Structural mechanism of TRPV3 channel inhibition by the plant-derived coumarin osthole

Abstract

TRPV3, a representative of the vanilloid subfamily of TRP channels, is predominantly expressed in skin keratinocytes and has been implicated in cutaneous sensation and associated with numerous skin pathologies and cancers. TRPV3 is inhibited by the natural coumarin derivative osthole, an active ingredient of Cnidium monnieri, which has been used in traditional Chinese medicine for the treatment of a variety of human diseases. However, the structural basis of channel inhibition by osthole has remained elusive. Here we present cryo-EM structures of TRPV3 in complex with osthole, revealing two types of osthole binding sites in the transmembrane region of TRPV3 that coincide with the binding sites of agonist 2-APB. Osthole binding converts the channel pore into a previously unidentified conformation with a widely open selectivity filter and closed intracellular gate. Our structures provide insight into competitive inhibition of TRPV3 by osthole and can serve as a template for the design of osthole chemistry-inspired drugs targeting TRPV3-associated diseases.

Keywords: TRP channels; competitive inhibitor; cryo-EM; osthole; single-channel recordings.

Figure 1. Osthole inhibition and cryo‐EM

1. A

   Representative single‐channel recordings at 80 mV membrane potential from mTRPV3 incorporated into a planar lipid bilayer, in response to 5 μM 2‐APB alone, or in combination with 5, 25, or 50 μM osthole.

2. B

   mTRPV3 open probability (P_o ) at 80 mV in the presence of agonist 2‐APB and inhibitor osthole (0 vs. 5 μM osthole, ****P = 0.27 × 10⁻⁵; vs. 25 μM osthole ****P = 0.79 × 10⁻⁵; vs. 50 μM osthole ****P = 0.7 × 10⁻⁵, n = 10 independent experiments). Shown are also the chemical structures of 2‐APB and osthole. Statistical significance was calculated using one‐way ANOVA followed by Fisher’s least significant difference test. The data presented are mean ± SEM.

3. C, D

   3D cryo‐EM reconstruction of mTRPV3‐Y564A_Osthole viewed from the side (C) or top (D), with TRPV3 subunits colored in green, yellow, purple, and cyan. Osthole densities at sites 2 and 3 are colored in red and labeled.

Figure EV1. Comparison of mTRPV3 and mTRPV3‐Y564A single‐channel activity and osthole inhibition

1. Representative single‐channel recordings at 80 mV membrane potential from mTRPV3‐Y564A incorporated into a planar lipid bilayer, in response to 5 μM 2‐APB alone or in combination with 50 μM osthole.

2. Open probability for mTRPV3 and mTRPV3‐Y564A at 80 mV in the presence of agonist 2‐APB and osthole (0 vs. 50 μM osthole: ****P = 0.7 × 10⁻⁵ for mTRPV3 and ****P = 0.4 × 10⁻⁹ for mTRPV3‐Y564A, n = 9 independent experiments). Statistical significance was calculated using one‐way ANOVA followed by Fisher’s least significant difference test. The data presented are mean ± SEM.

Figure EV2. Overview of cryo‐EM data for mTRPV3‐Y564A

1. A–F

   Representative micrographs with example particles circled in red (A, D), reference‐free 2D class averages (B, E), and Euler angle distribution of particles contributing to the final reconstructions with larger red cylinders representing orientations comprising more particles (C, F) for mTRPV3‐Y564A_osthole (A–C) and mTRPV3‐Y564A_2APB+osthole (D–F).

Figure EV3

3D reconstruction workflow for mTRPV3‐Y564A_osthole

Figure EV4. Resolution of mTRPV3‐Y564A cryo‐EM reconstructions

1. A–D

   FSC curves (A‐B) and the local resolution presented as coloring of the cryo‐EM maps (C‐D) for mTRPV3‐Y564A_osthole (A, C) and mTRPV3‐Y564A_2APB+osthole (B, D).

Figure 2. Structure and osthole binding sites

1. A, B

   mTRPV3‐Y564A_Osthole structure viewed from the side (A) or top (B), with TRPV3 subunits colored in green, yellow, purple and cyan. Red mesh shows cryo‐EM densities for osthole.

2. C, D

   Close‐up views of site 2 (C) and site 3 (D), with red mesh showing densities for osthole. Osthole molecules and residues surrounding them are shown as sticks. Positions of residue substitutions in functional experiments (E) are labeled in red.

