Biochimica et Biophysica Acta 1813 (2011) 616–622

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Review

Is mPTP the gatekeeper for necrosis, apoptosis, or both?☆

Kathleen W. Kinnally ⁎, Pablo M. Peixoto, Shin-Young Ryu, Laurent M. Dejean ⁎New York University College of Dentistry, Dept. Basic Sciences, 345 East 24th Street, New York, NY 10010, USA

Abbreviations: CsA, cyclosporine A; ROS, reactive oxdrial permeability transition; MOMP,mitochondrial outeiMACs, inhibitors of MAC☆ This article is part of a Special Issue entitled Mitoch⁎ Corresponding authors. Tel.: +1 212 998 9445; fax

E-mail addresses: kck1@nyu.edu (K.W. Kinnally), lm

0167-4889/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbamcr.2010.09.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 August 2010Received in revised form 22 September 2010Accepted 23 September 2010Available online 1 October 2010

Keywords:mPTP, mitochondrial permeability transitionporeMAC, mitochondrial apoptosis-inducedchannelVDAC, voltage-dependent anion-selectivechannelBcl-2 family proteinsPatch clamppharmacology

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. Thisphenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signalingcascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP)has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can leadto matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembranespace proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recentobservations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linkedto events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization duringcell death, involving three different mitochondrial channels, have been postulated. These include the mPTP inthe innermembrane, and themitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, andthe involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell deathare explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

ygen species; MPT, mitochon-r membrane permeabilization;

ondria: the deadly organelle.: +1 212 995 4087.d4@nyu.edu (L.M. Dejean).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

In recent years, the role of mitochondria in both apoptotic andnecrotic cell death has received considerable attention. An increase inmitochondrial membrane permeability is one of the key events inapoptotic and necrotic death, although the details of the mechanismsinvolved remain to be elucidated. Unlike the resting potentials ofneurons and cardiomyocytes, which are largely governed by basal K+

conductance of their plasma membranes, the value of the mitochon-drial resting potential is controlled by the very high resistance of theinner membrane, i.e., low permeability to all ions. While the innermembrane contains several channels, their opening is tightlyregulated in order to prevent dissipation of the membrane potentialand proton gradient that is the electrochemical energy reservoir forATP-synthesis and transport. These channels include the putativeK+

ATP channel and mitochondrial Centum-picoSiemen (mCS), whichare cation- and anion-selective channels that monitor metaboliclevels and aid in volume regulation, respectively [1–3]. There are alsoinnermembrane channels within the protein import complexes calledTIM22 and TIM23 [4,5]. The inner membrane also expresses multiplecalcium channel activities that range from the mitochondrial

ryanodine receptor (mRyR) of cardiomyocytes to the more genericcalcium uniporter [6,7]. Uncontrolled opening of any one of thesechannels may unleash havoc resulting in cell death. For example,exogenous targeting peptides putatively open the pore of the TIM23complex and induce a rapid and high-amplitude swelling ofmitochondria [8].

The mitochondrial permeability transition pore, or mPTP, ishowever the most notorious of all the inner membrane channels.mPTP has not only been linked to a rupture of mitochondrial outermembrane during cell death but also to a myriad of pathologies(Fig. 1) [9–11]. More recently, outer membrane channels such as themitochondrial apoptosis-induced channel (MAC) and the voltage-dependent anion channel (VDAC) were also shown to be directly orindirectly involved in mitochondrial permeabilization during apopto-sis and/or necrosis (Fig. 1) [12–15]. This review summarizes ourcurrent understanding of the role of the mPTP during mitochondrialmembrane permeabilization and how this channel may collaboratewith MAC and VDAC in order to regulate cell death.

