mitoBK single-channel recordings were reported for the first time in mitoplasts from glioma human cell line LN229 mitochondria (Siemen et al., 1999). Since then, patch-clamp experiments from mitoBK have been carried out not only using mitoblasts (Xu et al., 2002; Ohya et al., 2005; Gu et al., 2007; Cheng et al., 2008, 2011; Bednarczyk et al., 2013a,b; Soltysinska et al., 2014; Frankenreiter et al., 2017; Walewska et al., 2018; Balderas et al., 2019) but also lipid bilayers (Skalska et al., 2009; Fahanik-Babaei et al., 2011a,b). Overall, it has been found that mitoBK shares similar behavior to pmBK (see Table 1), with a unitary conductance (γ) around 282±23 pS in multiple K⁺ conditions, a voltage-dependent open probability (P_o) (Siemen et al., 1999; Bednarczyk et al., 2013b) which shows a leftward shift in the P_o – Voltage curves when increasing Ca²⁺ levels (Xu et al., 2002; Balderas et al., 2019) and sensitivity to negative hydrostatic pressure (Walewska et al., 2018). Interestingly, mitoBK P_o increases under hypoxic conditions (Gu et al., 2007; Cheng et al., 2008). BK localization in mitochondria is a result of VEDEC splice variant from KCNMA1 gene, which has been described with the aforementioned properties (Singh et al., 2013; Gałecka et al., 2021).

The first identified calcium- and voltage-activated potassium channel (nBK) in nucleus was characterized in rat pancreatic acinar cells at the single-channel level using the patch-clamp technique (Maruyama et al., 1995). Although there was no pharmacology approaches or microscope imaging, this study was pioneer in the search of BK channels in other intracellular organelles membranes. It has been found that nBK shares similar behavior to pmBK (Singh et al., 2012).

Almost 20years later, functional nBK channels were described using a set of different techniques, including immunoelectron microscopy and confocal fluorescence. Most importantly, single-channel recordings in isolated nuclei showed a P_o of 0.3 using 5μM Ca²⁺, increasing to ~0.8 at 10 uM Ca²⁺, indicating the presence of a Ca²⁺−activated channel with a γ=217 pS (Table 1; Li et al., 2014).

Likewise, as in pmBK, the accessory β1 subunit can assemble with the α subunit of mitoBK and nBK (Ohya et al., 2005; Li et al., 2014; Balderas et al., 2020). This subunit modify the pharmacological characteristics and gating of the channel. Recent findings have revealed the presence of mitoBK channels formed by the β1/α complex in mammalian myocyte mitochondria (Ohya et al., 2005; Testai et al., 2017; Balderas et al., 2020). β1 regulates expression and targets mitoBK to the IMM and changes the channel voltage sensitivity (Balderas et al., 2020). These results could explain how is possible to activate mitoBK in the mitochondrial environment (ΔΨ~−200mV, [Ca²⁺]_mit≈200μM). Under these conditions, the β1/α mitoBK conductance–voltage curve is leftward shifted and the channel shows an appreciable P_o (Ohya et al., 2005; Bautista et al., 2009; Balderas et al., 2020). The accessory β1 subunit detected in mitoplast from rat ventricular myocytes interacts with the cytochrome C oxidase (CCO), confirming the mitoBK-β1 complex association with the respiratory electron transport chain in heart mitochondria (Ohya et al., 2005). In addition, the mitoBK-β1 complex was also found in cultured pulmonary artery smooth muscle mitochondria (Loot et al., 2012).

mitoBK was characterized in other human cell lines from the endothelium, fibroblast, and glioblastoma where multiple splice variants were found that co-assemble with different types of auxiliary β subunits that may affect the complexity of the mitoBK channel pharmacology and gating (Wawrzkiewicz-Jałowiecka et al., 2021).

At present, only β4 expression has been reported in nuclear membranes co-localizing with B-type lamin (Li et al., 2014.). However, there are many questions that still need to be answered regarding the detailed mechanism of how accessory subunits are directed and assembled in IMM and ONM, as well as the modulatory effect they exert on mitoBK and nBK. Regarding γ subunits, the association with the mitoBK-α and nBK-α is still to be addressed (González-Cota et al., 2021).

