Scorpion Toxin Inhibits the Voltage-Gated Proton Channel Using a Zn 2 -Like Long-Range Conformational Coupling Mechanism
Scorpion Toxin Inhibits the Voltage-Gated Proton Channel Using a Zn 2 -Like Long-Range Conformational Coupling Mechanism
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蝎毒素利用类 Zn 2 长程构象耦合机制抑制电压门控质子通道
DOI：
10.1111/bph.14984
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发表时间：
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影响因子：
7.3
通讯作者：
Zhonghua Liu
中科院分区：
文献类型：
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作者：
Tang Dongfang;Yang Yuqing;Xiao Zhen;Xu Jiahui;Yang Qiuchu;Dai Han;Songping Liang;Tang Cheng;Hao Dong;Zhonghua Liu
背景和目的阻断电压门控质子通道HV1是治疗脑缺血、中风和癌症等疾病的一种很有前途的策略。然而，关于HV1通道拮抗剂的报道很少。在此，我们从蝎毒中分离出一种新的HV1通道拮抗剂，并阐明了其作用机制。实验方法在哺乳动物细胞系中异源表达了HV1和NAV通道，并用全细胞膜片钳记录了它们的电流。采用定点突变的方法产生突变体。用分子动力学方法模拟AGAP/W38F-HV1相互作用。关键结果蝎子毒素AGAP(抗肿瘤止痛肽)对HV1电流有明显的抑制作用。一个AGAP突变体降低了NaV通道的活性，但保持了HV1的活性(AGAP/W38F)。AGAP/W38F通过捕获处于失活状态的S4电压传感器来抑制HV1通道的激活，并抑制HV1电流，其pH依赖性比锌离子小。突变分析表明，AGAP/W38F与HV1通道中的锌离子结合口袋部分重叠(共同的位点为His140和His193)。与锌离子一样，HV1通道细胞内库仑网络(ICN)的E153A突变显著降低了对AGAP/W38F的抑制作用。实验数据和分子动力学模拟表明，AGAP/W38F通过类锌离子的长程构象耦合机制抑制了HV1通道。结论和意义我们的结果表明，HV1通道中的锌离子结合口袋可能是调节剂的热点，对设计HV1通道配体具有重要意义。此外，AGAP/W38F是研究HV1通道的有用分子探针和药物开发的先导化合物。
Background and PurposeBlocking the voltage‐gated proton channel HV1 is a promising strategy for the treatment of diseases like ischaemia stroke and cancer. However, few HV1 channel antagonists have been reported. Here, we have identified a novel HV1 channel antagonist from scorpion venom and have elucidated its action mechanism.Experimental ApproachHV1 and NaV channels were heterologously expressed in mammalian cell lines and their currents recorded using whole‐cell patch clamp. Site‐directed mutagenesis was used to generate mutants. Toxins were recombinantly produced inEscherichia coli.AGAP/W38F‐HV1 interaction was modelled by molecular dynamics simulations.Key ResultsThe scorpion toxin AGAP (anti‐tumour analgesic peptide) potently inhibited HV1 currents. One AGAP mutant has reduced NaVchannel activity but intact HV1 activity (AGAP/W38F). AGAP/W38F inhibited HV1 channel activation by trapping its S4 voltage sensor in a deactivated state and inhibited HV1 currents with less pH dependence than Zn2+. Mutation analysis showed that the binding pockets of AGAP/W38F and Zn2+in HV1 channel partly overlapped (common sites are His140 and His193). The E153A mutation at the intracellular Coulombic network (ICN) in HV1 channel markedly reduced AGAP/W38F inhibition, as observed for Zn2+. Experimental data and MD simulations suggested that AGAP/W38F inhibited HV1 channel using a Zn2+‐like long‐range conformational coupling mechanism.Conclusion and ImplicationsOur results suggest that the Zn2+binding pocket in HV1 channel might be a hotspot for modulators and valuable for designing HV1 channel ligands. Moreover, AGAP/W38F is a useful molecular probe to study HV1 channel and a lead compound for drug development.