Redox Regulation of Mitochondrial Potassium Channels Activity

Abstract

Redox reactions exert a profound influence on numerous cellular functions with mitochondria playing a central role in orchestrating these processes. This pivotal involvement arises from three primary factors: (1) the synthesis of reactive oxygen species (ROS) by mitochondria, (2) the presence of a substantial array of redox enzymes such as respiratory chain, and (3) the responsiveness of mitochondria to the cellular redox state. Within the inner mitochondrial membrane, a group of potassium channels, including ATP-regulated, large conductance calcium-activated, and voltage-regulated channels, is present. These channels play a crucial role in conditions such as cytoprotection, ischemia/reperfusion injury, and inflammation. Notably, the activity of mitochondrial potassium channels is intricately governed by redox reactions. Furthermore, the regulatory influence extends to other proteins, such as kinases, which undergo redox modifications. This review aims to offer a comprehensive exploration of the modulation of mitochondrial potassium channels through diverse redox reactions with a specific focus on the involvement of ROS.

1. Introduction

A redox (reduction-oxidation) reaction is a chemical process in which the oxidation state of atoms or their groups changes. These reactions play a crucial role in various fundamental cellular functions. Both photosynthesis and cellular respiration involve complex redox reactions. In cellular respiration, redox reactions occur as glucose, fatty acids, and proteins undergo oxidation to form carbon dioxide, while oxygen is concurrently reduced to water [1,2,3,4].

Redox reactions also manifest when reactive oxygen species (ROS) or nitrogen reactive species (RNS) impact molecules within cells. Moreover, redox reactions have the capacity to regulate multiple enzymatic pathways in cells, which is a phenomenon referred to as redox signaling [5,6,7]. This review aims to provide a comprehensive overview of the impact of redox reactions on mitochondrial potassium (mitoK) channels. Additionally, it will describe examples of cellular redox processes wherein mitoK channels play a pivotal role. The localization of these channels within the mitochondria, in conjunction with prevalent redox reactions occurring within these organelles, offers a novel and intriguing perspective on the regulatory mechanisms governing these proteins [5,8].

1.1. Mitochondrial Membrane Permeability

The low permeability of the inner mitochondrial membrane (IMM) to ions, especially to protons, is the basis for maintaining mitochondrial energy homeostasis, transmembrane potential, functioning of the respiratory chain, oxidative phosphorylation (OxPhos) and overall mitochondrial efficiency [9]. The vast majority of substances, with the exception of O₂, CO, CO₂, NO^• and H₂O₂, among others, are unable to freely cross the inner mitochondrial membrane. In order to overcome this limitation related to the tight regulation of their transport, IMM contains a variety of membrane transport proteins that play a key role in mitochondrial functioning [10].

Potassium ions are an example of such limited diffusion, which cross the IMM barrier thanks to the presence of various potassium channels (previously known as potassium uniport) and the K⁺/H⁺ antiporter. This process is called the potassium ions cycle. In this context, the potassium ions cycle enables the coordinated flow of K⁺ between cytosol and the mitochondrial matrix, which is important for maintaining both their functional and structural homeostasis [11]. The phenomenon of potassium cycle plays a crucial role in mitochondrial functions, IMM remodeling and cell energy processes [12].

1.2. Mitochondrial Potassium Channels and Channels’ Pharmacology

In recent years, researchers have demonstrated that the family of mitoK channels is among the most numerous and diverse classes of mitochondrial channel proteins [13,14]. It includes calcium-activated channels such as large conductance (mitoBK_Ca), intermediate conductance (mitoIK_Ca), and small conductance (mitoSK_Ca). Additionally, there are ATP-sensitive potassium (mitoK_ATP) channels, voltage-regulated potassium (mitoKv1.3, mitoKv1.5 and mitoKv7.4) channels, mitochondrial hyperpolarization-activated cyclic nucleotide-gated (mitoHCN) channels, mitochondrial sodium-activated potassium (mitoSlo2) channels and mitochondrial two-pore domain potassium (mitoTASK) channels [14]. The activity of potassium channels undergoes changes under the influence of various external and interorganellar stimuli, such as alterations in pH, concentrations of calcium and sodium ions, temperature, ROS or the differential expression of regulatory proteins associated with potassium channels proteins [13,15,16]. It is noteworthy that potassium channels constitute not only a highly diverse group but are also widely distributed at both the cellular and tissue levels. Analogues of almost all these channels were discovered in the cell membrane and other organelles, including the endoplasmic reticulum (ER) or nucleus of various cell types [17].

