Structural mechanism of TRPV5 inhibition by econazole

The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recent advances in cryo-electron microscopy (cryo-EM) have provided profound insights into the intricate modulation of TRPV5 by both exogenous and endogenous agents. However, the precise mechanisms underlying TRPV5 inhibition by the antifungal agent econazole (ECN) have remained elusive, primarily due to the limited resolution of currently available structures. In this study, we employ cryo-EM to comprehensively investigate how the ECN inhibits TRPV5. Through a comprehensive approach including our structural findings, site-directed mutagenesis, calcium measurements, electrophysiological assessments, and molecular dynamics simulations, we determined the binding site for ECN. The ECN binding site was found to be consist of three key residues: F472 and L475, both residing on the S4 helix, and W495, situated on the S5 helix. Furthermore, our structural analysis of TRPV5 in the presence of both ECN and PI(4,5)P2, which does not show the bound activator, provides insight into a potential mechanism of TRPV5 inhibition. This pathway entails a series of conformational changes within the S5 and S6 helices that ultimately leading to the disruption of the PI(4,5)P2 interaction and subsequent closure of the channel pore.