Ildiko Szabo

Matthias Soddemann

Luigi Leanza

Mario Zoratti

Dr Erich Gulbins
email: erich.gulbins@uni-due.de

Ildikò Szabò

Dr Ildiko Szabò

Single point mutations of a lysine residue change function of Bax and Bcl-x L expressed in Bax-and Bak-less mouse embryonic fibroblasts -novel insights into the molecular mechanisms of Bax-induced apoptosis

Keywords: ion channels, Bax, Bcl2-like proteins, apoptosis GRIVTLISFGAFVAKHLKTINQESCIEPLAESITDVLVRT-KRDWLVKQRGWDGFVEF Bcl-2 P10415 GRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRH-LHTWIQDNGGWDAFVEL Bcl-xL Q07817 GRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDH-LEPWIQENGGWDTFVEL Bax Q07812

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Single point mutations of a lysine residue change function of Bax and Bcl-xL expressed in Bax-and Bak-less mouse embryonic fibroblasts -novel insights into the molecular mechanisms of Bax-induced apoptosis

Introduction

Mitochondria mediate apoptosis by releasing proapoptotic factors such as cytochrome c, apoptosis-inducing factor (AIF), second mitochondria-derived activator of caspase (Smac, also known as Diablo), Htr2a/Omi, and endonucleases [START_REF] Wyllie | More than one way to go[END_REF] . The release of these factors is the direct consequence of the activation of pro-apoptotic proteins from the Bcl-2 (B-cell CLL/lymphoma 2) family, in particular Bax (Bcl-2-associated X) and Bak (Bcl-2 antagonist/killer-1). Various proteins in the Bcl-2 family are key positive or negative regulators of apoptosis. They share conserved regions called Bcl-2 homology domains (BH1, BH2, and BH3). Biochemical and subcellular fractionation studies localized the antiapoptotic members, such as Bcl-2 and Bcl-x L (Bcl-2-like 1), at the outer mitochondrial membranes (OMM) and, to a lesser extent, at the endoplasmic reticulum and nuclear membranes. Proapoptotic factors either reside at the OMM, as is true for Bak, or are cytosolic proteins in unstimulated cells, as is the case for Bax, Bid (BH3-interacting-domain death agonist), and Bad (Bcl-2-associated death promoter), at least in most cells.

Although it is very well known that Bax and Bak are crucial for the induction of apoptosis in many systems, the molecular details of Bax-mediated apoptotic events are still unclear (e.g., 2) . A diversity of stimuli have been suggested as initiators of the activation of Bax and Bak [START_REF] Chipuk | Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis[END_REF][START_REF] Nie | Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H 2 O2-induced apoptosis[END_REF] , including direct stimulation by BH3-only proteins such as Bid or Bim [START_REF] Gavathiotis | BAX activation is initiated at a novel interaction site[END_REF][START_REF] Kim | Stepwise activation of Bax and Bak by tBid, Bim and Puma initiates mitochondrial apoptosis[END_REF] . Bax activation is believed to be a highly regulated, multi-step process involving mitochondrial translocation and oligomerization, and this process ultimately leads to mitochondrial dysfunction and apoptosis (7,[START_REF] Annis | Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis[END_REF] . Upon the induction of apoptosis, Bax migrates to the mitochondria, where it is integrated into the outer membrane as a monomer. Bax monomers then oligomerize and may form large pores in the outer mitochondrial membrane [START_REF] Annis | Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis[END_REF] . In its inactive form, Bak is already constitutively present in mitochondrial membranes and is released from inhibitory binding partners upon apoptotic stimulation. Bcl-2 and Bcl-x L antagonise the activity of proapoptotic members at the OMM, at least partly through direct interactions [START_REF] Minn | Bcl-xL regulates apoptosis by heterodimerization-dependent and -independent mechanisms[END_REF] . Bax and Bid, as well as Bcl-x L and Bcl-2 form pores in synthetic lipid vesicles and membranes (e.g. [START_REF] Schendel | Ion channel activity of the BH3 only Bcl-2 family member, BID[END_REF][START_REF] Antonsson | Inhibition of Bax channel-forming activity by Bcl-2[END_REF][START_REF] Schlesinger | Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic Bcl-2[END_REF][START_REF] Minn | Bcl-x(L) forms an ion channel in synthetic lipid membranes[END_REF] . The ability of Bax to form ion-conducting pores in synthetic and mitochondrial membranes suggests that such pores are involved in the release of cytochrome c [START_REF] Saito | Bax-dependent transport of cytochrome c reconstitued in pure liposomes[END_REF][START_REF] Kuwana | Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane[END_REF][START_REF] Lovell | Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax[END_REF][START_REF] Martinez-Caballero | Assembly of the mitochondrial apoptosis-induced channel, MAC[END_REF] . A recent hypothesis envisions Bax working as a twin-arginine translocation (Tat)-like protein of the proteintransporting Tat system rather than as an always-open ion-conducting pore in the OMM [START_REF] Antignani | How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?[END_REF] . Bcl-2like proteins have also been shown to interact with some mitochondrial components. Evidence points to interactions between Bax and the machinery involved in mitochondrial fission and fusion, in particular mitofusin 1 (Mfn1), Mfn2, and dynamin-related protein 1 (Drp1) (e.g., 18, 19 and references therein) . Bcl-x L interacts with the adenine nucleotide translocator, the most abundant protein in the inner mitochondrial membrane and an important component of the permeability transition pore complex, and it has been proposed that this interaction prevents cell death [START_REF] Brenner | Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator[END_REF] . It has also been suggested that Bcl-2 as well as Bax interact with the voltage-dependent anion channel [START_REF] Vander Heiden | Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival[END_REF][START_REF] Shimizu | Bcl-2 proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC[END_REF] .