3. E

   Dose–response curves for inhibition of wild‐type and mutant TRPV3 channels by osthole. The changes in the fluorescence intensity ratio at 340 and 380 nm (F ₃₄₀/F ₃₈₀) evoked by addition of 200 μM (wild type, R487A, Y540A) or 25 μM (Y564A) of 2‐APB or 5 mM of camphor (H426A) after pre‐incubation with various concentrations of osthole were normalized to their maximal values in the absence of osthole. Data points are mean ± SEM. Curves through the points are logistic equation fits, with the mean ± SEM values of the half‐maximal inhibitory concentration (IC₅₀ ) and the Hill coefficient (n _Hill), 20.5 ± 0.5 μM and n _Hill = 1.84 ± 0.14 (n = 4 technical replicates) for mTRPV3, 44.3 ± 7.4 μM and n _Hill = 0.76 ± 0.11 (n = 4 technical replicates) for mTRPV3‐Y540A, 54.3 ± 1.5 μM and n _Hill = 1.66 ± 0.07 (n = 3 technical replicates) for mTRPV3‐R487A, 88.2 ± 11.0 μM and n _Hill = 0.86 ± 0.09 (n = 8 technical replicates) for mTRPV3‐Y564A, and 141 ± 5 μM and n _Hill = 2.44 ± 0.21 (n = 3 technical replicates) for mTRPV3‐H426A.

4. F

   Double logarithmic Schild plot for 2‐APB concentration dependencies of TRPV3 activation in the presence of 10 µM (green squares, n = 6 technical replicates) and 30 µM (red circles, n = 6 technical replicates) osthole. Data points are mean ± SEM. Lines are linear fits.

Figure EV5. Comparison of mTRPV3‐Y564A_osthole with mTRPV3‐Y564A_{2APB‐osthole} and mTRPV3 structures and pore radii

1. A, B

   Superposition of mTRPV3‐Y564A_osthole (green) and mTRPV3‐Y564A_{2APB‐osthole} (purple) viewed parallel to the membrane (A) and extracellularly (B) with the osthole molecules shown as space‐filling models.

2. C

   Superposition of the pore‐forming S6 and P‐loop domains with the pore‐lining residues shown as sticks.

3. D

   Superposition of a single pore‐forming domain in the closed apo (blue) and antagonist osthole‐bound (green) TRPV3. Movement of the N‐terminal end of the TRP helix and P helix in mTRPV3 compared to mTRPV3‐Y564A_osthole is indicated by green arrows. The kink in S5 of mTRPV3‐Y564A_osthole is indicated.

4. E

   Superposition of the pore‐forming S6 and P‐loop domains with the pore‐lining residues shown as sticks.

5. F

   Pore radius for mTRPV3 (blue), mTRPV3‐Y564A_2APB (orange), mTRPV3‐Y564A_osthole (green), and mTRPV3‐Y564A_2APB+osthole (purple). The vertical dashed line denotes the radius of a water molecule, 1.4 Å.

Figure 3. Comparison of 2‐APB‐bound open and osthole‐bound closed TRPV3 structures

1. A, B

   Superposition of transmembrane domains in mTRPV3‐Y564A_2APB (orange) and mTRPV3‐Y564A_osthole (green) viewed parallel to the membrane (A) and extracellularly (B), with molecules of 2‐APB and osthole shown as space‐filling models. In (A), only two of four TRPV3 subunits are shown, with the front and back subunits omitted for clarity. In (B), rotation of the pore‐forming domains in mTRPV3‐Y564A_osthole compared to mTRPV3‐Y564A_2APB is indicated by green arrows.

2. C

   Superposition of a single pore‐forming domain in mTRPV3‐Y564A_2APB and mTRPV3‐Y564A_osthole. Movement of TRP and P helices in mTRPV3‐Y564A_osthole compared to mTRPV3‐Y564A_2APB is indicated by green arrows. The region of S6 that undergoes α‐to‐π transition is highlighted in red. The kink in S5 of mTRPV3‐Y564A_osthole is indicated.

3. D, E

   Pore‐forming domains in mTRPV3‐Y564A_2APB and mTRPV3‐Y564A_osthole with residues lining the pore shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profiles are shown as space‐filling models (gray).

Figure 4. Comparison of binding site 2–4 regions in 2‐APB‐ and osthole‐bound structures

1. A–C

   Superposed regions of site 2 (A), 3 (B), and 4 (C) in mTRPV3‐Y564A_2APB (orange) and mTRPV3‐Y564A_osthole (green) structures, with molecules of 2‐APB and osthole shown as ball‐and‐stick models. Residues contributing to ligand binding are shown as sticks. Arrows in C show movement of domains in mTRPV3‐Y564A_osthole compared to mTRPV3‐Y564A_2APB. Residues mutated in functional experiments (Fig 2E) are labeled in red.