Structural mechanism of TRPV3 channel inhibition by the anesthetic dyclonine

Abstract

Skin diseases are common human illnesses that occur in all cultures, at all ages, and affect between 30% and 70% of individuals globally. TRPV3 is a cation-permeable TRP channel predominantly expressed in skin keratinocytes, implicated in cutaneous sensation and associated with numerous skin diseases. TRPV3 is inhibited by the local anesthetic dyclonine, traditionally used for topical applications to relieve pain and itch. However, the structural basis of TRPV3 inhibition by dyclonine has remained elusive. Here we present a cryo-EM structure of a TRPV3-dyclonine complex that reveals binding of the inhibitor in the portals which connect the membrane environment surrounding the channel to the central cavity of the channel pore. We propose a mechanism of TRPV3 inhibition in which dyclonine molecules stick out into the channel pore, creating a barrier for ion conductance. The allosteric binding site of dyclonine can serve as a template for the design of new TRPV3-targeting drugs.

Fig. 1. Dyclonine inhibition and cryo-EM.

a Representative ratiometric Fura-2-based fluorescence measurements of changes in intracellular Ca²⁺ for HEK 293 GnTI⁻ cells expressing wild-type mouse TRPV3. The changes in the fluorescence intensity ratio at 340 and 380 nm (F₃₄₀/F₃₈₀) were monitored in response to the addition of 200 µM 2-APB (arrow) after pre-incubation of cells with various concentrations of dyclonine. The experiment was repeated independently seven times with similar results. b Dose–response curve for TRPV3 inhibition by dyclonine. The changes in the F₃₄₀/F₃₈₀ ratio were normalized to its maximal value in the absence of dyclonine and fitted by the logistic equation (red line), with IC₅₀ = 29.8 ± 5.3 µM and n_Hill = 1.61 ± 0.27 (n = 7 independent experiments). The values are mean ± SEM. The inset shows the chemical structure of dyclonine. Source data are provided as a Source Data file. c, d 3D cryo-EM reconstruction of TRPV3_Dyc viewed from the side (c) or top (d), with subunits colored green, yellow, purple, and cyan. e, f The same cryo-EM density as in (c, d), but cut off along the dashed lines in (d) and (c), respectively. Putative densities for dyclonine and sodium ions are shown in red and green.

Fig. 2. TRPV3_Dyc structure and dyclonine binding sites.

a, b TRPV3_Dyc structure viewed from the side (a) or top (b), with subunits colored green, yellow, purple, and cyan. Red mesh shows densities for dyclonine. Dyclonine molecules are shown as sticks. c, d Close-up views of all four dyclonine binding sites (c) and only one of them (d). e Representative ratiometric fluorescence measurements of changes in intracellular Ca²⁺ for HEK 293 GnTI⁻ cells expressing F666A mutant TRPV3 channels. The changes in the fluorescence intensity ratio F₃₄₀/F₃₈₀ were monitored in response to the addition of 200 µM 2-APB (arrow) after pre-incubation of cells with various concentrations of dyclonine. The experiment was repeated independently three times with similar results. f Dose–response curves for inhibition of wild-type and mutant TRPV3 channels by dyclonine. The changes in the fluorescence intensity ratio F₃₄₀/F₃₈₀ evoked by addition of 200 µM 2-APB after pre-incubation with various concentrations of dyclonine were normalized to their maximal values in the absence of dyclonine. Curves through the points are logistic equation fits, with IC₅₀ = 29.8 ± 5.3 µM and n_Hill = 1.61 ± 0.27 (n = 7 independent experiments) for wild-type TRPV3, IC₅₀ = 673 ± 37 µM and n_Hill = 2.07 ± 0.08 (n = 3 independent experiments) for F666A, IC₅₀ = 31.5 ± 1.6 µM and n_Hill = 1.63 ± 0.07 (n = 3 independent experiments) for I663W, IC₅₀ = 31.5 ± 1.0 µM and n_Hill = 2.33 ± 0.11 (n = 4 independent experiments) for Y564A and IC₅₀ = 9.8 ± 0.7 µM and n_Hill = 1.68 ± 0.19 (n = 6 independent experiments) for F633A. Source data are provided.

Fig. 3. Two possible orientations of dyclonine in the portal site.

Pore domain in TRPV3_Dyc with dyclonine in the head towards the pore (blue, a, b) or tail towards the pore (green, c, d) orientations, viewed parallel to the membrane (a, c) or extracellularly (b, d). The F666 side chains and dyclonine molecules are shown in sticks. Only two of four TRPV3 subunits are shown in (a) and (c), with the front and back subunits omitted for clarity.

Fig. 4. Control functional experiments.

a Dose–response curves for the F666A mutant and wild-type TRPV3 activation by 2-APB. 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 and EC₅₀ = 27.4 ± 4.5 µM and n_Hill = 1.19 ± 0.09 (n = 4 independent experiments) for wild-type TRPV3 and EC₅₀ = 20.7 ± 0.9 µM and n_Hill = 0.80 ± 0.02 (n = 3 independent experiments) for F666A. The data for wild-type TRPV3 have been published before. b Dose–response curves for inhibition of the F666A mutant and wild-type TRPV3 by osthole. The changes in F₃₄₀/F₃₈₀ were normalized to their maximal values in the absence of osthole. Curves through the points are fits with the logistic equation and IC₅₀ = 20.5 ± 0.5 µM and n_Hill = 1.84 ± 0.14 (n = 4 independent experiments) for wild-type TRPV3 and IC₅₀ = 33.1 ± 3.8 µM and n_Hill = 1.37 ± 0.19 (n = 3 independent experiments) for F666A. The data for wild-type TRPV3 have been published before. c Double logarithmic Schild plot for 2-APB concentration dependencies of TRPV3 activation in the presence of 10 μM (pink circles, n = 3 independent experiments) and 60 μM (green circles, n = 3 independent experiments) of dyclonine. Lines through the points of the corresponding color are linear fits. For all panels, data points are presented as mean ± SEM and source data are provided.

Fig. 5. Conformational changes that accompany dyclonine binding.

Superposition of the pore domains in TRPV3_Dyc (blue) and apo-state TRPV3 (PDB ID: 7MIN; orange) viewed parallel to the membrane (a) or extracellularly (b). The F666 side chains and dyclonine molecules are shown in sticks. Only two of four TRPV3 subunits are shown in (a), with the front and back subunits omitted for clarity. Blue arrows indicate flipping of the F666 side chain, movement of P-loop extracellularly and of dyclonine towards the pore center upon binding in the transmembrane portals.

Fig. 6. TRPV3_Dyc pore and its radius in comparison with the closed and open states.

a Pore-forming domain in TRPV3_Dyc with the residues contributing to pore lining in the dyclonine-bound (TRPV3_Dyc), closed (TRPV3_Closed) and open (TRPV3_Open) states shown as sticks. Only two of four subunits are shown, with the front and back subunits omitted for clarity. The pore profile is shown as a space-filling model (green). The region that undergoes α-to-π transition in S6 is highlighted in pink. b Pore radius for TRPV3_Dyc (blue), TRPV3_Closed (orange, PDB ID: 7MIN) and TRPV3_Open (green, PDB ID: 7MIO) calculated using HOLE. The vertical dashed line denotes the radius of a water molecule, 1.4 Å.