Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+.
Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+.
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DOI：
10.1529/biophysj.104.042671
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发表时间：
2004-11
影响因子：
3.4
通讯作者：
O. Vergun;I. Reynolds
中科院分区：
文献类型：
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作者：
O. Vergun;I. Reynolds
在这项研究中，我们研究了线粒体膜电位（DeltaPsim）的波动，在单个分离的脑线粒体荧光成像。将线粒体连接到盖玻片上，并用含有20 μ M EDTA的基于K+的缓冲液灌注，补充有苹果酸盐和谷氨酸盐，以及罗丹明123用于DeltaPsim测定。DeltaPsim波动触发的线粒体Ca 2+的摄取，因为它们被抑制钌红，一个Ca 2 +-单向转运体阻滞剂，和高浓度的EGTA。需要非常低浓度的Ca 2+（约30 nM）来引发波动。ATP和ADP都可逆地抑制DeltaPsim波动，最大作用发生在100 μ M。核苷酸的影响不能解释的线粒体ATP-合成酶的逆转模式，因为寡霉素是无效的和不可水解的类似物的ATP和ADP没有停止的波动。腺嘌呤核苷酸的影响被废除的腺嘌呤核苷酸转运与羧基糖苷的封锁，但不敏感的另一种抑制剂，bongkrekic酸。ATP敏感性K+通道不参与DeltaPsim波动的机制，因为抑制剂5-羟基癸酸酯或激活剂二氮嗪不影响DeltaPsim的动力学。我们建议DeltaPsim波动在脑线粒体不是自发的，但由Ca 2+触发，并由腺嘌呤核苷酸调制，可能从线粒体内膜的基质侧。
In this study we investigated fluctuations in mitochondrial membrane potential (DeltaPsim) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K+-based buffer containing 20 microM EDTA, supplemented with malate and glutamate, and rhodamine 123 for DeltaPsim determination. DeltaPsim fluctuations were triggered by mitochondrial Ca2+ uptake since they were inhibited by both ruthenium red, a Ca2+-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca2+ (approximately 30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited DeltaPsim fluctuations, with maximal effects occurring at 100 microM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K+-channels are not involved in the mechanism of DeltaPsim fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of DeltaPsim. We suggest DeltaPsim fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca2+ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane.