Targeting the Mitochondrial Potassium Channel Kv1.3 to Kill Cancer Cells: Drugs, Strategies, and New Perspectives:

Cancer is the consequence of aberrations in cell growth or cell death. In this scenario, mitochondria and ion channels play a critical role in regard to cell proliferation, malignant angiogenesis, migration, and metastasis. In this review, we focus on Kv1.3 and specifically on mitoKv1.3, which showed an aberrant expression in cancer cells compared with healthy tissues and which is involved in the apoptotic pathway. In recent years, mitoKv1.3 has become an oncological target since its pharmacological modulation has been demonstrated to reduce tumor growth and progression both in vitro and in vivo using preclinical mouse models of different types of tumors

Mitochondrial Kv1.3: a New Target in Cancer Biology?

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Recombinant Human Voltage Dependent Anion Selective Channel Isoform 3 (hVDAC3) Forms Pores with a Very Small Conductance

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Properties of the permeability transition of pea stem mitochondria

In striking analogy with Saccharomyces cerevisiae, etiolated pea stem mitochondria did not show appreciable Ca2+ uptake. Only treatment with the ionophore ETH129 (which allows electrophoretic Ca2+ equilibration) caused Ca2+ uptake followed by increased inner membrane permeability, membrane depolarization and Ca2+ release. Like the permeability transition (PT) of mammals, yeast and Drosophila, the PT of pea stem mitochondria was stimulated by diamide and phenylarsine oxide and inhibited by MgADP and Mg-ATP, suggesting a common underlying mechanism; yet, the plant PT also displayed distinctive features: (i) as in mammals it was desensitized by cyclosporin A, which does not affect the PT of yeast and Drosophila; (ii) similarly to S. cerevisiae and Drosophila it was inhibited by Pi, which stimulates the PT of mammals; (iii) like in mammals and Drosophila it was sensitized by benzodiazepine 423, which is ineffective in S. cerevisiae; (iv) like what observed in Drosophila it did not mediate swelling and cytochrome c release, which is instead seen in mammals and S. cerevisiae. We find that cyclophilin D, the mitochondrial receptor for cyclosporin A, is present in pea stem mitochondria. These results indicate that the plant PT has unique features and suggest that, as in Drosophila, it may provide pea stem mitochondria with a Ca2+ release channel

F-ATPase ofDrosophila melanogasterForms 53-Picosiemen (53-pS) Channels Responsible for Mitochondrial Ca2+-induced Ca2+Release

Mitochondria of Drosophila melanogaster undergo Ca2+-induced Ca2+ release through a putative channel (mCrC) that has several regulatory features of the permeability transition pore (PTP). The PTP is an inner membrane channel that forms from F-ATPase, possessing a conductance of 500 picosiemens (pS) in mammals and of 300 pS in yeast. In contrast to the PTP, the mCrC of Drosophila is not permeable to sucrose and appears to be selective for Ca2+ and H+. We show (i) that like the PTP, the mCrC is affected by the sense of rotation of F-ATPase, by Bz-423, and by Mg2+/ADP; (ii) that expression of human cyclophilin D in mitochondria of Drosophila S2R+ cells sensitizes the mCrC to Ca2+ but does not increase its apparent size; and (iii) that purified dimers of D. melanogaster F-ATPase reconstituted into lipid bilayers form 53-pS channels activated by Ca2+ and thiol oxidants and inhibited byMg(2+)/gamma-imino ATP. These findings indicate that the mCrC is the PTP of D. melanogaster and that the signature conductance of F-ATPase channels depends on unique structural features that may underscore specific roles in different species

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (K(Ca)3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoK(Ca)3.1) of various cancer cell lines. The role mitoK(Ca)3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity K(Ca)3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the mu M range, depending on channel expression. In contrast, inhibition of the plasma membrane K(Ca)3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoK(Ca)3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoK(Ca)3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-kappa B (NF-kappa B) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-kappa B and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial K(Ca)3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits

Background/Aims: The permeability transition pore (PTP) is an unselective, Ca2+-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca2+-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu2+ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu2+ treatment and show PTP-dependent mitochondrial Ca2+ release but not swelling. Cu2+ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase

Functional characterization of potassium channels in the cyanobacterium synechocystis SP.PCC 6803

