Neurology Innovation Centre, Hatfield Research Laboratories, Eisai Ltd., Hatfield, UK.
Department of Cell and Developmental Biology, Consortium for Mitochondrial Research, University College London, London, UK.
Sci Rep. 2017 Sep 5;7(1):10492. doi: 10.1038/s41598-017-10673-8.
Mitochondrial Ca uptake has a key role in cellular Ca homeostasis. Excessive matrix Ca concentrations, especially when coincident with oxidative stress, precipitate opening of an inner mitochondrial membrane, high-conductance channel: the mitochondrial permeability transition pore (mPTP). mPTP opening has been implicated as a final cell death pathway in numerous diseases and therefore understanding conditions dictating mPTP opening is crucial for developing targeted therapies. Here, we have investigated the impact of mitochondrial metabolic state on the probability and consequences of mPTP opening. Isolated mitochondria were energised using NADH- or FADH-linked substrates. The functional consequences of Ca-induced mPTP opening were assessed by Ca retention capacity, using fluorescence-based analysis, and simultaneous measurements of mitochondrial Ca handling, membrane potential, respiratory rate and production of reactive oxygen species (ROS). Succinate-induced, membrane potential-dependent reverse electron transfer sensitised mitochondria to mPTP opening. mPTP-induced depolarisation under succinate subsequently inhibited reverse electron transfer. Complex I-driven respiration was reduced after mPTP opening but sustained in the presence of complex II-linked substrates, consistent with inhibition of complex I-supported respiration by leakage of matrix NADH. Additionally, ROS generated at complex III did not sensitise mitochondria to mPTP opening. Thus, cellular metabolic fluxes and metabolic environment dictate mitochondrial functional response to Ca overload.
线粒体钙摄取在细胞钙稳态中起着关键作用。过多的基质钙浓度，特别是在与氧化应激同时发生时，会促使线粒体内膜高电导通道（线粒体通透性转换孔，mPTP）开放。mPTP 的开放已被认为是许多疾病中细胞死亡的最终途径，因此，了解决定 mPTP 开放的条件对于开发靶向治疗至关重要。在这里，我们研究了线粒体代谢状态对 mPTP 开放的概率和后果的影响。使用 NADH 或 FADH 连接的底物对分离的线粒体进行供能。通过荧光分析和同时测量线粒体钙处理、膜电位、呼吸率和活性氧物种 (ROS) 的产生，评估 Ca 诱导的 mPTP 开放的功能后果。琥珀酸诱导的、依赖于膜电位的逆向电子传递使线粒体对 mPTP 开放敏感。随后，mPTP 诱导的去极化在琥珀酸存在下抑制逆向电子传递。mPTP 开放后，复合物 I 驱动的呼吸减少，但在存在与复合物 II 连接的底物时仍能维持，这与基质 NADH 漏出抑制复合物 I 支持的呼吸一致。此外，在复合物 III 处产生的 ROS 不会使线粒体对 mPTP 开放敏感。因此，细胞代谢通量和代谢环境决定了线粒体对钙过载的功能反应。