2. Separating fact from fiction on the structure andfunction of mPTP

The mPTP was originally observed in swelling experiments onisolated mitochondria reported in landmark studies by Hunter andHaworth in 1979 [16–18]. The mPTP has a large caliper pore withlow ion selectivity. Opening of the mPTP increases the permeabilityof the inner membrane for molecules up to 1.5 kDa that leads toorganelle swelling and mitochondrial depolarization. The mPTP can

Fig. 1. Mitochondrial ion channels in apoptosis and necrosis. Left, apoptotic stimuli induce relocation of Bax from the cytosol into the mitochondrial outer membrane (MOM). Bax,Bak, and possibly other unidentified protein(s) oligomerize and formMAC to release cytochrome c. VDAC oligomerization upon apoptotic stimuli was also reported to be involved incytochrome c release. Right, necrotic stimuli lead to exacerbated calcium uptake and reactive oxygen species generation bymitochondria. High levels of calcium and reactive oxygenspecies (ROS) induce a cyclophilin-D (Cyp D)-sensitive opening of mPTP that leads to swelling of the matrix and release of calcium. Swelling disrupts the outer membrane, whilereleased calcium activates proteases, phosphatases, and nucleases that lead to necrotic degradation. Adapted from Ref. [112]. TIM23/22, translocases of the inner membrane mRyR,mitochondrial ryanodine receptor; TOM, translocase of the outer membrane; MCU, mitochondrial calcium uniporter; MIM, mitochondrial inner membrane.

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be activated in cells and metabolizing isolated mitochondria by amyriad of effectors but, most notably, calcium plus phosphate andreactive oxygen species (see an extensive review in Ref. [19] and amore recent review in Ref. [20]). Reversible closure of mPTP occursupon removal of calcium with EGTA or by the addition of ADP,magnesium, or cyclosporine A (CsA). The principal trigger for mPTPopening is matrix calcium in the presence of phosphate, but theactivating concentrations of both are thought to rely on severalother factors. For example, higher calcium levels are needed formPTP opening in the presence of any one of several mPTP inhibitorsincluding high negative membrane potential, low matrix pH,accumulation of adenine nucleotides like ADP, and other divalentcations like magnesium and strontium. Conversely, the calciumlevels needed for mPTP activation are lower if adenine nucleotidesare depleted or a mitochondrial uncoupler is added to depolarizethe membrane potential after the uptake of calcium. Opening of themPTP in response to oxidative stress has been linked to ischemia–reperfusion injury in the heart [9] and more recently to neurode-generative diseases, like Alzheimer's and multiple sclerosis [10,11].Hence, mPTP has become an important pharmacological target forboth cardio- and neuroprotection.

The mPTP can also temporarily open or “flicker” leading totransient membrane potential variations in response to certainintracellular signals unrelated to cell death. Then, mPTP closureleads to a “resealing” of the inner membrane and a restoration of theability to synthesize ATP [21]. This mode of action of the mPTPunderlies a mitochondrial version of “calcium-induced calciumrelease” between the cytosol and the mitochondrial matrix [22–24].That is, transient opening of mPTP could provide the pathway formitochondrial Ca2+ extrusion under relatively normal conditions. Therapid removal of calcium from the cytosol and its subsequent releasefrom mitochondria through mPTP flickering could prevent calciuminactivation of channels essential to refilling calcium stores as well asallow for signaling in these microdomains. Thus, mPTP flickeringcould impact processes as diverse as muscle contraction and salivasecretion [25,26].

Furthermore, brief opening of mPTP causes a transient mito-chondrial depolarization and a short burst of ROS production andreveals a potential role for mPTP in ROS signaling [27]. ROSgenerated by one mitochondrion might then interact with neigh-boring mitochondria, perhaps once again transiently opening mPTP.The ensuing depolarization could produce additional ROS. Mito-

chondria-generated ROS has putative roles in a myriad of cellsignaling as evidenced by the redox sensitivity of chaperones,kinases, and even gene expression [28]. Hence, mPTP can, at leastindirectly, impact many cellular processes that are not linked to celldeath. Nevertheless, flickering of the mPTP can result in sufficientproduction of ROS leading to sustained mPTP opening andeventually cell death [29].

Even though the phenomenon of mitochondrial permeabilitytransition was originally described in the late 1970s, the molecularcomposition of the mPTP remains a mystery today. The biochemicalstudies of mitochondrial complexes by Brdiczka's group [30] and theelectrophysiological studies of Zoratti's group [31] were among thefirst to propose that the complexes responsible for mPTP activityspanned both the inner and outermembranes of mitochondria. In fact,they were proposed to be in contact sites, or close junctions, betweenthe two mitochondrial membranes.

