the function of complexes between the outer mitochondrial membrane

Review

The function of complexes between the outer mitochondrial

membrane pore (VDAC) and the adenine nucleotide translocase

in regulation of energy metabolism and apoptosis

Mikhail Y. Vyssokikh1 and Dieter Brdiczka2�

1A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow,

Russia;2Department of Biology, University of Konstanz, Konstanz, Germany

Received: 9 May, 2003; accepted: 28 May, 2003

Key words: hexokinase, creatine kinase, free energy, permeability transition pore, Bax, cytochrome c

The outer mitochondrial membrane pore (VDAC) changes its structure either volt-

age-dependently in artificial membranes or physiologically by interaction with the ade-

nine nucleotide translocase (ANT) in the c-conformation. This interaction creates con-

tact sites and leads in addition to a specific organisation of cytochrome c in the

VDAC–ANT complexes. The VDAC structure that is specific for contact sites generates

a signal at the surface for several proteins in the cytosol to bind with high capacity, such

as hexokinase, glycerol kinase and Bax. If the VDAC binding site is not occupied by

hexokinase, the VDAC–ANT complex has two critical qualities: firstly, Bax gets access

to cytochrome c and secondly the ANT is set in its c-conformation that easily changes

conformation into an unspecific channel (uniporter) causing permeability transition.

Activity of bound hexokinase protects against both, it hinders Bax binding and employs

the ANT as anti-porter. The octamer of mitochondrial creatine kinase binds to VDAC

from the inner surface of the outer membrane. This firstly restrains interaction be-

tween VDAC and ANT and secondly changes the VDAC structure into low affinity for

hexokinase and Bax. Cytochrome c in the creatine kinase complex will be differently or-

ganised, not accessible to Bax and the ANT is run as anti-porter by the active creatine

kinase octamer. However, when, for example, free radicals cause dissociation of the

octamer, VDAC interacts with the ANT with the same results as described above:

Bax-dependent cytochrome c release and risk of permeability transition pore opening.

Eukaryotic porins are membrane proteinsthat form aqueous channels in the cell mem-

brane and the mitochondrial outer mem-brane. In contrast to bacterial porins that

Vol. 50 No. 2/2003

389–404

QUARTERLY

�To whom correspondence should be addressed: Department of Biology, University of Konstanz, D 78457Konstanz, Germany; e-mail: [email protected]

Abbreviations: ANT, adenine nucleotide translocase; CK, creatine kinase; HK, hexokinase; VDAC, volt-age-dependent anion channel.

have a similar function, the structure ofeukaryotic porins is not known at a useful res-olution. The mitochondrial outer membranepore was first characterised by MarcoColombini (1979) as a voltage dependentanion channel (VDAC). The properties ofVDAC have been widely investigated in recon-stituted systems by several groups (Colom-bini, 1979; Benz, 1994) exploring the main dif-ferences compared to bacterial porins. Theseare: sensitivity to voltage already at 30 mVand to polarity of the applied voltage. In gen-eral, bacterial and mitochondrial outer mem-branes have segregating functions and poreproteins in the membranes control limited ex-change. However, in bacteria the outer mem-brane has more protective functions, whereasin mitochondria communicative functionsstand in the foreground.VDAC plays an important role in coordina-

tion of communication. A substantial aspectof this management is a transient formationof complexes with other proteins. This will bethe topic of the present article.

STRUCTURE OF VDAC

Although the VDAC amino-acid sequence isvery different compared to the bacterialporins, it is assumed the mitochondrial outermembrane pore may form a �-barrel com-posed of 16 �-strands analogous to the knowstructure in bacteria (Casadio et al., 2002).Three different isotypes of VDAC are ex-pressed in cells (Sampson et al., 1997). Thefunction of them will not be described in de-tails. All experiments mentioned here relateto VDAC isotype I, which is the main speciesin the outer mitochondrial membrane.

