由磷脂酰肌醇4,5-双磷酸[PI(4,5)P2]激活的钙选择性TRPV5通道参与钙稳态。最近,低温电子显微镜(cryo-EM)提供了外源和内源分子对TRPV5调节的分子细节。然而,抗真菌剂益康唑(ECN)抑制TRPV5的细节仍然难以捉摸,因为目前可用结构的分辨率较低.在这项研究中,我们使用cryo-EM来全面检查ECN如何抑制TRPV5。通过将我们的结构发现与定点诱变相结合,钙的测量,电生理学,和分子动力学模拟,我们确定S4螺旋上的残基F472和L475,还有S5螺旋上的W495残基,共同构成ECN结合位点。此外,TRPV5在ECN和PI(4,5)P2存在下的结构,没有显示结合的激活剂,揭示了一种潜在的抑制机制,其中ECN与PI(4,5)P2竞争,阻止后者结合,最终孔闭合。
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.