These watershed studies also led to the hypothesis that the mPTPcontains the outer membrane channel VDAC, which is also oftenreferred to as mitochondrial porin [30,31]. More recent studies inwhich the three Vdac isoforms are knocked out have raised doubtsabout the essential nature of the involvement of outer membranecomponents in mPTP as these knockouts continue to express mPTPactivity [32]. Nevertheless, VDAC continues to be purported as part ofthe mPTP mechanism in normal cells [33–35]. In other studies, VDAColigomers were even proposed to be directly responsible forcytochrome c release (Fig. 1) [15,36].

The adenine nucleotide translocator, or ANT, has long beenproposed to be an inner membrane component of mPTP. Some ofthese studies were biochemical, while others were pharmacological[30]. ANT inhibitors like bongkrekic acid and atractyloside lock thetranslocator in opposite conformations to prevent or induce amitochondrial permeability transition, respectively [37]. ANT is themost abundant inner membrane protein on a molar basis and isfrequently the target of misfolding. In this scenario, ANT undergoeslarge conformation changes and the atractyloside-stabilized confor-mation appears to be particularly vulnerable to damage leading tomisfolding and mPTP formation (reviewed in Refs. [38,39]). However,the seminal studies of Wallace's group clearly showed the mPTP wasin fact still present in Ant knockouts [40]. Hence, Ant does not providethe essential inner membrane permeability pathway for mPTP.Nevertheless, the adenine nucleotide translocator likely plays aregulatory role as the sensitivity of mPTP to certain pharmacological

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effectors was modified in mitochondria lacking all three isoforms ofAnt. Furthermore, knockouts of just Ant1 are more resistant toglutamate-induced excitotoxicity, a well-known mPTP-mediatedprocess [41]. Finally, the up-regulation of the uncoupling protein-3was shown to induce a sensitization of the mPTP to calcium [42,43],suggesting that innermembrane transporters other than the ANTmayalso regulate mitochondrial permeability transition.

More recently, the phosphate carrier was proposed to play thecentral role of inner membrane pore of mPTP [20]. While intriguing,the role of the phosphate carrier in the elusive mPTP awaits molecularstudies in which the effects of eliminating the various isoforms of thiscarrier on the mPTP characteristics are determined. Other putativecomponents have been identified including misfolded proteins [44]and polyphosphates [45]. Finally, other mPTP pore candidates couldaccount for the occurrence of mitochondrial permeabilization in ANT-deficient mitochondria. That is, other anion transporters, such as theaspartate-glutamate exchanger, were also found to form largeconductance pores [46–48].

Cyclophilin-D is a known activator of the mPTP as its inhibition byCsA leads to closure of the pore [49,50]. Cyclophilin-D-depletedmitochondria still undergo a calcium-induced permeability transition,but this phenomenon requires higher calcium concentrations and isinsensitive to CsA [51]. These observations reinforce the notion thatcyclophilin-D is an important regulator of themPTP but also refute thenotion that this protein might be a structural component of the pore.Finally, it was recently postulated that cyclophilin-D favors the openconformation of mPTP by masking a specific site for inorganicphosphate (Pi); the occupancy of this site by Pi leads to adesensitization of the mPTP to calcium [52].

3. mPTP and mechanisms of mitochondrial permeabilizationduring cell death

Apoptosis is a process of cell suicide that occurs in multi-cellularorganisms in response to developmental, homeostatic, or internaldamage signals. This programmed cell death involves extrinsic and/orintrinsic apoptotic pathways that converge in a series of biochemicalevents such as activation of caspases that leads to cell shrinkage andplasma membrane blebbing. On the other hand, necrosis is oftencaused by external factors, such as infection, toxins, trauma, orischemia–reperfusion injury that leads to cell swelling and eventuallyrupture of the plasma membrane. This scenario is in contrast toapoptosis, which is a naturally occurring cause of cellular death [53].Nevertheless, mitochondria compartmentalize a myriad of death-evoking signaling factors, whichwreak havoc upon their release to thecytosol and eventually cause either apoptotic or necrotic cell death.