VDAC CONDUCTANCE AND ION

SELECTIVITY

Analysis of the isolated VDAC after reconsti-tution in artificial bilayer membranes re-

sulted in the calculation of the pore diameterof 4 nm at a voltage smaller than 30 mV. Inthis high conductance state (4 nS at 1 M KClin the bathing fluid) the pore is anion selec-tive. Above 30 mV the diameter of the pore isreduced to 2 nm. The conductance decreasesto 2 nS and ion selectivity changes to cat-ion-selectivity (Colombini, 1979; Benz, 1994).The voltage-dependent conductance varia-tions are linked to large structural modifica-tion of VDAC. So far no information about thenature of this changes is available. It has beenpostulated that a positively charged loopmoves out of the channel (Song et al., 1998).But it is also possible that negative chargesmove into the mouth of the channel as it hasbeen observed for bacterial porins (Welte et

al., 1995). Macromolecules such as dextran,that can not penetrate the pore, have an os-motic effect on the aqueous interior of thechannel (Zimmerberg & Parsegian, 1986) andby that increase voltage sensitivity. In thepresence of dextran the low conductance cat-ion selective state is adopted already at 10 mV(Gellerich et al., 1993). It has been postulatedthat the pore acquires this state in the contactsites where the inner membrane potential ex-tends to the outer membrane by capacitativecoupling (Benz et al., 1990). Recently a newidea of generation of a potential at the outermembrane was proposed on a basis of meta-bolic cycling and translocation of ATP, ADP,and Pi across the inner membrane and differ-ent resistance in the contact sites (Lemeshko,2002).

PHYSIOLOGICAL SIGNIFICANCE OF

THE VOLTAGE DEPENDENCE

VDAC in the low conductance, cation-se-lective state is not permeable for ATP, ADPand other negatively charged small molecules.This has been demonstrated by Colombiniand co-worker investigating VDAC reconsti-tuted in artificial membranes (Rostovtseva &Colombini, 1997). In isolated mitochondria,

390 M. Y. Vyssokikh and D. Brdiczka 2003

enzymes in the inter-membrane space, such asadenylate kinase, have no access to externaladenine nucleotides in the presence of theKönig’s polyanion (König et al., 1977) that in-duces the low conductance state of VDAC(Benz et al., 1988). If we convey this results tothe physiological situation where more con-tact sites are formed and macromolecules arepresent, we have to assume that most VDACsare in the low conductance cation selectivestate. Indeed, mitochondria in situ of perme-abilised cardiomyocytes or skinned red mus-cle fibres have a 10 times higher apparent Kmfor ADP compared to isolated mitochondriafrom the same tissue. The high Kmcould be re-duced to normal values by generating porousouter membranes with digitonin or by thepro-apoptotic protein Bax suggesting that in

situ the outer membrane was not freely per-meable for ADP (Saks et al., 1995). Besidespure voltage dependent regulation, specificproteins in the intermembrane space or at-tached to the surface have been described thatphysiologically may also be involved in regula-tion of the pore permeability (Liu & Colom-bini, 1992; Saks et al., 2001).

PORINS AS SPECIFIC BINDING

SITES

In bacteria porins are receptors for variousphages. VDAC at the mitochondrial peripheryacts in a similar way as binding site for en-zymes such as hexokinase (Fiek et al., 1982)and glycerol kinase (Östlund et al., 1983). Ashift in transmembrane topology in the mem-brane also changes the surface-exposed loopsbetween the � strands. Considering the volt-age dependence of the transmembrane topol-ogy, this means that VDAC in the contact sitesmight have the role to reflect functionalchanges of the inner membrane potential atthe mitochondrial surface. Alternatively, suchsignalling to the surface of the outer mem-brane may be generated by interaction ofVDAC with the ANT (see below) if we assume

that the ANT would interact exclusively with acertain VDAC state.

CONTACT SITE CHARACTERISATION

AND ISOLATION

In support of this idea, it has been observedby immune electron microscopy and bindingstudies that hexokinase binds with higher ca-pacity to the outer membrane pore in the con-tact sites compared to pores beyond contacts(Weiler et al., 1985). Freeze fracturing analy-sis of isolated liver mitochondria revealedthat contact sites could be induced by dextranor atractyloside and suppressed by glycerol orDNP (Wicker et al., 1993; Bücheler et al.,1991). Thus, hexokinase binding to isolatedouter membrane or mitochondria in state ofinduced or suppressed contact sites was stud-ied. Hexokinase showed sigmoidal type ofbinding to mitochondria with contact sites,while binding to pure outer membrane or mi-tochondria with suppressed contact sites ledto hyperbolic binding curves (Wicker et al.,1993). This suggested a co-operative bindingof hexokinase to the pore in the contact sitearea. In agreement with this, hexokinase, bybinding, was activated and bound hexokinasewas found to form tetramers (Xie & Wilson,1990). In a recent investigation Hashimotoand Wilson (2000) were analysing differentepitops at the surface of bound hexokinase byspecific monoclonal antibodies. The authorsobserved a variation of the bound hexokinasestructure that correlated with functionalchanges of oxidative phosphorylation in theinner membrane. This direct reflection of in-ner membrane functions at the mitochondrialperiphery can be explained by the interactionsbetween VDAC in the outer and ANT in the in-ner mitochondrial membranes (Bühler et al.,1998; Beutner et al., 1996; Vyssokikh et al.,2001).Because of the high capacity of contact sites

for hexokinase binding, the enzyme was usedas a marker to isolate this membrane fraction

