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Pharmacological Research

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ell death disguised: The mitochondrial permeability transition pores the c-subunit of the F1FO ATP synthase

lizabeth A. Jonasa,∗,1, George A. Porter Jrb,1, Gisela Beutnerb, Nelli Mnatsakanyana,ambiz N. Alavianc

Department of Internal Medicine, Section of Endocrinology, Yale University, New Haven, CT, USADepartment of Pediatrics (Cardiology), University of Rochester Medical Center, Rochester, NY, USADivision of Brain Sciences, Department of Medicine, Imperial College London, UK

r t i c l e i n f o

rticle history:eceived 12 February 2015eceived in revised form 9 April 2015ccepted 20 April 2015vailable online xxx

a b s t r a c t

Ion transport across the mitochondrial inner and outer membranes is central to mitochondrial function,including regulation of oxidative phosphorylation and cell death. Although essential for ATP productionby mitochondria, recent findings have confirmed that the c-subunit of the ATP synthase also housesa large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), thepersistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane and

eywords:itochondria

alcium dysregulationell deathetabolism

cell death. This review will discuss recent advances in understanding the molecular components of mPTP,its regulatory mechanisms and how these contribute directly to its physiological as well as pathologicalroles.

© 2015 Elsevier Ltd. All rights reserved.

on channels

. Mitochondria at the center of cell metabolism and celleath

Mitochondria are complex organelles responsible for producingnergy in the form of ATP for most eukaryotic cells. They reg-late several other essential processes including calcium (Ca2+)omeostasis, heme and steroid biosynthesis. In addition, the mito-hondrion lies at the center of the cellular response to stress andhe control of cell death.

To produce energy in the form of ATP, mitochondria utilize sub-trates produced in the cytosol by carbohydrate, lipid and proteinetabolic pathways. These products, particularly acetyl co-enzyme, enter the tricarboxylic acid cycle. Turns of the TCA cycle synthe-ize NADH and FADH2 that donate their electrons to the electronransport chain. The energy of the bonds of NADH and FADH2 is usedo pump H+ ions out of the matrix by the NADH dehydrogenase and

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

ther electron transport complexes, creating a proton motive forcehat in turn drives the F1FO ATP synthase [1]. Upon kinetic repo-itioning of the ATP synthase rotor, ATP is synthesized from ADP

∗ Corresponding author at: Department of Internal Medicine (Endocrinology),ale University School of Medicine, P.O. Box 208020, New Haven, CT 06520, USA.el.: +1 203 785 3087.

E-mail address: Elizabeth.jonas@yale.edu (E.A. Jonas).1 These authors contributed equally to the work.

ttp://dx.doi.org/10.1016/j.phrs.2015.04.013043-6618/© 2015 Elsevier Ltd. All rights reserved.

and Pi [2]. The machinery required for ADP/ATP exchange betweenthe cytoplasm and the matrix including the outer membrane volt-age dependent anion channel (VDAC) and the adenine nucleotidetransporter (ANT) at the inner membrane are intimately linked tothat of the ATP synthase [3].

2. Mitochondrial inner membrane leak: regulator ofmetabolic rate and uncoupling

There are two currents that complete the current loop of theproton pumping activity of the electron transport complexes. First,ATP is formed by hydrogen ion (H+) translocation through theATP synthase in the opposite direction to that of the electrontransport complexes. Second, an apparently wasteful leak in theinner mitochondrial membrane provides a pathway for uncou-pling of oxidation from phosphorylation as H+ ions enter thematrix through channels independent of ATP production. Classi-cally, uncoupling proteins carry out this role. Known physiologicalfunctions of uncoupling proteins are to generate heat for organismswith large surface to volume ratio, to depolarize mitochondriain order to temper oxidative damage and to regulate metabolic

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

rate during hibernation and at other times [4–7]. In addition touncoupling proteins, however, intrinsic uncoupling exists withinother inner mitochondrial membrane channels and transportersand within the F1FO ATP synthase [8,9].

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. Mitochondrial inner membrane Ca2+ cycling regulatesellular Ca2+ dynamics: the example of neuronal short termynaptic plasticity

Mitochondrial inner membrane depolarization occurs not onlyhrough proton movement but also via the flux of other ions includ-ng Ca2+ across mitochondrial membranes. Ca2+ movement into the

itochondrial matrix is a physiological event that takes place inesponse to increased cytosolic Ca2+ levels. Ca2+ buffering is fre-uently employed by mitochondria in cells that experience rapidlyhanging cytosolic Ca2+ levels.

Mitochondria regulate cytosolic levels of Ca2+ and the release ofa2+ and metabolites through an intricate system involving several

on channels. The discovery of the molecular structure for the Ca2+

niporter ion channel (MCU) at the mitochondrial inner membraneas generated increasing interest in mechanisms of Ca2+ manage-ent within the cell body of many types of cells and also in the

resynaptic terminals of neurons [10–13]. Additional isoforms ofCU and its helper MICU that confer tissue specificity and other

ehaviors have added to our understanding of the mechanismsf activity dependent energy production by mitochondria [14,15].itochondrial Ca2+ release also appears to be highly regulated,

nvolving both exchangers and channels, but, unlike the MCU, theolecular components of a Ca2+ release channel were only recently

iscovered and form the main focus of this review.Ca2+ re-release from mitochondria determines short term

ynaptic plasticity in certain neuronal synapses [16]. During neu-onal activity, Ca2+ influx across the plasma membrane occurshrough glutamate receptors and voltage gated Ca2+ channels. Aftera2+ enters the cytosol, Ca2+ clearance is performed by the actionsf Ca2+ ATPases at the plasma membrane and by buffering throughptake by intracellular stores including the endoplasmic reticulumER) and mitochondria [12,17]; these processes reset the normallyow Ca2+ levels present in resting neurons or neuronal synapses.he Ca2+ that is buffered by intracellular stores is eventually re-eleased, providing, for example, for residual Ca2+ in presynapticndings. Residual Ca2+ increases the amount of Ca2+ available forynaptic vesicle fusion, enhancing the amount of neurotransmittereleased for a given stimulus [18,19].

