Costantini P, Chernyak B V, Petronilli V, Bernardi P
Consiglio Nazionale delle Ricerche Unit for the Study of Physiology of Mitochondria, University of Padova Medical School, Via Trieste 75, I-35121 Padova, Italy.
J Biol Chem. 1996 Mar 22;271(12):6746-51. doi: 10.1074/jbc.271.12.6746.
After accumulation of a Ca2+ load, the addition of uncoupler to respiring rat liver mitochondria is followed by opening of the permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A. The channel's voltage threshold is profoundly affected under conditions of oxidative stress, with a shift to more negative values that may cause MTP opening at physiological membrane potentials. In this paper we further clarify the mechanisms by which oxidative agents affect the apparent voltage dependence of the MTP. We show that two sites can be experimentally distinguished. (i) A first site is in apparent oxidation-reduction equilibrium with the pyridine nucleotide (PN) pool (NADH/NAD + NADPH/NADP); PN oxidation is matched by increased MTP open probability under conditions where the glutathione pool is kept in the fully reduced state; this site can be blocked by N-ethylmaleimide but not by monobromobimane, a thiol-selective reagent. (ii) A second site coincides with the oxidation-reduction-sensitive dithiol we have recently identified (Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638-16642); dithiol cross-linking at this site by arsenite or phenylarsine oxide is matched by increased MTP open probability under conditions where the PN pool is kept in the fully reduced state; at variance from the first, this site can be blocked by both N-ethylmaleimide and monobromobimane and is probably in equilibrium with the glutathione pool. Based on these findings, we reassess the mechanisms by which many oxidative agents affect the MTP and resolve conflicting reports on the relative role of PN and glutathione oxidation in the permeability transition within the framework of MTP (dys)regulation at two separate sites.
在积累了钙离子负荷后，向呼吸状态的大鼠肝脏线粒体中添加解偶联剂，随后通透性转换孔（MTP）开放，这是一种对环孢菌素A敏感的电压依赖性通道。在氧化应激条件下，该通道的电压阈值会受到深刻影响，向更负的值偏移，这可能导致在生理膜电位下MTP开放。在本文中，我们进一步阐明了氧化试剂影响MTP表观电压依赖性的机制。我们表明，有两个位点可以通过实验区分。（i）第一个位点与吡啶核苷酸（PN）池（NADH/NAD + NADPH/NADP）处于明显的氧化还原平衡状态；在谷胱甘肽池保持完全还原状态的条件下，PN氧化与MTP开放概率增加相匹配；该位点可被N - 乙基马来酰亚胺阻断，但不能被硫醇选择性试剂单溴代二苯并咪唑阻断。（ii）第二个位点与我们最近鉴定的氧化还原敏感二硫醇重合（Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., and Bernardi, P. (1994) J. Biol. Chem. 269, 16638 - 16642）；在PN池保持完全还原状态的条件下，亚砷酸盐或苯砷酸氧化物在此位点进行二硫醇交联与MTP开放概率增加相匹配；与第一个位点不同，该位点可被N - 乙基马来酰亚胺和单溴代二苯并咪唑都阻断，并且可能与谷胱甘肽池处于平衡状态。基于这些发现，我们重新评估了许多氧化试剂影响MTP的机制，并在MTP（失调）调控的两个独立位点的框架内，解决了关于PN和谷胱甘肽氧化在通透性转换中相对作用的相互矛盾的报道。