During early intrinsic apoptosis, permeabilization of the mito-chondrial outer membrane releases pro-apoptotic factors, likecytochrome c, from the intermembrane space into the cytosol[12,54–56]. Importantly, this event is highly regulated throughspecific interactions between proteins of the Bcl-2 family [12,54–60]. In this family, effector proteins such as Bax and Bak are essentialto the machinery allowing membrane permeabilization. On the otherhand, anti-apoptotic members such as Bcl-2 and Bcl-xL inhibit thisprocess by directly binding to the pro-apoptotic effectors [57]. Finally,BH3-only proteins such as Bad or truncated Bid relay apoptotic stimulito the mitochondria by interacting with both effectors and anti-apoptotic Bcl-2 family members [54,55,57]. Therefore, when chal-lenged by an apoptotic stimulus, the combined signaling within theBcl-2 family dictates the immediate fate of the cell, i.e., whether or notto induce permeabilization of the outer membrane [54]. Thisphenomenon has been coined mitochondrial outer membranepermeabilization, or MOMP, and the released cytochrome c leads toan activation of the executioner caspases by proteolysis andeventually to plasma membrane blebbing [55,61].

Cytochrome c release during MOMP typically occurs through theouter membrane channel MAC. MOMP and MAC formation have beenconsistently reported in a variety of cell lines during at least twodifferent apoptotic insults, deprivation of interleukin-3 [62] andkinase inhibition [63]. MAC activity is observed before mitochondrialdepolarization, is exquisitely regulated by Bcl-2 family proteins, andcan initiate release of apoptotic mediators from mitochondria tocommit the cell to die [3,62–65]. Such release can in turn beprevented by overexpressing Bcl-2 or Bcl-xL or using inhibitors ofMAC (iMACs) [62,66]. Bax oligomers have been identified as part ofthis channel and knockout studies have shown that MAC containsneither Vdac1 nor Vdac3 [62,67]. On the other hand, Vdac2 wasshown to regulate the pro-apoptotic protein Bak, which is anotherputative component of MAC [67–69]. Finally, CsA does not preventMAC-induced cytochrome c release indicating that MAC and mPTPare two separate molecular entities [70]. However, some observa-tions suggest that MAC and mPTP may participate in an amplificationloop during cell death and this putative crosstalk is discussed belowin Section 5 [3,56].

Opening of the mPTP leads to a transitory permeabilization of theinner membrane also called mitochondrial permeability transition(MPT). Sustained opening of mPTP has catastrophic consequences forthese organelles. Mitochondria seem to pop as they rapidly swell;this swelling occurs because the high concentration of proteins in thematrix space exerts a large colloidal osmotic pressure. The cristaeunfold as the matrix swells so that the outer membrane ruptures.Depolarized mitochondria become a major liability for the cell as ATPis hydrolyzed in efforts to restore the membrane potential.Furthermore, this unspecific loss of outer membrane integrity alsocauses a spillage of mitochondrial death factors, like cytochrome c,from the intermembrane space to the cytosol. Finally, the collapse ofthe membrane potential combined with the leakage of pyridinenucleotides, e.g., NADH, can lead to the generation of reactive oxygenspecies via the direct transfer of electrons to molecular oxygen[20,71].

The outer membrane channel VDAC may act indirectly to facilitatemPTP opening. For example, VDAC1 was shown to regulate thetransport of calcium, an mPTP activator, across the outer membrane[72]. Intriguingly, partial closure of the channel, but not its opening,increases the calcium permeability of VDAC1 [14,73]. Interestingly,superoxide but not hydrogen peroxide induces a mitochondrialpermeabilization, which is blocked by VDAC1 antibodies [74].Therefore, VDAC1 may under certain circumstances act as an mPTPsensitizer even if it may not be a component of the pore [32].