Subtle modulation of the activity of mitoK channels affects the activity of the respiratory chain complexes. Moreover, mitoK channels also take part in the readjustment of mitochondrial matrix volume, preventing uncontrolled matrix shrinkage or swelling [18]. It has also been shown that opening of the mitoK channels has a cytoprotective effect [19,20,21]. Similar to the heart, the metabolism of brain tissue relies on the proper functioning of the respiratory chain, making it highly susceptible to hypoxia and damage during reperfusion [22]. The involvement of mitoK channels such as mitoBK_Ca, mitoIK_Ca, mitoK_ATP or mitoSK_Ca channels in cardio- and neuroprotective mechanisms has been demonstrated in many experimental animal models, including rats [23,24], guinea pig [25,26,27], rabbit [28,29], beef heart [30,31] and in isolated atrial trabeculae [32]. The detailed mechanism of this phenomenon will be discussed in the second section of this review. Conversely, the inhibition of mitoK channels not only reduces cell proliferation but also induces cell death [17,33,34]. The well-known mitoBK_Ca channels inhibitor, paxilline, has been demonstrated to eliminate the malignancy-promoting effects in murine and human models of breast cancer [35]. Thus, mitoK channels could serve a dual role: as potential therapeutic targets for ischemia/reperfusion injuries, such as heart attacks and strokes, and in the context of anticancer therapies [36,37]. Hence, ongoing research is focused on elucidating the in vivo mechanisms that regulate the activity of mitoK channels. In order to exert an exogenous influence on mitoK channel activity, numerous research groups have continually explored novel compounds characterized by high specificity for various molecular potassium channels [38]. The existing literature already reports positive protective effects on ischemia/reperfusion (I/R) processes through the activation of mitoK_ATP channels by the potassium channel opener, diazoxide, and the large-conductance Ca²⁺-activated potassium (mitoBK_Ca) channels by NS1619, a benzimidazolone analogue, and its follower NS11021, which is more specific and potent channel opener [39]. Nevertheless, it is noteworthy that these compounds exhibit limited specificity toward mitoK channels [40,41,42]. In contrast, toxins isolated from the venom of various scorpion species such as iberiotoxin or charybdotoxin inhibit the activity of this channel [43,44]. There are substances that modulate most of the detected Ca²⁺-activated mitoK channels, including mitoSK_Ca and mitoIK_Ca channels which are activated by NS309 and DCEBIO and inhibited by TRAM34 [38,45]. Additionally, the activity of mitoSK_Ca channels is inhibited by apamin, a component of bee venom, and the activity of mitoIK_Ca channels is inhibited by clotrimazole. The mitoK_ATP channel is activated also by isoflurane, nicorandil, and (3R,4S)-4-[(4-Chlorophenyl) (1H-imidazol-2-ylmethyl) amino]-3,4-dihydro-3-hydroxy-2,2-dimethyl-2H-1-benzopyran-6-carbonitrile monohydrochloride (BMS-191095); and it is blocked by 5-hydroxydecanoic acid (5-HD) and glibenclamide [38].

