University of Groningen The role of small conductance calcium-activated potassium channels in mitochondrial dysfunction Krabbendam, Inge

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University of Groningen

The role of small conductance calcium-activated potassium channels in mitochondrial dysfunction

Krabbendam, Inge DOI:

10.33612/diss.144370526

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Publication date: 2020

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Krabbendam, I. (2020). The role of small conductance calcium-activated potassium channels in mitochondrial dysfunction: Targeting metabolic reprogramming and calcium homeostasis. University of Groningen. https://doi.org/10.33612/diss.144370526

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Preface

Neurodegenerative diseases affect millions of people worldwide and seriously degrade the quality of life. Alterations of potassium channels in the brain have been linked to the pathogenesis of neurodegeneration. For instance, genetic evidence links calcium-activated potassium (KCa) channels to Alzheimer’s

Disease (AD) (1,2). KCa channels also gained attention in the field of ischemia and

cancer. In neurons, plasma membrane KCa channels regulate excitability, while

mitochondrial located KCa (mitoKCa) channels play a role in balancing levels of

mitochondrial reactive oxygen species (ROS) and mitochondrial calcium (Ca2+_),

leading to protection against oxidative stress. Oxidative stress and mitochondrial dysfunction serve nowadays as pathological markers for neurodegeneration, next to neuronal hyperexcitability and Ca2+_{deregulation, all}

contributing to cell death (3–9). Several types of cell death can be related to neurodegeneration, including excitotoxicity, oxytosis, ferroptosis and inflammaging (10–12). As a response mechanism to protect against defects in cellular energy metabolism and cell death, compounds that are able to mediate metabolic (re)programming gained interest also in the field of neuroscience, besides long-standing studies in oncology (13,14). Interestingly, previous data of our group showed that small conductance Ca2+_{-activated K}+_{(SK) channels}

modulate mitochondrial Ca2+_{levels and protect neurons against glutamate}

excitotoxicity and glutamate-induced oxytosis, of which the latter is a well-established in vitro model to study mitochondrial dysfunction associated with oxidative stress. Moreover, pharmacological SK channel modulators also attenuated inflammation. It is not yet understood precisely how SK channel modulators affect mitochondrial function and inflammation and how they are able to confer cellular protection. The objective of this thesis was to investigate the role of SK channels on mitochondrial dysfunction and inflammation, thereby focusing on Ca2+_{, ROS and mitochondrial metabolism.}

Mitochondrial calcium-activated potassium channels

As reviewed in chapter 2, KCa channels are found in the central nervous system

(CNS), in the cardiovascular system, particularly in the heart, and in several other organs. KCa channels are expressed at the plasma membrane and in membranes

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(BK, IK and SK) at the inner mitochondrial membrane (IMM) of several cell types has gained a lot of interest in cardiovascular, neurological and cancer research (15–19), attributed to the involvement of the channels in mitochondrial function and Ca2+_{homeostasis. K}

Ca channels located at the IMM facilitate K+ entry into

the mitochondrial matrix, leading to depolarization of the mitochondrial membrane and a reduced driving force for Ca2+_{influx into mitochondria (19–}

21). Under oxidative stress conditions, mitochondrial dysfunction and subsequent cell death is a consequence of elevated mitochondrial calcium (mito[Ca2+_{]) levels, higher probability of mitochondrial permeability transition}

pore (mPTP) formation, enhanced ROS production, and inhibition of the respiratory chain in the mitochondria. A mechanism that obtained interest in the search for protection against diseases that involve oxidative stress, is mitochondrial preconditioning or mitohormesis, a process where cells undergo adaptation to oxidative stress. This process is attributed to a mild stressor that increases the resistance of both mitochondria and the entire cell to subsequent insults (22,23). Notably, pharmacological activation of mitoKCa channels

contribute to cell survival by lowering mito[Ca2+_{], attenuating formation of}

mitochondrial ROS, and restoring the mitochondrial membrane potential (MMP) (24,25). Moreover, mitoKCa activation also prevents the loss of ATP

production in the mitochondrial electron transport chain. Several studies identified mitoKCa channels as central players in the molecular mechanisms

underlying preconditioning in cardiac ischemia and in different neurodegenerative conditions. Preconditioning and postconditioning mechanisms promoted by activation of mitoBK and mitoSK channels decreased the infarct size in conditions of ischemic stress via attenuation of mitochondrial ROS generation, attenuating mito[Ca2+_{] and inhibiting mPTP formation (26–28).}

