Modulation of inner mitochondrial membrane channel activity

Three classes of inner mitochondrial membrane (IMM) channel activities have been defined by direct measurement of conductance levels in membranes with patch clamp techniques in 150 mM K Cl. The 107 pS activity is slightly anion selective and voltage dependent (open with matrix positive potentials). Multiple conductance channel (MCC) activity includes several levels from about 40 to over 1000 pS and can be activated by voltage or Ca²⁺. MCC may be responsible for the Ca²⁺-induced permeability transition observed with mitochondrial suspensions. A low conductance channel (LCC) is activated by alkaline pH and inhibited by Mg²⁺. LCC has a unit conductance of about 15 pS and may correspond to the inner membrane anion channel, IMAC, which was proposed from results obtained from suspension studies. All of the IMM channels defined thus far appear to be highly regulated and have a low open probability under physiological conditions. A summary of what is known about IMM channel regulation and pharmacology is presented and possible physiological roles of these channels are discussed.     

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking

We have previously shown that mitochondrial membrane potential () drop promoted by prooxidants and Ca²⁺ can be reversed but not sustained by ethylene glycol-bis(-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993).Arch. Biochem. Biophys. 307, 1–7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after was collapsed. Total recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during drop continues even after is already eliminated. Titration with 5,5-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.     

Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane

The mitochondrial permeability transition pore allows solutes with a m.w. 1500 to equilibrate across the inner membrane. A closed pore is favored by cyclosporin A acting at a high-affinity site, which may be the matrix space cylophilin isozyme. Early results obtained with cyclosporin A analogs and metabolites support this hypothesis. Inhibition by cyclosporin does not appear to require inhibition of calcineurin activity; however, it may relate to inhibition of cyclophilin peptide bond isomerase activity. The permeability transition pore is strongly regulated by both the membrane potential () and pH components of the mitochondrial protonmotive force. A voltage sensor which is influenced by the disulfide/sulhydryl state of vicinal sulfhydryls is proposed to render pore opening sensitive to . Early results indicate that this sensor is also responsive to membrane surface potential and/or to surface potential gradients. Histidine residues located on the matrix side of the inner membrane render the pore responsive to pH. The pore is also regulated by several ions and metabolites which act at sites that are interactive. There are many analogies between the systems which regulate the permeability transition pore and the NMDA receptor channel. These suggest structural similarities and that the permeability transition pore belongs to the family of ligand gated ion channels.     

Properties of channels in the mitochondrial outer membrane

Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6±1 subunits.     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Permeability transition pore of the inner mitochondrial membrane can operate in two open states with different selectivities

Prooxidants induce release of Ca²⁺ from mitochondria through the giant solute pore in the mitochondrial inner membrane. However, under appropriate conditions prooxidants can induce Ca²⁺ release without inducing a nonspecific permeability change. Prooxidant-induced release of Ca²⁺ isselective. Presumably, this is the result of the operation of a permeability pathway for H⁺ coupled to the reversal of the Ca²⁺ uniporter, the latter generating the selectivity. The solute pore and prooxidant-induced Ca²⁺-specific pathways exhibit common sensitivities to a set of inhibitors and activators. It is proposed that the pore can operate in two open states: (1) permeable to H⁺ only and (2) permeable to solutes of M_r<1500. Under some conditions, prooxidants induce the H⁺-selective state which, in turn, collapses the inner membrane potential and permits selective loss of Ca²⁺ via the Ca²⁺ uniporter.     

Purification and characterization of the voltage-dependent anion channel from the outer mitochondrial membrane of yeast