1.3. From Redox Homeostasis to Cellular Redox Stress

Reduction–oxidation (redox) reactions play a pivotal role in numerous biochemical pathways with oxygen serving as the archetypal oxidant in these intricate processes. This phenomenon is prominently exemplified in the functionality of mitochondria. It is noteworthy that redox reactions extend beyond oxygen, encompassing other essential elements such as nitrogen and sulfur [46]. The delicate balance between reducing and oxidative species gives rise to what is termed redox homeostasis, and a lack of it leads to redox stress. This imbalance is a critical aspect of cellular processes [47]. Living organisms employ a plethora of reactions to sustain their physiological functions, many of which involve the intricate interplay of oxidation and a reduction in substrates. Tretter et al. provide comprehensive insights into these processes in their publication [48]. The significance of reactive species in cellular processes regulation is well established. O-Uchi et al. present compelling evidence that the controlled production of low to moderate levels of reactive species is pivotal for the proper regulation of basic cellular processes [5]. These reactive species act as messengers, facilitating signaling mechanisms without causing undue tissue damage [48,49]. To avoid an excessive synthesis of reactive species and oxidative stress, cells have evolved cellular antioxidant enzymes and small molecules that undergo reversible oxidation. When the amount of reactive species exceeds the limit of adaptation of a cell or its compartment to the changed redox homeostasis, tissue damage occurs, which is usually in a non-specific manner. Very delicate cellular redox systems, which vary in subcellular compartments in terms of expected and safe concentrations and depending on the physiological or pathological state of cells and organelles, have emerged to cause more difficulties than previously expected in the therapeutic targeting of oxidative stress in disease states. Antioxidant enzymes catalyze the degradation of harmful oxidative species into non-reactive species, but it is also important to note that one reactive species is often converted to another with different reaction specificity (Figure 1).

Mitochondrial ion channels and transporters are considered as both sensors and regulators of cellular redox signaling [5]. Potassium channels are not the exception; they are modified as other cellular proteins (Figure 2). These modifications could be signaling or pathological if the stimulus overcomes cellular hormesis. That possibility is particularly seen in an ischemia/reperfusion injury.