Vol. 50 VDAC–ANT complexes in regulation of energy metabolism and apoptosis 391

from osmotically disrupted mitochondria bydensity gradient centrifugation (Ohlendieck et

al.,1986; Adams et al., 1989). Recent applica-tion of the contact site isolation revealed inkidney mitochondria that hexokinase activity

in the contacts was decreasing when the ANTwas shifted to the m-conformation by pre-treatment with bongkrekic acid, whereas itwas increasing after induction of thec-conformation by pre-incubation withatractyloside (Fig. 1).We do not know how the structure of the

VDAC changes and whether the alterationsare induced by the membrane potential or bythe interaction with the ANT. However, wecan summarise the observations of volt-age-dependent conductance changes and stud-ies of hexokinase binding as showing thatVDAC alters its transmembrane topology cor-related to functional mitochondrial states. Be-cause it affects the affinity and capacity ofhexokinase binding, we can define a tensedand relaxed state of the pore in analogy to theregulation of allosteric enzymes. The tensedstate would be the low conductance pore in acomplex with the ANT, whereas the relaxedstate would be characterised by high conduc-tance without complex formation (Fig. 2).

CYTOCHROME c IS A COMPONENT

OF THE CONTACT SITES

It was observed that not only hexokinase butalso cytochrome c was a component of the con-tact sites (Wiêckowski et al., 2001) and its con-centration increased in the presence ofatractyloside and decreased upon treatmentwith bongkrekic acid (Fig. 1). Thus, similarlyas observed for hexokinase, the structure ofthe ANT was responsible for the cytochrome c

distribution at the surface of the inner mem-brane.

ISOLATION AND CHARACTERISATION

OF VDAC–ANT COMPLEXES

Complexes between VDAC and ANT weregenerated in vitro with isolated porin and ANTproteins (Bühler et al., 1998). But they werealso isolated from brain or kidney mem-


Figure 1. Variation of cytochrome c and hexo-

kinase in the contact site fraction of kidney mito-

chondria by perturbing porin–ANT complexes.

Distribution of inner membrane and contact site frac-tions in a sucrose density gradient separating sub-frac-tions of rat kidney mitochondria after osmotic shock.The gradient was divided into 55 fractions (FractionNo.) starting from 54.3% sucrose, left to 30.8%, right.Succinate dehydrogenase (SDH, dashed line) and hex-okinase (HK, solid bold line) were used as markers forthe inner and the outer membranes, respectively.Cytochrome c concentration (empty circles) in the dif-ferent fractions was determined by specific antibodiesand varied significantly in 500 �M atractyloside-treated (panel A), compared to 250 �M bongkrekate-treated (panel B) mitochondria.

branes, solubilized in Triton X-100, by bindingthe attached hexokinase to an anion ex-changer. It was found that the bound hexo-kinase complex contained VDAC and ANTisotype I (Vyssokikh et al., 2001). From thesame membrane extracts a second complexcomposed of porin, creatine kinase, and ANTcould be isolated (Beutner et al., 1996;Beutner et al., 1998). Based on the separationand purification of the different complexestwo types of VDAC–ANT aggregates could bedefined: one complex where VDAC and ANTinteracted directly and VDAC achieved higheraffinity for hexokinase (Fig. 2) and a secondcomplex where VDAC interacted indirectlywith the ANT through mitochondrial creatine

kinase octamer. In this case VDAC exposed adifferent structure at the surface that had lowaffinity for hexokinase (Fig. 2) as hexokinaseactivity was always absent in the creatinekinase complexes.Determination of the molecular mass of the

isolated complexes suggested that in bothcases tetramers of the active kinases were

present (Beutner et al., 1996). Hexokinase isactive as a monomer of molecular mass 100kDa, whereas the functionally active mito-chondrial creatine kinase unit is a dimer withmolecular mass 85 kDa. Four dimers of mito-chondrial creatine kinase form a cubic struc-ture with identical top and bottom faces thatwere able to connect two membranes (Rojo et

al., 1991). It is known from in vitro studiesthat the octamer of mitochondrial creatinekinase interacts directly with the outer mem-brane pore (Brdiczka et al., 1994), while the in-teraction with the ANT may be indirectthrough cardiolipin (Schlattner et al., 2001)that is known to be tightly bound to the ANT(Beyer & Klingenberg, 1985) (Fig. 2).

