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Contents lists available at ScienceDirect

Cell Calcium journal homepage: www.elsevier.com/locate/ceca

Review

Targeting a mitochondrial potassium channel to fight cancer Luigi Leanza a , Elisa Venturini b , Stephanie Kadow b , Alexander Carpinteiro b , Erich Gulbins b,∗∗ , Katrin Anne Becker b,∗ a b

Department of Biology, University of Padova, Viale G. Colombo 3, 35131 Padova, Italy Department of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany

a r t i c l e

i n f o

Article history: Received 8 July 2014 Received in revised form 10 September 2014 Accepted 11 September 2014 Available online xxx Keywords: Potassium channel Mitochondria Cell death Bax Toxins

a b s t r a c t Although chemotherapy is able to cure many patients with malignancies, it still also often fails. Therefore, novel approaches and targets for chemotherapeutic treatment of malignancies are urgently required. Recent studies demonstrated the expression of several potassium channels in the inner mitochondrial membrane. Among them the voltage gated potassium channel Kv1.3 and the big-potassium (BK) channel were shown to directly function in cell death by serving as target for pro-apoptotic Bax and Bak proteins. Here, we discuss the role of mitochondrial potassium channel Kv1.3 (mitoKv1.3) in cell death and its potential function in treatment of solid tumors, leukemia and lymphoma. Bax and Bak inhibit mitoKv1.3 by directly binding into the pore of the channel, by a toxin-like mechanism. Inhibition of mitoKv1.3 results in an initial hyperpolarization of the inner mitochondrial membrane that triggers the production of reactive oxygen species (ROS). ROS in turn induce a release of cytochrome c from the cristae of the inner mitochondrial membrane and an activation of the permeability transition pore, resulting in opening of the intrinsic apoptotic cell death. Since mitoKv1.3 functions downstream of pro-apoptotic Bax and Bak, compounds that directly inhibit mitoKv1.3 may serve as a new class of drugs for treatment of tumors, even with an altered expression of either pro- or anti-apoptotic Bcl-2 protein family members. This was successfully proven by the in vivo treatment of mouse melanoma and ex vivo human chronic leukemia B cells with inhibitors of mitoKv1.3. © 2014 Published by Elsevier Ltd.

1. Introduction Chemotherapeutic drugs target malignant tumors by a variety of mechanisms, including inhibition of proliferation, direct induction of tumor cell death by any form of apoptosis or necrosis, by activation of the immune system and by altering the niche of the tumor, in particular the tumor stem cell niche. Death of tumor cells is an important mechanism to contribute to a successful chemotherapy. Thus, it is of great clinical interest to define molecular mechanisms that mediate cell death. Since, unfortunately, chemotherapy still often fails and many patients die, it is also very important to identify novel targets for chemotherapy and, thereby, potential novel chemotherapeutic drugs. Here, we will focus on the role of potassium channel of the Shaker family, Kv1.3, in mitochondria (mitoKv1.3), its role in apoptosis and its potential function as targets in chemotherapy.

∗ Corresponding author. Tel.: +49 201 723 1949; fax: +49 201 723 5974. ∗∗ Corresponding author. Tel.: +49 201 723 3118; fax: +49 201 723 5974. E-mail addresses: [email protected] (E. Gulbins), katrin.becker-fl[email protected] (K.A. Becker).

Mitochondria are in the center of many pathways that induce apoptosis or necrosis [1,2]. Death receptors, but also exogenous stress stimuli such as irradiation, converge to the activation of the pro-apoptotic Bcl-2 family proteins Bax and Bak. The critical role of Bax and Bak in apoptosis has been shown in numerous studies (for a recent review see Ref. [3]). Activation of Bax by pro-apoptotic stimuli results in its translocation and integration into the outer mitochondrial membrane. The molecular details of this process are still unknown. Bak proteins seem to be loosely associated with the outer mitochondrial membrane [4]. Activation of Bak results in integration of the protein into the outer mitochondrial membrane [4]. Activated Bax assembles in oligomers that seem to form pores although these pores were detected at low pH and their in vivo relevance remains to be established [5–9]. Structural studies indicated that after incorporation of Bax into the outer mitochondrial membrane, only two amino acids stick out of the outer mitochondrial membrane and face the inner mitochondrial membrane, i.e. amino acids 127 and 128 located between the 5th and 6th helices of Bax [10]. The amino acid at position 128 is a positively charged lysine. However, it is well established that the integration of Bax mediates the release of cytochrome c and other pro-apoptotic molecules such as APAF1

http://dx.doi.org/10.1016/j.ceca.2014.09.006 0143-4160/© 2014 Published by Elsevier Ltd.