Before Pre-Paleozoic, the Earth's atmosphere had a composition different from today; in fact the organisms are not able to live in aerobic condition. The advent of cyanobacteria brought significant innovations, in fact, these bacteria unlike other phototrophs existing at that time, had chlorophyll molecules and protein complexes that allowed the use of water as electron donor to produce oxygen gas. This innovation developed over millions of years to get the current atmosphere. All these changes led to an inevitable biochemical and metabolic evolution of organisms. In Proterozoic or in early Cambrian, cyanobacteria began to reside within certain eukaryote cells. According to the endosymbionthic theory, chloroplasts evolved from a small primitive cyanobacterium settled within eukaryotic cells. Today, cyanobacteria are found throughout the Earth's environment, from oceans to fresh water and soil in the arctic areas, deserts and hot springs. Our attention is focused on cyanobacterium Synechocystis sp. PCC 6803. This strain was isolated for the first time from fresh water in California and now is considered a good model for scientific studies. It is spontaneously transformable, is able to integrate foreign DNA into its genome by homologous recombination (allowing targeted gene replacement) and can grow in the absence of photosynthesis if a suitable fixed-carbon source such as glucose is provided. Moreover it is the first photosynthetic organism for which the complete genome was sequenced (Kaneko et al., 1996). In the proteome of Synechocystis several putative ion channels can be identified (Kuo et al., 2005). However, none of them have been characterized from the functional point of view and their physiological role is still unknown. Ion channels are ubiquitous membrane proteins that control the passage of ions through biological membranes. These proteins, in all prokaryotes and eukaryotes, allow the correct ion distribution necessary to cellular functions. The basic features of the channels are selectivity and gating, the first is the property that controls the kind of ion that flows across the membrane and the second is the process of opening and closing the ion pathway. In fact the passage through the pore is governed by a "gate," which may be opened or closed by chemical, mechanical or electrical signals (Hille, 2001). Potassium (K+) is the most abundant cation in organisms and plays a crucial role in the survival and development of cells, by regulating enzyme activity and tuning membrane potential. This is one of the reasons for which potassium channels are one of the most studied among classes of channels. The field of prokaryotic potassium channels underwent a rapid development over the past years thanks to the application of a combination of bioinformatics and molecular biology, beside electrophysiology and structural studies. Understanding of their structure and actual mechanism of ion conduction allow to obtain more information about the function of potassium channels in general. A bioinformatic screening of Synechocystis sp. PCC 6803 proteome identified two putative proteins on which we focused our attention. The first one was named SynK and it displays sequence homology with KvAP (a voltage gated potassium channel of A. pernix) (Jiang et al., 2003). The second one, SynCaK, displays sequence homology to MthK, a Ca2+-dependent potassium channel from M. thermoautotrophicum (Jiang et al., 2002). Our goal was to understand whether they actually function as ion channels and to reveal their roles in the physiology of cyanobacteria. The characteristics and function of these proteins were studied through an integrated approach involving molecular biology techniques, biochemistry, electrophysiology and microscopy. SynK was initially identified in the genome of Synechocystis sp. PCC 6803 using the selectivity filter amino acid sequence (TMTTVGYGD) as a query sequence. This protein of unknown function shows six membrane spanning segments (S1-S6) and a pore region between S5 and S6 helixes. Before starting my Ph.D, SynK gene has been cloned and expressed in mammalian cells (Chinese Hamster Ovary, CHO) in fusion with EGFP (a fluorescent protein). Subsequent Western-blotting analysis showed that the fusion protein was correctly expressed. Confocal microscopy studies demonstrated its membrane localization and patch-clamp analysis revealed an activity of voltage-gated outwardly rectifying potassium selective channel in CHO cells. In addition, the double location of SynK in plasma and thylakoid membrane of cyanobacteria was shown by immunogold electron microscopy and Western blot on isolated membrane fractions. During my P.h.D, I performed the construction of two different mutants of SynK channel. The first SynK mutant, corresponding to the protein with a single amminoacid mutation