Does VDAC play a role in cell death outside of modulation of themPTP? Oligomers of VDAC1 have been proposed to form channelstructures to allow the release of cytochrome c through the outermembrane during apoptosis [36]. However, even though VDAC1oligomers have been observed during apoptosis, their role ascomponents of a functional cytochrome c release channel remainsto be determined. VDAC activity is also known to be regulated by anti-apoptotic Bcl-2 family proteins [75]. The NMR solution structure ofhuman VDAC1 identified strands 17 and 18 as a putative binding sitefor Bcl-xL [76]. VDAC2 complexes with pro-apoptotic Bak andmediates its availability to form the cytochrome c release channelMAC [68,69,77]. Interestingly, pro-apoptotic Bax does not change thechannel activity of VDAC but the truncated BH3-only protein Bidcloses VDAC. This closure could limit metabolite transport and leadmitochondrial dysfunction and death [78,79].

The single channel behaviors of MAC and mPTP, as well as VDAC,are quite different as shown in the current traces of Fig. 2 and thesummary of their channel characteristics provided in Table 1. Asexpected, blocking MAC with iMACs [66,80] and mPTP with CsA alsoimpacts onset of cell death. Below, we will further discuss the role ofmPTP in apoptosis and necrosis and the possibility that mPTP andMAC may act synergistically during apoptosis.

Fig. 2. The channel activities of VDAC, mPTP, and MAC. Current traces were recordedfrom patches excised from either reconstituted outer (MAC and VDAC) or native innermembranes (mPTP) under symmetrical 150 mM KCl. Current traces are represented inthe same scale for comparison. Current levels of open (O) channels are shown withdownward transitions to closed (C) states in all the traces. Mouse MAC and VDAC traceswere adapted from Ref. [62], while mouse mPTP was from Ref. [56].

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4. mPTP opening: suicidal, criminal, or both?

In themid-1990s, themPTP attracted the attention of investigatorsin the cell death field, because it was reported that at least some formsof apoptosis could be inhibited by CsA [81,82]. Also, the suppression ofthe calcium activation of the mPTP by overexpression of Bcl-2supports a role for this channel in apoptosis [83,84]. However, theswelling of the matrix as proposed in the standard model of mPTPactivation is not observed in all scenarios of apoptosis [85]. Moreover,the permeabilization of the inner membrane is sometimes seen onlyin late apoptosis, after the release of cytochrome c and caspaseactivation [86]. Neither extrinsic nor intrinsic pathways of apoptosiswere altered in cells deficient in the mPTP activator cyclophilin-D[51,87,88]. These findings suggest that mPTP opening does not initiateapoptosis and that this pore is instead central to necrosis [89–91].These observations suggest that a more selective mechanism ofpermeabilization such asMAC formationmay typically be operating atleast during early apoptosis [29,62,65,92,93].

Even if the pore is not a principal player in apoptotic cell suicide,mPTP is certainly criminal in other ways. The involvement of themPTP in necrosis and ischemia–reperfusion injury of the heart is aparticularly well documented case [20]. The lack of oxygen supply totissues does not immediately lead to cell death during ischemia.However, the subsequent reperfusion triggers an oxidative stress thatcauses the demise of the cells. More particularly, it seems thatischemia initiates changes in the cells, such as the increase of free ADP,

Table 1Comparison of the channel activities of mPTP, MAC, and VDAC.a

mPTP MAC VDAC

Peak conductance (nS) 1.1±0.1 1–5 0.7±0.1Transition size (nS) 0.3–1.0 0.3–2.0 0.36±0.04Ion selectivity Sl. Cation Sl. Cation AnionPK/PCl 7 4.7±1.3 0.7±0.1Voltage-dependent Yes No YesPore diameter (nm) 2.8±0.1 2.7–6.0 2.2±0.05Physiologicalmodulators

Ca2+, Pi, ADP[16–18]

tBid, Cyt. c[65,67]

NADH [14,109]

Pharmacologicalagents

CsA [49] iMACs, Bcis[66,80,110]

Koenig'spolyanion, G3139,DIDS [36,111]

Sl, slightly; tBid, truncated Bid; Cyt. c, cytochrome c; CsA, cyclosporine A; iMACs,inhibitors of MAC; Bcis, Bax channel inhibitors; DIDS, 4,4′-diisothiocyanatostilbene-2,29-disulfonic acid.

a The description of the effects on mPTP, MAC, and VDAC of the physiologicalmodulators and pharmacological agents cited is given in Sections 2 and 3.