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and caspase 9 from mitochondria into the cytoplasm (for a review see [11]). The function of Bak seems to be homologous to that of Bax and they are able to replace each other in genetic knock-out models [12]. Cytochrome c release from mitochondria is a key step in mitochondrial apoptosis and it was shown to occur in two independent steps [13,14]: first, cytochrome c, which physiologically binds to cardiolipin at the inner mitochondrial membrane, is released into the inter-membrane space [13,14]. The detachment of cytochrome c from cardiolipin has been shown to be mediated by reactive oxygen species (ROS) [13,14]. However, it is unknown if ROS intermediates directly change the oxidation of cardiolipin and whether that results in the release of cytochrome c, or whether oxygen radicals indirectly act to trigger the release of cytochrome c. Cytochrome c is released from the mitochondria into the cytoplasm by a Bax/Bak-dependent mechanism to execute cell death [15]. ROS also target the permeability transition pore (PTP) [16], which will be discussed below in the present review. 2. Kv1.3 Kv1.3 is a potassium channel belonging to the Shaker family of voltage-gated channels [17,18]. Voltage gated potassium channels (KV ) are a large family of potassium (K+ ) channels expressed in both excitable and non-excitable cells. In humans, they represent the largest family of K+ channels, comprehending at least 40 genes. KV channels are divided in 12 subfamilies (KV 1-KV 12). As all K+ channels, they have a homo- or hetero-tetrameric structure constituted by 4 subunits, named ␣ subunits, and other additional subunits, i.e. ␤, ␥ and ␦, which associate around the ion conducting central pore. The selectivity filter for K+ is characterized by a distinctive P-loop structure formed by 5 conserved amino acidic residues (GYGD) located in the subunits [19]. These residues expose an electronegative carbonyl oxygen atom, which mimics the hydration sphere when the ions get access to the filter [19,20]. Kv channel ␣ subunits possess 6 transmembrane helices (S1–S6) with both the N-terminus and C-terminus on the intracellular side of the membrane [21]. The first 4 helices form the voltage sensor, while the last two helices, together with the loop, form the pore region [22]. The S4 helix contains four positively charged arginine residues and acts as the voltage-sensor domain [23]. Plasma membrane Kv1.3 and the other members of the Kv family control resting and action potential in excitable cells, while in non-excitable tissues regulate cell volume and proliferation, but also cell death [24–28]. In particular, it has been demonstrated that plasma membrane Kv1.3 participates in controlling proliferation [18], apoptosis [29,30] and neurotransmitter release by excitable cells [31]. Kv1.3-deficient mice were generated [32] and show an increase in the platelet count, a finding that would be consistent with a role of Kv1.3 in apoptosis of platelets or precursors [33]. However, the lack of Kv1.3-expression seems to be compensated in most cells of these mice by up-regulation of Kv1.1 and of a chloride channel [34]. 3. Kv1.3 inhibitors Non-permeant Kv1.3 inhibitors can be peptide inhibitors or organic compounds [35]. Among peptide Kv1.3 inhibitors there are margatoxin, Stichodactyla heliantus toxin (ShK) and charybdotoxin [36]. Although the use of charybdotoxin demonstrated the crucial role of K+ channels in lymphocytes activation, this compound, at low concentrations, also inhibits IKCa subfamily channels, therefore other inhibitors are preferred. Stichodactyla heliantus toxin is the most potent inhibitor of Kv1.3, blocking the channel at high affinity (Kd = 11 pM) and showing a 1000-fold higher selectivity