RECONSTITUTION OF VDAC–ANT

COMPLEXES

The hexokinase–porin–ANT complex and theporin-creatine kinase–ANT complex were iso-lated and functionally reconstituted in vesicles(Beutner et al., 1996). ATP loaded into the vesi-cles did not leak out, although VDAC was a


Figure 2. Different configurations of the VDAC–ANT complex connected to either hexokinase or creatine

kinase.

Each of the three components of the two kinase complexes can exist in different configurations that support or in-hibit complex formation. The adenine nucleotide translocator (ANT-c, induced by atractyloside) is facing the cytosoland interacts with the outer membrane pore (VDAC), whereas ANT-m (induced by bongkrekate) is orientated to thematrix and does not interact with VDAC. The green ring represents cardiolipin that might change from outer to in-ner leaflet of the membrane correlated to the ANT structure. Active ANT is a dimer. VDAC-t (tensed) is a dimerwhen it interacts with the ANT-c. VDAC-r (relaxed) is not bound to ANT and may also exist as monomer. A thirdstructure of VDAC, unable to bind hexokinase, is present in the complex with the octamer of creatine kinase (CK).Hexokinase (HK) associates to oligomers preferentially in the contact sites formed by the VDAC–ANT-c complex.The oligomerisation of hexokinase leads to activation of the enzyme. Monomers of hexokinase bind to VDAC beyondthe contacts with less affinity and activity. Creatine kinase (CK) binds to VDAC as octamer. The octamer preferen-tially associates with cardiolipin that is bound to ANT.

component of the complexes. However, the kin-ases in the complexes had access to the inter-nal ATP through the ANT. This was shown byeither glucose-6-phosphate formation from ex-ternal glucose via hexokinase or creatine phos-phate generation by creatine kinase from ex-ternal creatine. Both reactions were inhibitedby atractyloside, a specific inhibitor of theANT. The results suggested that the kinases,after reconstitution, were functionally coupledto the ANT. The ANT was controlling the ex-change of adenine nucleotides through porinand was working as anti-porter (Krämer &Palmieri, 1989). However, the functional stateof the ANT in the reconstituted complexescould be converted into an unspecific uni-por-ter (resembling the permeability transitionpore) by addition of Ca2+ as has also been dem-onstrated for several related mitochondrialtransport systems by Dierks et al. (1990).

IMPORTANCE OF METABOLIC

CHANNELLING IN REGULATION OF

ENERGY METABOLISM

The tight functional coupling of peripheralkinases to the ANT is important because of tworeasons: firstly, it regulates the activity of oxi-dative phosphorylation by ADP, and secondly,it increases the free energy (�G) in the ATPsystem. To explicate the first point we mayimagine the situation after eating a carbohy-drate-rich meal: High glucose concentration en-ters the resting muscle cell that must bephosphorylated and converted to UDP-glucoseto accomplish incorporation into glycogen. TheATP for this process is more efficiently pro-vided by oxidation of pyruvate in the mitochon-dria than by lactate formation. Hexokinasebound to the VDAC–ANT complex induces theoxidative pathway by converting intra-mito-chondrial ATP into ADP and by that it acti-vates oxidative phosphorylation. To describethe second advantage of metabolic channelling,we have to consider that all ATP en-ergy-consuming processes in the cell (including

ion pumps) depends on the transfer of the �phosphoryl group of ATP according to the reac-tion: ATP + X = X-P + ADP. In a subsequentstep X-P dissociates when, for example, ionsare pumped. This means that the efficiency ofATP to drive these phosphorylation reactionsdepends on the level of ADP and inorganicphosphate (Pi) during the reaction according tothe equation of �G = �Go’ + RT ln ([ADP][Pi]/[ATP]). Thus, the power (�G) of ATP andthe flux through the ATP-dependent reactionwould increase, if ADP and Pi would be low orbe continuously withdrawn from the reaction.In the complex between mitochondrial

