Vanilloid site-mediated TRPV3 activation by cannabidiol and potent inhibition by...

TRPV3 is a heat- and ligand-gated calcium-permeable ion channel, which is mostly expressed in the skin and plays important roles in skin physiology. Targeting TRPV3 with selective ligands represents a promising therapeutic strategy for the correction of cutaneous pathological processes. For the rational drug design and prognosis of clinical outcomes, it is essential to understand the mechanisms of ligand action. Here, we study the mechanism of TRPV3 activation by cannabidiol (CBD) and inhibition by 14-deoxy-11,12-didehydroandrographolide (DDA). Pharmacological data were obtained via calcium imaging in TRPV3-expressing CHO cells and two-electrode voltage-clamp recordings in Xenopus oocytes. We show that CBD activates human TRPV3 with the EC₅₀ of 12.8 µM, while DDA potently inhibits CBD-evoked activation with the IC₅₀ of 7 nM. DDA acts as a modality-specific inhibitor: a competitive reversible antagonist for CBD activation, a negative allosteric modulator for 2-aminoethoxydiphenyl borate (2-APB) activation, and is neutral to heat activation. Using molecular docking and site-directed mutagenesis, we show that both CBD and DDA bind to the vanilloid site, an important regulatory pocket in the transmembrane region of TRPV3. Thus, single-residue substitutions in the vanilloid site disrupt activation by CBD (A560L) or increase its activation potency (L664S), and abrogate TRPV3 inhibition by DDA (L557A, L664S). Our results expand the pharmacological profiles of CBD and DDA and highlight their potential as drug candidates for TRPV3-targeting therapeutics.