with respect to other Kv and IK channels [36–40]. It contains 35 amino acid residues, bound together by three disulfide bounds [36–40]. This structure interacts with the negative charged residues of Kv1.3 (e.g. Asp386 ) in the channel pore vestibule through its positive residues (e.g. His19 , Ser20 , Lys22 , Tyr23 ) [36,37]. The substitution of a critical Lysine (ShK-Lys22 ) with neutral residues substantially reduces the affinity of the toxin for the channel, indicating that other residues do not bind efficiently to the channel pore vestibule [38,40]. Permeant inhibitors are blockers that are able to cross the plasma membrane and have an effect on a mitochondrial K+ channel. Among them there are: Psora-4, PAP-1 and clofazimine. The most potent Kv1.3 inhibitor is Psora-4 (EC 50 = 3 nM) [35]. It is a small molecule isolated from the plant Ruta graveolens and belongs to the group of psoralen-molecules. Psoralens are a class of photosensitive molecules used in the therapy of different skin diseases such as psoriasis [41,42]. Since Psora-4 also inhibits Kv1.5, a new derivative has been synthesized, which is more selective than the former, PAP-1 (EC 50 = 2 nM) [43]. Both Psora-4 and PAP-1, however, can act, at higher concentrations, also on other Kv family members. The third membrane permeant inhibitor, clofazimine, is a highly lipophilic compound of riminophenazine family [44], and it is currently used in the treatment of different dermatological diseases such as leprosy [45]. Its use is under investigation in several infectious and non-infectious diseases, like antibiotic resistant tuberculosis, due to its apparent anti-mycobacterial and antiinflammatory activity, although the precise mechanism is still unclear [46]. Clofazimine appears to inhibit bacterial proliferation by binding guanine bases of bacterial DNA, disrupting the cell cycle finally killing the bacterium [47].

4. Mitochondrial Kv1.3 and K+ fluxes across the mitochondrial membrane Besides the plasma membrane localization, Kv1.3, as well as other Kv channels (Kv1.1 and Kv1.5), was also found in the inner mitochondrial membrane [48–51]. Kv1.3 seems to be active in the inner mitochondrial membrane even at negative resting potential (−180 mV) as demonstrated by the hyperpolarization induced after incubation of isolated mitochondria with its specific inhibitors, such as margatoxin and Stichodactyla heliantus toxin [29]. In normal conditions, K+ channels located to the inner mitochondrial membrane should mediate an inward K+ flux from the cytosol to the mitochondrial matrix, following the electrochemical gradient of this ion [52] (Fig. 1). This positive flux is compensated by the efflux of protons (H+ ) mediated by the respiratory chain and by the K+ /H+ antiporter, to avoid volume changes and depolarization [52,53]. Patch clamp experiments on the inner mitochondrial membrane of lymphocyte mitochondria demonstrated that the inner mitochondrial membrane located Kv1.3 was active and showed the same electrophysiological properties as the correlated plasma membrane channel, i.e. slope conductance of 25 pS in 150 mM KCl, K+ selectivity, slight rectification and inhibition by margatoxin and Psora-4, suggesting that both plasma membrane and mitochondrial channels are encoded by the same gene [48,49]. Since no targeting sequence is present in the Kv1.3 protein, the molecular mechanisms of mitochondrial targeting of Kv1.3 are still unknown. As postulated by Mitchell in the 1960s, the mitochondrial ATPase activity is coupled to H+ translocation across the inner mitochondrial membrane, characterized by a low permeability to H+ , cations and anions, and is mediated by the respiratory chain complexes [54]. This process is associated to a substratespecific exchange-diffusion carrier system that leads to reversible transmembrane shuffle of anions and cations [54,55] (Fig. 1).

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Fig. 1. Mitochondrial potassium cycle. Potassium ions (K+ ) cannot pass the impermeable inner mitochondrial membrane (IMM), but nevertheless they can enter mitochondria through specific potassium channels. To control mitochondrial volume homeostasis, a specific K+ /H+ antiporter exploits the proton electrochemical gradient produced by the activity of the respiratory chain to pump out K+ from the matrix.