Ca2+-sensitive ligand gated mitochondrial channels, which areidely conserved and found in species from invertebrates toammals, open in response to elevated Ca2+ within the mito-

hondrial matrix. In the squid presynaptic terminal, opening of aa2+-activated mitochondrial channel is correlated with enhancedeurotransmitter release [20]. Electrophysiological recordings [21]emonstrate that within the resting presynaptic terminal, the con-uctance of mitochondrial membranes is low [20]. In contrast,uring high frequency electrical stimulation of the presynapticerve, a large increase in mitochondrial membrane ion channelctivity takes place [20]. The delay in onset of the mitochon-rial activity and the persistence of the mitochondrial activityfter stimulation are in keeping with the role of a channel and/orxchanger in re-releasing Ca2+ from mitochondria for short termlasticity [20,22,23]. Furthermore, mitochondrial activity and shorterm increases in post stimulation synaptic transmitter releasere both abrogated by applying the uncoupler FCCP (carbonylyanide p-trifluoromethoxyphenylhydrazone), which depolarizesitochondria, preventing Ca2+ handling [20].

. Bcl-2 family proteins: regulators of mitochondrial outerembrane permeability and cell death

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In contrast to the normal physiological role for Ca2+ releasehannels and exchangers in mitochondrial membranes, one ofhe main regulators of pathological mitochondrial permeability

PRESSesearch xxx (2015) xxx–xxx

or leakiness are the proteins of the Bcl-2 family. Programmedcell death (apoptosis) in vertebrate cells may be initiated bysignaling at the plasma membrane or by intracellular pathwaysthat lead to changes in mitochondria [24]. The final common path-way for programmed cell death in many systems is mitochondrialouter membrane permeabilization (MOMP) [25–28]. Pro-apoptoticBcl-2 family members such as Bax regulate MOMP by induc-ing the formation of large outer membrane pores comprised ofactivated oligomerized proteins, aided by other pro-apoptotic moi-eties [25,27,29]. In their canonical role, the anti-apoptotic Bcl-2family proteins such as Bcl-xL protect cells against MOMP by inter-acting with, and inhibiting the pore forming properties of, thepro-apoptotic family members [28,30].

MOMP leads to the release of several inter-membrane spaceproteins such as cytochrome c [31,32]. The resultant decreasein cytochrome c levels compromises the ability of mitochondriato maintain the mitochondrial inner membrane potential and toproduce ATP [33]. In addition, cytochrome c released into the cyto-plasm activates downstream cytosolic enzyme pathways includingeffector caspases that execute cell death [34].

5. The mitochondrial permeability transition andpathophysiology

In some cases, an increase in mitochondrial outer membranepermeability may also be triggered by an acute inner membranedepolarization [35], particularly after cytosolic and mitochondrialCa2+ overload. Although Ca2+ uptake and re-release from mitochon-dria is a normal physiological event in cells, accumulation of Ca2+

in the matrix can have detrimental effects, including diminutionin energy production by the ATP synthase [36]. Ca2+ overload canproduce an uncoupling process described historically as a rapidincrease in permeability of the mitochondrial inner membraneto solutes and the halting of ATP production [37–39]. This phe-nomenon is termed permeability transition (PT).

PT can be reversible or irreversible [35,37,40–45]. If notreversed, PT leads to an even more extreme form of catastrophicPT associated with structural breakdown of the mitochondrialmatrix accompanied by outer mitochondrial membrane ruptureand cell death [35]. The interaction of this kind of mitochondrial celldeath with apoptotic death produced by MOMP has been debated.Although the two types of cell death seem to be overlapping, itis safe to say that pathological PT is associated with necrotic celldeath such as is found in ischemia or injury whereas MOMP occur-ring in the presence of sufficient amounts of ATP may have a moreimportant role in developmental and genetically predetermineddeath [46,47]. Inter-membrane space pro-apoptotic factors suchas cytochrome c and Smac/DIABLO are released during both formsof cell death. In MOMP, outer membrane permeabilization aloneleads to release of these factors, whereas in prolonged PT, ruptureof the outer membrane after inner membrane swelling releasespro-apoptotic factors into the cytosol [35].

6. Physiological functions of the permeability transitionpore

PT has been extensively studied for its role in ischemic injuryin brain, heart and other organs as well as in neurodegenerativeconditions [48]. In the heart, data suggest that opening of themPTP during early reperfusion after ischemia is a harmful event

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

that precipitates further damage to the myocardium [49]. Howeveradditional data also suggest that transient mPTP opening duringpreconditioning can be protective, thus serving a physiological roleeven during injury [43].

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Further evidence of physiological opening of the mPTP hasmerged over the last few years [40–45]. Flickering of the mPTPas described in the late 1990s [50,51], and an association

etween transient mPTP opening and “superoxide flashes” has beenescribed in striated muscle mitochondria [44]. It has been hypoth-sized that transient opening of the mPTP releases mitochondrialatrix Ca2+ to maintain mitochondrial homeostasis [52] although

his function of the mPTP has recently be questioned [53], ands mentioned above, mitochondrial matrix Ca2+ re-release regu-ates synaptic transmission [20]. Finally, as discussed in more detailelow, we have observed physiological, long-term opening of thePTP in the early embryonic heart [54].

. Regulation of the permeability transition pore

The mPTP is induced by elevated mitochondrial matrix Ca2+,OS, inorganic phosphate, and intracellular acidification [55,56].

n contrast, it is inhibited by ATP/ADP and Mg2+ [40,57]. Recenteports have also confirmed increased activity of PT by polyphos-hates, chains of 10s to 100s of repeating phosphates linked byTP-like high energy bonds [58–61]. The actions of Ca2+ may alsoequire polyhydroxybutyrate (PHB), which enters mitochondriand enhances the ability of Ca2+ to induce PT [62].