We have recently shown that OMM-integrated Bax directly interacts with and inhibits an inner membrane-located mitochondrial potassium channel, Kv1.3 [START_REF] Szabò | A novel potassium channel in lymphocyte mitochondria[END_REF] , in lymphocytes [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] . Kv1.3 has been shown to be expressed in different tissues and cell types, namely in brain, lung, thymus, spleen, lymph node, fibroblasts, B lymphocytes, T lymphocytes, tonsils, macrophages, microglia, oligodendrocytes, osteoclasts, platelets, liver, skeletal muscle, and brown and white fat [START_REF] Gutman | International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels[END_REF] . The absence of Kv1.3 in lymphocytes prevents Bax-induced release of cytochrome c, as well as changes in mitochondrial membrane potential and the production of reactive oxygen species (ROS). In a previous study we showed that wild-type Bax inhibits Kv1.3 and induces the release of cytochrome c from isolated mitochondria, whereas Bax with a single point mutation (K128E) does not induce this effect [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] . According to a model of the structure of the membrane-integrated Bax monomer, at least amino acids 127 and 128, located between the 5th and 6th helices of Bax, protrude from the outer mitochondrial membrane into the intermembrane space [START_REF] Annis | Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis[END_REF] . The amino acid at position 128 is a highly conserved, positively charged lysine that mimics the action of the crucial lysine in Kv1.3blocking toxins, probably by binding to the ring of four aspartate residues of the channel vestibule, -5which faces the intermembrane space in mitochondria. We also determined that Bax binds to Kv1.3 in a toxin-like manner, i.e. to the vestibule region of the channel and preincubation of Bax with recombinant Kv1.3 prevents its pro-apoptotic effects in isolated mitochondria [START_REF] Gulbins | Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes[END_REF] . The physiological relevance of Kv1.3 for apoptosis was illustrated by the facts that knock-down of Kv1.3 expression in human peripheral blood lymphocytes impaired apoptosis in these cells, and expression of mitochondria-targeted Kv1.3 was sufficient to sensitize apoptosis-resistant CTLL-2 T lymphocytes, which lack Kv channels.

Here, we test the function of Bax(K128E) in a cell system and analyse the mechanism of Bax-Kv1.3 interactions also by using a mutant of Bcl-x L as a model. We report that mutation of lysine 128 in Bax to glutamate (Bax K128E) abrogates the pro-apoptotic function of Bax in a cellular context. The critical role of lysine 128 in Bax for the induction of apoptosis is demonstrated by the finding that exchange of Glu158 of Bcl-x L (corresponding to K128 in Bax) with lysine converts this anti-apoptotic protein into a pro-apoptotic protein. Our findings reveal that a conserved lysine in Bax plays an important role in the induction of apoptosis in vivo.

Results

Previous findings [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] demonstrated that Bax requires lysine 128 to induce apoptotic events in isolated mitochondria. The mutation of lysine 128 in Bax prevents the release of cytochrome c and the depolarisation of the mitochondrial membrane, events that were observed after the incubation of isolated mitochondria with wild-type Bax. These findings indicate that the positively charged lysine of Bax plugs the pore of the mitochondrial potassium channel Kv1.3, and thereby initiates mitochondrial changes during apoptosis. A detailed sequence analysis of the anti-apoptotic proteins Bcl-2 and Bcl-x L in various species revealed that these proteins contain a negative charge in the position corresponding to that of K128 in Bax (based on amino acid sequence alignment): aa 158 for Bcl-x L (Fig. 1). In these anti-apoptotic proteins, a conserved lysine/arginine precedes this glutamate. Thus, whereas in pro-apoptotic Bcl-2-like proteins an isolated positive charge is found at aa 128, in anti-apoptotic Bcl-2-like proteins the positive charge at aa 157 is counterbalanced by a negative charge at position 158, and this counterbalancing results in a net charge of zero in antiapoptotic proteins at this site. The absence of a net positive charge at this site is expected to prevent inhibition of Kv1.3 by the anti-apoptotic Bcl-2-like proteins, even if they are in close contact with each other.

On the basis of these considerations, and to gain insight into the mechanism of Kv1.3-Bax interaction, we replaced glutamate 158 in Bcl-x L with a lysine and tested whether the mutant protein inhibits Kv1.3 and triggers pro-apoptotic events. First we used patch clamp experiments on Jurkat T lymphocytes to determine the effect of the E158K mutant of Bcl-x L on Kv1.3. Bcl-x L E158K caused a marked inhibition of Kv1.3 (Fig. 2A), comparable to that caused by wild-type Bax (Fig. 2B) (IC 50 of 5 nM for Bcl-x L E158K; IC 50 of 4 nM for wild-type Bax). In contrast, neither wild-type Bcl-x L nor mutant K128E Bax or GST altered Kv1.3 currents (Figs. 2C-E, and [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] ). Bax and Margatoxin (MgTx) competed for the same binding site on Kv1.3 (Fig. 2F) further supporting a specific interaction of Bax with the pore region of Kv1.3 and the notion that pro-apoptotic Bcl-2-like proteins mimic the interactions of Kv1.3 toxins [START_REF] Gutman | International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels[END_REF] . These results indicate the importance of this particular lysine for the inhibition of Kv1.3 by pro-apoptotic Bcl-2-like proteins.

To show that mutant Bcl-x L induces changes in mitochondria similar to those induced by wild-type Bax, we incubated isolated, purified Jurkat mitochondria with recombinant GST-Bax (lacking the C-terminal transmembrane domain and therefore able to insert into the membrane [START_REF] Antonsson | Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria[END_REF] ), GST-Bcl-x L , GST-Bcl-x L E158K, or GST as a control. Recombinant GST-Bax and full length Bax induced very similar effects on isolated mitochondria [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] . Recombinant GST-Bax triggers a hyperpolarization followed by a decrease of the potential of the inner mitochondrial membrane (Fig. 3A). The electrochemical gradient for K + predicts that K + should enter the matrix through a potassium channel located in the inner mitochondrial membrane (IMM). If the influx of K + becomes inhibited, hyperpolarization is expected. In our experiments, depolarization, which followed hyperpolarization, was inhibited by cyclosporin A and, thus, was probably mediated by the opening of the mitochondrial permeability transition pore (PTP) (24Suppl. Mat.) . The transmembrane potential of Kv1.3-containing Jurkat mitochondria was not altered by incubation with GST alone, GST-Bax K128E [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] , or GST-Bcl-x L (Fig. 3A). In contrast, marked hyperpolarization followed by depolarization was observed when mitochondria were treated with GST-Bcl-x L E158K (Fig. 3A).