The chemiosmotic hypothesis is now accepted considering the existence of the mitochondrial membrane potential and the dependency of energy conservation on charge separation at the inner mitochondrial membrane. Intermediates of the metabolism provide electrons that are transported through the respiratory chain complexes to the oxygen. This is correlated to the pumping of H+ from the matrix to the inter membrane space, that produces an electrochemical gradient ␮, defined by the equation ␮ =  + pH, where  represents the membrane potential difference. The present manuscript discusses the role of mitochondrial Kv1.3 in the organelle physiology and bioenergetics and, therefore, we will focus on K+ fluxes (Fig. 1). Channels for other cations, e.g. Na+ and Ca2+ , were reviewed by others [52,56,57]. Considering the cytosolic K+ concentration (150 mM) and the mitochondrial membrane potential (−180 mV, negative inside), the electrochemical equilibrium should be reached by a very high K+ concentration inside the organelle [58]. Instead, mitochondrial K+ concentration depends on a continuous equilibrium between K+ influx and efflux mediated by different transport systems [59]. Mitochondrial K+ accumulation is also prevented by organelle swelling induced by osmotic water influx through aquaporins, that induces release of cytochrome c by rupture of the outer mitochondrial membrane and loss of mitochondrial function, e.g. ATP production. This problem, already underlined and solved by Mitchell, is bypassed by the presence of a specific antiporter that couples cation excretion with H+ re-entry into the matrix. All these observations determine the K+ futile cycle, that dissipates both energy and mitochondrial membrane potential: indeed, on one hand, the presence of the K+ /H+ antiporter solves the K+ distribution and the mitochondrial volume homeostasis, on the other hand, induces energy dissipation through the inner mitochondrial membrane. The chemiosmotic hypothesis was based on the presence of antiporters and on the low permeability of the inner mitochondrial membrane to cations. Today, we know that even if K+ channels (like mitoKv1.3) are present in the inner mitochondrial membrane, there is a very slow transport of K+ inside the mitochondria, because of the fine regulation of these channels, to reduce energy dissipation during the K+ futile cycle. To compensate for charge movement, the respiratory chain must increase the rate of proton transfer from the matrix to the mitochondrial inter-membrane space.

The K+ cycle participates in the modulation of coupling between ATP synthesis and mitochondrial respiration, thus contributing to the regulation of different processes including mitochondrial volume, mitochondrial structural integrity and production of ROS [60] (Figs. 1 and 2). According to the chemiosmotic model, the electrochemical proton gradient ␮H (composed mainly of ) must decrease to increase the respiratory chain activity. Thus, passive charge flow and ␮H () are coupled, and the opening of K+ channels in the inner mitochondrial membrane will induce depolarization. Certainly, this system can also function in the opposite way: blocking cation inward flux through the closure of selective channels, is expected to lead to an increase of a ␮H (), causing a hyperpolarization of the inner mitochondrial membrane. Regarding K+ fluxes, experiments based on measuring mitochondrial swelling had demonstrated the presence of a non-specific and inducible K+ /H+ exchanger, that uses the energy accumulated by the proton gradient and that can transport also Na+ and Li+ [61]. Changes in matrix pH and Mg2+ concentration, linked to fluctuations of mitochondrial volume, regulate the K+ /H+ antiporter [61]. As suggested by Brierley in the 1970s (for review [62]) mitochondrial K+ fluxes are controlled by an interplay between selective K+ channels, that mediate an inward K+ transport, which is in turn regulated by K+ /H+ exchangers, in order to finely control mitochondrial volume homeostasis. While the role of exchangers in avoiding mitochondrial swelling due to K+ entry is obvious, the presence and the activity of K+ channels is apparently not helpful for mitochondrial physiology. One possible function could be found during organelle biogenesis, since K+ is the main intra-mitochondrial cation, but the most interesting idea is that mitochondrial volume control could have an effect on the respiratory chain, causing an increase in fatty acid oxidation as a consequence of changes in matrix volume [63]. In this scenario, mitoKv1.3 is a voltage-dependent K+ channel that mediates an inward K+ flux to the mitochondrial matrix and participates to the K+ cycle. As discussed above, inhibition of mitochondrial potassium channels blocks the K+ entrance into the mitochondrial matrix leading to hyperpolarization of the inner mitochondrial membrane [49,64] (Fig. 2). Electrophysiological observations by patch clamp on mitoplasts, started by the group of Inoue and colleagues with the ATP-regulated K+ channel

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Fig. 2. MitoKv1.3 and ROS production. Inhibition of the mitochondrial Kv1.3 by membrane permeant inhibitors or by interaction with Bax, inhibits the inward potassium flux mediated by the channel. The reduced flux of K+ triggers mitochondrial membrane hyperpolarization, that in turn acts to uncouple either or both complex I and/or III of the respiratory chain causing an increase in reactive oxygen species (ROS) production.