Pi, and of cytosolic calcium, which ultimately leads to superoxideproduction during reperfusion [9]. Such a scenario would lead to avicious cycle. The subsequent mPTP opening would further dysregu-late calcium homeostasis and inhibit the respiratory chain, resultingin further reactive oxygen species production and mPTP opening.Numerous experimental evidence supports the involvement of themPTP in ischemia–reperfusion. For example, CsA is the classicalinhibitor of the mPTP, and this agent can reduce the occurrence ofnecrosis in cardiomyocytes subjected to anoxia–reperfusion. CsA wasalso effective in protecting these same cells against reperfusion-induced mitochondrial membrane potential collapse [94]. Anotherexample of mPTP involvement during necrosis can be found in strokemodels of primary brain cells [95].

Examination of cyclophilin-D-deficient mice has provided evenmore compelling evidence that the mPTP plays a crucial role innecrosis [87,88,96]. The pore is still present in mitochondria and cellsobtained from these mice. However, cyclophilin-D ablation increasesthe amount of calcium required for mPTP opening and abolishes thesensitivity to CsA [97]. This phenotype has also been associated withthe resistance of cyclophilin-D knockout cells to necrotic stimuli suchas A23187-induced calcium overload, and to the decrease of heart andbrain injury following ischemia–reperfusion [87,88,96,97]. However,cells isolated from these same animals still died in response totreatments with classical apoptotic inducers such as staurosporine oretoposide [88].

Taken together, these data indicate that mPTP opening is chieflyinvolved in necrosis rather than in triggering cytochrome c releaseduring early apoptosis. Nevertheless, MAC and mPTP opening may actalone or in combination, depending on cell type and death stimulus, toamplify the death signals. In this context, the swelling precipitated bymPTP opening would cause a remodeling of the cristae, which couldfacilitate a more complete release of cytochrome c and other pro-apoptotic factors from the mitochondria [29,98].

5. Crosstalk between mPTP and MAC links necrosis and apoptosis

A synergistic relationship between apoptosis and necrosis couldenhance removal of damaged cells and minimize inflammation, whilerestoring tissue homeostasis. Crosstalk signaling between mPTPopening resulting in MPT and MAC formation resulting in MOMPcould provide such a platform. It is known that MAC formationprecedes mPTP opening in many cases, e.g., interleukin-3 withdrawalfrom FL5.12 cells [3,62]. However, MAC assembly may also occur aftermPTP opening in other situations, e.g., hepatitis in mouse caused bylipopolysaccharide through the tumor necrosis factor receptorpathway [99]. Recently, other modes of interaction between MACand mPTP have been identified.

As discussed above, mechanisms that block mPTP, like CsA orknocking out cyclophilin-D, reduce necrosis and, in some cases, alsosuppress apoptosis [3]. The reversible events leading up to necroticcell death often include a reduction in available energy sources likeATP and a switch from oxidative phosphorylation to glycolysis as themajor source of ATP synthesis. Cells swell as Na+ accumulates in theface of reduced Na+/K+ ATPase activity. Low levels of calcium entryinitiate limited mPTP opening as well as activation of proteases andphosphatases. If the irritant is removed, the cells may recover.Persistent irritation or massive harmful stimuli may cause irreversibledamage.When cells can no longer cope, plasmamembrane integrity islost and the cells are considered dead by necrosis.

Cellular stresses, which if sustained will result in mPTP openingand necrosis, may also activate intrinsic apoptosis. Clearly, calciumoverload opens mPTP and depolarizes mitochondria, which thenjeopardizes buffering of cytosolic calcium transients. The resultingelevated levels of cytosolic calcium may then lead to MAC formationthrough activation of proteases and phosphatases like calpain andcalcineurin (Fig. 3). Calpain is a calcium-dependent cysteine protease

Fig. 3. Crosstalk between MAC and mPTP amplifies cell death by apoptosis and necrosis. Elevated cytosolic Ca2+ activates calpain and calcineurin, which facilitate MAC formationthrough activation of Bid, Bad, and Bax. Mitochondrial calcium overload in the matrix induces mPTP opening. Cytochrome c released after either MAC formation or mPTP openingfacilitates apoptosome formation and caspase activation. Released cytochrome c causes further dysregulation of calcium homeostasis by interacting with the ER calcium releasechannel IP3 receptors (IP3R). Anti-apoptotic Bcl-2 suppresses while ROS signaling enhances the formation/activation of MAC, mPTP, and IP3R. Thus, opening of MAC/mPTP,cytochrome c release, and activation of IP3R may form a positive feedback loop to amplify the cell death signal. ER and MITO indicate endoplasmic reticulum and mitochondria,respectively. Adapted from Ref. [3].

