MODULATION OF THE MITOCHONDRIAL CYCLOSPORINE A-SENSITIVE PERMEABILITY TRANSITION PORE BY MATRIX PH - EVIDENCE THAT THE PORE OPEN CLOSED PROBABILITY IS REGULATED BY REVERSIBLE HISTIDINE PROTONATION
MODULATION OF THE MITOCHONDRIAL CYCLOSPORINE A-SENSITIVE PERMEABILITY TRANSITION PORE BY MATRIX PH - EVIDENCE THAT THE PORE OPEN CLOSED PROBABILITY IS REGULATED BY REVERSIBLE HISTIDINE PROTONATION
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DOI：
10.1021/bi00067a039
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发表时间：
1993-04-27
期刊：
影响因子：
2.9
通讯作者：
BERNARDI, P
中科院分区：
文献类型：
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作者：
NICOLLI, A;PETRONILLI, V;BERNARDI, P
在蔗糖介质中的线粒体采取了钙离子脉冲，但不显示开放的渗透性转换孔（MTP）膜去极化解偶联剂。这是由于基质酸化和膜去极化后快速的Ca 2+流出将孔锁定在闭合构象中（Petronilli，V.，可乐角，和Bernardi P.（1993）J.Biol.Chem.268，1011-1016）。在这里，我们表明，除了焦碳酸二乙酯（DPC）之前，膜去极化恢复解偶联剂诱导MTP开放的能力。由于DPC不改变基质酸化的速率和程度以及加入解偶联剂后Ca 2+释放的速率和程度，其对孔开放的影响似乎是由于调节孔开闭概率的组氨酰残基的修饰。这一假设在硫氰酸钾培养基中孵育的断电线粒体的研究中得到证实。虽然在酸性pH值下孔开放被阻止，但DPC在pH 6.5下允许Ca 2+依赖性孔开放，该过程保持对环孢菌素A的完全敏感性。DPC的孔诱导可以被羟胺完全阻止和部分逆转，这表明DPC的作用可以特异性地追溯到组氨酰残基的乙氧羰基化，而不是与酪氨酰或巯基的反应，同时不能排除赖氨酰残基的可能参与。由于DPC增加孔开放的概率，即使在基质pH值在7.0和7.7之间，我们建议，可逆质子化的一个或多个组氨酰残基的基质侧的MTP起着生理调节孔开放的作用。
Energized mitochondria in sucrose medium take up a Ca2+ pulse but do not show opening of the permeability transition pore (MTP) upon membrane depolarization by uncoupler. This is due to locking of the pore in the closed conformation by matrix acidification and fast Ca2+ efflux following membrane depolarization (Petronilli, V., Cola, C., & Bernardi P. (1993) J. Biol. Chem. 268, 1011-1016). Here we show that addition of diethyl pyrocarbonate (DPC) prior to membrane depolarization restores the ability of uncoupler to induce MTP opening. Since DPC does not modify the rate and extent of matrix acidification and the rate and extent of Ca2+ release following addition of uncoupler, its effects on pore opening appear to be due to modification of histidyl residues regulating the pore open-closed probability. This hypothesis was confirmed in studies with deenergized mitochondria incubated in potassium thiocyanate medium. While at acidic pH values pore opening is otherwise prevented, DPC allows Ca2+-dependent pore opening at pH 6.5 in a process that maintains full sensitivity to cyclosporin A. Pore induction by DPC can be completely prevented and partially reversed by hydroxylamine, indicating that the effect of DPC can be specifically traced to carbethoxylation of histidyl residue(s) rather than to reaction with tyrosyl or sulfhydryl groups, while the possible involvement of lysyl residues cannot be excluded. Since DPC increases the pore open probability even at matrix pH values between 7.0 and 7.7, we propose that reversible protonation of one or more histidyl residues on the matrix side of the MTP plays a role in the physiological modulation of pore opening.