Likewise, the mutant GST-Bcl-x L E158K, similarly to GST-Bax and t-Bid-pre-activated full-length Bax (Fig. 3B), triggered a massive release of mitochondrial cytochrome c, while GST-Bcl-x L had no effect (Fig. 3C, see Fig. 3D for purity control). Importantly, the proapoptotic effects of Bax and GST-Bcl-x L E158K were abrogated in mitochondria isolated from CTLL-2/pJK cells lacking Kv1.3 (Fig. 3B andC). This finding indicates that the Bcl-x L E158K, similarly to Bax, exerts its action by regulating Kv1.3 activity in mitochondria isolated from CTLL-2/Kv1.3 cells.

To test the function of the mutant Bcl2-family proteins in intact cells, we transfected MEFs deficient for both Bax and Bak (DKO MEFs) with expression vectors for wild-type Bax, Bax K128E, or Bcl-x L E158K. Bax/Bak-deficient cells are widely used for determining the effect of mutant Bcl-2 proteins in a cellular context and on isolated mitochondria (see e.g. [START_REF] Nie | Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H 2 O2-induced apoptosis[END_REF][START_REF] Gavathiotis | BAX activation is initiated at a novel interaction site[END_REF] . The transfection of DKO MEFs with wild-type Bax or Bcl-x L E158K restored apoptosis induced by staurosporine, etoposide or sphingomyelinase, whereas transfection with Bax K128E failed to restore apoptosis in these cells (Figs. 4A, B andC). The cytochrome c release induced by staurosporine in Bax or mutant Bcl-x L E158K expressing MEF DKO cells was inhibited by Cyclosporin A (Fig. 4D), suggesting a possible involvement of the permeability transition pore in this experimental setup. To avoid a possible pro-apoptotic effect of protein overexpression per se, the stoichiometry of transfection was adjusted to yield amounts of protein close to those found in untransformed wildtype cells. The protein levels were determined in the transfected and stimulated cells by Western blot and FACS (which permits us to determine the expression of Bax in the population also investigated for apoptosis) and were similar to the levels of Bax endogenously expressed by wildtype MEFs (Figs. 4A andB).

To confirm that Bax interacts with Kv1.3 via lysine 128, we co-transfected Kv1. To exclude that the lack of pro-apoptotic effects of mutant K128E Bax in DKO MEFs (Fig. 4) and in CTLL-2 cells [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] may be caused by alterations of the membrane-insertion capability and/or of the pore-forming properties of Bax by the mutation, we addressed whether the Bcl-2 family proteins used in our studies can efficiently insert into mitochondria. Figure 5A illustrates that mutant recombinant Bax and Bcl-x L are able to insert into MEF DKO isolated mitochondria as efficiently as the wild-type recombinant Bax and Bcl-x L , as assayed by alkaline extraction.

Activation and oligomerization of Bax can be detected by analyzing resistance of Bax to trypsin [START_REF] Lucken-Ardjomande | Bax activation and stress-induced apoptosis delayed by the accumulation of cholesterol in the mitochondrial membranes[END_REF] .

In particular, following these events, aminoacids at the N-terminus of Bax become susceptible to trypsin digestion: the rest of the protein is protected, giving rise to truncated Bax of 15 kDa. In our system, in mitochondria isolated from DKO MEF cells, the recombinant Bax WT and Bax mutant equally give rise to such a truncated form (revealed by using an antibody against aa. 1-171 of Bax (Δ21)), indicating that the mutated version of Bax is still able to form higher order oligomers (Fig. 5B). In accordance, K128E Bax is able to induce the release of Smac/Diablo from these mitochondria, similarly to Bax WT, while the release of cytochrome c is impaired when using mutant Bax (Fig. 5C). Smac/Diablo is released not only from isolated mitochondria using recombinant proteins, but also in MEF DKO cells transfected with mutant Bax (Fig. 5D; see Fig. 5E for purity control of cytosolic fraction). Importantly, the effects of recombinant GST-Bax and full length Bax [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] on isolated mitochondria and those of Bax expressed in MEF cells are the same (Fig. 3 and4). These results further indicate the ability of Bax and of K128E Bax to form oligomers in the outer membrane and to allow the exit of Smac/Diablo, but also point to another event, i.e. inhibition of Kv1.3 by Bax, necessary for the release of cytochrome c. This compulsory proapoptotic event does not take place with K128E Bax (see Figure 4A), or in the absence of Kv1.3 [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] .

The notion that interaction of Kv1.3 with Bax is important for the release of cytochrome c but not for the oligomerization of Bax, is further indicated in Figs. 6A and6B, where in isolated mitochondria, Smac/Diablo and HtrA2/Omi release occurs independently of the presence of Kv1.3 upon incubation with recombinant Bax, suggesting that interaction of the channel with Bax is involved mainly in cytochrome c mobilization rather than in its translocation through the mitochondrial outer membrane. We tested pro-apoptotic factor release also in intact CTLL-2/pJK and CTLL-2/Kv1.3 cells challenged with staurosporine. In accordance with data of Figs. 3, 6A and 6B, cytochrome c is released in a Kv1.3-dependent manner, while Smac release occurred also in the absence of the channel. The release of Smac was insensitive to CSA (Fig. 6C). In contrast, the release of apoptosis-inducing factor (AIF) and endoG required expression of Kv1.3 and was absent in CTLL-2-pJK treated with staurosporine (Fig. 6 C).