2. Mitochondrial Potassium Channels in Ischemia/Reperfusion Injury

Ischemic heart and brain diseases are leading causes of death globally. Ischemia manifests as a restriction of blood flow to any tissue, resulting in a deficiency of oxygen necessary for cellular metabolism as well as a reduced availability of nutrients and an inadequate removal of metabolic waste products [50]. In the context of ischemic tissue damage, a more detrimental factor is the abrupt restoration of circulation and, consequently, oxygen. This leads to numerous ischemia/reperfusion (I/R) injuries. The I/R process affects various cellular components, particularly the organelles, such as mitochondria. Mitochondria play a pivotal role in initiating a cascade of events that activate intracellular processes, ultimately leading up to cellular apoptosis or necrosis [51]. Due to ischemia, OxPhos is stopped and, consequently, mitochondrial ATP synthesis is also stopped. A lack of oxygen causes an accumulation of hypoxia-inducible factor-α (HIF-1α) and causes the cell to switch to anaerobic metabolism, which is less efficient in ATP synthesis than in OxPhos [52]. Due to glycolytic metabolism, lactate is generated, leading to the acidification of the cytosol and cellular environment. Furthermore, the electron transport chain ceases to pump protons out of the matrix, resulting in a decrease in both mitochondrial membrane potential (ΔΨm) and ΔpH. The reduction in ATP concentration carries several implications. Primarily, it disrupts ion homeostasis in both mitochondria and the entire cell, as numerous systems responsible for ion homeostasis fail to function properly. Inhibiting Na⁺/K⁺-ATPase results in an elevated extracellular concentration of K⁺ and the accumulation of Na⁺ intracellularly. This elevation of Na⁺ within the cell reduces the activity of the Na⁺/Ca²⁺ exchanger. Consequently, due to the diminished electrochemical gradient, transporting Ca²⁺ outside the cell leads to Ca²⁺ overload in the cytosol and an influx of Ca²⁺ into the mitochondrial matrix [53,54]. Concerning ROS, they may be generated partially even during the ischemic period, as there is a residual amount of oxygen that remains unreduced by cytochrome c oxidase (COX). However, a significant burst of ROS occurs primarily during reperfusion, originating mainly from complex I and III. This is attributed to non-specific electron leakage and reverse electron transport (RET), respectively. The overload of mitochondria with ROS and Ca²⁺ contributes to the opening of the mitochondrial permeability transition pore (mPTP), resulting in the loss of ΔΨm [55,56,57,58]. These events are followed by apoptosis-inducing factors and cytochrome c release from mitochondria triggering cell death. Studies have demonstrated that the activation of mitoK channels exerts a cytoprotective effect, offering a potential avenue for preventing cell death. In the context of I/R, mitoK channels emerge as pivotal players and, consequently, potential therapeutic targets. The activation of mitoK channels proves instrumental in averting severe cell damage and altering cellular fate through various mechanisms. Primarily, the influx of K⁺ ions facilitated by mitoK channels induces a mild uncoupling of mitochondria. This restoration of electron flow through the respiratory chain serves to inhibit the massive formation of ROS, particularly by complex I [59,60,61]. Secondly, a moderate depolarization of IMM slows Ca²⁺ ion accumulation and prevents mPTP opening [62] averting membrane disruption and cell death signaling [18,63,64,65,66,67,68]. MitoK channels are able to react to negative phenomena occurring at several stages of ischemia/reperfusion injury (I/R injury). Since decreased ATP levels is an early symptom of an ischemic insult, the first activated mitoK channel is the mitoK_ATP channel, which is inhibited during normoxia. Next, the dissipation of ΔΨm could lead to the opening of the voltage-sensitive mitoKv7.4 channel [69]. Moreover, an increase in Ca²⁺ concentration leads to the activation of channels such as mitoSK_Ca, mitoIK_Ca and mitoBK_Ca. However, an increasing of Na⁺ concentration impacts the activity of the mitoSlo2 channel. Nevertheless, during the reperfusion phase, the concomitant presence of ROS may influence the activation of both mitoK_ATP and mitoBK_Ca channels [54,69,70].

3. Direct and Indirect Redox Regulation of Mitochondrial Potassium Channels

The mitoK channels, situated in the IMM, are exposed to reactive species generated in the mitochondrial environment. Additionally, there are reports suggesting that the activity of mitoK channels can also be subject to modification through the enzymatic action of protein kinases (see Section 3.2). As a result, the redox-induced alteration of protein kinases mediated by ROS, NO^• or H₂S can significantly impact their enzymatic activity, consequently modulating the functionality of mitoK channels.

3.1. Potassium Channels Regulation via Gasotransmitters: Nitric Oxide and Hydrogen Sulfide

The concept of “gasotransmitters” was introduced for the regulation of cellular function over two decades ago [71]. Since then, there has been substantial evidence demonstrating the diverse signaling effects of low concentrations of nitric oxide (NO^•) and hydrogen sulfide (H₂S) [72]. These effects are evident in alterations of cellular respiration, modifications in ATP synthesis, and an enhancement of cellular cytoprotection. The observed positive outcomes associated with “gasotransmitters” align with the benefits derived from the activation of numerous mitoK channels. Various research centers have concentrated their efforts on elucidating the potential connections between mitoK channels and the subtle doses of NO^• or H₂S as “gasotransmitters” [73]. It has been revealed that NO^• and H₂S possess the capability to directly and indirectly modulate the activity of mitoK channels (

Table 1. Effect of redox species on the activity of mitoK channels.

Channel	Effect	Reactive Species	Reference
mitoKATP	activation	H2S	[70]
		S-nitrosothiols	[75]
		nitroxyl	[76]
		nitrolinoleate	[76]
		NO	[75,76,77]
		diamide	[70,78]
		phenylarsine oxide	[70,78]
		O2•− *	[79]
		H2O2 **	[75,76]
	inhibition	NADPH	[76]
mitoBKCa	activation of the hemin-inhibited channels	H2S	[80]

* contrary results—Costa and Garlid (2008) [75] claim that superoxide anion was found not to open mitoK_ATP, and superoxide-dependent opening of this channel was shown to be due to superoxide dismutation to H₂O₂; ** contrary results—Chiandussi et al. (2002) [78] showed that H₂O₂ inhibited mitoK_ATP-dependent swelling.