in the selectivity filter of the pore (mutation Y181A), was used for expression in CHO cells. In accordance to the literature, this mutant protein loses its potassium channel activity. I also produced a ∆SynK Synechocystis mutant strain. Its functional analysis allowed to understand the physiological role of SynK in cyanobacteria. In order to characterize the function of the SynK protein, we initially verified that the deletion mutant did not express Synk, using Western blot technique. To evaluate the physiological role of the SynK protein, we initially compared growth of the wild type (WT) and mutant strain in different conditions. Characterization of the mutant phenotype was investigated by comparing photosynthetic activity in WT and mutant strains. Using a similar approach we have identified in the genome of Synechocystis sp. PCC 6803 a second protein classified as a putative potassium channel that displays sequence homology with MthK, a calcium dependent potassium channel from the archeon Methanobacterium thermoautophicum. Using several structural prediction programs, we analyzed the primary sequence of the protein translated from sll0993 and we observed that this protein (that we called SynCaK), like MthK, is predicted to contain two membrane spanning segments, a recognizable K+ channel signature sequence, with only conservative substitutions, and a regulatory sequence for K+ conductance (RCK). Also in the case of sll0993, we cloned and expressed the protein in fusion with GFP in CHO cells and studied their activity by patch clamp. Moreover, in order to study the role of SynCaK in cyanobacteria physiology we produced a SynCaK-deficient Synechocystis mutant. To gain further information about the activity of the channel, we have expressed and started the purification of the protein in another heterologous system, E. coli. Purified recombinant channel proteins are often studied by incorporating them into an artificial planar bilayer system (Ruta et al., 2003). During my Ph.D, I also continued the work begun during my thesis in Biotecnology on the study of ion channels in mitochondria of Graminaceae. Classical bioenergetics techniques reveal activities compatible with the presence of a potassium channel in durum wheat mitochondria, but the study of channels in mitochondria of plant systems is a still unexplored field in the world. To this end, we started a study through the parallel use of different techniques, which allowed a more complete characterization of the activity of channels present in wheat mitochondria. In particular, we followed two approaches. First, biochemical studies on isolated mitochondria, through the use of SDS-PAGE and immunoblotting, allowed the evaluation of the sample used in terms of enrichment and purity (data completely absent in the literature to date). Second, preparations of mitochondria from roots of durum wheat were suitable for electrophysiological studies in particular patch clamp technique, applied for the first time on plant mitochondria. Finally, I was involved in collaboration with the laboratory of Professor Nobuyuki Uozumi at Tohoku University in Japan. This group obtained a mutant for Synechocystis aquaporin. Aquaporins are membrane proteins embedded in the cell membrane that regulate the flow of water. I contributed to the characterization of the acquaporin-less mutant by performing experiments measuring photosynthetic activity. In particular, we performed several experiments of oxygen evolution demonstrating that the photosyntetic efficiency is higher in the mutant with respect to the WT when the organisms are incubated in hyperosmotic medium. The next step is to clarify how exactly a hyperosmotic stress and the absence of aquaporin are correlated with the photosynthesis and what is the underlying mechanism.Prima del Pre-Paleozoico, l'atmosfera terrestre aveva una composizione diversa rispetto a quella quella di oggi, infatti, gli organismi non erano in grado di vivere in condizioni aerobiche. L'avvento dei cianobatteri ha portato rilevanti innovazioni, infatti, questi a differenza di altri batteri fototrofi esistenti a quel tempo, presentavano molecole di clorofilla e complessi proteici che permisero di utilizzare l’acqua come donatore di elettroni per la produzione dell’ossigeno. Questa modificazione ha permesso, lungo milioni di anni, di ottenere l'attuale atmosfera. Tutti questi cambiamenti portarono ad una inevitabile evoluzione biochimica e metabolica degli organismi. Nel Proterozoico o agli inizi del Cambriano, i cianobatteri iniziarono a risiedere all'interno di alcune cellule eucariotiche. Secondo la teoria endosimbiotica, i cloroplasti evolsero da un piccolo cianobatterio primitivo presente all'interno delle cellule eucariotiche. Oggi, i cianobatteri si trovano in diversi ambienti terrestri, da oceani ad acque