Both neuronal mitoBK- and mitoSK channels provoke protection by maintaining mitochondrial function. Importantly, however, mitoSK channels were shown to directly influence mitochondrial respiration (24). Among the different subcellular localizations of SK channels, in particular the mitochondrial-located SK2 channels are conferring protection against oxidative stress (24). With respect to SK channels, we investigated the impact of SK channel activation on mitochondrial Ca2+_{regulation in oxidative stress paradigms (chapter 3, 4 and 5).}

In addition, we studied whether the channels are involved in mitochondrial preconditioning and ROS production, mitochondrial complex activity and metabolic shifts, as a part of potential protective mechanisms against

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ferroptosis in neurons and inflammatory processes in macrophages (chapter 5

and 6).

SK channel activation and mitochondrial calcium in oxidative stress

The impact of ER – mitochondria coupling on mitochondrial Ca2+_homeostasis

and oxytosis in neuronal cell survival

Neuronal cell survival is impacted by Ca2+ _{regulation, since Ca}2+ _{ions are involved}

in action potential transduction, neurotransmitter release, and several downstream signalling pathways such as mitochondrial metabolism (29). Intracellular Ca2+ _{stores including those in the ER and mitochondria play a critical}

role in maintaining the intracellular Ca2+ _{homeostasis. Moreover, dysfunctional}

Ca2+ _{signalling has been linked to neurodegeneration (30). The physical}

connection between ER and mitochondria is established by ER-located IP3R,

GRP75 and OMM-located VDAC. These mitochondria-associated ER membranes (MAMs) were found to be critical for intracellular calcium [Ca2+_]

i signalling and

regulation of metabolism. Under healthy conditions, Ca2+_{is transferred from the}

ER along the MAM into the mitochondrial matrix where it facilitates ATP production through promotion of the TCA cycle and mitochondrial respiration (31–33). Disrupted Ca2+_{homeostasis can lead to mito[Ca}2+_{] overload, and}

subsequently to impaired mitochondrial metabolism and respiration (34,35). In addition, enhanced MAM-mediated mito[Ca2+_{] uptake leads to MMP loss, ROS}

production and eventually cell death (36,37). Several studies have proven that enhanced or weakened ER-mitochondrial coupling (EMC) and associated mitochondrial damage is detrimental. Moreover, different diseases either have decreased or enhanced EMC (40,41), which can be explained by the fact that both diminishing basal mito[Ca2+_{] uptake as well as inducing mito[Ca}2+_{] overload}

can lead to cell death. Using genetically encoded linkers sensitive to rapamycin, we provided evidence that EMC strengthening increased the vulnerability of HT22 cells to glutamate-induced oxytosis and cell death, through an increase in mito[Ca2+_{] uptake and impaired mitochondrial respiration (chapter 3). The}

linkers used in our study consist of ER-and OMM targeted anchors whose endogenous function is not related to EMC and which rapidly form a bridge upon rapamycin treatment (39). Although this type of physical connection is artificial,

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similar endogenous mechanisms regulating EMC are observed in neurodegenerative diseases.

Recent research showed that chronic incubations with high glucose of either insulin-secreting cells or pancreatic islets reduced ER Ca2+_{store, increased basal}

mito[Ca2+_{], and reduced ATP-stimulated ER-mitochondria Ca}2+_{exchanges, in}

conditions of increased organelle interactions (38). Under our experimental conditions, we enhanced EMC and proved that SK channel activation still mediated protection against glutamate challenge which induced cellular damage in the presence of ER-mitochondrial linkers. Interestingly, overexpression of either SK2 or mitoSK2 together with pharmacological positive modulator CyPPA treatment further enhanced cell survival compared to the GFP/mitoGFP overexpression, and this was independent of any influence on IP3R

– VDAC1 interaction (chapter 3). Thus, these data indicate that the overexpression of SK2 channels increased CyPPA-mediated protection against glutamate toxicity. In addition, these neuroprotective actions of SK2 channel activation were independent of the physical EMC.