The basic pharmacology properties of mitochondrial potassium channels like mitoK_ATP, mitoBK, and mitoKv1.3 are similar to their equivalents in plasma membrane from different cell types (Szewczyk et al., 2006; Laskowski et al., 2016). Therefore, activators and inhibitors previously described for the pmBK channel can exert the same effect on mitoBK (O’Rourke, 2007; Szewczyk et al., 2010). Different reports indicate that nonspecific interactions of potassium channel modulators may occur, indicating that these compounds may influence cell and mitochondrial function regardless of their main targets (Szewczyk et al., 2010; Laskowski et al., 2016; Augustynek et al., 2018).

CGS7181(ethyl2-hydroxy-1-[[(4-methylphenyl)amino]oxo]-6-trifluoromethyl-1H-indole carboxylate) is an indole carboxylate derivative that, just as its analog CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate), activates mitoBK in single-channel recordings from astrocytoma (Augustynek et al., 2018). Using the inside-out patch-clamp configuration, they report that the open probability (NPo) increases from 0.09 in the control to 0.55 in the presence of 1μM of CGS7181. This activity was subsequently inhibited by adding 10 μM of paxilline to the bath (mitochondrial matrix; Augustynek et al., 2018). Augustynek et al. (2018) proposed that activation of mitoBK by CGS7184 induces an influx of potassium ions into the negatively charged mitochondrial matrix and promotes a light uncoupling of mitochondria. This uncoupling stimulates the activity of the mitochondrial respiratory chain to restore the potential of the mitochondrial membrane by pumping protons from the matrix into the mitochondrial intermembrane space (Augustynek et al., 2018). The agonistic effect observed in the presence of CGS7184 is dependent on potassium and charybdotoxin, indicating that the target of this compound is mitoBK (Augustynek et al., 2018).

Sakamoto demonstrated that 12,14-dichloro dehydroabietic acid (diCl-DHAA) activates mitoBK (Sakamoto et al., 2008) similarly as it activates the pmBK channel (Ohya et al., 2005). Additionally, adding 3 μM of paxilline eliminates channel opening events, allowing the authors to confirm that diCl-DHAA activates the mitoBK channel, likewise the pmBK channel (Sakamoto et al., 2008). Finally, they evaluated the protective effects of diCl-DHAA against ischemic cell death in cardiomyocytes by using the simulated ischemia procedure. diCl-DHAA has protective effects on cardiac myocytes against ischemic injury through the opening of mitoBK channels, supporting the idea that the opening of mitoBK is a novel way to protect cardiac myocytes from ischemic and reperfusion injury (Sakamoto et al., 2008).

Ohya et al. (2005) showed that 17β-estradiol could increase the mitoBK channel P_o, activation that is inhibited by paxilline (Ohya et al., 2005). Importantly, in the presence of 17β-estradiol, cell death decreased significantly during simulated ischemia, and that this cardioprotective effect was eliminated by 3μM paxilline (Ohya et al., 2005). They concluded that this cardioprotective effect is due to the activation of mitoBK by 17β-estradiol, and since 17β-estradiol activates BK only in the presence of the β1 subunit (Valverde et al., 1999; Granados et al., 2019), this result confirms the presence of mitoBK-β1 in rat ventricular myocytes.

The benzimidazole derivatives BK activator family includes NS1619, NS004, NS1604, NS11021, and NS1643 that can activate mitoBK (Skalska et al., 2009; Szewczyk et al., 2010). NS1619 activates mitoBK at micromolar concentrations (Szewczyk et al., 2006). Moreover, the activation of mitoBK by NS1619 has a cytoprotective effect in guinea pig heart before simulated ischemia; this effect was antagonized by paxilline (Xu et al., 2002; Stowe et al., 2006; Singh et al., 2013). MitoBK activators have been reported to protect the heart against ischemic injury (Shintani et al., 2004). Furthermore, like the effect of mitoK_ATP activation, mitoBK opening has been implicated in preconditioning. For example, preconditioning of hearts with mitoBK activators such as NS1619 or NS11021 reduced myocardial infarction and this beneficial effect could be antagonized by co-administration with paxilline (Bentzen et al., 2009). The activation of mitoBK by NS1619 leads to cytoprotection of cardiomyocytes during ischemia/reperfusion or treatment with ouabain (Augustynek et al., 2018). However, it should considered that NS1619, like NS004, may present non-mitoBK-dependent effects in the mitochondria (Debska et al., 2003; Heinen et al., 2007b).