Many signaling cascades have been proposed to cause or pro-ect against ischemia reperfusion injury by targeting the mPTP. TheISK (Reperfusion Injury Salvage Kinase) pathway, involving PI3K,kt, and Erk 1/2; SAFE (Survivor Activating Factor Enhancement)athway, involving TNF� and STAT-3; and PKA, PKC, and PKG mayll converge on inactivation/phosphorylation of GSK3�, which isnown to regulate the mPTP [63–65]. However, the importance ofhese signaling cascades, in comparison to simple oxidative stress,as been questioned [64,66].

Cellular metabolic pathways regulate the PT. First, electronransport chain activity regulates the mPTP; an increase in mito-hondrial energization (membrane potential) inhibits the PT, whilee-energization/depolarization (a fall in membrane potential)nhances it [37,38,64]. Components of the glycolytic pathway maylso control opening of the mPTP, as the binding of hexokinase IIo the OMM in cardiac myocytes inhibits MOMP and PT [67]. Fur-hermore, a complex of ANT, VDAC, hexokinase, and mitochondrialreatine kinase (mtCK) regulates PT [68,69]. Combined, these datauggest that mitochondrial function and metabolic channeling ofubstrates and products (high energy phosphate bonds in the formf creatine kinase) into and out of mitochondria play a large role inhe regulation of the mPTP. Finally, the major pharmacologic agentssed to manipulate the mPTP target these metabolic pathways.or example, inhibitors of ANT can either attenuate (bongkrekiccid) or enhance (atractyloside) mPTP opening [38,70,71]. In addi-ion, the pharmacological agent most efficient in inhibiting PT isyclosporine A (CsA), an immunosuppressant drug which binds toyclophilin D (CypD) and inhibits the channel activity associatedith PT. CyPD binds to ANT, F1FO ATP synthase, and the phosphate

arrier (discussed below) further suggesting a role for metabolicathways in the regulation of the PTP.

. The quest for the PT pore

.1. Electrophysiologic properties of the mPTP

Thus, PT is an important event that performs both physio-ogic and pathophysiologic functions. Most current studies make

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

he assumption that PT begins as the opening of a Ca2+ sensitiveon channel in the inner mitochondrial membrane similar to theon channel activity initiated by mitochondrial Ca2+ influx occur-ing during physiological mitochondrial Ca2+ cycling. Such a Ca2+

PRESSesearch xxx (2015) xxx–xxx 3

release channel is heavily regulated; therefore it is assumed thatonly after prolonged opening does pathological PT (with MOMP)occur [72]. The conversion of a physiological Ca2+ extrusion mech-anism into a pathological channel opening is perhaps correlatedwith energy failure as a result of arrest of ATP synthesizing activ-ity and slowing of energy dependent Ca2+ extrusion mechanisms.Despite these hypothetical models, the factors that regulate thetransition from physiological to pathophysiological events are notcompletely understood. Therefore, identification of the molecularstructure of the pore will provide the target for regulatory activi-ties, allowing for a greater understanding of PT modulation duringhealth and disease.

Description of the biophysical properties of the pore that opensin the inner membrane during PT (the mPTP) provided the ear-liest indication that PT was initiated by the opening of an ionchannel. The first patch clamp recordings of mitochondrial innermembrane were published in 1987. This early report highlighteda ∼100 pS channel recorded by patch-clamping giant mouse livermitochondria produced by cuprizone application [73]. In the late1980s, a putative mPTP was recorded by patch-clamping mito-chondrial inner membrane or mitoplast preparations [74]. Theactivity occurred at positive potentials of the patch pipette and wasfound either in whole organelle mode or in single channel recor-dings in the organelle-attached configuration. The activity wasslightly anion over cation selective with multiple sub-conductancestates ranging from 30 pS to a peak single channel conductanceof 1.3 nS. Lower conductances were attributed to substates of thelarger channel openings because of long periods lacking activityfollowed by periods of multi-conductance behavior [74]. Conduc-tances of 550 pS were frequently observed at positive potentials.Gating was less common at negative potentials but this observationwas consistent with the presence of prolonged openings and fewersub-conductance steps at negative patch potentials contrasted withincreased flickering at positive potentials. The authors concludedthat conductance levels were not sharply defined, consistent withthe existence of many varied conductance levels of the channel.

Also in 1989, Kinnally et al. [75] recorded a similar mitochon-drial multiconductance channel (MMC) in mouse liver mitoplasts.This channel changed over time, with low activity at the onset of therecording followed by progressively higher activity at later timesduring the recording. The channels were sometimes open more fre-quently at negative potentials but at times rectification occurred inthe opposite direction (more frequently open at positive poten-tials). Channel activity displayed multiple conductances rangingfrom 10 to 1000 pS and was weakly cation-selective. These earlystudies began to establish expected criteria for activity of mPTP.

Shortly after the first recordings of the putative mPTP were per-formed, similar inner membrane activity was found to be inhibitedby CsA. In patch clamp experiments performed in liver mitochon-dria, channel activity was rapidly inhibited by submicromolarconcentrations of CsA in a manner consistent with the expres-sion of the binding site on the matrix side of the inner membrane.Ca2+-activated large conductance channel activity up to 1.3 nS wasinhibitable, but a 107 pS inner membrane conductance similar tothe first recorded inner mitochondrial membrane channel was alsoobserved in the recordings. This smaller conductance was resistantto CsA, suggesting that this activity might be due to a separate ionchannel [70]. The large conductance channel was sensitive to Mg2+,Mn2+, Ba2+ and Sr2+ in that order, which inhibited the activity in acompetitive manner with Ca2+, the main activator of the channel[55].

