Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstr. 76, 24118 Kiel, Germany.
Cells. 2023 Jun 28;12(13):1742. doi: 10.3390/cells12131742.
(1) Background: Several members of the ubiquitous aquaporin family, AQP, of water and neutral solute channels carry a cysteine residue in the selectivity filter region. Traditionally, toxic mercury-containing compounds are used to bind to the cysteine as covalent AQP inhibitors for physiological studies or analysis of structure-function relationships. (2) Methods: We tested thiol-reactive methylthiosulfonate reagents, MTS, as alternative Cys modifiers for AQP inhibition. Three MTS reagents transferring S-alkyl moieties of increasing size, i.e., S-methyl, S--propyl, and S-benzyl, were used with yeast-expressed water-selective AQP1 and the aquaglyceroporin AQP9. Respective Cys-to-Ala variants and mouse erythrocytes that naturally express AQP1 and AQP9 served as controls. (3) Results: Both wildtype AQP isoforms were inhibited by the Cys modifiers in a size-dependent manner, whereas the Cys-to-Ala-variants exhibited resistance. Sub-millimolar concentrations and incubation times in the minute range were sufficient. The modifications were reversible by treatment with the thiol reagents acetylcysteine, ACC, and dithiothreitol, DTT. (4) Conclusions: MTS reagents represent a valid alternative of low toxicity for the inhibition of mercurial-sensitive AQPs.
(1) 背景：水通道蛋白家族（AQP）中的几个成员，AQP，是水和中性溶质通道，在选择性滤过区带有一个半胱氨酸残基。传统上，含汞的有毒化合物被用来与半胱氨酸结合，作为生理研究或分析结构-功能关系的共价 AQP 抑制剂。
(2) 方法：我们测试了巯基反应性的甲基硫磺酸酯试剂（MTS），作为替代 Cys 修饰剂的 AQP 抑制剂。三种将 S-烷基部分转移到更大尺寸的 MTS 试剂，即 S-甲基、S-丙基和 S-苄基，用于酵母表达的水选择性 AQP1 和水甘油通道蛋白 AQP9。相应的 Cys-to-Ala 变体和天然表达 AQP1 和 AQP9 的小鼠红细胞作为对照。
(3) 结果：两种野生型 AQP 同工型都被 Cys 修饰剂以尺寸依赖性方式抑制，而 Cys-to-Ala 变体则表现出抗性。亚毫摩尔浓度和分钟范围内的孵育时间就足够了。修饰物可以通过用巯基试剂乙酰半胱氨酸（ACC）和二硫苏糖醇（DTT）处理来逆转。
(4) 结论：MTS 试剂是抑制汞敏感 AQP 的低毒性有效替代品。