[65], identified the presence of selective K+ channels of the inner mitochondrial membrane; among them our group discovered the mitochondrial counterpart of Kv1.3, mitoKv1.3 [52]. 5. Mitochondrial K+ fluxes and ROS production The superoxide anion is the precursor of most ROS. Mitochondrial superoxide, besides contributing to mitochondrial damage, has an effect on the redox signaling on the cell. Radicals are produced via one-electron transfer to oxygen from sites of the respiratory chain [66,67] and, under appropriate circumstances, by diversion from cytochrome c to oxygen with the intermediary of p66Shc [68–70] or as a product of monoamino oxidase activity [70]. The major sites of ROS formation in mitochondria are complexes I and III of the respiratory chain [71], even if complex II was recently also highlighted as a molecule producing superoxide or hydrogen peroxide [72]. An increased or diminished supply of electrons to complex I increases ROS production (Fig. 2). The membrane potential , the ATP/ADP ratio and phosphorylation of the complexes I and IV [73] regulate the mitochondrial respiratory chain. Hyperpolarization of the inner mitochondrial membrane changes the redox state of the respiratory chain complexes, thereby increasing single electron leakage at complexes I and III to molecular oxygen and increasing superoxide anion production [73–75]. Complex III-dependent ROS formation can be activated by depolarization of inner mitochondrial membrane under conditions of a highly reduced coenzyme Q pool [76]. The membrane impermeable superoxide anion is released to either the mitochondrial matrix space or to the inter-membrane space when produced at complex I or III, respectively. 6. Regulation of mitochondrial Kv1.3 in apoptosis As pointed out above, several studies in the last years have shown the presence of multiple potassium channels in mitochondria: calcium-dependent potassium channels [77], ATP-regulated potassium channels [65,78], BK-channels [79,80], and the Kv

channels (Kv1.1, Kv1.3 and Kv1.5) [48–50,81–83]. The expression of these channels in mitochondria was shown by patch clamping of mitoplasts, western blotting of purified mitochondria, confocal microscopy and, at least for Kv1.3 and Kv1.5, also by electron microscopy [48–50,81–83]. In particular, patch clamping of the inner mitochondrial membrane and electron microscopy unambiguously proved the presence of these channels. In the case of Kv1.3, mitoKv1.3 expression was observed in mitochondria of freshly isolated peripheral blood lymphocytes, macrophages, non-malignant and cancer cell lines [48–50,81–83]. Moreover, cell lines were genetically modified to study Kv1.3 expression/activity [48,49,82]. In particular, CTLL-2 cells, which lack Kv1.3, were transfected to express Kv1.3 in the cells or specifically in mitochondria [48,49,82]. On the contrary, Jurkat cells or freshly isolated peripheral blood cells, that normally express Kv1.3, were used to downregulate Kv1.3 with Kv1.3-specific siRNA [48,49,82]. Patch clamp analysis and western blot studies on the expression level of Kv1.3-transfected CTLL-2 cells revealed Kv1.3 activities, expression levels, biophysical characteristics and pharmacological properties comparable to that found in genetically non-manipulated Jurkat cells, indicating the suitability of CTLL-2 cells as genetic model to analyze the function of Kv1.3 at least in vitro [48,49]. Functional studies using Kv1.3-deficient and Kv1.3reconstituted CTLL-2 cells or lymphocytes transfected with Kv1.3-targeting siRNA revealed that Kv1.3 activity is involved in cell death induced by TNF␣, CD95, staurosporine, sphingomyelinase or C6 -ceramide [48,49] (Fig. 3). Further, membrane-permeable inhibitors of Kv1.3 such as Psora-4, PAP-1 or clofazimine induced cell death in Kv1.3-expressing cells, while membraneimpermeable inhibitors of Kv1.3 such as margatoxin did not (Fig. 3). Most importantly, transfection of Kv1.3-deficient cells with a mitochondrial-targeted Kv1.3 construct was sufficient to reconstitute cell death proving that mitoKv1.3 is required for the induction of cell death by a variety of stimuli [49]. Structural analysis of Kv1.3 toxins indicates that a lysine in these toxins is required to inhibit Kv1.3 [49,82]. As stated above, only two amino acids of Bax face the inner mitochondrial membrane and one of them is the lysine 128. This lysine is evolutionarily highly