Modulation of mitochondrial calcium uptake in ferroptosis

IP3R-mediated release of Ca2+ from the ER is subsequently transferred by the

mitochondrial Ca2+_{uniporter (MCU) to facilitate mitochondrial Ca}2+_uptake.

Located in the IMM, MCU is the true entry point of Ca2+ _{into the mitochondrial}

matrix. Therefore, we hypothesized that besides modulating ER – mitochondria contact points, antagonism of MCU may influence the cells’ vulnerability to oxidative stress or ferroptosis in HT22 cells (chapter 4). We demonstrated that inhibition of mito[Ca2+_{] uptake using ruthenium red (RR) and mitoxantrone (MX)}

restored cell viability in ferroptosis and attenuated mitochondrial Ca2+ _levels

following ferroptosis. Both compounds attenuated lipid peroxidation levels induced by erastin, an inducer of ferroptosis, and co-treatment with RR reduced mitochondrial ROS levels similar to ferrostatin, a classical inhibitor of ferroptosis. While both RR and MX reduced mitochondrial Ca2+_{overload, they}

were not able to rescue the decrease in mitochondrial respiration due to ferroptosis. Interestingly, Arduino and colleagues postulated that MX-mediated inhibition of MCU Ca2+_{currents did not influence oxidative phosphorylation}

(OXPHOS) (42). However, their experiment was performed using acute treatment. In our study, MX treatment alone resulted in a reduction in cell

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proliferation, a decrease in OXPHOS and an increase in early-apoptosis. Interestingly, co-treatment with SK channel activator, CyPPA, potentiated the protective effect of RR but demonstrated no significant additive effect to the protective action of MX. For further understanding of this additive effect, future research could investigate possible physical proximity or interaction between the two proteins. Notably, it has been suggested that opening of ATP-sensitive potassium (mitoKATP) channels by diazoxide in heart causes a drop in Ψm, thereby

limiting mitochondrial Ca2+_{entry through MCU and consequent Ca}2+_overload,

providing protection against ischemia-reperfusion injury (43,44). Furthermore, we showed that MICU1 knockdown cells were more sensitive to erastin treatment compared to control. In conclusion, we demonstrated that MCU antagonism and mito[Ca2+_{] reduction is protective against ferroptosis, and that}

potentially MICU1 plays a regulatory role.

The differential impacts of ROS on cell survival Mitohormesis or mitochondrial preconditioning

Apart from preservation of mito[Ca2+_{] levels, mitochondrial K}

Ca channels have

been shown to modulate mitochondrial ROS production, thereby inducing mitochondrial preconditioning or mitohormesis. By mildly increasing ROS production at the level of mitochondrial complexes, these channels have been shown to provide neuro- and cardioprotection, effects that could be abrogated by ROS scavengers (chapter 2). There is an increasing number of studies showing the ROS-dependent BK channel-mediated protection in for example ischemia reperfusion (I/R) (45,46). Moreover, mitoKATP opening evoked protective effects

against I/R and cerebral ischemia (47,48), with both ROS and nitric oxide (NO) production participating in the observed protection (49).

Interestingly, a study by Dai and colleagues found that simultaneous administration of cell-permeant superoxide dismutase (SOD) mimetic MnTBAP

([5,10,15,20-Tetrakis(4-carboxyphenyl)-21H,23H-porphine]manganese(III)chloride) with BK channel opener NS1619, reduced the NS1619-induced protective effect against ischemic preconditioning (45), which is in line with our observation described in chapter 5. Notably, mucosal

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permeability-sparing and anti-inflammatory effects triggered by NS1619 to prevent I/R was found to be ROS-dependent (45).