NS11021 exerts other protective effects by activating mitoBK channels, which are abolished in the presence of paxilline. For example, nanomolar concentrations of NS11021 improve the bioenergetic performance of the mitochondria of the heart (Aon et al., 2010; Testai et al., 2014). NS11021 also protects against ischemic injury when applied prior to ischemia or when applied immediately after reperfusion. These findings support the idea that ischemia and reperfusion-induced tissue damage can be reduced by pharmacological activation of cardiac mitoBK channels (Bentzen et al., 2009).

The following compounds are mitoBK inhibitors: charybdotoxin (Gu et al., 2007; Skalska et al., 2009; Augustynek et al., 2018), iberiotoxin (Cheng et al., 2011), and paxilline (Xu et al., 2002; Ohya et al., 2005; Sakamoto et al., 2008; Augustynek et al., 2018; Balderas et al., 2020) y Ba²⁺ (Xu et al., 2002). These compounds have been characterized previously in the pmBK having similar effects to those found in mitoBK (Szabo and Zoratti, 2014). MitoBK is inhibited by the blockers charybdotoxin, iberiotoxin, and paxilline at concentrations in the nanomolar range (O’Rourke, 2007; Singh et al., 2012). Adding 100nM of paxilline to the bath in the inside-out configuration decreases the P_o of the mitoBK and increases the mean close time with no effects on the mean open time. These results suggest that paxilline decreases the probability of opening by stabilizing the closed state of the channel (Balderas et al., 2020). We note here that the Lingle group who proposed that paxilline binding is state-dependent binding preferentially to the closed state of the pmBK (Zhou and Lingle, 2014).

Recently, Kravenska et al. (2020) reported in human astrocytoma cell mitoplasts that different forms of Aβ (a self-aggregating peptide) produced by cleavage of a transmembrane glycoprotein (the amyloid precursor protein involved in Alzheimer’s disease), including monomers, oligomers, and fibrils, inhibit mitoBK in a concentration-dependent manner. Five μM of Aβ fibrils, oligomers or monomers produced 80, 70, and 50% inhibition, respectively. All forms of Aβ inhibited mitoBK channel activity when applied to both sides of the membrane, indicating an indirect effect on the channel (Kravenska et al., 2020).

Paxilline- and iberiotoxin-specific pmBK channel inhibitors block nBK. nBK is activated by NS1619, a specific activator of pmBK. Therefore, nBK channels share similar pharmacological properties with the pmBK and mitoBK channels, targeting the same compounds (Gobeil et al., 2002; Singh et al., 2012; Li et al., 2014; Du et al., 2020).

Experiments in isolated nuclei of brain endothelial cells using NS1619 as an activator of nBK and iberiotoxin as a blocker showed that nBK is coupled to the activity of perinuclear prostaglandin receptors (EP3). Iberiotoxin abolished K⁺-dependent membrane potential changes and the expression of eNOS transcription induced by the activation of agonists of the prostanoid EP₃-receptor, M&B 28767, while NS1619 produced Ca²⁺ transients and alterations in the perinuclear membrane potential (Gobeil et al., 2002; Singh et al., 2012).

Li et al. (2014) showed that nBKs in the nuclear envelope of hippocampal cells are functional and sensitive to pharmacological inhibition by paxilline. This compound’s blockage of nBK causes transient increases in Ca²⁺ and depolarization of the nucleoplasm relative to the perinuclear lumen, thus affecting the transcription of calcium-dependent genes, neuronal activity, and dendritic arborization in these neurons (Li et al., 2014).

On the other hand, treatment with paxilline, both in isolated RAW264.7 macrophage nuclei and whole cells, resulted in a dose-dependent increase in the phosphorylation of CREB in the nucleus (Selezneva et al., 2021). We recall here that treatment of the nucleus with high concentrations of Ca²⁺ also causes CREB phosphorylation. These results do not exclude a role for the BK channels located in other cell membranes, due to the high membrane permeability of paxilline, which would allow it to block the BK channels of both the plasma membrane and intracellular organelles (Selezneva et al., 2021).

The functions of the mitoBK channel can be easily studied using isolated mitochondria. However, we cannot apply these studies’ results directly to intact cells (Li and Gao, 2016). It has been hypothesized that the activity of this channel is essential for mitochondrial function and homeostasis. mitoBK is expressed in IMM, in which they could regulate ion and protein movement involved cell apoptosis and the electron transport chain (ETC), respectively (Szabo and Zoratti, 2014; Li and Gao, 2016). Most studies have mainly focused on the cytoprotective effect on cardiac and neuro ischemia of mitoBK channels. Still, they have also shown significant evidence regarding mitochondrial structure and function, reactive oxygen species (ROS) regulation, mitochondrial Ca²⁺ retention capacity, and permeability transition pore (mPTP) activation in cellular respiration and cancer as well (see Figure 1A).