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Fig. 3. Apoptotic cell death induced by mitoKv1.3 inhibition. MitoKv1.3 mediates an inward potassium flux from the cytosol to mitochondrial matrix. Direct interaction of mitoKv1.3 with Bax, translocated into the outer mitochondrial membrane after induction of apoptosis, blocks the flux of potassium ions into the matrix, thus triggering a hyperpolarization of the mitochondrial membrane. Inhibition of mitoKv1.3 by specific membrane permeant inhibitors, Psora-4, PAP-1 and clofazimine, mimics the effects induced by Bax-mitoKv1.3 interaction. Mitochondrial membrane hyperpolarization triggers uncoupling of the respiratory chain complexes causing increase in ROS production. ROS activate the permeability transition pore (PTP), that induces depolarization of mitochondrial membrane and release of several mitochondrial proteins, as well as favours the detachment of the cytochrome c from the cristae. Cytochrome c is released both by outer mitochondrial membrane ruptures and by Bax oligomers, and once in the cytosol, triggers the activation of the intrinsic apoptotic pathway.

conserved among all pro-apoptotic members of the Bax/Bak family, while absent in the non-apoptotic members of this protein family [49,82]. Bcl-2 and Bcl-xL contain a negatively charged glutamate at position 158 that corresponds to that of amino acid 128 in Bax and a conserved lysine/arginine preceding this glutamate at amino acid 157 [49,82]. These analogies between Kv1.3 toxins and Bax led to studies that tested whether Bax and Bak bind to Kv1.3 and induce cell death in a toxin-like fashion. Initial studies indicated that cells lacking Kv1.3 are resistant to Bax-induced apoptosis triggered by transfection of Bax and that isolated mitochondria resisted the pro-apoptotic effect of recombinant Bax [49,82]. Kv1.3-deficient isolated mitochondria failed to release cytochrome c upon treatment with Bax, an effect that Bax rapidly induced in Kv1.3-positive mitochondria [49,82]. Further studies using recombinant Bax, Bak or Kv1.3 as well as co-immunoprecipitation experiments of Bax and Kv1.3 from isolated mitochondria revealed that Kv1.3 and Bax physically interact upon induction of apoptosis, but not in resting cells [49,82]. Most importantly, patch clamp studies revealed a functional interaction of Kv1.3 with Bax [49,82]. These studies showed that Bax inhibits Kv1.3 with an IC50 in the low nM range whereas recombinant Bcl-2 or Bcl-xL were without effect. The interaction of Bax and Kv1.3 was pH-dependent and the IC50 of Bax increased from 4 to 12 nM when the bath solution pH was lowered to pH 6.7 and to a value 50 nM at pH 6.0. Since protonation of Histidine 404 of Kv1.3 has been shown to reduce the interaction of the pore with toxins, it is assumed that Bax binds to and inhibits Kv1.3 in a very similar manner as highly specific Kv1.3 inhibitor toxins (e.g. margatoxin), which dock in the outer-facing vestibule of Kv1.3 [38–40]. Specifically, this lysine in Kv1.3-blocking toxins and presumably also in Bax binds to the ring of four aspartate residues of the channel vestibule, which faces the inter membrane space [49,82] (Fig. 3).

This mechanism was further proven by several mutation studies, i.e. a mutant of lysine 128 in pro-apoptotic Bax to a negatively charged glutamate and, vice versa, a mutant of glutamate 158 in the anti-apoptotic Bcl-xL [82]. While the mutant K128E Bax still integrated into the mitochondrial membrane and formed ion channels when reconstituted into black lipid bilayer indicating its functionality, it did not induce ROS release, hyperpolarization/depolarization of the mitochondrial membrane potential or release of cytochrome c from isolated mitochondria [82]. Moreover, it also failed to inhibit Kv1.3 in patch clamp experiments. In contrast, mutation of Bcl-xL to E158K converted the protein into a pro-apoptotic protein inducing ROS-release, hyperpolarization/depolarization of the mitochondrial membrane, release of cytochrome c from isolated mitochondria and inhibition of Kv1.3 in patch clamp experiments [82]. Transfection of Bax/Bak double knock-out MEF cells with expression vectors for wildtype Bax, K128E Bax or E158K Bcl-xL confirmed the suggested mechanism of Kv1.3-Bax interactions and showed that transfection of wildtype Bax or E158K Bcl-xL , but not of K128E Bax, restored apoptosis and release of cytochrome c induced by staurosporine [82]. Inhibition of mitoKv1.3 by Bax results in several dramatic changes of mitochondrial physiology, in particular the release of cytochrome c from mitochondria, an initial hyperpolarization of the inner mitochondrial membrane that is followed by a depolarization and the production of ROS [48,49,81–84]. The initial hyperpolarization of the inner mitochondrial membrane is consistent with the inhibition of Kv1.3 and results in an increased release of ROS that oxidize the permeability transition pore (PTP) finally resulting in depolarization. ROS are also involved in the release of cytochrome c, possibly by a change of the oxidation state of cardiolipin that binds to Kv1.3 [13,14], but other, yet unknown mechanisms seem

