Daphnetin ameliorates skin inflammation by selectively inhibiting transient receptor potential vanilloid 3 channels

Chemical irritants or ultraviolet exposure can lead to an imbalance in skin barrier homeostasis and trigger various inflammatory skin diseases. Daphnetin, a bioactive coumarin compound extracted from the traditional Chinese medicine Cortex Daphnes (Zushima), has been shown to suppress skin lesions induced by chemical irritants such as DNCB or DNFB in mice. However, the molecular mechanism underlying the action of daphnetin remains unclear. Here, we report that daphnetin alleviates skin inflammation by directly targeting the calcium-permeable and warmth-selective TRPV3 channel. Subcutaneous administration or topical application of daphnetin efficaciously suppresses DNFB- or UVB-induced dermatitis in wild-type mice but confers no additional benefit in Trpv3 knockout mice. In whole-cell patch-clamp recordings, daphnetin selectively inhibits human and mouse TRPV3 channel currents induced by 2-APB in a concentration-dependent manner, with IC₅₀ values of 19.91 ± 2.83 μM (hTRPV3) and 24.10 ± 2.20 μM (mTRPV3), respectively. The single-channel patch-clamp results show that daphnetin significantly reduces the open probability and open frequency without significantly altering the unitary conductance of TRPV3 channel. Molecular docking and site-directed mutagenesis indicate that residue Q580 on the S4-S5 linker and residue T665 on the S6 segment of TRPV3 are critical for daphnetin binding to TRPV3. Taken together, our results demonstrate that daphnetin exerts its anti-inflammatory effects by selectively inhibiting TRPV3 channel via a novel dual-binding-site mechanism. Thus, daphnetin not only provides a valuable tool compound for understanding TRPV3 channel gating mechanisms but also serves as a lead compound for further modification of specific TRPV3 channel inhibitors.