dolce, in terre artiche, in deserti ed in sorgenti termali. La nostra attenzione è focalizzata sul cianobatterio Synechocystis sp. PCC 6803. Questo ceppo è stato isolato per la prima volta da una sorgente di acqua dolce in California e ora è considerato un buon organismo modello per studi scientifici. È spontaneamente trasformabile, è in grado di integrare DNA estraneo nel suo genoma attraverso ricombinazione omologa (consentendo la sostituzione mirata dei geni) e può crescere in assenza di fotosintesi se viene fornita un'adeguata fonte di carbonio, come il glucosio. Inoltre è il primo organismo fotosintetico per il quale il genoma è stato sequenziato (Kaneko et al., 1996). Nel proteoma di Synechocystis sono stati identificati diversi putativi canali ionici (Kuo et al., 2005). Tuttavia, nessuno di essi è stato caratterizzato da un punto di vista funzionale e il loro ruolo fisiologico rimane ancora sconosciuto. I canali ionici sono proteine di membrana che controllano il passaggio degli ioni attraverso esse. Queste proteine, in tutti i procarioti e gli eucarioti, permettono la corretta distribuzione ionica necessaria per le funzioni cellulari. Le caratteristiche base dei canali sono la selettività ed il gating, la prima è la proprietà che controlla il tipo di ioni che attraversa la membrana, la seconda è il processo di apertura e chiusura del percorso degli ioni. In realtà il passaggio attraverso il poro è regolato da un gate, che può essere aperto o chiuso da segnali chimici, meccanici o elettrici (Hille, 2001). Il potassio (K+) è il catione più abbondante negli organismi viventi e svolge un ruolo cruciale per la sopravvivenza e lo sviluppo delle cellule, regolando l'attività enzimatica e il potenziale di membrana. Questo è uno dei motivi per i quali i canali del potassio sono una delle classi di canali più studiate. Il campo dei canali del potassio procariotici ha subito un rapido sviluppo negli ultimi anni grazie all'applicazione di una combinazione di tecniche di bioinformatica e biologia molecolare, affiancate a studi di elettrofisiologia e studi strutturali. La comprensione della loro struttura e del meccanismo di conduzione degli ioni permette di ottenere ulteriori informazioni sulla funzione dei canali di potassio in generale. Uno screening bioinformatico del proteoma di Synechocystis sp. PCC 6803 ha individuato due proteine putative su cui abbiamo concentrato la nostra attenzione. La prima è stata chiamata SynK e mostra omologia di sequenza con KvAP (un canale del potassio voltaggio di A. pernix) (Jiang et al., 2003). La seconda, SynCaK, mostra omologia di sequenza con MthK, un canale del potassio Ca2 +-dipendente di M. thermoautotrophicum (Jiang et al., 2002). Il nostro obiettivo era quello di capire se effettivamente queste proteine funzionano come canali ionici e di comprendere il loro ruolo nella fisiologia dei cianobatteri. Le caratteristiche e la funzione di queste proteine sono state studiate attraverso un approccio integrato comprendente tecniche di biologia molecolare, biochimica, elettrofisiologia e microscopia. Il gene SynK è stato inizialmente identificato nel genoma di Synechocystis sp. PCC 6803 utilizzando la sequenza amminoacidica del filtro di selettività (TMTTVGYGD) come sequenza query. Questa proteina di funzione sconosciuta mostra sei segmenti transmembrana (S1-S6) ed una regione del poro tra le eliche S5 e S6. Prima di iniziare il mio Dottorato, SynK è stato clonato ed espresso in cellule di mammifero (Chinese Hamster ovary, CHO) in fusione con la EGFP (una proteina fluorescente). Una successiva analisi western-blotting ha dimostrato che la proteina di fusione è correttamente espressa. Studi di microscopia confocale hanno dimostrato la sua localizzazione nella membrana di cellule CHO e l'analisi patch-clamp ha rivelato un'attività di canale outwardly rectifying selettivo per il potassio. Inoltre, è stata dimostrata per SynK, in frazioni di membrana isolate da cianobatteri, mediante microscopia elettronica (attraverso la tecnica dell’immunogold) e tecniche di western blot, una doppia localizzazione nella plasmamembrana e nelle membrane tilacoidi.Durante il mio Dottorato, è stata eseguita la costruzione di due diversi mutanti del canale SynK. Il primo mutante corrisponde alla proteina con una mutazione puntiforme nel filtro di selettività del poro (mutazione Y181A) e utilizzato per l'espressione in cellule CHO. In base alla letteratura, questa proteina mutante perde la sua attività di canale del potassio. Inoltre, è stato prodotto un ceppo mutante knock-out (ΔSynK) in Synechocystis. La sua analisi funzionale ha permesso di capire il ruolo fisiologico di SynK nei cianobatteri. Al fine di caratterizzare la funzione della proteina SynK, abbiamo inizialmente verificato, attraverso western blot, che il ceppo mutante effettivamente non esprimesse