Finally, we tested whether recombinant Bax WT and Bax K128E may give rise to channel activity by incorporating these proteins into a planar lipid bilayer. The K128E Bax was able to insert into liposomes and oligomerize as assayed by cross-linking with disuccinimidyl suberate (Fig. 7A). Both WT and mutant Bax proteins formed channels in this system only when added at very high (up to 16 nM; wt: n=28; mutant: n=20) concentrations. Although the activities differed, the differences were within the range of the variability displayed by Bax channels from one experiment to the other. Note in Fig. 7B that the amplitude of the concentration gradient-driven current passing through the pores at zero applied voltage (the condition believed to prevail at the outer mitochondrial membrane; I-V plots), and the kinetic behaviour (current traces) are similar for the wild-type and mutant channels.

Discussion

In the present work we provide insights into the mechanisms of action of the pro-apoptotic Bcl-2 family protein Bax, namely the inhibition of mitochondrial Kv1.3. We also show that an inactive mutant of Bax forms ion-conducting channels similar to those formed by wild-type Bax, a finding indicating that Bax does not exert its action during apoptosis merely by forming ionconducting oligomers.

The data obtained with Bcl-x L E158K are consistent with the model we recently proposed for the action of Bax [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] . Wild-type Bax inhibits Kv1.3, a potassium-selective channel of the inner mitochondrial membrane, and to exhibit actions similar to those exerted by margatoxin and charybdotoxin, which block the channel with high specificity [START_REF] Gulbins | Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes[END_REF] . These toxins possess a crucial lysine that blocks the channel by binding to the ring of four aspartate residues of the channel vestibule. Position 128 in Bax, which folds out of the OMM after insertion [START_REF] Annis | Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis[END_REF] , corresponds to a highly conserved lysine. Bax K128E mutants did not inhibit Kv1.3 and exerted no pro-apoptotic effect on isolated mitochondria. If lysine 128 were important for the pro-apoptotic action of Bax via inhibition of Kv1.3, we expect that introducing a positive charge at the corresponding position in Bcl-x L turns this anti-apoptotic protein into a blocker of Kv1.3 with proapoptotic effects. Our results confirm this hypothesis and show that the addition of Bcl-x L E158K to isolated mitochondria efficiently blocks Kv1.3 and induces hyperpolarization and the release of cytochrome c. DKO MEFs were used to verify the effect of these mutations in a cellular context. The fact that mutant Bcl-x L E158K inhibits Kv1.3 and is sufficient to restore cell death in cells lacking Bax and Bak strongly indicates that the crucial lysine residue mediates inhibition of the channel; it may also be important for the physical interaction between Bax and Kv1.3. A single amino acid residue seems to be sufficient to mediate strong protein-protein interactions as previously shown by the observation that, for instance, a point mutation of K27 in agitoxin reduces the affinity of the toxin for the KcsA K + channel 130-fold [START_REF] Mackinnon | Structural conservation in prokaryotic and eukaryotic potassium channels[END_REF] . In agreement, data shown in Fig. 4E indicate that Bax K128E does not interact with Kv1.3.

The existence of various potassium channels in the IMM of different tissues has been reported (e.g., 30 and references therein) . Mitochondrial potassium channels permit K + transport into the mitochondrial matrix, regulating thus mitochondrial volume homeostasis, changes in mitochondrial membrane potential, respiration, pH gradient and synthesis of ROS [START_REF] Szewczyk | Pharmacology of mitochondrial potassium channels: the dark side of the field[END_REF] . In various systems, open potassium channels seem to keep the PTP closed [START_REF] Cheng | Interaction of the mitochondrial potassium channels with the permeability transition pore[END_REF] . Kv1.3 was recently found in mitochondria of prostate cancer and breast cancer cell lines PC3 and MCF-7 [START_REF] Gulbins | Role of Kv1.3 mitochondrial potassium channel in apoptotic signalling in lymphocytes[END_REF] , in hippocampal neurons [START_REF] Bednarczyk | Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria[END_REF] and in astrocytes [START_REF] Cheng | Interaction of the mitochondrial potassium channels with the permeability transition pore[END_REF] . Furthermore, BK, a high-conductance calcium-activated potassium channel, is present and functionally active in mitochondria from cardiac tissues [START_REF] Gu | Hypoxia increases BK channel activity in the inner mitochondrial membrane[END_REF] . A mitochondrial intermediate-conductance calcium-dependent potassium channel (IK) is also present at least in a colon carcinoma cell line [START_REF] Marchi | Intermediate conductance Ca 2+ -activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells[END_REF] . However, we have recently shown that Bax does not inhibit IK [START_REF] Sassi | An investigation of the occurrence and properties of the mitochondrial intermediateconductance Ca 2+ -activated K + channel mtKCa3.1[END_REF] .

Activity resembling that produced by K ATP channels has also been recorded by path clamp in the IMM of various cells (37 and references therein) . The observation that the ring of negatively charged amino acids in the pore of potassium channels is structurally highly conserved in all voltage-gated potassium channels (e.g., [START_REF] Rauer | Structural conservation of the pores of calcium-activated and voltage-gated potassium channels determined by a sea anemone toxin[END_REF] suggests that the action of Bax may not be limited to Kv1.3.