creatine kinase and the ANT these require-ments are perfectly fulfilled. The equilibrium ofthe reaction will not be reached, as ATP, just ex-ported, is directly utilised and ADP produced isimmediately taken up into the matrix by theANT while Pi is excluded by complex formation.Thus, metabolic channelling through functionalcoupling between ANT and creatine kinasepushes the balance of the reaction betweencreatine and ATP far to the side of phos-phocreatine production. A further advantage ofthis coupling to the ANT is that the higher freeenergy of ATP at the surface of the inner mem-brane is preserved in appearance of a higherphosphocreatine/creatine quotient. ATP iselectrogenically exported by the ANT at the ex-pense of the membrane potential. It has beencalculated that this process increases the freeenergy of ATP by additional 12 kJ/mol (Heldt et

al., 1972). Thus the phosphocreatine/creatineratio could be increased by this quantitythrough coupling between mitochondrialcreatine kinase and the ANT.

THE VDAC–ANT COMPLEX AS

PERMEABILITY TRANSITION PORE

Mitochondria contain an unidentified struc-ture that forms a large unspecific pore underconditions of high matrix Ca2+, Pi and oxida-tive stress (Haworth & Hunter, 1980). Open-ing of this so called permeability transition


pore was fully reversible upon withdrawal ofCa2+ (Crompton & Costi, 1988). Electrophy-siological measurements revealed propertiesof the pore related to VDAC, suggesting that itmay be a component of the whole structure(Zoratti & Szabó, 1995; Kinally et al., 1993).The permeability transition pore could be spe-cifically blocked by cyclosporin A, which bindsto cyclophilin D, a peptidyl-prolyl-cis-trans iso-merase present in the mitochondrial matrix(Crompton & Costi, 1988; Halestrap &Davidson, 1990). By employing a cyclophilinD affinity matrix, a complex between VDACand ANT was isolated by Crompton et al.

(1998) after extracting heart mitochondrialmembranes with the detergent Chaps. Thiscomplex was reconstituted in vesicles andpore opening by Ca2+ and Pi addition was reg-istered in cyclosporin A-sensitive manner.Comparable results were obtained with theisolated hexokinase–porin–ANT complex.Cyclophilin was co-purified through isolationof the complex (Beutner et al., 1998). The com-plex was reconstituted in phospholipid vesi-cles that were loaded with malate. The inter-nal malate was released, correlated to increas-ing Ca2+ concentrations. The process was in-hibited by cyclosporin A, suggesting that thereconstituted hexokinase–porin–ANT com-plex had properties of the permeability transi-tion pore (Fig. 3A).Two questions are still discussed: firstly,

whether ANT is the only structure that causespermeability transition, and secondly, whetherANT forms the pore only as a complex withVDAC. Concerning the first question, it is pos-sible that all related antiporters such asPi/OH– or Glut/Asp exchangers can form apermeability transition pore depending on theactual condition. As to the second question, itis possible to claim that also the purified ANTforms unspecific pores in the presence ofCa2+. This was shown by reconstitution ofANT in bilayer membranes (Brustovetsky &Klingenberg, 1996) or in vesicles (Rück et al.,

1998). Yet these pores were not sensitive tocyclosporin A, as cyclophilin D was missing.

Cyclophilin D is thus a second important regu-latory component of the permeability transi-tion pore (Crompton et al. 1988; Halestrap &Davidson, 1990). The idea that VDAC mayhave as well regulatory functions was sup-ported by the analysis of the reconstitutedporin–creatine kinase–ANT complex. In con-trast to the reconstituted hexokinase com-plex, there was no release of enclosed malatefrom the vesicles by addition of Ca2+ (Fig. 3B).As shown schematically in Fig. 3B, VDAC islinked to the octamer of creatine kinase thathinders interaction with the ANT. However,direct interaction between VDAC and ANTwas possible after dissociation of the creatinekinase octamer. In this case malate was liber-ated dependent on Ca2+ addition (Fig. 3B).The release was inhibited by cyclosporin A.The results suggested that VDAC, similarly ascyclophilin D, might regulate of the permeabil-ity transition pore. A possible role of VDACmight be to keep the ANT in thec-conformation, exposing the ATP/ADP bind-ing site to the cytosolic surface of the innermembrane according to the re-orientating car-rier model proposed by Klingenberg (Klingen-berg et al., 1971). In this conformation, that isinduced by atractyloside, the conversion ofthe ANT into an unspecific pore by Ca2+ is fa-cilitated and leads to permeability transition.However, if VDAC and ANT interact with theoctamer of mitochondrial creatine kinase,conversion of ANT to the permeability transi-tion pore-like state would be suppressed (Fig.3B). In fact, this has been observed in a trans-genic mouse model expressing mitochondrialcreatine kinase in liver mitochondria(O’Gorman et al., 1997; Dolder et al., 2003).The permeability transition pore was openedin isolated mouse liver mitochondria byatractyloside and Ca2+. In contrast, in livermitochondria from transgenic mice, contain-ing creatine kinase, the permeability transi-tion was inhibited by substrates that stabi-lised the octamer of creatine kinase, such ascreatine and cyclocreatine.