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

8.2. Characterization of a molecular complex regulating the pore

The recent identification of the molecular structure match-ing the biophysical properties of mPTP was aided by seemingly

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nrelated sets of findings. One was that Bcl-xL enhances metabolicfficiency (decreases uncoupling) by binding to the �-subunit ofhe ATP synthase. The second finding was that CypD, which hadeen known for many years to regulate PT, binds to the stator armf ATP synthase, specifically on the OSCP subunit. The third findinguggested that closure of the mPTP is related to the level of CypDctivity in a developmentally regulated manner as activity falls athe onset of respiration in mammalian heart. The final project foundhat ATP synthase assembles into a very large complex with otherroteins that may regulate the mPTP. These findings will now beiscussed in greater detail.

.3. Bcl-xL regulates metabolic efficiency by binding to the-subunit of the ATP synthase

Inefficiency of metabolism is correlated with cell death underonditions of neurodegeneration or acute cellular injury such asccurs during PT [76–78]. In contrast, a highly efficient state ofetabolism requires maximally decreased uncoupling of the innerembrane, and it has been found recently that Bcl-2 family pro-

eins regulate this efficiency by binding directly to the ATP synthase79–81]. It was known previously that Bcl-2 family proteins reg-late mitochondrial outer membrane permeability to produce or

nhibit synaptic degeneration and cell death [30,82–84]. There-ore, it seemed possible that Bcl-2 family proteins could formart of a large protein complex that regulates mPTP and celleath.

During the initiation of cell death an important function of Bcl-L, in addition to protection from MOMP, is to increase the releasef ATP through enhanced VDAC opening. This decreases the prob-bility of MOMP in cancer cell lines by providing extra ATP tovercome cell death stimuli [33,85]. In the neuronal synapse, injec-ion of either Bcl-xL or ATP enhances synaptic transmitter release86], suggesting that Bcl-xL increases ATP levels in the synapse [87].hese findings raise the possibility that Bcl-xL might regulate notnly the release but also the production of ATP.

In support of a role for Bcl-xL in the manufacture ofTP, hippocampal neurons overexpressing Bcl-xL show a large

ncrease in cytoplasmic ATP levels. Surprisingly, this increasen ATP accompanies a decrease in neuronal oxygen uptake anderobic glycolysis, consistent with the notion that Bcl-xL overex-ression increases mitochondrial bioenergetic efficiency [80,81].cl-xL depletion reverses these effects on metabolism, decreasingTP production and increasing oxygen uptake by resting cells

80,88].Direct interaction of Bcl-xL with the �-subunit of the ATP

ynthase maximizes the efficiency of ATP production [80,81]y decreasing a leak conductance in the mitochondrial innerembrane. This was first measured by patch clamp recordings

f submitochondrial vesicles (SMVs) enriched in ATP synthase80].

Closure of the leak within the inner membrane in the presencef Bcl-xL aids actively firing neurons to increase neurotransmitterelease [88,89], consistent with a correlation between the increasen metabolic efficiency and the long-term higher efficacy of synap-ic transmission found in Bcl-xL expressing neurons. In contrast,pening of the Bcl-xL-regulated leak decreases metabolic efficiencynd predisposes neurons to death, providing yet another clue thathe Bcl-xL-regulated inner membrane leak could be mPTP. Further-

ore, neurons lacking Bcl-xL display a fluctuating mitochondrial

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

nner membrane potential and a marked depolarization in the pres-nce of the ATP synthase inhibitor oligomycin [81]. These datauggest that Bcl-xL may regulate inner membrane coupling and celleath via direct effects on F1FO ATP synthase.

PRESSesearch xxx (2015) xxx–xxx

8.4. Cyclophilin D binds to ATP synthase and regulatespermeability transition

Another piece of the puzzle that helped determine the molecularcomponents of mPTP was the discovery of the interaction betweenCypD and ATP synthase [90]. It has long been known that CypD,a chaperone protein and peptidyl-prolyl cis-trans isomerase thatresides in the mitochondrial matrix, regulates the mPTP by enhanc-ing its sensitivity to Ca2+. In keeping with this, once it was foundthat CsA inhibits PT, CsA rapidly became one of the major tools tostudy the mPTP. Later, it was found that CsA attenuates mPTP byinhibiting CypD [91–93].

In 2005, experiments using CypD null mice demonstrated thatCypD was not itself the pore of the mPTP but that it played animportant regulatory role, particularly in the modulation of themPTP by Ca2+ [94–97]. Four groups showed that deletion of CypDdecreased sensitivity to ischemia–reperfusion injury in the heartand brain [94–96] and the Molkentin group suggested that mPTP’sphysiologic function is to maintain “homeostatic mitochondrialCa2+ levels to match metabolism with alterations in myocardialworkload” [52].

CypD expression varies widely among cell types. CypD is morehighly expressed in aged hearts, and these changes in expres-sion may regulate its association with a complex of proteinsthat increases mPTP opening during reperfusion [63]. Further-more, CypD activity appears to be regulated by cell signaling andmetabolic pathways [64,98–101] and by developmental cues indifferentiating myocytes (see below).

