Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, Georgia, United States of America.
PLoS Pathog. 2012;8(6):e1002750. doi: 10.1371/journal.ppat.1002750. Epub 2012 Jun 7.
We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of a novel cation channel (TcCat) from Trypanosoma cruzi, the etiologic agent of Chagas disease. This channel is potassium permeable and shows inward rectification in the presence of magnesium. Western blot analyses with specific antibodies indicated that the protein is expressed in the three main life cycle stages of the parasite. Surprisingly, the parasites have the unprecedented ability to rapidly change the localization of the channel when they are exposed to different environmental stresses. TcCat rapidly translocates to the tip of the flagellum when trypomastigotes are submitted to acidic pH, to the plasma membrane when epimastigotes are submitted to hyperosmotic stress, and to the cell surface when amastigotes are released to the extracellular medium. Pharmacological block of TcCat activity also resulted in alterations in the trypomastigotes ability to respond to hyperosmotic stress. We also demonstrate the feasibility of purifying and reconstituting a functional ion channel from T. cruzi after recombinant expression in bacteria. The peculiar characteristics of TcCat could be important for the development of specific inhibitors with therapeutic potential against trypanosomes.
我们从克氏锥虫（恰加斯病的病原体）中鉴定、表达、纯化、重建并分析了一种新型阳离子通道（TcCat），对其进行了电生理特性鉴定。该通道对钾离子具有通透性，在镁离子存在的情况下表现为内向整流。使用特异性抗体的 Western blot 分析表明该蛋白在寄生虫的三个主要生活周期阶段均有表达。令人惊讶的是，当寄生虫暴露于不同的环境压力时，它们具有快速改变通道定位的前所未有的能力。当游离体被酸性 pH 值处理时，TcCat 迅速易位到鞭毛的顶端；当滋养体被高渗胁迫处理时，TcCat 易位到质膜；当无鞭毛体被释放到细胞外介质时，TcCat 易位到细胞表面。TcCat 活性的药理学阻断也导致游离体对高渗胁迫的反应能力发生改变。我们还证明了在细菌中重组表达后，从 T. cruzi 中纯化和重建功能离子通道的可行性。TcCat 的特殊特性对于开发针对锥虫的具有治疗潜力的特异性抑制剂可能很重要。