Figure 3. The VDAC-ANT complex as permeability transition pore.

A, left panel: The scheme depicts the presumable arrangement of the hexokinase–porin–ANT complex in the con-tact sites of mitochondria. Hexokinase (HK) binds as a tetramer to the VDAC–ANT complex. Glycerol kinase (GK)and the mitochondrial benzodiazepin receptor (mBr) bind to the same pore configuration. A, right panel: The com-plex was isolated from a Triton X-100 extract of brain membranes and was reconstituted in phospholipid vesiclesthat were loaded with malate or ATP. Cyclophilin was co-purified with the complex and could be extracted from thecomplex with phosphate buffer, pH 4.5. The entrapped malate was released from the vesicles by Ca2+ between 100and 600 �M. Malate release was inhibited either after cyclophilin extraction (-Cyp D) or by incubation of the vesicleswith 0.5 �M cyclosporin A (CSA) when 200 or 500 �M Ca2+ were added. According to Brustovetsky & Klingenberg(1996) Ca2+ interacts with cardiolipin (DPG) around the ANT, thus changing the structure of the ANT fromATP/ADP antiporter to unspecific uniporter state. The uniporter state of ANT may act as permeability transitionpore (PTP) in intact mitochondria. B, left panel: The scheme shows a possible arrangement of the complex be-tween VDAC, the octamer of mitochondrial creatine kinase and the ANT in the contact sites of mitochondria. B,

right panel: The complex was isolated from the Triton X-100 extract of brain membranes and was reconstituted inegg yolk liposomes that were loaded with malate. Malate was not released by increasing Ca2+ concentrations(Octamer). When the octamer was dissociated into dimer by 20 min incubation of the liposomes with 5 mM MgCl2,20 mM creatine, 50 mM KNO3 and 4 mM ADP, porin–ANT complexes could be formed. In these complexes Ca2+

could shift the ANT structure to the unspecific PTP and the entrapped malate was liberated (Dimer). Malate perme-ability of PTP was inhibited by pre-incubation of the vesicles with 100 nM cyclosporin A (dimer + CSA). Theoctamer-dimer equilibrium of the mitochondrial creatine kinase can be shifted to dimer by reactive oxygen species(ROS) (Dolder et al., 2001).

B

A

It should be mentioned here that mitochon-drial creatine kinase, besides its importantrole in organisation of the peripheral mito-chondrial compartment, is also localised inthe cristae (Kottke et al., 1994) where it hascertainly different functions and is not linkedto VDAC.

THE VDAC–ANT COMPLEX AS A

TARGET FOR Bax DEPENDENT

CYTOCHROME c RELEASE

There are several signal- and tissue-specificpathways to induce apoptosis. One of them isa release of cytochrome c from mitochondria(Yang et al., 1997; Liu et al., 1996; Kluck et al.,

1997) that activates caspases (Liu et al., 1996)by binding to a cytoplasmic protein Apaf-1 inpresence of dATP (Saleh et al., 1999). Themechanism by which external Bax releasescytochrome c is still controversial and mayalso depend on the actual localisation ofcytochrome c besides the binding and incorpo-ration of Bax at the mitochondrial surface.VDAC and ANT interact preferentially

when adopting a specific molecular confor-mation. The ANT has to be in the c-conforma-tion, as atractyloside induces the complexwith the outer membrane pore (Vyssokikh et

al., 2001), while bongkrekate shifts the ANTto the m-conformation and by that repressesthis interaction. VDAC in the complex withthe ANT appears to be in the low conduc-tance cationically selective, tensed state.This can be deduced from the observationthat heart muscle mitochondria in situ showa reduced response to external ADP (Saks et

al., 1995). The structural change of the outermembrane pore achieved by interaction withthe ANT is recognised at the mitochondrialsurface and leads to higher affinity ofhexokinase. Recent observations suggestthat this tensed pore structure is the pre-ferred target of Bax molecules. Pastorino et

al. (2002) found that hexokinase and Baxcompete for the same binding site and

Capano and Crompton (2002) co-precipitatedVDAC and ANT when Bax was immuno-pre-cipitated from extracts of cardiomyocytes.

