Structural mechanism of TRPV5 inhibition by econazole

Abstract

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P₂, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P₂, preventing the latter from binding, and ultimately pore closure.

Keywords: ECN; TRP; Transient receptor potential channel; activation by PI(4,5)P(2); cryo electron microscopy; cryo-EM; gating mechanism inhibition; inhibition by econazole.

Figure 1.. TRPV5 channel in complex with econazole in presence and absence of PI(4,5)P₂.

Density maps and econazole (ECN) binding site formed by S1, S4, and S5 helices of TRPV5 in the presence of PI(4,5)P₂ and ECN (A, E) and without ECN (PDB 8FFO) (B, F), and TRPV5 in the absence of PI(4,5)P₂ with (C, G) and without ECN (D, H). Insets in panel A depict the chemical structure of the ECN. Maps were contoured at 0.14 (A, B, C), and 0.16 (D) on chimeraX. For E-H panels, S1, S4, and S5 helices densities were selected using the respective coordinates model within the 2.5–3 Å range at same contour level than A-D, and splitted using splitbyzone chimera command. Horizontal grey lines delineate the membrane region. Lipid are represented in yellow, ECN in green, and PI(4,5)P₂ in orange. Hole generated-pore profile of TRPV5_PIP2+ECN (I), TRPV5_PIP2 (J), TRPV5_ECN (K), TRPV5_Apo (L), and pore radii along the pathway (M). Dotted line at 1.1 Å signifies the radius of the dehydrated calcium ion. See also Figures S1–S3.

Figure 2.. Econazole binding site of TRPV5.

Econazole-protein interaction determine using LigPlot in TRPV5_PIP2+ECN (A), TRPV5_ECN (B), and the lipid tails in TRPV5_Apo (C), with overlaid transparent density contoured at the same contour level than Figure 1 (σ=3). Map densities were splited as was described in Figure 1. Dotted blue lines connect two atoms used to calculate the distance (blue). See also Figures S3, S4, S5.

Figure 3.. Conformational changes of TRPV5 inhibited by econazole.

Comparative structural analysis of the TRPV5 activated with PI(4,5)P₂ (orange) and inhibited by ECN (blue): view from extracellular (A), parallel to the membrane (B, C), and from intracellular side (D) of S4, S4-S5 linker, S5, and S6 helices. The pore region is represented by grey dots. Structural rotation and displacements are indicated by curved and straight arrows, respectively. Blue dash lines link atoms employed for distance calculation. See also Figures S4

Figure 4.. TRPV5 inhibition by econazole.

(A) Intracellular Ca²⁺ concentration measurements were performed in HEK293 cells transfected with the Ca²⁺ sensor GCaMP6 and wild-type or mutant rbTRPV5 channels, as described in the Methods section. Cells were pretreated with various concentrations of econazole in a Ca²⁺ free extracellular solution, and the increases of fluorescence in response to increasing extracellular Ca²⁺ to 1 mM were plotted, normalized to the response evoked in cells without econazole pretreatment, n=3 different transfections. See Figure S5 for representative traces and further details. All data plotted as mean ± SEM. (B) Representative currents from HEK293 cells transfected with wild-type or mutant rbTRPV5. Whole cell patch clamp experiments were performed as described in the Methods section. At the beginning of the experiment the cells were kept in a nominally Ca²⁺ free solution containing 1 mM Mg²⁺. In this solution, currents are largely blocked by Mg²⁺ and the trace amounts of Ca²⁺. Monovalent currents were initiated by removing Mg²⁺ and chelating Ca²⁺ with EGTA (0 Ca²⁺ 0 Mg²⁺). Top traces show currents at 100 mV, bottom traces at −100 mV, the dashed line indicates zero current. The application of 10 μM econazole (ECN) is indicated by the horizontal lines. (C) The percent inhibition evoked by 10 μM econazole is plotted. Statistical significance was calculated with one-way analysis of variance and Tukey’s post hoc test. The bar height is the mean ± SEM (D) Dynamic configuration of ECN molecules (purple) within each subunit (ECN1-ECN4) sampled at 10 ns intervals from every of the five independent replicas of 500 ns all-atom molecular dynamics simulation. These conformations are compared to the position observed in TRPV_PIP2+ECN cryo-structure (green). (E) Top, the violin plot showcases the distribution of distances from the center of mass of the ECN molecules within each of the 4 subunits (ECN1-ECN4) to the key residues constituting the binding site, recopilated from the 2.5 μs simulation total (5×500 ns): bottom, a heatmap representation of mean binding energies per residue, calculated at 10 ns intervals across each of the five simulations (detoted as 1–5) and for each ECN molecule (ECN1-ECN4) employing the g_mmpbsa tool and the solvent-accessible surface are (SASA) model. Residues that contribute most significantly to the binding energy are depicted in green. See also Figures S5.