Mutations at K128 in Bax and at E158 in Bcl-x L have not been previously reported, but other point mutations have been shown to alter the activity of Bax and Bcl-x L . Recently, Gavathiotis and coworkers described a point mutation of Bax that identifies a novel structural location of Bax activation [START_REF] Gavathiotis | BAX activation is initiated at a novel interaction site[END_REF] . To determine whether this single amino acid mutation affects the activation of Bax in cells, those authors reconstituted DKO MEFs with either wild-type Bax or Bax (K21E) and monitored the induction of apoptosis in response to BIM SAHB BH3 stapled helix. BIM SAHB induced the time-dependent apoptosis of Bax-reconstituted MEFs, whereas the single K21E point mutation within Bax reduced the incidence of BIM SAHB-triggered apoptosis [START_REF] Gavathiotis | BAX activation is initiated at a novel interaction site[END_REF] . Cysteine 62 of Bax was shown to be critical for its conformational activation and proapoptotic activity in response to H 2 O 2 -induced apoptosis [START_REF] Nie | Cysteine 62 of Bax is critical for its conformational activation and its proapoptotic activity in response to H 2 O2-induced apoptosis[END_REF] . These results, like ours, point to the importance of a single amino acid for protein-protein interaction and for the proapoptotic efficiency of Bax. Lysine 128 in Bax and the glutamate 158 in Bcl-xL are not located within the BH3 domains making and it is, therefore, unlikely that these aminoacids are involved in the dimerization process of Bax or Bcl-xL.

With regard to the possibility that Bax mutations may cause alterations in the release of cytochrome c, Heimlich and colleagues reported that the Bax-induced release of cytochrome c from mitochondria depends on α-helices 5 and 6 [START_REF] Heimlich | Bax-induced cytochrome c release from mitochondria depends on alpha-helices-5 and -6[END_REF] , a region comprising K128. However, the effect of these deletions in intact cells has not been investigated. Regarding Bcl-x L , a G138A mutation abrogates the ability of Bcl-x L to dimerize with Bax and prevents the inhibition of cell death induced by deprivation of IL-3 or dexamethasone in the murine prolymphocytic IL-3-dependent cell line F5.12 (40,41, ) . It should be noted that we mutated Bcl-x L to analyse the role of a critical lysine in Bax for the induction of apoptosis. Our studies demonstrate that a point mutation in Bcl-x L converts the protein into a pro-apoptotic protein, whereas the mutation of Bax at K128 prevents the induction of apoptosis by Bax. Thus, these mutation studies strongly support the notion that lysine 128 in Bax plays a crucial role in the induction of apoptosis in a cellular model. The findings further support the notion that Bax induces apoptosis via the direct inhibition of a mitochondrial potassium channel by plugging the pore with K128. We would like to point out that we do not propose Bcl-x L to interact with Kv1.3 in vivo; the Bcl-x L mutant was used as a tool to reinforce the evidence concerning the importance of the lysine residue in Bax.

Recombinant Bax can directly insert into membranes with the α-helices 5 and 6, and it can also spontaneously oligomerize (e.g. [START_REF] Antonsson | Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria[END_REF] . In vitro experiments have shown that Bax oligomers are able to conduct cytochrome c [START_REF] Saito | Bax-dependent transport of cytochrome c reconstitued in pure liposomes[END_REF][START_REF] Antonsson | Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria[END_REF][START_REF] Epand | Direct evidence for membrane pore formation by the apoptotic protein Bax[END_REF] . These findings, and the observation that some proteins from the Bcl-2 family form ion-conducting pores upon reconstitution in artificial membranes [START_REF] Schendel | Ion channel activity of the BH3 only Bcl-2 family member, BID[END_REF][START_REF] Antonsson | Inhibition of Bax channel-forming activity by Bcl-2[END_REF][START_REF] Schlesinger | Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic Bcl-2[END_REF][START_REF] Minn | Bcl-x(L) forms an ion channel in synthetic lipid membranes[END_REF] suggest that cytochrome c may cross the OMM through an aqueous channel formed by assembled Bax monomers, Bak monomers, or hetero-oligomers of both proteins. Our findings do not contradict this hypothesis, but indicate that a Kv1.3-dependent event is necessary for the release of cytochrome c upon the interaction of Bax with mitochondria. Formation of Bax oligomers and pore formation by Bax in the OMM may well occur independently of Kv1.3, as suggested by the release of Smac/Diablo and HtrA2/Omi occurring independently of Kv1.3. On the other hand, the Bax mutant is efficiently inserted and activated in mitochondria (Fig. 5), is able to form oligomers and ion channels and mediate the release of Smac/Diablo and HtrA2/Omi (Figs. 567), yet, cytochrome c is not released with mutant Bax and cell death is impaired (Fig. 4). The notion that Bax itself is not sufficient alone for mediating the efflux of cytochrome c from mitochondria has been also demonstrated by other investigations on the roles of cardiolipin [START_REF] Ott | Role of cardiolipin in cytochrome c release from mitochondria[END_REF] and Opa-1 [START_REF] Pellegrini | A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis[END_REF] in controlling the release of cytochrome c from mitochondria. Similarly to cytochrome c, the release of apoptosisinducing factor and EndoG are Kv1.3-dependent (Fig. 6). AIF and EndoG have been proposed to be released in a ROS-dependent way from mitochondria [START_REF] Norberg | Mitochondrial regulation of cell death: Processing of apoptosis-inducing factor (AIF)[END_REF][START_REF] Kim | Reactive oxygen species-dependent EndoG release mediates cisplatin-induced caspase-independent apoptosis in human head and neck squamous carcinoma cells[END_REF] . Given that Kv1.3 inhibition by Bax leads to an increase in ROS release [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] , one possible explanation for our findings is that Kv1.3 is only involved in ROS-mediated mobilization/release of pro-apoptotic factors. Further work is required to prove this hypothesis.

Our data showing that Bax and mutant Bcl-x L expressed in MEF DKOs induce a CSAsensitive cytochrome c release point to a possible involvement of PTP in this process, at least in our experimental setup. CSA may have additional effects in intact cells and data by Schinzel et al [START_REF] Schinzel | Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia[END_REF] obtained in Cyclophilin-D-less Bax and Bak containing MEFs argue against the involvement of PTP in staurosporine-induced apoptosis. Thus, the role of PTP in MEF death is controversial and further work is required to definitively exclude or prove its role.