la proteina. Mentre per valutare il ruolo fisiologico della proteina SynK, abbiamo confrontato la crescita del ceppo wild-type (WT) e mutante in diverse condizioni. La caratterizzazione del fenotipo mutante è stata studiata confrontando l’attività fotosintetica nel WT e nel mutante. Utilizzando un approccio simile abbiamo identificato nel genoma di Synechocystis sp. PCC 6803 una seconda proteina classificata come putativo canale del potassio che mostra omologia di sequenza con MthK, un canale del potassio calcio dipendente di Methanobacterium thermoautophicum. Attraverso l’utilizzo di vari programmi di predizione strutturale, abbiamo analizzato la sequenza primaria della proteina tradotta e abbiamo osservato che questa (che abbiamo chiamato SynCaK), come MthK, contiene due segmenti transmembrana, un filtro di selettività tipico dei canali del K+, con sostituzioni conservative, e un dominio di regolazione della conduttanza del potassio (RCK domain). Anche in questo caso, abbiamo clonato ed espresso la proteina in fusione con EGFP in cellule CHO e studiato la loro attività tramite patch clamp. Inoltre, al fine di studiare il ruolo di SynCaK nella fisiologia dei cianobatteri abbiamo prodotto un mutante knock-out per SynCaK. Per ottenere ulteriori informazioni sull’attività del canale, abbiamo espresso e iniziato la purificazione della proteina in un altro sistema eterologo, E. coli. Le proteine canale-ricombinanti sono spesso studiate mediante la loro integrazione in doppi strati artificiali (Ruta et al., 2003). Durante il mio Dottorato, ho anche continuato il lavoro iniziato durante la mia tesi di laurea in Biotecnologie Industriali sullo studio dei canali ionici nei mitocondri delle Graminaceae. Tecniche classiche di bioenergetica hanno rivelato attività compatibili con la presenza di un canale di potassio nei mitocondri di grano duro, ma lo studio dei canali nei mitocondri di sistemi vegetali è un campo ancora inesplorato nel mondo. A tal fine, è stato iniziato uno studio attraverso l'utilizzo parallelo di diverse tecniche, che hanno consentito una caratterizzazione più completa delle attività dei canali presenti nei mitocondri di grano. In particolare, sono stati seguiti due approcci. In primo luogo, studi biochimici sui mitocondri isolati, attraverso l'uso di SDS-PAGE e immunoblotting, che hanno permesso la valutazione del campione utilizzato in termini di arricchimento e di purezza (dati del tutto assenti in letteratura fino ad oggi). In secondo luogo, sono state definite preparazioni di mitocondri da radici di grano duro adatte per studi elettrofisiologici. In particolare, per la prima volta è stata applicata la tecnica di patch clamp su mitocondri vegetali. Infine, ho svolto una collaborazione con il laboratorio del Professor Nobuyuki Uozumi presso la Tohoku University in Giappone. Questo gruppo ha ottenuto un mutante per l’acquaporina di Synechocystis. Le acquaporine sono proteine di membrana incorporate nelle membrane cellulari che regolano il flusso dell'acqua. Ho contribuito alla caratterizzazione del mutant-less acquaporin attraverso esperimenti di misura dell'attività fotosintetica. In particolare, sono stati eseguiti diversi esperimenti di evoluzione di ossigeno che dimostrano che l'efficienza fotosintetica è più alta nel mutante rispetto al WT quando gli organismi vengono incubati in un mezzo iperosmotico. Il passo successivo sarà quello di chiarire esattamente come uno stress iperosmotico e l'assenza di acquaporina sono correlati con la fotosintesi e quindi il meccanismo sottostante

Mitochondrial potassium channels in cell death

Mitochondria are intracellular organelles involved in several processes from bioenergetics to cell death. In the latest years, ion channels are arising as new possible targets in controlling several cellular functions. The discovery that several plasma membrane located ion channels have intracellular counterparts, has now implemented this consideration and the number of studies enforcing the understanding of their role in different metabolic pathways. In this review, we will discuss the recent updates in the field, focusing our attention on the involvement of potassium channels during mitochondrial mediated apoptotic cell death. Since mitochondria are one of the key organelles involved in this process, it is not surprising that potassium channels located in their inner membrane could be involved in modulating mitochondrial membrane potential, ROS production, and respiratory chain complexes functions. Eventually, these events lead to changes in the mitochondrial fitness that prelude to the cytochrome c release and apoptosis. In this scenario, both the inhibition and the activation of mitochondrial potassium channels could cause cell death, and their targeting could be a novel pharmacological way to treat different human diseases