So far, the physiological role of mitoBK has been reported mainly by pharmacology or using genetic models (Szabo and Zoratti, 2014). Most studies have primarily focused on the cytoprotective effect on cardiac ischemia and reperfusion (I/R) injuries, to which mitoBK has been associated after the pioneering work of Xu et al. (2002). Using pharmacological agents to open and block the channel, mitoBK shows to be involved in such cardioprotection (Wang et al., 2004; Stowe et al., 2006; Bentzen et al., 2009, 2010; Borchert et al., 2013; Singh et al., 2013; Testai et al., 2013; Schmitt et al., 2014).

The cardioprotective effect mediated by the mitoBK channel is attributed mainly to (a) an increase in K⁺ in the mitochondrial matrix, (b) the retention of Ca²⁺, (c) a decrease in ROS, and (d) closure of the mPTP (Hermann et al., 2015). The flux of K⁺ from the cytosol to the negatively charged mitochondrial matrix is caused by the opening of mitoBK channels, which depolarizes the organelle (Szewczyk et al., 2006). The opening of mitoBK reduces the influx of Ca²⁺, decreasing the Ca²⁺ overload in the mitochondria (Xu et al., 2002; Du et al., 2020). Therefore, the functional effect of mitoBK channel activators is to reduce ROS production and Ca²⁺ overload, improving homeostasis and mitochondrial redox status after I/R as seen in isolated guinea pig hearts (Heinen et al., 2007a,b; Bentzen et al., 2009).

Due to nonspecific effects of drugs, the role of mitoBK in protection against I/R injury has been questioned, invoking biochemical and molecular reasons (see Gaspar et al., 2009; Szewczyk et al., 2009; Wojtovich et al., 2011, 2013). Conclusive evidence for the role of mitoBK in cardioprotection comes from studies using BK knockout mouse models (Kcnma1 −/−). The hearts of these mice are not protected from ischemic injury under treatment with NS1619 or NS11021. This lack of protection is revealed by measurements of cardiac function and infarct size in isolated perfused hearts (Singh et al., 2013; Wojtovich et al., 2013; Soltysinska et al., 2014). These experiments demonstrate that BK activator-mediated cardioprotection requires KCNMA1 expression and that mitoBK activation protects cardiomyocytes from ischemia and reperfusion injury (Singh et al., 2013; Goswami et al., 2019). Nonetheless, in vascular smooth muscle myocytes, the evidence suggests that mitoBK channels are not involved in protection against I/R injury (Frankenreiter et al., 2017).

Strong evidence shows that mitoBK channels located in IMM in neurons are associated with neuroprotective effects (Kulawiak and Szewczyk, 2012; Singh et al., 2013; Wojtovich et al., 2013; Soltysinska et al., 2014; Li and Gao, 2016; Krabbendam et al., 2018; Du et al., 2020). Kulawiak et al. (2008) demonstrated that opening of mitoBK located in IMM of rat brain, stimulated by CGS7184 and NS1619, inhibits hydrogen peroxide production by 20%. This effect is sensitive to BK channel blockers iberiotoxin and charybdotoxin. These results suggest that the opening of mitoBK inhibits ROS, promoting neuronal survival and neuroprotection (Kulawiak et al., 2008). However, Gaspar et al. (2009) proposed that the protective effect of NS1619 may not be mediated by mitoBK in every system. They studied primary rat cortical neurons and found that preconditioning with NS1619 caused mitochondrial depolarization, an effect that was displayed even with preincubation with paxilline (Gaspar et al., 2009). A possible explanation for this negative result could be that paxilline takes longer to diffuse through the plasma membrane and reach mitoBK in IMM (Balderas et al., 2015).

Subsequently, NS11021 was used to evaluate its cytoprotective effect on primary cortical neurons of rats with glutamate-induced excitotoxicity. On the one hand, due to the suppression of glutamate excitotoxicity, attenuation of oxidative stress, and preservation of mitochondrial function, mitoBK-dependent neuroprotection is induced (Borchert et al., 2011). On the other hand, the mitochondrial division inhibitor, mdivi-1, exhibited protective effects in ischemic injury by regulating the activation of mitoBK in mitochondria of cardiac neurons due to an increase in BK channel expression levels and attenuation of oxidative stress, mitochondrial dysfunction, and neuronal apoptosis (Liu et al., 2012). This neuroprotective effect is associated with the increase of mitochondrial Ca²⁺ and the decrease in ROS production mediated by mitoBK (Kulawiak and Szewczyk, 2012; Krabbendam et al., 2018; Du et al., 2020).