) [74]. The review above elucidated the impact of gaseous signaling molecules on the activity of mitoK channels.

In indirect regulation, NO^• stimulates soluble guanylate cyclase (sGC), leading to the synthesis of cyclic guanosine monophosphate (cGMP). Subsequently, cGMP activates protein kinase G (PKG). PKG, in turn, demonstrates the capability to phosphorylate the mitoBK_Ca channels, thereby enhancing the probability of its opening. Compounds that elevate cGMP levels, along with ischemic preconditioning, confer cardiac protection against ischemia and reperfusion injury by modulating the activity of cardiomyocyte-specific BK_Ca channels [81].

It has additionally been postulated that a thiol/disulfide switch mechanism exists through which BK channel activity can promptly and reversibly respond to alterations in the redox state of the cell, particularly during transitions between hypoxic and normoxic conditions [82]. Second of the gasotransmitters involved in redox reactions is H₂S, which is also endogenously produced in cells. The hydrogen sulfide influences cell bioenergetics and mitochondrial function. The effects of H₂S depend on the dose used, with lower concentrations revealing beneficial effects, while higher concentrations manifest cytotoxicity [83,84]. In order to properly fulfill their task at physiological conditions, adequately react during stress conditions and finally adapt to a constantly changing environment, proteins including ion channels continually undergo modifications, which alter their properties and functions. Both mitoBK_Ca and mitoK_ATP may be upregulated due to S-sulfhydration by H₂S [85,86,87].

The S-glutathionylation is an oxidative and reversible modification that involves the attachment of glutathione to the cysteine residue of the protein of interest [88]. This process is abundant in mitochondria. For S-glutathionylation within mitochondria, glutathione-dependent oxidoreductase 2 (GRX2) and glutathione-S-transferases (GST Alpha, Kappa, Mu, Pi and Zeta) are responsible [88,89]. However, non-enzymatic S-glutathionylation has been observed also, especially under oxidative stress, such as I/R injury. Under hypoxia, S-glutathionylation may be upregulated, leading to a hyper-glutathionylation of proteins, which activates oxidative stress signaling. On the other hand, the S-glutathionylation of mitochondrial proteins under normoxic conditions is reversible, dependent upon redox fluctuations, and an enzymatically driven modification [90]. It has been shown that enzymes of the tricarboxylic acid cycle, SLC proteins (UCP2, UCP3, ANT), all complexes of OxPhos and many other proteins within the inner mitochondrial membrane are S-glutathionylated in parallel. The S-glutathionylation of K_ATP has been reported. As was demonstrated by Yang and colleagues, the S-glutathionylation of a cysteine residue of the Kir6.1 channels subunit leads to K_ATP channels activity inhibition [91,92,93].

Given the structural similarities between plasmalemmal and mitochondrial potassium channels, the potential regulation of the mitoK_ATP channels [94] through S-glutathionylation appears highly plausible. Unfortunately, as of the present day, there is an absence of direct evidence to substantiate the occurrence of S-glutathionylation on mitoK_ATP channels.

