瞬时受体电位香草酸 2 (TRPV2) 是一种 Ca2 渗透性非选择性阳离子通道。尽管TRPV2在免疫反应、癌症进展和心脏发育中发挥重要作用，但TRPV2的药理学探针仍有待鉴定。我们的目的是发现 TRPV2 抑制剂并阐明其作用的分子机制。在表达鼠 TRPV2 的 HEK-293 细胞中进行基于荧光的 Ca2 测定，用于鉴定白花丹素作为一种新型 TRPV2 抑制剂。应用膜片钳、计算机对接和定点诱变来研究白花丹素相互作用的关键分子机制。 ELISA 和 qPCR 分别用于评估一氧化氮释放和炎症介质的 mRNA 水平。 si-RNA 干扰用于敲低 TRPV2 表达，并通过蛋白质印迹法进行验证。神经学和组织学分析用于检查小鼠大脑中动脉闭塞/再灌注 (MCAO/R) 后的脑损伤。Plumbagin 是一种有效的 TRPV2 负变构调节剂，IC50 值为 0.85μM，选择性是 TRPV1 的 14 倍以上。 TRPV3 和 TRPV4。白花丹素通过降低通道开放概率来抑制 TRPV2 活性，而不影响单一电导。此外，白花丹素与TRPV2跨膜螺旋S5和S6之间的孔螺旋和柔性环形成的细胞外袋结合。白花丹素可有效抑制 LPS 诱导的 BV-2 小胶质细胞炎症，并改善 MCAO/R 小鼠的脑损伤。白花丹素是研究 TRPV2 病理生理学的新型药理学探针。 TRPV2 是治疗神经炎症和缺血性中风的新型分子靶标。© 2024 英国药理学会。

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable non-selective cation channel. Despite the significant roles of TRPV2 in immunological response, cancer progression and cardiac development, pharmacological probes of TRPV2 remain to be identified. We aimed to discover TRPV2 inhibitors and to elucidate their molecular mechanism of action.Fluorescence-based Ca2+ assay in HEK-293 cells expressing murine TRPV2 was used to identify plumbagin as a novel TRPV2 inhibitor. Patch-clamp, in silico docking and site-directed mutagenesis were applied to investigate the molecular mechanisms critical for plumbagin interaction. ELISA and qPCR were used to assess nitric oxide release and mRNA levels of inflammatory mediators, respectively. si-RNA interference was used to knock down TRPV2 expression, which was validated by western blotting. Neurological and histological analyses were used to examine brain injury of mice following middle cerebral artery occlusion/reperfusion (MCAO/R).Plumbagin is a potent TRPV2 negative allosteric modulator with an IC50 value of 0.85 μM, exhibiting >14-fold selectivity over TRPV1, TRPV3 and TRPV4. Plumbagin suppresses TRPV2 activity by decreasing the channel open probability without affecting the unitary conductance. Moreover, plumbagin binds to an extracellular pocket formed by the pore helix and flexible loop between transmembrane helices S5 and S6 of TRPV2. Plumbagin effectively suppresses LPS-induced inflammation of BV-2 microglia and ameliorates brain injury of MCAO/R mice.Plumbagin is a novel pharmacological probe to study TRPV2 pathophysiology. TRPV2 is a novel molecular target for the treatment of neuroinflammation and ischemic stroke.© 2024 British Pharmacological Society.