Deciphering the mitochondrial proton leak

摘要： 线粒体功能是一系列健康和疾病的基础，从新陈代谢到 心率调节，产热，先天遗传疾病，癌症，衰老，心脏和肌肉功能障碍， 糖尿病和肥胖症。利用氧化磷酸化，线粒体将质子从基质泵到 膜间空间产生质子梯度。质子通过ATPase复合体返回基质 以产生支持有机体生命的三磷酸腺苷。大约20%-50%的质子泄漏回母体，但不 推动三磷酸腺苷的产生，这被称为质子泄漏或去偶联。传统的质子泄漏测量方法 克拉克型氧探针法和海马法采用耗氧率(OCR)作为读数。这些OCR 基于质子泄漏的测量不能排除“质子滑动”，它被定义为呼吸链活动 消耗氧气并传递电子，而不会将质子挤出膜外。有一个知识鸿沟 如何评估特定的线粒体质子泄漏和滑动。为了解决这个差距，我建立了一个直接的 一种精确测量质子泄漏和通过将线粒体暴露在pH中消除质子滑移的方法 线粒体靶向pH敏感指示剂mt-cpYFP使细胞在梯度应激下通透性增强。 缓冲液缺乏新陈代谢底物，因此使细胞处于不活跃状态，从而允许分离 线粒体内膜的物理性质来自能量活跃的过程，如离子泵。 使用这种方法，我揭示了腺嘌呤核苷酸转运体1(ANT1)被低估了 在支持过多的质子泄漏方面的作用。利用这种新的线粒体质子泄漏测量方法，在 R35 Mira建议，我们建议1)比较不同细胞类型之间的线粒体质子泄漏 Mt-cpYFP转基因小鼠；2)解剖ANT1介导的线粒体质子泄漏的分子基础；3) 筛选防止或增强线粒体质子泄漏的新药/化合物。完成这项研究 将提供线粒体质子泄漏的关键机制，并将提供一组新的药物 线粒体功能参与了这些不同健康过程的代谢动态平衡。

Abstract: Mitochondrial function underlies a broad spectrum of health and disease conditions that range from metabolic rate regulation, thermogenesis, to inborn genetic disorders, cancer, aging, cardiac and muscle dysfunction, diabetes and obesity. Using oxidative phosphorylation, the mitochondria pump protons from the matrix to the intermembrane space to generate a proton gradient. The protons return to the matrix via the ATPase complex to generate ATP that supports the life of the organism. About 20-50% of protons leak back to matrix but do not drive ATP production and this is called proton leak or uncoupling. The traditional proton leak measurement by Clark type oxygen probe and Seahorse assay use oxygen consumption rate (OCR) as a readout. These OCR based proton leak measurements cannot exclude “proton slip”, which is defined as respiratory chain activity that consumes O2 and transfers electrons without extruding protons out of the membrane. There is a knowledge gap of how to assess the specific mitochondrial proton leak and slip. To address this gap, I established a direct method for measuring the accurate proton leak and eliminating proton slip by exposing mitochondria to a pH gradient stress in saponin permeabilized cells with mt-cpYFP, a mitochondrial targeted pH sensitive indicator. The buffers lack metabolic substrates, thus making the cells energetically inactive, allowing separation of the physical property of the mitochondrial inner membrane from energetically active processes such as ion pumping. Using this approach, I have revealed that the adenine nucleotide transporter 1 (ANT1) has an under-appreciated role in supporting an excessive proton leak. With this novel mitochondrial proton leak measuring method, in this R35 MIRA proposal, we propose to 1) Compare mitochondrial proton leak between different cell types from the mt-cpYFP transgenic mouse; 2) Dissect the molecular basis of ANT1-mediated mitochondrial proton leak; 3) Screen for new drugs/compounds to prevent or augment the mitochondrial proton leak. Completion of this study will provide key mechanisms of mitochondrial proton leak and will provide a novel group of drugs that modulate mitochondrial function involved in the metabolic homeostasis of these diverse health processes.