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that can cleave the BH3-only protein Bid. Newly formed truncated Bidcan then trigger Bax oligomerization and MAC assembly, leading toMOMP [100]. Similarly, calcineurin is a protein phosphatase that canactivate another BH3-only protein Bad. Dephosphorylated Badinterferes with the anti-apoptotic function of Bcl-2 or Bcl-xL, whichagain facilitates MAC formation [101–103]. Interestingly, depho-sphorylated Bad can also directly activate mPTP opening in theabsence of truncated Bid, thereby auto-amplifying mPTP-inducedmitochondrial permeabilization [103]. Hence, activation of calpainand calcineurin by calcium may amplify mPTP activation, facilitateMAC assembly, and commit cells to die by intrinsic apoptosis. Asimilar scenario follows excitotoxic injury resulting in necrosis viamPTP opening and then delayed apoptosis through MAC assemblyresulting from AMP-kinase activation of the BH3-only protein Bim[104].

Positive feedback between MAC formation and mPTP openingmay enhance cytochrome c release and progression of cell deathprocesses. In fact, there are several means to release cytochrome c[105]. One report indicates that released cytochrome c binds to andrelieves the calcium-dependent inactivation of IP3R in the ER, whichcauses further Ca2+ release during apoptosis [106]. This additionalcalcium release may activate mPTP, which would cause matrixswelling. The ensuing cristae remodeling and eventual rupture ofthe outer membrane caused by mPTP opening would enhancecytochrome c release from the folds of the inner membrane andultimately caspase activation. These findings suggest that a smallrelease of cytochrome c via MAC may form a positive feedback loopto amplify the cell death signals through further calcium dysregula-tion and opening of mPTP (Fig. 3). This loop utilizes many of thesame players recently identified as important in the transfer ofcalcium from the ER to mitochondria, which is needed to maintainnormal bioenergetics for survival and prevent autophagy [107]. Onecan imagine that inappropriate generation of IP3 could similarlyinduce opening of mPTP. Another such amplification loop is based

on oxidation of cardiolipin by ROS, which is often associated withischemia–reperfusion injury and early apoptosis in some cell types.Peroxidation of cardiolipin decreases the binding of cytochrome c tothe inner membrane, which increases its availability for release tothe cytosol [105]. Furthermore, remodeling of the cristae throughdisruption of OPA1 oligomers by truncated Bid also facilitatescytochrome c release [108]. Finally, caspase degradation of therespiratory chain might also generate ROS. In contrast, cells alsocontain the means to suppress apoptosis and necrosis, e.g., throughproteins like Bcl-2 and HSP70, which are now being explored fornovel therapeutics [105].

6. Future perspectives

The permeability of the mitochondrial membranes is key todecisions regarding survival and death and whether apoptosis ornecrosis will take place. Understanding the relationship between thesecell death pathways will provide insight into their regulation and mayreveal novel therapeutic targets. mPTP opening and MAC formationprovide opportunities for crosstalk signaling between apoptosis andnecrosis. One compelling problem in this field is lack of informationregarding the molecular basis of mPTP and its relationship with othermitochondrial channels like MAC and VDAC. Even though our directstudies of mitochondrial channels are somewhat limited, thesechannels are, nevertheless, emerging as promising therapeutic targetsfor aging and diseases including cancer and neurodegenerativediseases, like Parkinson's and Alzheimer's diseases.

Acknowledgments

This work was supported by the National Institutes of Health[grant GM57249] to K.W.K.We apologize that many important paperswere not cited here because of space constraints; many can be foundin the cited reviews.

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