THE VDAC–ANT COMPLEXES

CONTAIN CYTOCHROME c

As described above, it was observed that thecontact sites contained cytochrome c. As theVDAC–ANT complexes are derived from thecontact sites, it was not surprising that thecomplexes contained a significant amount ofcytochrome c (Wiêckowski et al., 2001; Vysso-kikh et al., 2002). This was found, although thecomplex was eluted from anion exchanger col-umn by 200 mM KCl, suggesting that cyto-chrome c was not bound by ionic interaction.To investigate whether Bax would be able to in-teract with the cytochrome c within theVDAC–ANT complexes, the latter were recon-stituted in phospholipid vesicles as describedabove (Fig. 4A). After reconstitution, the vesi-cles were loaded with malate. Bax liberated theendogenous cytochrome c but did not releasethe internal malate (Fig. 4A). The Bax-depen-dent liberation of endogenous cytochrome c

was abolished when the VDAC–ANT complexwas dissociated by bongkrekate (Fig. 4B).

THE IMPORTANCE OF KINASES IN

REGULATION OF APOPTOSIS

Release of cytochrome c from mitochon-dria proceeds in two steps: the first one in-volves the release of a small fraction ofcytochrome c from the compartment be-tween the outer and the peripheral innermembranes and the contact sites. It is in-duced by Bax at low concentrations(Pastorino et al., 1999) in the early phase(Capano & Crompton, 2002). The secondstep includes release of additional fractionsof cytochrome c located at the surface of thecristae membranes and results from open-ing of the permeability transition pore, fol-



Figure 4. Bax-dependent release of cytochrome c from the hexokinase–VDAC–ANT complex.

A, left side, schematic representation of the reconstituted hexokinase(HK)–VDAC(P)–ANT complex in multi-layervesicles. Cytochrome c (red circles), that was still attached to the complexes, could be released by Bax as shown onthe right side. However, malate loaded into the vesicles was not liberated by Bax but by 200–500 �M Ca2+ throughopening of ANT as permeability transition pore (Fig. 3A). This suggested that Bax specifically interacted withcytochrome c in the porin–ANT complex. The sigmoidal curve of cytochrome c release might indicate that Bax is ac-complishing this process as oligomer. B, left side, schematic representation of the association/dissociation of thehexokinase(HK)–VDAC(P)–ANT complex by atractyloside (ATR) or bongkrekate (BA) after reconstitution inmulti-layer vesicles. Treatment of the complexes with bongkrekate changed ANT into the m-conformation (ANT-m).This led to dissociation of the complex and a correlated structural change of VDAC, with decreasing affinity forhexokinase and Bax. Cytochrome c might have redistributed as well. As shown on the right side, Bax was unable torelease cytochrome c after the structural change of the complex by bongkrekate. All cytochrome c remained in thesediment (BA sed) in contrast to the control, without bongkrekate treatment, whereas no cytochrome c appeared inthe supernatant (BA sup).

A

B

lowed by mitochondrial swelling and mem-brane disruption (Halestrap et al., 2002).The massive cytochrome c release can alsobe a consequence of VDAC closure preced-ing membrane disruption (Vander Heiden et

al., 1997). The observed failure of mitochon-dria to exchange adenine nucleotides withthe cytosol may be a consequence of thetransformation of VDAC into the tensedstate. The two described kinases bound toVDAC are involved in the regulation of bothprocesses of cytochrome c liberation.