There is extensive evidence that preconditioning effects in the CNS mediate neuroprotection, for instance shown by one study of an in vitro model of oxygen-glucose deprivation/re-oxygenation in neuron-astrocyte co-cultures subjected to ischemic preconditioning (50). To our knowledge, we are the first to show SK channel-induced preconditioning effects in neurons against oxidative stress. In chapter 5 we elucidated that SK channel activation by CyPPA slightly reduced mitochondrial complex I and II activity, which is in line with a mild increase in mitochondrial ROS production that was observed after long-term treatment with CyPPA. Similarly, CyPPA used at low concentrations promoted cell survival against H2O2 damage, and opening of SK channels induced a mild

reduction of the mitochondrial membrane potential (MMP) and a slight increase in mitochondrial ROS formation (51). In our study, CyPPA rescued neuronal cells from ferroptosis, while scavenging mitochondrial ROS with MnTBAP reduced both mitochondrial ROS production induced by CyPPA and also its protection. Thus, our findings indicate that SK channel activation provoked preconditioning effects that play a role in the neuronal protection against ferroptosis. This is in line with previous observations showing that increasing superoxide levels to a threshold level that does not affect cell survival protects SH-SY5Y neuroblastoma cells against oxidative stress through a mitohormetic process (52). Mitohormesis has been shown to be a process also occurring in vivo (53), for example SOD2 depletion in embryonic mice resulted in mitochondrial adjustments and enhanced antioxidant capacity, leading to resistance to subsequent oxidant challenges in later life (54). Furthermore, deletion of SOD2 extended lifespan in Caenorhabditis elegans (C. elegans) by altering mitochondrial function (55). We found an SK channel-mediated complex I activity reduction in HT22 cells and increased mean lifespan in C. elegans (chapter 5). In line with these findings, mild complex I inhibition with low rotenone concentrations reversed aging-related regulation of gene expression and prolonged lifespan in killifish (56). Future studies should investigate whether indeed SK channel activation leads to mild mitochondrial ROS production in vivo, for instance using longevity models in C. elegans, killifish or

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Reverse electron transport

The current literature indicates that low ROS levels are beneficial, mediating adaptation to stress via signalling, while high levels of ROS are detrimental because they evoke oxidative stress. Recent studies suggest that the site at which ROS are produced is also important in assessing effects on cellular homeostasis. An established mechanism of site-specific ROS signalling is reverse electron transport (RET). RET occurs when electrons from coenzyme Q are transferred back to complex I, generating a significant amount of ROS (57,58). RET has been shown to stimulate lifespan in D. melanogaster (59), and is crucial for the activation of macrophages in response to bacterial infection (60), since blocking mitochondrial ROS arising from complex I abrogated the energy switch from OXPHOS to glycolysis in macrophages (61). In chapter 6, we aimed to elucidate potential effects of SK channel activation on inflammation and whether it affects ROS-RET. SK channel activation reduces ROS, potentially also ROS induced by RET, via inhibiting complex I. Future experiments would further substantiate this hypothesis. We demonstrated a reduction in cellular ROS with CyPPA upon LPS stimulation. In line, activation of mitochondrial BK channels attenuated ROS-RET in cardiac mitochondria (62). The effect of SK channels and RET in inflammation needs more investigation in the future. In addition, it would be interesting to study mitochondrial KCa channels and ROS-RET in other disease

models, such as diabetes type 2, where RET could be a potential target (63), and also in the context of neuronal- and cardiac function.

Novel strategy to improve ROS-targeted therapy in brain cancer

Oxidative stress plays an important role in cancer cell proliferation. Recently, mitohormesis was also identified in cancer cells, where it stimulated a subpopulation of cancer cells to basally upregulate mitochondrial stress responses, providing an adaptive metastatic advantage (64). Whereas mitohormesis is linked to longevity in non-disease conditions, it is thus used by cancer cells to contribute to tumour development. Although cancer cells are equipped with enhanced antioxidant defence capacities to benefit from these higher ROS levels, it has been observed that these cells are sensitive to further increases in ROS levels (65,66). Even though many recent studies focus on targeting ROS (67–70), resistance is still a big problem in the treatment of

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glioblastoma.