Although the exact mechanism of CypD’s action in the mito-chondrial matrix has not been clarified, recent data support theidea that this requires its binding to F1FO ATP synthase. First, thepropensity toward PT is regulated by ATP hydrolysis and synthesisin as much as this regulates the membrane potential. Therefore,PT requires twice the Ca2+ load in mitochondria that are in theprocess of hydrolyzing ATP (making a membrane potential) versussynthesizing ATP (dissipating the membrane potential) [38,39,56].Second, CypD binds to proteins on the stator of ATP synthase(OSCP, b and d-subunits, [71]) as well as to the F1FO ATP synthasebinding partners ANT [102,103] and the phosphate carrier (PiC,[52,104,105]). Recent work on the binding of CypD to ATP synthaseindicates that this occurs exclusively with OSCP, and depletionof OSCP using siRNA also depletes CypD [56] suggesting that theassociation regulates the expression of each protein. Interestingly,CsA decreases the propensity toward PT to the same degree (half)that decreases in OSCP expression increase the propensity towardPT (double), suggesting an inhibitory effect of OSCP on PT [56].Third, purified ATP synthase dimers produce current consistentwith mPTP in artificial lipid membranes [56]. The single channelactivity has a maximal conductance of 1–1.3 nS with subconduc-tance states. The current is not stimulated by Ca2+ or inhibited byCsA presumably because CypD is not present in the purified dimerpreparation although the enzymatic activity of the F1FO ATP syn-thase is preserved. The ATPase dimer current is also not sensitiveto bongkrekic acid or atractyloside, agents that primarily affectANT activity, making it unlikely that ANT forms part of the poreof the channel [56]. These data emphasize the idea that the regula-tion of the mPTP may occur via the interaction of CypD and othermolecules with F1FO ATP synthase and its binding partners.

8.5. PT activity regulates cardiac development

A third line of evidence that helped unravel the identity of the

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

mPTP was a series of studies of mitochondrial function during car-diac development. Until recently, the general consensus was thatopening of the mPTP was a devastating event that triggers celldeath. However, as discussed above, over the last two decades, data

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ave emerged suggesting that transient opening of the mPTP coulderve a physiologic purpose.

In the heart, physiological variations in mPTP activity play aritical role in cardiac myocyte differentiation and cardiac devel-pment [106]. Interestingly, the mPTP is open in myocytes inhe early embryonic mouse heart, and this opening is not associ-ted with any form of cell death. However, by the mid-embryonictage, the mPTP is closed [106]. This closure coincides with acti-ation of complex I of the electron transport chain, assembly oflectron transport chain supercomplexes called respirasomes, andctivation of oxidative phosphorylation [54]. These changes cause

fall in mitochondrial-derived ROS that signals the myocyte tondergo further differentiation [106]. Furthermore, pharmacolog-

cally inhibiting or genetically deleting mPTP enhances myocyteifferentiation, while opening mPTP inhibits differentiation [106].hese findings have been confirmed in cardiac stem cells [107,108],nd various reports have stressed the importance of the mPTP dur-ng cardiac development and myocyte differentiation [109,110].

.6. Regulatory molecules do not form the pore of mPTP

The F1FO ATP synthase interacts with a large number of pro-eins many of which have been candidates for mPTP. ANT was anarly candidate to form the mPTP since atractyloside and bongkre-ic acid, which inhibit ANT, affect the mPTP [38] and ANT was foundo interact with CypD [92]. VDAC was also an early candidate toorm the mPTP due to its high conductance and its association withNT in immunoprecipitation experiments [102]. In addition, it washown that a complex of ANT, VDAC, hexokinase, and mitochon-rial creatine kinase (mtCK) could form high conductance poreshen reconstituted into membranes [68,69]. Finally, the PiC is aore recent candidate to form the mPTP [105].However, genetic deletion of ANT1 and 2 and of the PiC

emonstrated that these proteins were not essential to mPTP for-ation, although these studies still supported their regulatory roles

104,111,112]. Furthermore, deletion of VDAC did not affect poreormation [113]. Additional data suggest that the conformation ofNT may be important for regulation of the mPTP [114]. Atracty-

oside induces mPTP opening and is known to stabilize the “c”onformation of ANT, such that the adenine nucleotide transportite faces the cytoplasmic, or intermembrane space, side of theMM [114]. In contrast, bongkrekic acid prevents mPTP openingnd stabilizes ANT in its “m,” or matrix facing conformation [114].owever, as both ATR and BKA inhibit ANT, it is unlikely that spe-ific effects on ADP/ATP translocation regulate the mPTP and a moreikely scenario is that the conformation of ANT itself can regulatehe mPTP. This is supported by a report that Ca2+ stabilization ofNT’s “c” conformation is related to its proline isomerization andypD is a peptidyl-prolyl, cis-trans isomerase [115].

It remains unclear how these candidate molecules regulate thePTP, but evidence suggests that they form large macromolecu-

ar structures with F1FO ATP synthase in the inner mitochondrialembrane. ANT and PiC can form a complex with F1FO ATP syn-

hase called the synthasome [3]. In addition, the large complex ofNT, VDAC, hexokinase and mtCK is likely also involved in the reg-lation of ATP synthesis [68,69]. Therefore, each of these moleculesay regulate the structure and activity of F1FO ATP synthase, and,

n doing so, modulate the opening of the mPTP.

. The mPTP, a molecular definition

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

.1. The c-subunit of F1FO ATP synthase comprises the PT pore

These various reports all agree that F1FO ATP synthase is a majoractor in the formation of the mPTP, and recent evidence suggests

PRESSesearch xxx (2015) xxx–xxx 5

that the FO or membrane portion of F1FO ATP synthase in factforms the pore [48,116–120]. Mammalian F1FO ATP synthase is a∼600 kDa complex of 15 subunits. The membrane portion, or FO,contains a ring of 8 very hydrophobic c-subunits and subunits a, b,e, f, g, and A6L. A stalk composed of the �, �, and � subunits connectsthe c-subunit ring to the catalytic F1 component made of a hexamerof alternating � and � subunits, where ATP synthesis and hydroly-sis occur. Finally, a stator containing the b, d, F6, and OSCP subunitsconnects the lateral portion of FO to the top of the F1. Movement ofprotons between the c-subunit and the a-subunit causes rotationof the c-subunit ring, the energy of which is transferred to F1 tosynthesize ATP [121–125].

