TRPV3 activation by different agonists accompanied by lipid dissociation from the vanilloid site

Abstract

TRPV3 represents both temperature- and ligand-activated transient receptor potential (TRP) channel. Physiologically relevant opening of TRPV3 channels by heat has been captured structurally, while opening by agonists has only been observed in structures of mutant channels. Here, we present cryo-EM structures that illuminate opening and inactivation of wild-type human TRPV3 in response to binding of two types of agonists: either the natural cannabinoid tetrahydrocannabivarin (THCV) or synthetic agonist 2-aminoethoxydiphenylborane (2-APB). We found that THCV binds to the vanilloid site, while 2-APB binds to the S1-S4 base and ARD-TMD linker sites. Despite binding to distally located sites, both agonists induce similar pore opening and cause dissociation of a lipid that occupies the vanilloid site in their absence. Our results uncover different but converging allosteric pathways through which small-molecule agonists activate TRPV3 and provide a framework for drug design and understanding the role of lipids in ion channel function.

Fig. 1.. Apo-state structure in cNW30 nanodisc and TRPV3 activation by agonists.

(A) Cryo-EM density map of human TRPV3 in the closed, apo state at 2.46-Å resolution, viewed parallel to the membrane. Lipid densities are colored brown. The semitransparent surface represents the lipid nanodisc density. (B) Structural model of TRPV3_apo viewed parallel to the membrane. One of the four subunits is colored green, while the others are colored gray. (C and D) Representative ratiometric Fura-2AM–based fluorescence measurements of changes in intracellular Ca²⁺ for human embryonic kidney (HEK) 293S GnTI⁻ cells expressing wild-type human TRPV3. Changes in the fluorescence intensity ratio at 340 and 380 nm (F₃₄₀/F₃₈₀) were monitored in response to application of agonists THCV (C) or 2-APB (D) at different concentrations. (E and F) Concentration dependencies of TRPV3 activation by THCV (E) and 2-APB (F). Changes in the F₃₄₀/F₃₈₀ ratio were normalized to its maximal value at saturating agonist concentrations and fitted with the logistic equation (black lines), with half-maximal effective concentration (EC₅₀) = 6.1 ± 0.5 μM and n_Hill = 1.63 ± 0.13 for THCV (E; n = 3 independent experiments) and EC₅₀ = 93.0 ± 5.0 μM and n_Hill = 1.63 ± 0.11 for 2-APB (F; n = 6 independent experiments). Data are means ± SEM. The insets show chemical structures of THCV (E) and 2-APB (F). Source data are provided.

Fig. 2.. Binding of THCV to the vanilloid site.

(A) Structure of OPEN_THCV viewed parallel to the membrane (left) and extracellularly (right), with subunits colored light green, yellow, pink, and blue. The molecules of THCV are shown as space-filling models (dark green). Boxed is the region expanded in (B). (B) Close-up view of the vanilloid site in OPEN_THCV structure, with the molecule of THCV (dark green) and side chains contributing to THCV binding shown in sticks. (C to E) Close-up views of the vanilloid site in OPEN_THCV (C), INACT_THCV (D), and their superposition (E), with cryo-EM densities for the protein (semitransparent surface) and THCV (green mesh) shown in (C) and (D). (F) Concentration-response curves for THCV activation of wild-type (WT) TRPV3 (black, n = 3) as well as L557A (cyan, n = 3), L563A (pink, n = 3), A560L (purple, n = 3), A560M (orange, n = 3), and N561A (green, n = 3) mutant channels. Curves through the data points are the fits with the logistic equation. The EC₅₀ values are provided in table S2. n, the number of independent measurements. Data are means ± SEM.

Fig. 3.. Closed apo and THCV-bound open and inactivated states.

(A to C) Structures of APO (A), OPEN_THCV (B), and INACT_THCV (C), viewed parallel to the membrane, with the N and C termini colored blue and pink, respectively, and the molecules of THCV (dark green) shown as space-filling models. (D to F) Pore-forming domains in APO (D), OPEN_THCV (E), and INACT_THCV (F), with residues contributing to pore lining shown as sticks. Only two of the four subunits are shown, with the front and back subunits omitted for clarity. The pore profile is shown as a space-filling model (gray). The region that undergoes the α-to-π transition in S6 is highlighted in pink. (G to I) Intracellular view of the S6 bundle crossing in APO (G), OPEN_THCV (H), and INACT_THCV (I), with the surface shown in the corresponding color. (J to L) Interface between the neighboring subunits in APO (A), OPEN_THCV (B), and INACT_THCV (C), with the C terminus (pink) adapting coiled [(J) and (L)] or helical (K) conformations and N terminus (blue) appearing in the open state (K).