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to be also important in the release of cytochrome c by inhibition of Kv1.3. Several further studies demonstrated that Bax does not only interact with Kv1.3, but also with the homologs of Kv1.3, Kv1.5 and Kv1.1 [49,82]. Further, Siemen et al. showed that Bax interacts with BK-channels in the inner mitochondrial membrane and thereby triggers the release of cytochrome c from mitochondria also resulting in cell death [79]. It is possible that similar molecular mechanisms as identified for Kv1.3 apply to the interaction of Bax with these K+ channels, but the details need to be identified. In summary, the model of mitoKv1.3 and Bax interactions predicts that the positively charged lysine of Bax plugs the pore of the channel, and thereby initiates mitochondrial changes during apoptosis. The inactivation of mitoKv1.3 by Bax, toxins or pharmacological inhibitors results in an active change of mitochondria physiology, i.e. hyperpolarization of the mitochondrial membrane and ROS release (Fig. 3).

7. Kv1.3 and malignancies Kv1.3 has been shown to be expressed in brain, lung, thymus, spleen, lymph node, fibroblasts, lymphocytes [48], tonsils, macrophages [84], microglia, oligodendrocytes, osteoclasts, platelets, liver, skeletal muscle, in hippocampal neurons [85], astrocytes [86] and brown and white fat [17,57]. Furthermore, Kv1.3 was shown to be expressed in various cancers [87], such as lymphoma [88], melanoma [89], glioma [90], breast [91,92], prostate [93], gastric [94] and colon cancer [95]. Finally, Kv1.3 was shown to be expressed in the prostate and breast cancer cell lines PC3 and MCF-7, respectively, and lymphoma and leukemia cells even in mitochondria [83,96]. As described above, Kv1.3 is a direct target of Bax and mediates its pro-apoptotic effects. Thus, drugs that inhibit mitoKv1.3 should induce cell death mimicking Bax interaction, even in cells overexpressing anti-apoptotic Bcl-2-like proteins. Therefore, direct inhibitors of mitoKv1.3 are very attractive novel chemotherapeutic agents. This was exemplified for chronic lymphocytic leukemia (B-CLL), a common leukemia with limited treatment options: importantly, the effects where obtained independently of the prognostic factors (ZAP70, CD38 and hyper-somatic mutation) [83]. These studies used membrane-permeable inhibitors of Kv1.3, such as Psora-4 and PAP-1, and in particular clofazimine [83]. B-CLL cells showed higher levels of Kv1.3 than B-cells from healthy subjects [83], although the levels varied in individual patients and the comparison of tumor cells with normal, mature B-cells is of limited value. Psora-4 or PAP-1 applied together with inhibitors of the multidrug resistance pumps, and clofazimine very efficiently triggered death of B-CLL cells, while surprisingly the drugs had almost no effect on B or T cells isolated from healthy subjects or on T cells isolated from the same B-CLL patient [83]. The relatively high sensitivity of B-CLL cells compared with nonmalignant lymphocytes was explained by an increased, constitutive oxidative stress in cancer cells compared to non-malignant cells [83]. This hypothesis is consistent with the finding of increased ROS production and a marked mitochondrial depolarization in BCLL cells after treatment of the tumor cells and inhibition of Kv1.3 with Psora-4, PAP-1 and clofazimine [83]. The chemotherapeutic effect of Psora-4, PAP-1 and clofazimine is not restricted to hematological malignancies. It was shown that clofazimine also kills melanoma cells in vitro and reduced the size of an orthotopic melanoma in a mouse model in vivo by approximately 90% [81]. These studies indicate that inhibition of Kv1.3 is an attractive novel target to be explored for its potential to induce death of a variety of tumor cells. Since membrane permeable inhibitors of

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