Age-dependent structural and functional alterations of F1FO ATPsynthase in rat brain and heart mitochondria have been reported[126]. The FO portion is found to be present at 3 months in themembranes of heart mitochondria in the absence of an equivalentcomplement of F1. During the period from 3 to 12 months, the levelof F1 increases with an increase in ATPase activity and this accom-panies a decrease in proton leak consequent to binding of F1 to FO.Decreases of F1 content with respect to that observed for FO aredetected for aging heart in animals from 12 to 24 months suggest-ing the presence of lone FO leak channels in the membranes of theseaging mitochondria [126].

To determine if the membrane portion of the F1FO ATP synthasecomprises a cell death regulatory moiety that could produce PTunder cell stress, the Pinton group performed different measuresof cell death and PT after c-subunit expression levels were genet-ically manipulated either by depletion of all three isoforms of thec-subunit or by overexpression of a tagged version of the proteinin HeLa cells [116]. Given that proliferating cell lines rely on gly-colysis for ATP production in normal glucose-containing medium,depleting the c-subunit did not alter ATP levels in the cells. Deple-tion of the c-subunit, however, prevented both CsA-sensitive PTmeasured by the calcein-cobalt quench technique and by mito-chondrial morphological analysis. Cell death brought on by H2O2was also attenuated by c-subunit depletion as was cell death inneurons brought on by glutamate-induced excitotoxicity.

Although the above study indicated that the F1FO ATP synthasec-subunit was an important component of the mPTP [116], thiswork did not directly determine what portion of ATP synthasecould form the pore of the mPTP. Subsequent to that publication,work from the Bernardi group suggested that the mPTP could onlyform from dimers of ATP synthase [56], demonstrating mPTP-likechannel activity from purified dimers, and not monomers, of ATPsynthase. However, the regulation of such a purified mPTP by tra-ditional mechanisms has not been conclusively demonstrated, andthe actual pore of the mPTP in this model remained to be deter-mined. Furthermore, it was subsequently demonstrated by ourgroup that purified monomers and c-subunits (discussed below)can form the pore of mPTP [117]. Finally, the ability of ATP synthaseinhibitory factor 1 (IF1) to promote dimer formation yet prevent PTargues against the idea that ATP synthase dimers are necessary andsufficient for mPTP formation [127,128].

Membranous pores are usually formed by integral membraneproteins, and it is interesting that molecules that regulate PT areeither not membrane proteins, or, if they are integral membraneproteins (see above), they have been shown not to form the pore.For example, CypD and Bcl-xL regulate ATP synthesis by interactingwith the stator and � subunit of ATPase, respectively, and theseproteins are not embedded in the inner mitochondrial membrane.However, ATP synthase contains integral membrane proteins inits FO segment. 9 polypeptides form the FO and the stator, but

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

only three, a, b and c, are required for proton translocation andare evolutionarily highly conserved, like PT. In addition, �0 cellsthat lack mitochondrial DNA do not contain an a-subunit butdo undergo PT. Attention focused on the c-subunit, and not the

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-subunit, for a number of reasons. First, the structure of purifiedacterial c-subunit rings suggests that the center of the mammalian-subunit ring could form an ion conducting channel that wouldllow for uncoupling if the stalk partially or completely dissociatedrom it [129]. Furthermore, the c-subunit had been shown previ-usly to express ion channel activity [130]. As a matter of fact, theammalian c-subunit undergoes conformational changes from an-helix to a �-sheet when in contact with water, encompassingpaces that form the walls of ion channels [131]. These ion channelsave a diameter of 2.3 nm which allows molecules up to 1.5 kDa toass, similar to PT.

Homologues of ATP synthase c-subunit are also present in theO subcomplex of various vacuolar H+-ATPases, which are locatedoth on the plasma membrane and on intracellular membranes132,133]. It has been shown that transmembrane domain 1 ofOV1-ATPase c-subunits faces the center of the VO rotor structurend lines a water-accessible pore structure [134,135]. VO c-subunitsave been shown to be involved in formation of mega-channels inap junctions between cells [136]. Therefore pore-forming abilitys an important feature of all homologous c-subunits which shareimilar amino acid sequence [118].

.2. The c-subunit of ATP synthase creates the high conductancePTP pore

More recent experiments have directly tested the hypothe-is that the main membrane embedded portion of mammalian1FO ATP synthase, i.e. the c-subunit ring, forms the pore ofhe mPTP [118,117]. Indeed, electrophysiologic recordings of theurified mitochondrial c-subunit yielded a multi-conductance,oltage dependent channel with prominent subconductance states117]. Patches contained a ∼100-pS conductance, which appearedo be a subconductance state of the multiconductance activityather than a separate conductance. Peak single channel conduc-ances of ∼1.5–2 nS were similar to activity described previouslyor the mitochondrial multiple conductance channel (MCC) [75].lso consistent with MCC, channel activity often but not alwaysemonstrated negative rectification. At very positive patch pipetteotentials of over 100 mV, single channel conductances of ∼1.5 nSnd ∼2 nS were also consistently observed. There was a higherikelihood of observing single channel events or gating at posi-ive potentials, most likely because of the negative rectification,n keeping with similar activity observed previously for the mPTP74].

Regarding the voltage dependence of the c-subunit recor-ings, it should be pointed out that voltage dependence is an

nherent property of the channel [137] and is not dependentn the mitochondrial inner membrane potential or on the solu-ions used to record the currents. It can be measured by varyinghe voltage across the membrane (the command or holdingoltage). Early published current–voltage (I–V) curves of mPTP74,75] reveal that the channel shows both non-rectifying con-uctances and a rectification in the I–V that can be either inhe positive or negative voltage range. These data are consis-ent with I–V plots of recent recordings of the purified c-subunit117] (Fig. 1).

