Fig. 4: CHL binding contributes to SAF312 inhibition.

a, b CHL binds to the VBP pocket, and the corresponding cryo-EM densities are shown as blue and orange meshes. b The stick models of CHL and the side chains of its binding residues are displayed. The side chains of crucial residues involved in the interaction with CHL are shown in sticks and their distances to SAF312 and CHL are labeled. c Superposition of the CHL from hTRPV1_SAF312 (wheat) and TRPV2_CHL (light blue, PDB 7XEM). The ligands partially overlap. d, e I-V curves of hTRPV1^WT (d) and L515A (e) were generated from the 3 μM capsaicin-induced peak currents without (peak1, blue) or with 1 μM SAF312 (purple). f 3 μM capsaicin-induced current with 1 μM SAF312 at +80 mV for hTRPV1 ^WT and L515A was normalized to the first current of 3 μM capsaicin (peak1). Data were shown as mean ± SEM from five cells. g The residue contribution for CHL was calculated by MM/GBSA (n = 3), data are shown as mean ± SD. h, i Representative I-V curves were obtained from pH 5-induced peak currents without (peak1, blue) or with 1 μM SAF312 under the MβCD treatment with different concentrations (red) 0.5 mM (h) and 5 mM (i). j Dose-dependent recovery of MβCD on SAF312 inhibition to hTRPV1 current. The membrane CHL was depleted by different concentrations of MβCD for 15 min at 37 °C in the absence of serum before each whole-cell patch-clamp recording. The inhibition percentage of MβCD was calculated with I _{pH 5 + 1 μM SAF312}/I _{pH 5 (peak1)} %. Data were shown as mean ± SEM (n = 6, 5, 7, 6, and 5 for control, 0.5, 1, 3 and 5 mM MβCD). k The RMSD of CHL with and without SAF312 is plotted for three independent 400 ns MD simulations.