Instituto Carlos Chagas (Fiocruz), Rua Prof. Algacyr Munhoz Mader, PR, Brazil.
Int J Parasitol. 2009 Jul 15;39(9):955-61. doi: 10.1016/j.ijpara.2009.01.002.
Mammalian mitochondria, as well as rat, plant and Caenorhabditis elegans mitochondria, possess an ATP-sensitive K+ channel (mitoK(ATP)) that has been pharmacologically characterised. Opening of mitoK(ATP) and the subsequent K+ entry into the matrix was shown to have three effects on mitochondria physiology: (i) an increase in matrix volume (swelling), (ii) an acceleration of respiration, and (iii) an increase in reactive oxygen species (ROS) production. These effects on mitochondria bioenergetics have been shown to be part of distinct intracellular signalling pathways, to protect against cell death and to modulate gene transcription. To date, such a channel or its activity has not been described in trypanosomatids. In the present study, we show pharmacological evidence for the presence of a mitoK(ATP) in trypanosomatids. Cells were incubated in a hypotonic medium followed by mild detergent exposure to isolate mitoplasts from Trypanosoma cruzi and Crithidia fasciculata. Mitoplasts swelled when incubated in KCl medium due to respiration-driven K+ entry into the matrix. Swelling was sensitive to the presence of ATP when the mitoplast suspension was incubated in K+ -containing, but not in K+ -free, medium. The ATP inhibition of swelling was reversed by the mitoK(ATP) agonist diazoxide and the diazoxide-induced swelling was inhibited by the mitoK(ATP) blockers 5-hydroxydecanoate (5HD) or glibenclamide. Similar to mammalian and rat mitochondria, trypanosomatid mitoK(ATP) activity was modulated by the general protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate (PMA) and antagonist chelerythrine. As expected, the potassium ionophore valinomycin could also reverse the ATP-inhibited state but this reversal was not sensitive to 5HD or glibenclamide. Dose response curves for ATP, diazoxide and 5HD are presented. These results provide strong evidence for the presence of an ATP-sensitive K+ in trypanosomatid mitochondria.
哺乳动物线粒体以及大鼠、植物和秀丽隐杆线虫的线粒体都具有一种已被药理学鉴定的 ATP 敏感性钾通道（mitoK(ATP)）。现已证实，mitoK(ATP)的开放以及随后的钾进入基质会对线粒体生理学产生三种影响：（i）基质体积增加（肿胀）；（ii）呼吸加速；（iii）活性氧（ROS）产生增加。这些对线粒体生物能量学的影响已被证明是不同的细胞内信号通路的一部分，可防止细胞死亡并调节基因转录。迄今为止，在原生动物中尚未描述过这样的通道或其活性。在本研究中，我们提供了原生动物中存在 mitoK(ATP)的药理学证据。将细胞在低渗介质中孵育，然后用温和的去污剂处理以从克氏锥虫和福氏利什曼原虫中分离出线粒体。由于呼吸驱动的钾进入基质，线粒体在 KCl 介质中孵育时会肿胀。当线粒体悬液在含钾但不含钾的介质中孵育时，肿胀对 ATP 的存在敏感。ATP 抑制肿胀可被 mitoK(ATP)激动剂二氮嗪逆转，而二氮嗪诱导的肿胀可被 mitoK(ATP)抑制剂 5-羟癸酸（5HD）或格列本脲抑制。与哺乳动物和大鼠线粒体类似，原生动物 mitoK(ATP)的活性可被普遍的蛋白激酶 C（PKC）激动剂佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）和拮抗剂Chelerythrine 调节。正如预期的那样，钾离子载体缬氨霉素也可以逆转 ATP 抑制状态，但这种逆转对 5HD 或格列本脲不敏感。呈现了 ATP、二氮嗪和 5HD 的剂量反应曲线。这些结果为原生动物线粒体中存在 ATP 敏感性钾提供了有力证据。