Fig. 4.. Binding of 2-APB to the S1-S4 base and ARD-TMD linker sites.

(A and B) Structure of OPEN_2-APB viewed parallel to the membrane (A) and extracellularly (B), with subunits colored light green, yellow, pink, and blue. The molecules of 2-APB are shown as space-filling models (purple). Black rectangles indicate the regions expanded in (C) and (D). [(C) and (D)] Close-up views of the S1-S4 base (C) and ARD-TMD linker (D) sites in OPEN_2-APB structure, with the molecule of 2-APB (purple) and side chains contributing to 2-APB binding shown in sticks. (E and F) Concentration-response curves for 2-APB activation of wild-type (black) and mutant (other colors) TRPV3 channels. Curves through the data points are the fits with the logistic equation. The EC₅₀ values are provided in table S2. The number of independent measurements, n, equals to 6 for the wild-type channels and H426A mutant and equals to 3 for all other mutants. Data are means ± SEM. (G to L) Close-up views of the S1-S4 base [(G) and (I)] and ARD-TMD linker [(H) and (J)] sites in OPEN_2-APB [(G) and (H)], INACT_2-APB [(I) and (J)], and their superposition [(K) and (L)], with cryo-EM densities for 2-APB (purple mesh) shown in (G) to (J).

Fig. 5.. THCV and 2-APB bind to different sites but induce similar pore conformations.

(A and B) Superpositions of the TMDs in OPEN_2-APB (yellow) and OPEN_THCV (orange) (A) as well as INACT_2-APB (gray) and INACT_THCV (light blue) (B), viewed parallel to the membrane, with molecules of 2-APB (purple) and THCV (dark green) shown as space-filling models. Arrows show movement of domains in INACT_2-APB relative to INACT_THCV. (C and D) Superpositions of the pore-forming domains in OPEN_2-APB (yellow) and OPEN_THCV (orange) (C) as well as INACT_2-APB (gray) and INACT_THCV (light blue) (D), with residues contributing to pore lining shown as sticks. Only two of the four subunits are shown, with the front and back subunits omitted for clarity. The region that undergoes the α-to-π transition in S6 is highlighted in pink. (E and F) Superpositions of the intracellular pore entry in OPEN_2-APB (yellow) and OPEN_THCV (orange) (E) as well as INACT_2-APB (gray) and INACT_THCV (light blue) (F), with residues contributing to the gate shown as sticks.

Fig. 6.. Dissociation of the vanilloid-site lipid upon agonist binding.

(A) Cryo-EM density for TRPV3_apo (green), with the density for the vanilloid-site lipid colored in pink. Boxed is the region expanded in (B). (B) Close-up view of the vanilloid site in APO, with density for protein shown in green and resident lipid and its density in pink. (C and D) Close-up views of the vanilloid site in OPEN_THCV (C) and INACT_THCV (D), with density for protein shown in orange (C) and blue (D) and the molecule of THCV and its density in dark green. (E and F) Close-up views of the vanilloid site in OPEN_2-APB (E) and INACT_2-APB (F), with density for protein shown in yellow (E) and gray (F). The lack of the vanilloid-site lipid in (C) to (F) is indicated with asterisks.

Fig. 7.. Mechanism of agonist-induced TRPV3 activation.

Schematic representation of TRPV3 in the closed, open, and inactivated states, with molecules of THCV and 2-APB agonists and vanilloid-site lipid shown in orange, yellow, and pink colors, respectively. Arrows indicate the putative interstate transitions. Green circles represent cations. The pore-forming regions (violet) and TRP helix (blue) are shown as cylinders. The final place of the dislocated vanilloid lipid is chosen arbitrary. Note that APO and OPEN conformations have a π-bulge in the middle of S6, while S6 in INACT is entirely α-helical.