Fig. 1: Functional and cryo-EM characterization of TRPV6 inhibition by THCV.

a Whole-cell patch-clamp currents recorded from HEK 293 cell expressing hTRPV6 in response to −100 to 70 mV voltage ramp in the absence (black) and presence (red) of 50 µM THCV. Examples are representatives of currents recorded from 8 different cells in two independent transfections. Recordings were made in the absence of Ca²⁺, so the current was mainly carried by monovalent cations. The inset shows the chemical structure of THCV. b Representative ratiometric Fura-2 AM-based fluorescence measurements of changes in intracellular Ca²⁺ for HEK 293 GnTI⁻ cells expressing hTRPV6. The changes in the fluorescence intensity ratio at 340 and 380 nm (F₃₄₀/F₃₈₀) were monitored in response to application of 10 mM Ca²⁺ (arrow) after pre-incubation of cells with various concentrations of THCV. The experiment was repeated independently eight times with similar results. c Concentration–response curve for THCV inhibition of Ca²⁺ uptake. The background subtracted changes in the fluorescence intensity ratio at 340 and 380 nm (F₃₄₀/F₃₈₀) evoked by the addition of 10 mM Ca²⁺ after pre-incubation with various concentrations of THCV were normalized to the maximal change in F₃₄₀/F₃₈₀ after addition of 10 mM Ca²⁺ in the absence of THCV. Data shown as mean ± SEM. Red curve through the data points is a fit with the logistic equation, with the mean ± SEM values of the half-maximum inhibitory concentration (IC₅₀), 15.4 ±  2.3 µM (n = 8 independent experiments). Source data are provided as a Source Data file. d, e 3D cryo-EM reconstruction of TRPV6_THCV viewed from the side (d) or top (e), with subunits colored yellow, green, pink, and cyan. f Same cryo-EM density as in e but cut off along the dashed line in d. Putative densities for THCV and ions are shown in red and green.