Since in healthy brain cells, pharmacological SK channel activation with CyPPA, led to a moderate reduction in mitochondrial respiration and a mild increase in mitochondrial ROS (33 and chapter 5), we hypothesized that current cancer therapies targeting ROS could benefit from this. In chapter 7, we reported that combined treatment of auranofin, inhibiting thioredoxin reductases, and CyPPA, induced mitochondrial damage, ROS production and potentiated auranofin-induced toxicity in neuroblastoma cells, glioblastoma cells, and neurospheres generated from patient-derived glioblastoma cells. On the other hand, inhibition, and not activation, of mitochondrial K+_{channel (mitoKv1.3) was also}

leading to ROS-mediated apoptosis of cancer cells in vivo, via the same downstream events provoked by Bax. Inhibiting the depolarizing K+_{influx causes}

IMM hyperpolarization, thereby promoting increased ROS levels, loss of Ψm,

mPTP activation, and cytochrome c and further ROS release (71,72). In addition, several studies proposed IK channel inhibition as a target for cancer treatment (73,74). More studies are needed to investigate how inhibition of these ion channels differ from the treatment that we proposed in chapter 7.

Furthermore, we found that healthy (HT22) cells were less affected, which is in line with studies showing no toxicity in neurons and glia cells in the brain compared to cancer cells, with mitochondrial ROS being involved in this differential effect (75). Testing whether SK2/3 channel-specific inhibitors will protect against auranofin-induced cell death in cells overexpressing either SK2 or SK3 channels in cancer cells or using SK channel knockout cells might provide us more knowledge on understanding why cancer cells were found to be more vulnerable than HT22 cells, that only express SK2 channels (18).

SK channels mediate metabolic reprogramming Neuroprotection

Since mitochondrial dysfunction is a characteristic of neurodegeneration, we focused on exploring the possibility of metabolic reprogramming in neurons as a potential therapeutic action. As described before, we have found that SK channel activation attenuates mitochondrial respiration (chapter 5). Hence, we studied in addition whether SK channel activation could have concomitant effects on glycolysis that could contribute to the observed neuroprotection.

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Indeed, we found that an initial increase in glycolytic activity is crucial for CyPPA-mediated protection against oxidative stress in HT22 cells (chapter 5). Also in

vivo, CyPPA facilitated increased glycolysis and protected against paraquat and

slightly extended mean lifespan. Though mildly increasing ROS production at the level of mitochondrial complexes has been shown to be an established characteristic of all mitochondrial KCa channels, to our knowledge we are the

first to report that SK channel activation at the same time also influences glycolytic activity. We also showed that lactate plays an important role in neuroprotection, and this mechanism has also been demonstrated in CNS cells. For instance in neurons, the presence of L-lactate as an alternative source to drive ATP production, protected against glutamate-induced neuronal death (76,77). Interestingly, in skin fibroblasts it has been demonstrated that intermittent exposure to L-lactate triggered mitohormesis and prevented aged-related mitochondrial dysfunction and other aging-aged-related mechanisms (78). In alignment with what we observed in chapter 5, it was recently also shown in C.

elegans and in SH-SY5Y neuroblastoma cells that L-lactate mildly promoted ROS,

that triggered antioxidant defense- and pro-survival pathways (79). Similar to our data on SK channel-mediated complex I inhibition and metabolic shifts, more papers discuss the link between inhibition of mitochondrial OXPHOS and glycolysis. Interestingly, metformin has been shown to suppress mitochondrial respiration and promote glycolysis. However, only its effect on mitochondrial respiration, and not on glycolysis, played a role in the protection against hepatotoxicity (80). In addition, loss of mitochondrial function as a result of NO production in dendritic cells commits these cells to sustain enhanced glycolytic activity to meet their energy demands (81). In astrocytes, NO-induced upregulation of glycolysis through activation of energy-sensor AMP-activated protein kinase (AMPK) was shown to be protective (82). In contrast, promotion of longevity in C. elegans due to complex I inhibition occurred independently of AMPK activation (83). Further studies could include the investigation of SK channel effects on NO and AMPK expression and signalling in neurons.

