRPV3 (blue) and TRPV3(Y564A)2-APB (orange). 2-APB molecules and residues forming sites 2 and 3 are shown as sticks. Note that, while S5 and S6 show significant rearrangements during gating, sites 2 and 3 remain nearly intact

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Singh, A.K., McGoldrick, L.L. & Sobolevsky, A.I. Structure and gating mechanism of the transient receptor potential channel TRPV3. Nat Struct Mol Biol 25, 805–813 (2018). https://doi.org/10.1038/s41594-018-0108-7

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