Another group of compounds that post-translationally modified proteins in cells comprises RNS, which originate from NO^•, including the nitroxide anion (NO⁻), nitrosonium cation (NO⁺), peroxynitrite (ONOO⁻), S-nitrosothiols, nitrogen dioxide (NO₂), and higher nitrogen oxides. It is believed that RNS may exert a regulatory influence on physiological processes and could also contribute to the development of pathological disorders [95]. It has been demonstrated that one of the nitrox forms of RNS, namely nitroxyl (HNO), which is reduced by one electron and serves as the protonated form of NO^•, exhibits vasoprotective and vasodilating properties. The authors propose that the vasorelaxation and vasoprotective attributed to HNO may arise from its direct modulation of sarcolemmal K_ATP and K_V channels or through the activation of sGC and cGMP [96,97,98,99,100]. It has been elucidated that not only are potassium channels located in the plasma membrane activated, but also mitoK_ATP channels within the inner mitochondrial membrane undergo modulation by NADPH, NO^•, superoxide (O₂^•−), HNO, nitrolinoleate (LA-NO₂), and S-nitrosothiols. Some of these compounds activate mitoK_ATP channels directly, such as NADPH, or indirectly through intermediaries like LA-NO₂ and HNO, which are thought to mediate their effects via complex II. On the other hand, nitrolinoleate has also been shown to inhibit complex II, although an effective concentration of LA-NO₂ was significantly higher than that needed for mitoK_ATP channels’ upregulation. It is noteworthy that during ischemic preconditioning, the concentration of LA-NO₂ may be close to 1 μM, indicating that nitrated fatty acids, including LA-NO₂, can influence cell survival via mitoK_ATP channels. The molecular mechanism remains elusive, but it is presumed that thiol groups within cysteines are the primary targets for electrophilic LA-NO₂ [76,79,101]. It has been revealed that S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO^• donor, induces an increase in mitoK_ATP channels activity at low concentrations. However, this activation is inhibited by mitoK_ATP channels inhibitors such as 5-HD and glibenclamide. Conversely, high SNAP concentrations, and thus elevated NO^• levels, result in a decrease in mitoK_ATP channels activity. It is probable that heightened NO^• levels will inhibit cytochrome c oxidase, subsequently causing a reduction in the rate of electron flow through the respiratory chain [76,102].

Another oxidative protein modification is sulfhydration. As briefly mentioned earlier, sulfhydration upregulates the mitoK_ATP channels activity by converting the -SH group to -SSH. Additionally, this sulfhydration causes allosteric shift of the sulfonylurea receptor SUR2B subunit that enhances upregulation of potassium channel activity by its opener – levcromakalim.. Interestingly, nitration of the tyrosine residue in the Kir6.1 channel subunit was reduced as a result of sulfhydration of the SUR2B subunit. It is highly probable that C24S and C1455S within the SUR2B subunit undergo sulfhydration, as mutations in these sites prevented the cessation of nitration. This intriguing observation suggests that these protein modifications mutually influence each other within a single protein [85,103,104,105].

Mitochondria are also the main hub of reactive sulfur species (RSS), since they are places of oxidation of H₂S. The RSS required for post-translational modification mediated by protein persulfidation are generated from mitochondria through H₂S oxidation [106,107]. As a result of oxidation of methionine residue, the BK_Ca channels have been upregulated [108]. An enzyme, peptide methionine sulfoxide reductase (MsrA), which reduces methionine sulfoxides, partially abolished this effect. On the other hand, cysteine oxidation has been shown to inhibit the channel [109].

