Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons
Simultaneous monitoring of ionophore- and inhibitor-mediated plasma and mitochondrial membrane potential changes in cultured neurons
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DOI：
10.1074/jbc.m510916200
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发表时间：
2006-05-26
影响因子：
4.8
通讯作者：
Nicholls, David G.
中科院分区：
文献类型：
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作者：
Nicholls, David G.
虽然天然和合成的离子载体在细胞研究中被广泛利用，例如，影响细胞质游离钙浓度和原位线粒体脱钙，但它们固有的膜选择性缺乏意味着它们影响质膜和线粒体膜的离子渗透性。类似的模糊性影响了对荧光膜渗透阳离子（通常称为“线粒体膜电位指示剂”）信号的解释，因为这些探针的积累受到血浆和线粒体膜电位的影响。为了解决这些问题中的一些，开发了允许使用阳离子和阴离子荧光探针以单细胞分辨率同时监测血浆和线粒体膜电位的技术。描述了一种计算机程序，将荧光变化转化为血浆和线粒体电位变化的动态估计。利用这种技术，原代培养的大鼠小脑颗粒神经元显示离子霉素的浓度依赖性反应：低浓度模仿尼日利亚菌素超极化的线粒体，同时缓慢去极化质膜，并保持稳定的升高的细胞质钙。较高的离子霉素浓度诱导钙稳态的随机失败，这两个线粒体去极化和质膜去极化率提高之前。此外，质子载体羰基氰化物对三氟甲氧基苯腙仅在亚微摩尔浓度下选择性地使线粒体去极化。呼吸链抑制后的ATP合酶逆转使线粒体去极化26 mV。
Although natural and synthetic ionophores are widely exploited in cellstudies, for example, to influence cytoplasmic free calcium concentrations and to depolarize in situ mitochondria, their inherent lack of membrane selectivity means that they affect the ion permeability of both plasma and mitochondrial membranes. A similar ambiguity affects the interpretation of signals from fluorescent membrane-permeant cations (usually termed "mitochondrial membrane potential indicators"), because the accumulation of these probes is influenced by both plasma and mitochondrial membrane potentials. To resolve some of these problems a technique is developed to allow simultaneous monitoring of plasma and mitochondrial membrane potentials at single-cell resolution using a cationic and anionic fluorescent probe. A computer program is described that transforms the fluorescence changes into dynamic estimates of changes in plasma and mitochondrial potentials. Exploiting this technique, primary cultures of rat cerebellar granule neurons display a concentration-dependent response to ionomycin: low concentrations mimic nigericin by hyperpolarizing the mitochondria while slowly depolarizing the plasma membrane and maintaining a stable elevated cytoplasmic calcium. Higher ionomycin concentrations induce a stochastic failure of calcium homeostasis that precedes both mitochondrial depolarization and an enhanced rate of plasma membrane depolarization. In addition, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone only selectively depolarizes mitochondria at submicromolar concentrations. ATP synthase reversal following respiratory chain inhibition depolarizes the mitochondria by 26 mV.