SUPPRESSION OF Bax-DEPENDENT

CYTOCHROME c RELEASE AND

PERMEABILITY TRANSITION BY

HEXOKINASE

Hexokinase binds to the outer membranepore in its tensed state, when VDAC interactswith the ANT. In this state VDAC is not freelypermeable for adenine nucleotides. This hasbeen already observed in mitochondria in thepresence of dextran that increases hexokinasebinding and frequency of contacts betweenVDAC and ANT (Wicker et al., 1993). In thesemitochondria external pyruvate kinase hadless access to ADP produced by hexokinase(Gellerich et al., 2002). Although VDAC in thetensed state does not allow ATP transfer,hexokinase utilises mitochondrial ATP, sug-gesting a structural modification of VDACthrough binding of the enzyme.On the other side, the activity of mitochon-

drial bound hexokinase was found to be im-portant for protein kinase B-linked suppres-sion of cytochrome c release and apoptosis(Gottlob et al., 2001). The experiments sug-gested that Bax, following withdraw of growthhormone, liberated a small fraction of cyto-chrome c from the contact sites. In completeagreement with this, the Bax-dependent re-lease of cytochrome c bound in the VDAC–ANT complexes described above was inhibitedby activity of hexokinase in the presence ofglucose and ATP (Vyssokikh et al., 2002). This

was explained by stabilisation of hexokinasebinding to VDAC that is also the main targetof Bax. Besides this effect on Bax dependentcytochrome c release, activation ofhexokinase inhibited Ca2+-dependent openingof the permeability transition pore by ADPproduction (Beutner et al., 1998).

SUPPRESSION OF THE

PERMEABILITY TRANSITION BY

MITOCHONDRIAL CREATINE KINASE

Another kinase, which binds to VDAC, is mi-tochondrial creatine kinase. This enzyme in-teracts with porin exclusively in the octamericstate, while the dimer has only weak affinityto porin (Schlattner et al., 2001). Thus the as-sociation-dissociation equilibrium betweenoctamer and dimer determines the formationof octamer–VDAC complexes. Ligation of thecoronary artery in guinea pigs led to decreaseof octamer from 85% to 60% in the infarctedarea of heart (Soboll et al., 1999). Because ofthe dissociation of the creatine kinase octa-mer, the possibility of direct VDAC–ANT in-teraction was increased. As suggested above,the latter complexes may be the prerequisiteof Ca2+-induced permeability transition, fol-lowed by mitochondrial swelling, membranedisruption, cytochrome c release and apo-ptosis. Indeed, apoptotic cells encircle thearea of necrotic cells in infarcted heart. Heart,kidney and brain mitochondria contain twoANT isotypes. ANT isotype I is localised in theperipheral inner membrane, having higher af-finity to VDAC and cyclophilin, whereasisotype II is present in the cristae membranes(Vyssokikh et al., 2001). When ANT I wasover-expressed in cells, apoptosis was induced(Bauer et al., 1999). However, co-expression ofcyclophilin D or creatine kinase together withANT I abolished apoptosis induction. More-over, it has been observed that patients suffer-ing from dilated cardiomyopathy had signifi-cantly higher ANT I messenger RNA levels(Dörner et al., 1997). This phenomenon, such


as in the tissue culture experiments men-tioned above, may be responsible for the in-creased apoptosis rate observed in case ofcardiomyopathy. Considering that the ANT Iin the peripheral inner membrane would forma permeability transition pore, we assumethat proteins interacting with ANT I, such ascyclophilin and the octamer of mitochondrialcreatine kinase, would reduce the probabilityof pore opening and apoptosis induction.However, the chance of pore opening and per-meability transition would increase, if the ra-tio between the ANT I and its regulatory pro-teins is changing either by induction of ANT Ior by dissociation of the octamer of mitochon-drial creatine kinase.

CONCLUSION

The first electron microscopic observation ofthe dynamic behaviour of contact sites (Knoll& Brdiczka, 1983) reflected morphologicalperturbations in mitochondrial membranesinduced by association or dissociation of thedescribed kinase complexes. The differentcomponents of the complexes interact de-pending on their actual structures that areregulated by the mitochondrial membrane po-tential and the energy metabolism of the cell.Physiologically, the complexes are transientstructures that are subjected to regulation. Inthe isolated state they are stabilised becausealternatively interacting partners have beenremoved. Thus the reconstituted complexesrepresent certain functional states that thedifferent components have adopted. We haveshown how perturbation of a single compo-nent, such as ANT, changes the function ofthe whole complex. This means that physio-logical signals will change the complex archi-tecture as well, if they affect the properties ofindividual components, such as VDAC con-ductance or hexokinase or creatine kinases ac-tivity, with significant consequences for thecell.

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the function of complexes between the outer mitochondrial membrane

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