Immune cells

The switch from oxidative phosphorylation to glycolysis is a well-known mechanism involved in activation of immune cells via Toll-like receptors (TLRs), notably TLR4 (84). For example LPS that activates TLR4 strongly increased the

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TCA cycle intermediate succinate (85) that facilitated glycolysis via HIF-1α stabilisation (60). Glutamine-dependent anerplerosis was the major source of succinate accumulation and metabolic alterations (85). In bone marrow-derived macrophages, succinate strongly boosted IL-1β expression in the presence of LPS (60). Although as described in chapter 6 macrophage activation in terms of shape expansion and IL-1β gene expression was not enhanced by succinate in the presence of LPS compared to LPS alone in RAW macrophage cells, we did observe increases in glycolytic activity in these conditions. Though we studied yet RAW macrophages only, future studies should enrol for instance human microglia or other human immune cells.

In agreement with data revealing that SK3 channel activation inhibited inflammatory responses of primary microglial cells after exposure to LPS (86), we have shown here, in addition, that SK2/SK3 channel activation in macrophages reduced the increase in glycolysis facilitated by LPS and succinate (chapter 6). Notably, CyPPA reduces NO release in microglia (86). Since NO is known to upregulate glycolysis (82,87), it would be interesting to study CyPPA effects on NO related to a possible downregulation of glycolysis in inflammatory macrophages. Interestingly, in contrast to SK channels, inhibition, and not activation, of BK channels has been found to suppress LPS-induced microglia activation and contributed to NO production. Here, both plasma membrane- and nuclear BK channels were involved, of which the latter location was evident after long-term LPS treatment (88).

Cancer cells

Similar to immune cells, tumour cells make a switch from OXPHOS to glycolysis, a metabolic adaptation that facilitates tumour growth and cancer progression. Although it was not the focus of the investigation and auranofin is supposed to suppress tumour activity, in chapter 7 we observed slight increases in glycolytic activity upon the treatment with auranofin in neuroblastoma cells. However, we did demonstrate detrimental effects of auranofin on OXPHOS, that was further enhanced by SK channel activation to levels that would induce cell death. In our study, SK channel activation by CyPPA without auranofin had no detrimental effects on cell survival. In contrast, opening of mitochondrial BK channel alone was shown to induce glioma cell death by increasing mitochondrial respiration (89). Thus, pharmacological activation of SK channels in combination with

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auranofin induced brain cancer cell death, via suppressing mitochondrial function and potentiating ROS production.

Main conclusions

In this thesis, the role of small-conductance calcium-activated potassium channels in Ca2+_{homeostasis and oxidative stress in mitochondrial function has}

been studied. More specifically, we looked at the regulatory effects of SK channels on neurodegeneration, inflammation and brain cancer. Collectively, our studies have revealed the following findings:

Mitochondrial KCa channels mediate cellular protection against oxidative

stress through mitochondrial mechanisms of preconditioning, in several disease models including neurodegeneration and ischemia/reperfusion injury (chapter 2).

SK channel activation was neuroprotective against oxytosis in conditions of enhanced ER-mitochondrial coupling, that induced mitochondrial Ca2+_{overload, impaired bioenergetics and cell death (chapter 3).}

Attenuation of mitochondrial calcium uptake using ruthenium red (RR) and mitoxantrone (MX) restored cell viability in ferroptosis and decreased oxidative stress, while metabolism was not improved (chapter 4)

Co-treatment with SK channel activator CyPPA potentiated the protective effect of RR but demonstrated no significant additive effect to the protective action of MX (chapter 4).

Mild ROS induction by SK channel opening plays an important role in neuroprotection against oxidative stress in healthy cells but can promote anti-tumour capacities of thioredoxin reductase inhibitor auranofin in brain cancer cells (chapter 5 and chapter 7).

Neuroprotective effects by CyPPA against ferroptosis involve an initial, fast metabolic shift towards glycolysis (chapter 5)

Lower mitochondrial complex I activity by SK channel activation plays a role in cellular protection via reducing forward electron transfer but possibly also reverse electron transfer (chapter 5 and chapter 6)

Auranofin toxicity was potentiated by SK channel activation due to enhanced oxidative stress and mitochondrial dysfunction (chapter 7)

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