The activity of BK_Ca channels has been reported to be augmented by oxidants such as NAD⁺ and glutathione disulfide. Exemplify, in pulmonary arterial smooth muscle cells, 2 mM NAD⁺ and 5 mM glutathione disulfide (GSSG) have induced BK_Ca channels opening. On the contrary, reduced congeners—NADH and glutathione (GSH)—inhibited the channel [43,110]. Also, the sulfenylation and sulfinylation of BK_Ca channels have been reported. Transformation from -SOH to sulfinic (–SO₂H) and sulfonic (–SO₃H) acid have been shown to close the channel, considering modification to sulfonic (–SO₃H) acid irreversibly. Nevertheless, sulfenylation can be abolished via a reducing agent able to reinstate the thiol group (-SH) in the side chain of cysteine(s), such as GSH [111,112] or dithiothreitol (DTT). The activity of BK_Ca channels has been shown to be downregulated also by other compounds, for example NEM, DTNB and MTSEA, whose target is the thiol group [112,113,114,115,116,117]. These data suggest that sulfhydryl groups play a key role in regulating BK_Ca channels. Moreover, inhibition caused by NO^•-derived peroxynitrite in the cerebrovascular and coronary smooth muscle has been reported [118,119,120]. Finally, the redox sensitivity of BK_Ca channels was shown in mitoplasts obtained from mitochondria of the human endothelial cell line, EA.hy926. It has been demonstrated that a flavonoid, luteoline (LUT), has not altered mitoBK_Ca channels’ activity. However, in reducing conditions obtained by DTT, which is commonly known to break down disulfide bonds, LUT has activated mitoBK_Ca channels. This effect was hampered by the mitoBK_Ca channels inhibitor, paxilline, which is unexceptionable proof for mitoBK_Ca channels’ engagement. To our knowledge, this is the only scientific report confirming direct redox regulation of the mitoBK_Ca channel. Nevertheless, it could be assumed with a great dose of certainty that the mechanisms of redox regulation mentioned above also apply to the mitoBK_Ca channel and other congeners of BK_Ca channels [23,121].

3.2. Redox Reactions, Protein Kinases and Mitochondrial Potassium Channels Activity

The network of dependencies between the redox state of the cell, protein kinases activity and mitoK channels is very complex (Figure 3). Modifications of the structure and gating properties of the ion channels by their reversible phosphorylation is one of the most important mechanisms controlling their activity [70]. Some endogenous substances, like adrenomedullin, can confer cardioprotection via PKA- or PKC-mediated activation of mitoK_Ca or mitoK_ATP channels, respectively [5,122]. Since PKA and PKC enzymes are redox-sensitive kinases, the activities of both above-mentioned channels can be regulated by redox signaling [122]. For, instance some isoforms of PKC can be activated by H₂O₂ [123] and NO^• [77], which causes the higher activity of the mitoK_ATP channels. Interestingly, among different models that explain how diazoxide activates the K_ATP channel, there is also a theory that indirect activation occurs by an induction of PKC-ξ translocation [124]. PKC-ξ1 (mitochondrial PKC-ξ) is closely associated with mitoK_ATP channels [75]. Activation of this kinase causes opening of the mitoK_ATP channels. Moreover, the activator of PKC was shown to potentiate and accelerate the effect of diazoxide [16]. Potassium flux causes an increase in ROS production by complex I of the electron transport chain (ETC). These ROS activate both PKC-ξ1 (causing a positive feedback loop) and a second pool of PKC-ξ (PKCξ-2), which inhibits the mPTP. On the other hand, mitoK_ATP channels activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of ROS [125]. Interestingly, opening of the mitoK_ATP channels induced by PKG is not mediated by ROS [75]. Interestingly, a recent study claimed that limiting I/R injury by inhibiting the mPTP opening via PKCε activation is Independent of the mitoK_ATP channel [126], and some researchers doubt if mitoK_ATP channels exist at all [126,127].

4. Mitochondrial Potassium Channels and Respiratory Chain

The mitochondrial respiratory chain comprises a series of complex organized redox reactions, both functionally and structurally, culminating in the generation of a protonmotive force and, consequently, ATP synthesis. Certain redox centers, such as complexes I and III, can act as sources of ROS due to the release of leaky electrons, leading to the reduction in molecular oxygen. As discussed earlier, mitochondrial-generated ROS can influence the activity of mitochondrial potassium channels. There are indications proposing an alternative mechanism for the regulation of mitoK channels by the respiratory chain. Specifically, this involves the regulation of mitochondrial channels through potential interactions with respiratory chain proteins.