Summary The outer mitochondrial membranes of all organisms so far examined contain a protein which forms voltage-dependent anion selective channels (VDAC) when incorporated into planar phospholipid membranes. Previous reports have suggested that the yeast (Saccharomyces cerevisiae) outer mitochondrial membrane component responsible for channel formation is a protein of 29,000 daltons which is also the major component of this membrane. In this report, we describe the purification of this 29,000-dalton protein to virtual homogeneity from yeast outer mitochondrial membranes. The purified protein readily incorporates into planar phospholipid membranes to produce ionic channels. Electrophysiological characterization of these channels has demonstrated they have a size, selectivity and voltage dependence similar to VDAC from other organisms. Biochemically, the purified protein has been characterized by determining its amino acid composition and isoelectric point (pI). In addition, we have shown that the purified protein, when reconstituted into liposomes, can bind hexokinase in a glucose-6-phosphate dependent manner, as has been shown for VDAC purified from other sources. Since physiological characterization suggests that the functional parameters of this protein have been conserved, antibodies specific to yeast VDAC have been used to assess antigenic conservation among mitochondrial proteins from a wide number of species. These experiments have shown that yeast VDAC antibodies will recognize single mitochondrial proteins fromDrosophila, Dictyostelium andNeurospora of the appropriate molecular weight to be VDAC from these organisms. No reaction was seen to any mitochondrial protein from rat liver, rainbow trout,Paramecium, or mung bean. In addition, yeast VDAC antibodies will recognize a 50-kDa mol wt protein present in tobacco chloroplasts. These results suggest that there is some antigenic as well as functional conservation among different VDACs.     

Modulation of mitochondrial permeability transition pore affects multidrug resistance in human hepatocellular carcinoma cells

Multidrug resistance (MDR) is a critical problem in the chemotherapy of cancers. Human hepatocellular carcinoma (HCC) responds poorly to chemotherapy owing to its potent MDR. Chemotherapeutic drugs primarily act by inducing apoptosis of cancer cells, and defects in apoptosis may result in MDR. Mitochondrial permeability transition (mPT) is implicated as an important event in the control of cell death or survival and mPT represents a target for the development of cytotoxic drugs. This study aimed to investigate the effects of selective opener (Atractyloside glycoside, ATR) and inhibitor (Cyclosporine A, CsA) of mitochondrial permeability transition pore (mPTP) on a CDDP-resistant HCC cell line (SK-Hep1 cells). In this study, a stable MDR phenotype characterization of SK-Hep1 cell line (SK-Hep1/CDDP cells) was established and used to investigate the role of mPTP in MDR. Results suggested that ATR accelerated the decrease of mitochondrial membrane potential (ΔΨm), reduced the Bax activity, and increased the apoptosis of SK-Hep1/CDDP cells; while CsA inhibited mPTP opening, reduced and delayed the decline of mitochondrial membrane potential, and increased the Bax activity, leading to increased tolerance of SK-Hep1/CDDP cells to apoptosis induction. However, mPTP activity had no effect on the expression of MDR1 in cells,meanwhile the P-gp translocation to mitochondria was increased, and functionally activated. In conclusion, selective modulation of mPTP can affect MDR in human HCC cells. Therefore, activation of mPTP may provide a new strategy to sensitize cancer cells to chemotherapeutic drugs and to reverse the MDR in cancer cells.     

Importance of the mitochondrial outer membrane channel as a model biological channel

The channels of the mitochondrial outer membrane represent a useful model for studies into the mechanisms underlying phenomena of voltage-dependent gating and ion selectivity.     

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca^2?+?-dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca^2?+? concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD^? mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the “standard” PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca^2?+?, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.     

Two modes of activation of the permeability transition pore: The role of mitochondrial cyclophilin

Mitochondria possess an inner membrane channel, the permeability transition pore, which is inhibited by cyclosporin A (CBA) and by matrix protons. As suggested recently by our laboratory, pore closure by these inhibitors may be due to dissociation of mitochondrial cyclophilin (CyP-M), a matrix peptidyl-prolyl-cis-trans isomerase, from its putative binding site on the pore. Unbinding of CyP-M would follow a CsA-dependent or proton-dependent change in conformation of the CyP-M molecule. It is interesting that upon binding of CsA the enzymatic activity of CyP-M is inhibited, but it is not clear whether this event plays a role in pore inhibition. Here we report experiments designed to further test the role of CyP-M in pore function. Our results indicate that CyP-M-dependent and independent mechanisms of pore activation may exist, and that the peptidylprolyl-cis-trans-isomerase activity of CyP-M is not necessarily involved in pore modulation by CyP-M. (Mol Cell Biochem 174: 181–184, 1997)     