.3. F1 regulates biophysical characteristics of the purified-subunit

Purified c-subunit protein reconstituted into liposomes clearlyacks extrinsic regulatory moieties that are important for mPTPpening. The effects of Ca2+, a critical regulatory molecule, demon-

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

trate this property. Ca2+ binding sites have not been detected initochondrial FO, perhaps because mammalian c-subunits lack the

ormyl Met at the N-terminus to which Ca2+ binds to Escherichia coli

PRESSesearch xxx (2015) xxx–xxx

or chloroplast F1FO ATP synthase c-subunits [138]. In contrast, Ca2+

can bind to specific, low affinity and moderate capacity sites onthe �-subunit of mitochondrial F1 ATP synthase [139]. Other sitesin other ATP synthase-interacting molecules may also be impor-tant for regulation by Ca2+ and other agents [68,69]. Therefore,although the new models of the pore must account for all inducersand inhibitors, these molecules may not interact directly with the c-subunit pore itself, but may instead bind to sites in the F1 or to othermolecules such as ANT that undergo structural re-arrangements toopen and close the pore. Another example of this type of regulationis CypD.

In order to determine the location of the regulators, mitochon-drial recordings were carried out using purified mitochondrial andF1FO ATP synthase preparations. In these studies, the absence of aneffect of a modulator was taken as an indication that the ligand orbinding site for that modulator had been removed by the purifica-tion process. These studies demonstrated that the c-subunit leakchannel is regulated by the overlying F1 and peripheral regulatoryproteins [117]. In mitochondria or inner membrane preparationslacking the outer membrane such as submitochondrial vesicles(SMVs) [3], Ca2+ activates the c-subunit leak channel while CsAand ATP/ADP inhibit it, suggesting that the Ca2+ and CsA sensi-tive sites are present in these preparations. In contrast, removalof the F1 and other peripheral membrane proteins by urea treat-ment of the inner membrane or removal of CypD by purificationof ATP synthase monomers abrogates regulation of the c-subunitchannel by CsA and Ca2+ and greatly diminishes sensitivity toATP/ADP. These studies suggest that the CypD/Ca2+ binding site iscontained within or associated with the F1 portion of the ATP syn-thase and that a second, low-affinity ATP binding site exists in theFO. These results are consistent with reports identifying the bindingsite of CypD and benzodiazepine 423, an ATP synthase-inhibitoryand mPTP-sensitizing agent, on OSCP [140,141], and suggestthat the assembly of F1FO ATP synthase into monomers, dimers,and higher order oligomers can regulate the formation of themPTP.

Channel activity of the purified c-subunit is inhibited by thepurified �-subunit of F1, suggesting a structural rearrangementwhereby the stalk and F1 of the ATP synthase inhibits opening ofthe c-subunit channel, aided by ADP/ATP/Bcl-xL binding to the �-subunit and opposed by CypD/Ca2+ interaction with OSCP (Fig. 2). Inorder to demonstrate this further, mitochondria were treated withCa2+ following which the F1FO ATP synthase was immunoprecipi-tated using an antibody directed at the F1. These studies determinedthat Ca2+ exposure destabilizes the connection between the stalkand the c-subunit, disrupting protein/protein interaction betweenthe c-subunit and F1 (Fig. 3). The model suggests that the channelof the mPTP forms within the c-subunit ring itself upon reversibleCypD and Ca2+-dependent movement of the stalk away from thec-subunit [117].

It should be emphasized that the loss of protein/protein inter-action between F1 and FO does not require very drastic conditions;just 60 �M Ca2+ in the bath is enough to initiate what may indeedbe reversible PT [117]. This concentration is well within the rangeof physiological Ca2+ concentrations found within the mitochon-drial matrix [142–144] or concentrations measured adjacent to themitochondria in Ca2+ microdomains at the plasma membrane orER membrane [145,146]. That the loss of protein/protein interac-tion between F1 and FO is likely to be reversible has been shownupon chelation of Ca2+ in mitoplasts [68], intact mitochondria[147], intact neurons [20] as well as in reconstituted dimers of F-ATP synthase [148], suggesting that the F1 and the c-subunit canrecombine to close the mPTP, reforming intact F1FO ATP synthase

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

and re-initiating enzymatic function [149]. However, under cer-tain conditions, this separation may become irreversible, formingpathophysiological PT (with MOMP).

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Fig. 1. The current voltage relationship (I–V) of proteoliposome recordings of the purified c-subunit mimics that of mitoplast recordings of mPTP. (A–C) Graphs reproducedfrom Kinnally et al. [75] (with kind permission of Springer Science + Business Media) showing examples of the different I–V relationships for the mPTP found in differentmitoplast (isolated inner mitochondrial membrane) recordings. (D and E) Graphs reproduced from Alavian et al. [117] showing examples of the different I–V relationshipsfound in different proteoliposome recordings of the purified c-subunit. Shown in E are a low and high conductance mode of the channel.

FBa

Fi

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

ig. 2. Model of physiological mPTP activity: � subunit, which binds Bcl-xL and ATP/ADP, both Bcl-xL and CsA bind to F1 components during pore closure. The pore diameter also dend closed states.

ig. 3. Model of pathophysiological mPTP activity: Ca2+ induces separation of F1 from FO dun mPTP conductance. The components of F1FO that separate from the c-subunit during th

: The mitochondrial permeability transition pore as the c-subunit1016/j.phrs.2015.04.013

locks c-subunit conductance. CsA, inhibitor of CypD, blocks c-subunit conductance.creases during pore closure. The channel can switch reversibly between these open

ring mPTP opening. The pore diameter also expands during Ca2+-induced increasesis process are not yet completely known; the illustration here is schematic.