It is well known that mitochondrial potassium channels interact with various mitochondrial proteins [128,129,130], some of which are involved in the respiratory chain. For instance, it has been suggested that mitoK_ATP channels interact with succinate dehydrogenase [131,132]. In cardiac mitochondria, it was found that the β1 subunit of the mitoBK_Ca channels interacts with COX subunit I [133]. Furthermore, studies have demonstrated that other respiratory chain protein complexes interact with mitoBK_Ca channels in both cardiac [134] and brain mitochondria [135]. Additionally, mitochondrial tandem pore domain K⁺ channel TASK-3 interacts also with the respiratory chain [136]. A recent report revealed a similar interaction between the mitoKv1.3 channel and respiratory chain complex I [137]. The exact nature and functional implications of these interactions remain unclear. Is it possible that proteins comprising the respiratory chain directly interact with channel proteins, thereby regulating potassium channels activity through an unknown redox mechanism? This kind of direct functional coupling between the energy-generating system (respiratory chain) with the energy dissipation system (potassium channels) may lead to an interesting putative regulatory mechanism in mitochondria.

We found that the activity of mitoBK_Ca channels in glioblastoma U-87 MG cells is regulated by substrates and inhibitors of the respiratory chain [41]. This study suggested that cytochrome c oxidase (COX) is a key element of this kind of channel regulation [41]. Moreover, given that cytochrome c oxidase is the primary infrared-absorbing protein, it raises questions about the potential light regulation of mitoK channels [138]. Further research is imperative to clarify the functional consequences of these interactions. Undoubtedly, this form of regulation may prove to be distinctive and unique for mitoK channels.

5. Summary

Mitochondria contain the mitochondrial respiratory chain in the IMM and thus create an environment that serves as the primary source of ROS in the cell, particularly during I/R processes. Potassium channels situated in the IMM, like other proteins, undergo modification by ROS and RNS, significantly altering their activity. Studies suggest that mitoK channels not only passively encounter ROS but likely participate in numerous cellular processes. MitoK channels play a pivotal role by modulating the ΔΨm, which affects ROS synthesis, thus serving as both targets and regulators of redox reactions. Moreover, it appears that the mitoK and ROS interdependence extends; another potential site of mitoK interaction is its direct regulation of activity by respiratory chain complexes.

Interestingly, ROS play an important role in aging and lifespan [139]. Recently, it was demonstrated that BK_Ca channels are present in Drosophila melanogaster mitochondria, and channel mutants induce structural and functional defects in mitochondria, leading to an increase in ROS [140]. It also was found that the absence of BK_Ca channels reduced the lifespan of Drosophila, and the overexpression of human BK_Ca channels in flies extends their life. This suggested a potential role of mitochondrial potassium channels and ROS in regulating mitochondrial functional integrity and lifespan [140].

Taking into account changes in the activity of mitoK channels caused by redox reactions involving protein kinases, the regulation scheme becomes even more complex. It can be assumed that such a mechanism of regulating the activity of mitoK channels is related to subtle spatio-temporal regulation, the violation of which may lead to cell death. Furthermore, the pharmacological manipulation of mitoK channels activity through the use of inhibitors and potassium channel openers as influenced by redox reactions adds an extra layer of complexity [38].

Recent evidence strongly suggests that mitochondrial potassium channels play a significant role in inflammatory processes [141,142]. ROS synthesis in mitochondria takes place due to regulation by these proteins. It is known that mitochondria activate the inflammasome complex by releasing damage-associated molecular patterns (DAMPs) such as ROS, leading to the maturation of inflammatory molecules [141]. Additionally, inflammatory processes may be induced by an influx of potassium cations via mitoK and effects on mitochondrial structure/dynamics or effects on calcium ions overloading (followed by activation of the mitochondrial mega-channel, leading to the disruption of inner mitochondrial membrane and mitochondrial DNA release into the cytosol) [142].

Although the multidimensional nature of redox channel regulation presents considerable experimental challenges, it represents a crucial avenue for future research. Deciphering the complexities of redox processes in the context of mitoK channels holds promise for understanding their roles in physiological phenomena such as I/R injury, aging, inflammation, and cancer, where mitochondria play a substantial and multifaceted role.