6-Hydroxydopamine activates the mitochondrial apoptosis pathway through p38 MAPK-mediated, p53-independent activation of Bax and PUMA

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used toxin to study Parkinson's disease. In previous work, we have demonstrated that 6-OHDA increases mitochondrial membrane permeability leading to cytochrome c release, but the precise mechanisms involved in this process remain unknown. Herein we studied the mechanism of increased mitochondrial permeability of SH-SY5Y neuroblastoma cells in response to 6-OHDA. Cytochrome c release induced by 6-OHDA occurred, in both SH-SY5Y cells and primary cultures, in the absence of mitochondrial swelling or a decrease in mitochondrial calcein fluorescence, suggesting little involvement of the mitochondrial permeability transition pore in this process. In contrast, 6-OHDA-induced cell death was associated with a significant translocation of the pro-apoptotic Bax protein from the cytosol to mitochondria and with a significant induction of the BH3-only protein PUMA. Experiments in mouse embryonic fibroblasts deficient in Bax or PUMA demonstrated a role for both proteins in 6-OHDA-induced apoptosis. Although 6-OHDA elevated both total and nuclear p53 protein levels, activation of p53 was not essential for subsequent cell death. In contrast, we found that p38 mitogen-activated protein kinase (MAPK) was activated early during 6-OHDA-induced apoptosis, and that treatment with the p38 MAPK inhibitor SKF86002 potently inhibited PUMA induction, green fluorescent protein-Bax redistribution and apoptosis in response to 6-OHDA. These data demonstrate a critical involvement of p38 MAPK, PUMA, and Bax in 6-OHDA-induced apoptosis.     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.     

线粒体通透性转移孔与青蒿素抗疟机制研究

目的:为增进对青蒿素作用机制的了解,探讨参与调节线粒体体积的线粒体通透性转移孔在青蒿素抗疟机制中的作用.方法:分离线粒体,采用分光光度法检测青蒿素能否直接作用于离体线粒体导致线粒体体积变化;利用等效应图分析线粒体通透性转移孔抑制剂是否拮抗青蒿素的抗疟作用.结果:青蒿素可以直接导致离体疟原虫线粒体肿胀,而不会影响鼠肝线粒体体积;两种不同的线粒体通透性转移孔抑制剂均可拮抗青蒿素的抑疟效果.结论:青蒿素可以直接作用于离体疟原虫线粒体导致线粒体肿胀,且青蒿素导致线粒体肿胀的物种选择性与细胞毒性的物种选择性一致.此外,利用抑制剂阻断线粒体通透性转移孔的开放可以拮抗青蒿素的抗疟效果,证明线粒体通透性转移孔在青蒿素抗疟过程中起重要作用.     

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential ( _m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co²⁺ (CC) staining can be dissociated from the _m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent _m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.     

谷氨酰胺对心肌细胞线粒体膜PTP开放干预作用的实验研究

目的:探讨谷氨酰胺(Gln)对过度训练状态下心肌线粒体膜通透性转换孔(PTP)开放的干预作用及其可能机制。方法:30只SD大鼠随机分为3组(n=10):对照组(CG组)、过度训练组(OG组)和补充Gln+过度训练组(GOG组)。采用分光光度法检测大鼠心肌线粒体PTP开放程度,电化学法检测心肌丙二醛(MDA)、还原型谷胱苷肽(GSH)含量和磷脂酶A2(PLA2)活性。结果:OG组与GOG组比较,吸光度(A0)显著下降(P<0.05),吸光度变化(△A)值显著降低(P<0.05);荧光剂罗丹明123(Rh123)的荧光强度(F0)显著增强(P<0.05),Rh123荧光强度变化(△F)值明显降低(P<0.05)。与GOG组比较,线粒体GSH含量显著降低(P<0.05),PLA2活性显著增加(P<0.05);MDA含量显著升高(P<0.05)。结论:过度训练可导致心肌细胞线粒体PTP开放增加,过度训练状态下线粒体活性氧生成增多,PLA2活性增加及GSH的含量下降,补充外源性的Gln对这些变化有显著的干预作用。     

Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.