Li Shiliang, Sun Weijia, Li Shuang, Zhu Lili, Guo Shuai, He Jiaqi, Li Yuheng, Tian Chaoquan, Zhao Zhenjiang, Yu Tao, Li Jianwei, Zhang Yiqing, Hai Youlong, Wang Jiawen, Zheng Yongjun, Wang Rui, Hu Xiaoyong, Ling Shukuan, Li Honglin, Li Yingxian
Innovation Center for AI and Drug Discovery, School of Pharmacy, East China Normal University, Shanghai 200062, China.
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China.
Proc Natl Acad Sci U S A. 2025 Jan 7;122(1):e2407493121. doi: 10.1073/pnas.2407493121. Epub 2024 Dec 31.
TMEM16A, a key calcium-activated chloride channel, is crucial for many physiological and pathological processes such as cancer, hypertension, and osteoporosis, etc. However, the regulatory mechanism of TMEM16A is poorly understood, limiting the discovery of effective modulators. Here, we unveil an allosteric gating mechanism by presenting a high-resolution cryo-EM structure of TMEM16A in complex with a channel inhibitor that we identified, Tamsulosin, which is resolved at 2.93 Å. Tamsulosin wedges itself into a pocket within the extracellular domain of TMEM16A, surrounded by α1-α2, α5-α6, and α9-α10 loops. This binding stabilizes a transient preopen conformation of TMEM16A, which is activated by Ca ions while still preserving a closed pore to prevent Cl permeation. Validation of this binding site through computational, electrophysiological, and functional experiments, along with site-directed mutagenesis, confirmed the pivotal roles of the pocket-lining residues R605 and E624 on α5-α6 loop in modulating Tamsulosin binding and pore activity. Tamsulosin induces significant positional shifts in extracellular loops, particularly the α5-α6 loop, which moves toward the extracellular exit of the pore, leading to noticeable structural rearrangements in pore-lining helices. The hinges induced by P595 in α5 and G711 in α7 introduce flexibility to the transmembrane helices, orienting Y593 to collaborate with I641 in effectively gating the preopening pore. Notably, Tamsulosin demonstrates significant antiosteoporotic effects by inhibiting TMEM16A, suggesting potential for its repurposing in new therapeutic indications. Our study not only enhances our understanding of the gating mechanism of TMEM16A inhibition but also facilitates structure-based drug design targeting TMEM16A.
跨膜蛋白16A（TMEM16A）是一种关键的钙激活氯离子通道，对许多生理和病理过程至关重要，如癌症、高血压和骨质疏松症等。然而，人们对TMEM16A的调节机制了解甚少，这限制了有效调节剂的发现。在此，我们通过展示TMEM16A与我们鉴定的一种通道抑制剂坦索罗辛形成复合物的高分辨率冷冻电镜结构，揭示了一种变构门控机制，该结构的分辨率为2.93埃。坦索罗辛楔入TMEM16A细胞外结构域内的一个口袋中，该口袋被α1-α2、α5-α6和α9-α10环包围。这种结合稳定了TMEM16A的一种瞬时预开放构象，该构象被钙离子激活，同时仍保持封闭的孔以防止氯离子渗透。通过计算、电生理和功能实验以及定点诱变对该结合位点进行验证，证实了α5-α6环上口袋内衬残基R605和E624在调节坦索罗辛结合和孔活性方面的关键作用。坦索罗辛诱导细胞外环，特别是α5-α6环发生显著的位置变化，该环向孔的细胞外出口移动，导致孔内衬螺旋发生明显的结构重排。α5中的P595和α7中的G711诱导的铰链为跨膜螺旋引入了灵活性，使Y593与I641协同作用以有效地门控预开放孔。值得注意的是，坦索罗辛通过抑制TMEM16A表现出显著的抗骨质疏松作用，表明其在新治疗适应症中的重新利用潜力。我们的研究不仅增进了我们对TMEM16A抑制门控机制的理解，还促进了针对TMEM16A的基于结构的药物设计。