In summary, we demonstrate for the first time a single point mutation in Bcl-x L that converts the protein into a pro-apoptotic protein both in experiments with isolated mitochondria and in MEF cells. Our work addresses the mode of action of a single point mutant of Bax in a cellular context, and shows that the Bax mutant loses its ability to mediate staurosporine-induced cell death. Further work is required to understand how general this feature of Bax action is and to further investigate the role of Kv channels and/or other potassium channels in general in Bax-mediated apoptosis [START_REF] Szabò | Contribution of voltage-gated potassium channels to the regulation of apoptosis[END_REF] .

Materials and Methods

Cell cultures

Jurkat lymphocytes and CTLL-2 cells were grown in RPMI-1640 supplemented with 10% foetal calf serum, 10 mM HEPES (pH 7.4), 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acids, 100 units/ml penicillin, 100 μg/ml streptomycin (all Life Technologies, Karlsruhe, Germany), and 50 μM β-mercaptoethanol. Mouse interleukin-2 (IL-2) (4 units/ml, Roche Diagnostics, Mannheim, Germany) was added daily to CTLL-2 cells (American Type Culture Collection [ATCC], Manassas, VA, USA). DKO MEFs and control cells (both kindly provided by L. Scorrano) were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Milan, Italy) supplemented as above (without Hepes). If indicated, we added 2 µM cyclosporine A.

Transfections

The lipofectamine technique was used as described by the supplier (Invitrogen, Carlsbad, CA, USA) to co-transfect DKO MEFs with 2 μg/10 6 cells pJK/Kv1.3 and 5 μg each of the expression vectors pcDNA-Bax, pJK-K128E Bax, or pJK-E158-Bcl-x L so that the MEFs would express wild-type Bax, K128E Bax, or E158K Bcl-x L . Expression of Kv1.3 was confirmed by FACS analysis (not shown). The pJK plasmid contains a single chain antibody with a Myc-Tag.

The transfected MEF cells were sorted using anti-Myc-Tag antibodies and magnetic beads 36 hrs after transfection and then incubated for another 24 hrs. Alternatively, MEF cells were cotransfected with 2 μg pcDNA-EGFP-actin so that transfected cells could be detected or the cells were stained with FITC-coupled anti-Myc 9E10 antibodies to identify transfected cells (for transfection with pJK-E158K Bcl-x L ). Dead cells were removed after 12 hours, and the cells were then cultured in DMEM supplemented as above for an additional 36 hours and used for functional tests. For CTLL-2 cells we obtained stable clones by transfecting CTLL-2 cells with 40 μg/10 7 cells of pJK/Kv1.3 or pJK plasmids by electroporation and culturing them with 800 µg/ml G418 (Sigma, Deisenhofen, Germany). All experiments except for selection of clones were performed with the bulk cell population. All cultures were re-established from frozen stocks after 4 weeks of growth.

Cells were depleted of IL-2 before all experiments to avoid interference by signalling from the IL-2 receptor. To this end, cells were washed in 132 mM NaCl, 20 mM HEPES (pH 7.4), 5 mM KCl, 1 mM CaCl 2 , 0.7 mM MgCl 2 , and 0.8 mM MgSO 4 (H/S) and allowed to recover for 3 hours in cell culture medium without IL-2.

Recombinant proteins

Bax (amino acids 1-170) was cloned into pGEX-3X as GST fusion protein, expressed in BL21A1, and purified from bacterial lysates using glutathione-sepharose. Bacteria were lysed in 50 mL of 25 mM HEPES (pH 7.4), 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 125 mM NaCl, 10 mM each NaF, Na 3 VO 4 , and sodium pyrophosphate, 10 μM each aprotinin and leupeptin (A/L), and 1 mg/ml lysozyme. Samples were incubated on ice for 15 minutes so that lysis could be completed, and DNA was degraded with 5 μg/ml DNAaseI in 30 mM MgCl 2 for 30 minutes. After insoluble material had been pelleted by a 50-minute centrifugation at 11.000 × g at 4 o C, the GST fusion proteins in the supernatant were immobilised by incubation with 300 μl glutathione sepharose (GE Healthcare, Chalfont St. Giles, UK) for 1 hour at 4 o C. The beads were extensively washed so that any detergents could be eliminated and 20 mM glutathione was added to elute the fusion protein. Glutathione was removed by dialysis and the samples were concentrated to 1 ml by several centrifugation rounds through size-exclusion columns (cut-off, 10.000 Da; Viva Science/Sartorius, Göttingen, Germany). The K128E mutant of Bax and the E158K mutant of Bclx L were obtained by a site-directed mutagenesis PCR technique (Stratagene, La Jolla, CA, USA), cloned into pGEX-3X, and expressed and purified as a GST fusion protein as above. In the present study we used a GST-ΔC-Bax, which lacks the C-terminal transmembrane domain and does not require activation by truncated Bid (tBid) for insertion into the membrane and induction of cytochrome c release [START_REF] Antonsson | Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria[END_REF] .

Cellular apoptosis assays

To detect the release of mitochondrial cytochrome c and Smac, transfected MEF cells were either left untreated or treated with staurosporine for 12 hours, washed in cold HEPES/saline, incubated for 30 min at 4 o C in 0.3 M sucrose, 10 mM TES (pH 7.4), and 0.5 mM EGTA (TES buffer) and then Dounce-homogenized. Nuclei and unbroken cells were pelleted by centrifugation for 5 min at 600xg and 4 o C. Supernatants were centrifuged at 6000xg for 10 min at 4 o C. The supernatants were used to detect released cytochrome c or Smac/Diablo, the pellets were used to detect mitochondrial cytochrome c. Proteins were separated on 15% SDS-PAGE, blotted onto a nitrocellulose membrane, and developed with a monoclonal mouse anti-cytochrome c antibody (clone 7H8.2C12, BD Biosciences Pharmingen, San Diego, CA, USA) or with anti-Smac antibody and the Tropix ECL system (Bedford, MA, USA). Morphological signs of apoptosis, such as DNA condensation, nuclear fragmentation, and blebbing, were detected by staining (non-permeabilized) cells with 4 μg/ml propidium iodide and 4 μg/ml ethidium bromide. This method also differentiated apoptotic and necrotic cells. To detect cytochrome c, AIF, Smac, Omi and EndoG release in intact CTLL-2 cells, the cells were treated with 1 μM staurosporine with or without 2 μM CSA for 12 hours. After incubation, the cells were collected, washed with PBS, resuspended in 150 μL of TES buffer and incubated on ice. Cells were broken with an electric homogenizer (60 seconds).