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Additional regulation of the mPTP may be due to the associationf other molecules with F1FO ATP synthase. As discussed above,1FO ATP synthase can complex with ANT and PiC. In the studyf F1FO ATP synthase dimers, bongkrekic acid, which inhibits ANT,ails to attenuate the mPTP channel activity [56]; this is recapit-lated in studies of SMVs [80]. Therefore, the regulation of thePTP by ANT and PiC may occur through their association with

he peripheral membrane components of F1FO ATP synthase. Fur-hermore, the association of F1FO ATP synthase and ANT with PiC,

tCK, VDAC and hexokinase may explain why mPTP regulation canccur via these molecules. Therefore, the layers of regulation thatontrol formation of the c-subunit ring/mPTP are very complex anday involve many known and undiscovered proteins that asso-

iate directly or indirectly with F1FO ATP synthase, as suggested inrevious reports [3,150,151].

Regulators of the mPTP may also work directly on the F1FOTP synthase itself. F1 has binding sites that accommodate theffects of Ca2+, Mg2+, adenine nucleotides and Pi; and through CypDun)binding those of H+, CsA and possibly of oxidants [152]. There-ore, in summary, the new model of mPTP describes either direct orndirect interaction with all known inducers, inhibitors and modu-ators of pore function.

.4. Structural location of the pore within the c-subunit ring

The exact location of the pore within the c-subunit is becomingncreasingly understood. Although it has been proposed that theore of the ion channel sits between the two lateral stalks of a dimerf F1FO ATP synthases and not within the c-subunit ring [56], theres currently no electrophysiological evidence for the formation ofuch a channel, and regulation of the mPTP by components of F156,117,153] argue against this. If the pore were located betweenimeric membrane-associated subunits, then F1 would need to beositioned over the dimeric link, outside of the FO, directed to theide of the complex, in order to regulate channel activity. This seemsnlikely to happen, and, thus, a model of the pore forming in theegion between monomers of F1FO ATP synthase requires furtherypothesis testing.

Rather, it is likely that the leak is located either within the cen-ral portion of the c-subunit ring, between the individual c-subunit

onomers, or between the c-subunit and the other FO subunits,lthough the latter is less likely given the presence of PT in �0 cellshat lack both mitochondrial DNA and the a-subunit [116]. In twoeparate experiments, it has been demonstrated that the c-subuniting expands when it conducts ions, making it likely that the pores formed by the c-subunit ring. The first experiment used fluores-ent tetracysteine display with the placement of cysteine pairs onll c-subunit monomers within the ring. These studies showed thata2+ influx into cells causes a decrease in fluorescence consistentith expansion of the diameter of the c-subunit ring, while CsA

ncreases fluorescence consistent with a decrease in ring diame-er [117]. Mutagenesis to increase the diameter of the c-subuniting also demonstrated that ring expansion is a means to increaseonductance. Mutations targeted to four highly conserved glycinesithin the first (N terminus) alpha-helical region of the c-subunit

154] decrease the tight packing of the ring and increase aver-ge single-channel conductance compared with WT c-subunit rings117].

These findings support the hypothesis that the c-subunit is nec-ssary and sufficient to produce the pore of mPTP. When viewedrom the inter-membrane space, the denuded c-subunit oligomerppears as a ring with a central pore-like structure that is normally

Please cite this article in press as: E.A. Jonas, et al., Cell death disguisedof the F1FO ATP synthase, Pharmacol Res (2015), http://dx.doi.org/10.

bscured by the F1 stalk components gamma, delta and epsilon,uggesting that the pore may form within the center of the ringiven the proper hydrophilic conformation [129]. Although it haseen suggested that phosphlipids occupy the central cavity of the

PRESSesearch xxx (2015) xxx–xxx

c-subunit ring in F1FO ATP synthases from different species[155–157] other evidence provides for formation of a proteolipidor proteophospholipid channel structure within the central lipidregion [58,62,118,131,158]. Data suggest a working model wherebythe c-subunit pore forms within the proteolipid milieu upon acti-vation of mPTP (for example by elevated matrix Ca2+) whereuponthe ring expands and F1 shifts; the pore is closed by a decrease indiameter of the ring and inactivated by binding of the F1 compo-nents to the ring (Figs. 2 and 3). The details of these changes andtheir regulation remain a work in progress.

10. Conclusion

For many years investigators have sought the identity of themolecular structure underlying acute alterations in mitochondrialmorphology and inner membrane conductance known collectivelyas mitochondrial PT. Early evidence asserted that PT was caused byopening of an inner membrane ion channel. More recent data havesupported this idea and shown that the c-subunit of the F1FO ATPsynthase forms a channel with similar biophysical characteristics tomPTP but lacking regulation by Ca2+ or CsA and with reduced sen-sitivity to adenine nucleotides. Depletion of c-subunit isoforms incells blocks CsA-dependent PT and subsequent cell death. The tra-ditional regulation of mPTP by Ca2+, Mg2+, Pi, adenine nucleotides,CsA, CypD and recently Bcl-xL has now been assigned to sites on theF1 including the stator complex and the enzymatic portion of ATPsynthase. Inhibitors and activators may also work through periph-eral regulatory moieties such as ANT, PiC and VDAC that exist in alarge complex of proteins with the F1FO ATP synthase. Lipids andpolyphosphates also may play an important role in pore gating orformation.

In this model, activators of the mPTP open the pore in a gat-ing mechanism in which F1 moves away from the mouth of thec-subunit ring while the ring expands (Fig. 3). This process isreversible, perhaps due to binding of F1 components like the �-subunit to the ring or by the re-association of the entire F1 onto thering (Fig. 2). Although an amazing amount of information has cometo light recently regarding the molecular structure and regulation ofmPTP, there is still much to do to understand the details. In addition,the role of mPTP during development of oxidative phosphorylation,in aging and in supercomplex formation comprise rapidly changingfields.

Acknowledgments

NIH NS045876; NS081746 to EAJ; American Heart AssociationGrant 12GRNT12060233 to GAP.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.phrs.2015.04.013

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