(A) Overall view of the mTRPV3-dyclonine complex. Three putative binding modes (BMs) for dyclonine in the pore cavity of mTRPV3 channel (PDB ID code: 6DVZ) are denoted as BMA, BMB, and BMC (please find the details in the text), with the expanded view of BMA shown on the right. Four subunits of the tetramer are distinguished by different colors, and dyclonine in a schematic structure is shown in red. (B) (Left) Potential docking poses of dyclonine and TRPV3 channel. (Right) Cluster analysis showing all BMs distributed into three clusters, BMA, BMB, and BMC. (C) Representative whole-cell recordings show reversible blocking of 2-aminoethoxydiphenylborate (2-APB) (1 mM)-evoked responses by dyclonine (3, 10, or 30 μM) in HEK 293T cells expressing mutant TRPV3 channels as indicated, respectively. The combination of 3, 10, or 30 μM dyclonine and 2-APB was applied following the control currents evoked by a saturated concentration of 2-APB (1 mM, initial gray bar). Holding potential was –60 mV. Bars represent duration of stimuli. (D) Concentration-response curves of dyclonine on inhibition of the TRPV3 mutants. Solid lines represent fits by a Hill equation, with the half-maximal inhibitory concentration (IC50) shown in (E). For comparison, the dose-response curve of wile-type channel is displayed in gray. Four point mutations (L630W, N643A, I644W, and L655A) reduced the inhibitory efficiency of dyclonine, while the other two point mutations (L642A and I659A) enhanced the inhibitory effects of dyclonine on TRPV3 currents. (F) Average current responses of mutant channels compared with wild-type TRPV3 channels. Each substitution of putative residues except L639A retained their normal responses to 2-APB. Numbers of cells are indicated in parentheses. (G) Modulation of thiol-oxidizing and disulfide-reducing agents on the inhibitory effects of dyclonine. Whole-cell recordings from the wild-type TRPV3 and the mutants expressed in HEK 293 T cells, showing the effects of (2-(trimethylammonium) ethyl methanethiosulfonate, bromide) (MTSET) and dithiothreitol (DTT) on the responses to 2-APB with or without dyclonine after sensitization induced by 300 μM 2-APB. MTSET (1 mM) and DTT (10 mM) were locally applied for ~3 min to probe the accessibility, respectively. The responsiveness to 2-APB or 2-APB plus dyclonine was subsequently examined before and after treatments. Holding potential was –60 mV. (H) Summary of inhibition of relative currents elicited by 300 μM 2-APB, 300 μM 2-APB with dyclonine 10 or 1 μM. (I) Summary of inhibitory effects of dyclonine before and after treatments with MTSET and DTT. The dotted line indicates zero current level in all cases. Error bar represents SEM. N.S.: no significance; *p<0.05; **p<0.01; ***p<0.001.