Unbroken cells were separated by centrifugation at 600xg for 10 min at 4 °C. Mitochondria were separated from cytosol by centrifugation at 7000xg at 4 °C for 10 min. Mitochondria were resuspended in TES buffer. Both cytosolic fraction and mitochondria were analyzed by Western blot.

Alkaline extraction and trypsin treatment

Mitochondria were isolated as described by Szabò et al. [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] . 1 x 10 6 DKO MEFs cells/sample were washed with PBS and centrifuged 10 min at RT at 450 g. Then cells were resuspended in 1.5 ml of TES Buffer and incubated in ice for 30 min. After 150 strokes with a glass/teflon potter, intact cells were pelleted by centrifugation for 10 min at 4°C at 600xg. Mitochondria were separated by centrifugation of the resulting supernatant at 6000xg for 10 min at 4°C and washed in 500 μL of Buffer 1 (50 mM PIPES-KOH (pH 7.4), 50 mM KCl, 2 mM MgCl 2 , 2 mM EGTA, 10 μg/ml A/L, 2 mM ATP, 10 mM phosphocreatine, 5 mM succinate, and 50 μg/ml creatine kinase). Mitochondria were then incubated with GST-Bax, or GST-K128E Bax, or GST-Bcl-X L or GST-E158K Bcl-X L (20 nM each ) for 30 min on ice. Na 2 CO 3 (0.1 M final concentration) was added and samples were incubated for further 30 min on ice. Mitochondrial fraction and supernatant were separated by centrifugation at 20000xg for 10 min at 4°C. To assess Bax activation/oligomerization, isolated mitochondria were treated with trypsin (0.51 mg/ml final concentration) for 1.5 h at 30°C. Reaction was stopped by addition of equal volume of buffer 1 containing 2X protease inhibitors.

Mitochondrial fraction and supernatant were separated by centrifugation at 20000xg for 10 min at 4°C and the former was assayed for the presence of Bax by Western blot, using N-20 antibody (Santa Cruz) produced against a peptide mapping the N-terminus of Bax or Δ21 antibody (Santa Cruz) against an epitope corresponding to aminoacids 1-171 of Bax.

Cytochrome c and Smac/Diablo release from isolated mitochondria

Mitochondria (obtained from 1×10 6 CTLL-2 or MEF cells) were isolated as described above and were incubated for 30 minutes on ice in buffer 1 with 5 nM GST-Bax, or 5 nM K128E GST-Bax, or 5 nM GST, or 5 nM t-Bid, or 15 nM GST-Bcl-2, or 15 nM GST-Bcl-x L , or 15 nM E158K GST-Bcl-x L , or 0.1 nM t-Bid + 20 nM full length Bax (t-Bid and full length Bax were co-incubated for 15 min at 37 o C prior to addition to the mitochondria to achieve activation of Bax) so that chimeric proteins could bind to mitochondria. The very low concentration of t-Bid is sufficient to activate full length Bax in vitro, but too low to stimulate low amounts of Bax and Bak associating with the outer membrane of isolated mitochondria. The mitochondria were then pelleted and resuspended in prewarmed buffer 1 (37 o C) and incubated for 5 minutes. The reaction was terminated by the addition of one volume of ice-cold buffer 1, centrifugation at 20000 g at 4 o C. The supernatants transferred into new tubes. Reducing SDS sample buffer was added to the supernatants and the pellets. The samples (pellet and supernatant) were analyzed for the release of cytochrome c or Smac/Diablo by Western blotting.

Membrane potential in isolated mitochondria

The membrane potential of isolated mitochondria was determined by incubating purified mitochondria from Jurkat cells with 5 nM GST-Bax, or 5 nM GST-BaxK128E, or 5 nM GST, or 5 nM tBid, or 15 nM GST-Bcl-x L , or 15 nM GST-Bcl-x L E158K in buffer 1 supplemented with 10

Flow cytometry-studies

To identify cell death in pJK-K158E Bax-transfected cells, the cells were stained with FITCcoupled anti-Myc9E10 antibodies to detect the Myc-epitope present on the single chain antibody, which is also encoded by the pJK vector. Death was detected by staining with Cy3-labelled Annexin. Samples were analysed at a FACs-Calibur (Becton-Dickinson).

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Co-immunoprecipitation

For the co-immunoprecipitation experiments transfected MEFs were stimulated with staurosporine, lysed in 4% CHAPS 5 mM MgCl2, 137 mM KCl, 1 mM EDTA, 1 mM EGTA, 20 mM TrisHCl (pH 8.0) and 10 μg/ml aprotinin and leupeptin, and Bax/Bax K128E or Kv1.3 were immunoprecipitated from the lysates employing protein A/G agarose. The immunoprecipitates were washed 6-times in the lysis buffer, separated by 7.5% or 12.5% SDS-PAGE, blotted and developed with anti-Kv1.3 or anti-Bax antibodies. An aliquot of the immunoprecipitates was blotted with the immunoprecipitating antibody to test for similar amounts of protein in all samples. Control immunoprecipitates were performed with isotype-matched irrelevant antibodies. Specificity of immunoprecipitations was proven with CTLL-pJK cells lacking Kv1.3 expression (data not shown).

