Czirják Gábor, Enyedi Péter
Department of Physiology, Semmelweis University, Budapest, Hungary, H-1444.
Mol Pharmacol. 2006 Mar;69(3):1024-32. doi: 10.1124/mol.105.018556. Epub 2005 Dec 14.
TWIK-related spinal cord K+ channel (TRESK) is the most recently cloned two-pore-domain potassium (2PK+) channel, regulated by the calcium/calmodulin-dependent protein phosphatase calcineurin. Functional identification of endogenous TRESK and its distinction from the other 2PK+ channels, producing similar background K+ current, are impeded by the lack of specific inhibitors. Therefore, we searched for antagonists selective against TRESK among the mouse 2PK+ channels by screening more than 200 substances. Mibefradil, zinc, and mercuric ions inhibited TRESK expressed in Xenopus laevis oocytes with IC50 values lower than 10 microM. The specificity of the identified agents was determined by measuring their effects on mouse TALK-1, TASK-1, TASK-2, TASK-3, THIK-1, TRAAK, TREK-1, and TREK-2. Mibefradil failed to discriminate well among the functional 2PK+ channels; however, Zn2+ and Hg2+ exerted a significantly stronger inhibitory effect on TRESK than on the other channels. Sensitivity to zinc but insensitivity to ruthenium red were distinctive features of TRESK. Whereas both Zn2+ and Hg2+ were selective blockers of TRESK among the mouse 2PK+ channels, human TRESK was resistant to Zn2+; it was blocked only by Hg2+. His132 of mouse TRESK was partly responsible for this difference. Mouse TRESK expressed in COS-7 cells was also inhibited by Zn2+ and Hg2+, and TRESK single-channel current was diminished in outside-out patches, indicating that the action of the ions was membrane-delimited, most probably targeting the channel itself. Thus, both Zn2+ and Hg2+ are expected to inhibit endogenous TRESK in isolated mouse cells, and these ions can be applied to identify the calcineurin-activated 2PK+ channel in its natural environment.
TWIK相关脊髓钾通道（TRESK）是最近克隆的双孔结构域钾通道（2PK+通道），受钙/钙调蛋白依赖性蛋白磷酸酶钙调神经磷酸酶调控。由于缺乏特异性抑制剂，内源性TRESK的功能鉴定及其与其他产生相似背景钾电流的2PK+通道的区分受到阻碍。因此，我们通过筛选200多种物质，在小鼠2PK+通道中寻找对TRESK有选择性的拮抗剂。米贝拉地尔、锌离子和汞离子抑制非洲爪蟾卵母细胞中表达的TRESK，其半数抑制浓度（IC50）值低于10微摩尔。通过测量它们对小鼠TALK-1、TASK-1、TASK-2、TASK-3、THIK-1、TRAAK、TREK-1和TREK-2的影响来确定所鉴定药物的特异性。米贝拉地尔未能很好地区分功能性2PK+通道；然而，锌离子和汞离子对TRESK的抑制作用明显强于其他通道。对锌敏感但对钌红不敏感是TRESK的显著特征。虽然锌离子和汞离子都是小鼠2PK+通道中TRESK的选择性阻滞剂，但人TRESK对锌离子有抗性；它仅被汞离子阻断。小鼠TRESK中的组氨酸132部分导致了这种差异。在COS-7细胞中表达的小鼠TRESK也受到锌离子和汞离子的抑制，并且在外翻膜片中TRESK单通道电流减小，这表明离子的作用是膜限定的，很可能作用于通道本身。因此，锌离子和汞离子有望抑制分离的小鼠细胞中的内源性TRESK，并且这些离子可用于在自然环境中鉴定钙调神经磷酸酶激活的2PK+通道。