Patch clamp experiments

Whole-cell currents were recorded with an EPC 7 amplifier (List, Darmstadt, Germany (filter, 1 kHz; sampling rate, 5 kHz), as described in Szabò et al. [START_REF] Szabò | A novel potassium channel in lymphocyte mitochondria[END_REF] in Jurkat cells. Leak currents were not subtracted. The bath solution was composed of 150 mM NaCl, 5 mM KCl, 2. as well as that of pro-apoptotic Bax and Bak. The highly conserved lysine residue in Bax (K128) that is crucial for interaction with Kv1.3 [START_REF] Szabó | Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes[END_REF] is highlighted in yellow. In the corresponding position, Bak contains a glycine; however, adjacent to this G there is a histidine (in yellow). Furthermore, Bak molecules from other species are characterized by an arginine residue in the same position (not shown). The amino acid at position 158 is a highly conserved, negatively charged glutamate in both Bcl-x L (B) and Bcl-2 (C) from differing species. Accession numbers for sequences from the ExPASy databases are reported. panel). As a control for the efficiency of trypsinization, the same blot was re-probed with an antibody against Tom22, an outer membrane protein, which is anchored to the cytoplasm-facing side of the membrane only by a short helix (lower panel). C) Cytochrome c is released from isolated DKO MEF mitochondria by GST-Bax, but to much less extent by GST-K128E Bax, while Smac/Diablo is released equally as assessed by stripping and re-blotting of the same membrane with anti-Smac antibody. D) DKO MEF cells were transfected and treated as in Fig. 4C and then assessed for released Smac/Diablo content in the supernatant fraction. E) Mitochondria from DKO MEFs were purified and aliquots of the mitochondrial and cytosolic fractions were blotted with anti-Tom20 or anti-Tim23 antibodies and developed with alkaline phosphatase coupled secondary antibodies and ECL. The blots reveal the purity of the preparations and show that the cytosolic fractions do not contain the mitochondrial proteins Tom or Tim.

  3 and K128E Bax or wild type Bax into MEF DKO cells, induced apoptosis with staurosporine, lysed cells and determined co-immunoprecipitation of the proteins. The results (Fig. 4E) show that the K128E Bax mutant failed to bind Kv1.3, while wild-type Bax and Kv1.3 co-immunoprecipitated after induction of apoptosis. This finding further proves that aa 128 in Bax is critical for binding Kv1.3.

nM 3 ,

 3 3(')-dihexyloxacarbocyanine iodide (DioC 6 (3)), for 30 minutes at 4 o C. Mitochondria were then pelleted and resuspended in prewarmed buffer 2 containing 10 nM DioC 6 (3), and the transmembrane potential (ΔΨ m ) was determined by flow cytometry. Hyperpolarization increases the accumulation of DioC 6 (3) in mitochondria, as indicated by a right shift of the fluorescence signal; depolarization is indicated by a left shift. Complete depolarization of mitochondria was achieved by incubation with 1 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 10 minutes at room temperature and served as a control for the integrity of the mitochondria. The experiments were repeated with the ΔΨ m indicator tetramethylrhodamine ethyl ester (TMRE) and yielded very similar results.

  5 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES (pH 7.3). The intracellular solution contained 134 mM KCl, 1 mM CaCl 2 , 2 mM MgCl 2 , 10 mM EGTA, and 10 mM HEPES (pH 7.3). Intracellular voltages are reported, and outward currents are plotted upwards. Planar lipid bilayer A Warner Instruments (Hamden, CT, USA) BC-525C electrophysiological planar bilayer apparatus was used. Bilayers with a capacity of approximately 150 to 200 pF were prepared by painting a decane/chloroform solution of soybean asolectin (Sigma, Milan, Italy), partially purified by precipitation with cold acetone from a chloroform solution, across a 250-µm hole in a polystyrene cuvette. The inside of the cuvette constituted the trans compartment. The standard

Figure legends : Figure 1 :

 :1 Figure legends:

Figure 2 :

 2 Figure 2: Effect of Bax and Bcl-x L mutants on Kv1.3 current.

Fig. 3 :

 3 Fig. 3: Effect of Bax and Bcl-x L mutants on mitochondrial membrane potential and release of

Figure 4 :

 4 Figure 4: Bax and Bcl-x L E158K, but not Bax K128E, restore mitochondrial cytochrome c

Figure 6 .

 6 Figure 6. Kv1.3-dependent and -independent release of pro-apoptotic factors from

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Acknowledgements

The authors are grateful for help with some experiments to U. De Marchi, P. Cusin and M. Zenere.

The studies were supported by Italian Association for Cancer Research (AIRC to I.S. (grant 5118) and M.Z) and DFG grant Gu 335/13-3.

References:

experimental medium contained 100 or 150 mM KCl, 0.5 mM EGTA, and 8 mM HEPES (pH 7.5).

After the planar membrane had formed, salt concentration gradients were produced by adding equal volumes of the standard medium (in the trans compartment) and a medium with the same composition except for a higher concentration of KCl (in cis). The contents of both chambers were stirred by magnetic bars when necessary. Connections to the electrodes were provided by agar bridges. Protein was added to the cis side. Voltages reported are those of the cis chamber, and current is considered positive when carried by cations flowing from the cis compartment to the trans compartment. Output was recorded with a 10-KHz bandwidth on videotape by using a Medical Systems (New York, NY, USA) PCM-2 interface. The data were later analysed offline by using the pCLAMP program set (Molecular Devices, Sunnyvale, CA, USA).

Apoptosis was measured by Cy3-labelled Annexin V and FACS analysis. D) MEF DKO cells were transfected with constructs coding for the indicated proteins and were selected as described in the method section. Staurosporine-induced apoptosis resulted in cytochrome c release in Bax and E158K Bcl-x L expressing cells. Cytochrome c release was prevented by incubation of the cells with Where indicated, t-Bid was also added (5 nM) and increased insertion/activation of Bax (middle