Opening of mitoBK channels has been found to regulate the respiratory rate, mitochondrial depolarization, matrix volume, and ROS production (Heinen et al., 2007a,b; Kulawiak et al., 2008; Hermann et al., 2015).

In muscle mitochondria from Drosophila mutants slo1- / - severe defects were found in terms of the mitochondrial ultrastructure, aberrations in the arrangement of ridges, an increased size (swollen) of the organelle, and loss of continuity of IMM compared to wild type cells expressing mitoBK (Gaspar et al., 2009). Meanwhile, Du et al. (2020) analyzed mitochondria of HEK and PC12 cells transfected with mutant BK channels (BKG354S, mutation that affects the selectivity filter). This mutation caused a selective loss of BK channels in the mitochondrial membrane and the loss of mitochondrial content, ranging from the loss of voltage-gated anion channel (VDAC) proteins to a reduction in every component mitochondrial oxidative phosphorylation (OXPHOS). This led to depolarized and dysfunctional mitochondria and the loss of the cytoprotective effect due to the activation of mitoBK (Du et al., 2020). Therefore, the mitoBK channel plays a crucial role in maintaining mitochondrial structure, function, and content.

That said, it has been found that mitoBK present in IMM contributes to the regulation of volume of the mitochondrial matrix, influences uptake of K⁺, mitochondrial transmembrane potential, pH balance, Ca²⁺ transportation, ROS production, mitochondrial dynamics in general and it has also been proposed to participate in increasing bioenergetic efficiency (Aon et al., 2010; Leanza et al., 2019). These may be considered as the mechanisms proposed for the cytoprotection above (Testai et al., 2015; Wawrzkiewicz-Jałowiecka et al., 2020).

The cardio- and neuroprotection conferred by mitoBK activators appears to be associated with the modulation of the rate of mitochondrial reactive oxygen species (ROS) generation in brain and heart cells (Andrukhiv et al., 2006; Facundo et al., 2006; Heinen et al., 2007a; Kulawiak et al., 2008; Krabbendam et al., 2018; Kshatri et al., 2018; Gururaja et al., 2019). The conclusive evidence of the key role of mitoBK channels in ROS generation comes from the use of genetic models, which demonstrated that the absence of BK channels increases ROS production. However, Soltysinska et al. (2014) reported that the knockout of mitoBK channels increased postanoxic ROS production in ventricular mitochondrial cells. This result strongly suggests that mitoBK channels regulate the production of ROS, as well as the oxidative state in hypoxia and reoxygenation of mitochondria. Moreover, Gururaja et al. (2019) found that in Drosophila mitoplasts that genetically blocking mitoBK channels increases ROS production, the consumption of O₂ and the respiratory rate (Gururaja et al., 2019).

There is no consensus in the literature regarding the effect of mitoBK in the ROS production. Several reports demonstrate that MitoBK activation after I/R injury causes a reduction in ROS levels. ROS production increases when channel blockers are applied (Szewczyk et al., 2009; Cordeiro et al., 2015; Goswami et al., 2019). On the contrary, the activation of mitoBK in isolated and I/R injury-induced ventricular myocytes with NS11021, caused an increase in ROS levels. Addition of antioxidants, which decrease the open probability of mitoBK, abolished the increase in ROS production (Borchert et al., 2013). This increase in ROS production after mitoBK activation was also observed in a liver cancer cell line (Booth et al., 2016).

Ambivalence in responses after mitoBK activation could be related to some coupling between the channel and ROS generation sites. In the case that mitoBK is coupled to the mitochondrial complex I (reverse electron flow), the production of ROS should decrease upon mitoBK activation. However, if the channel is coupled to the mitochondrial complex III (direct electron flow), activation of mitoBK should lead to an increase in ROS (Krabbendam et al., 2018). In this regard, Stowe et al. (2006) showed in isolated mitochondria from cardiac cells that the succinate and rotenone-dependent H₂O₂ production that blocked reverse electron flow increased slightly after the activation of the mitoBK channel. On the other hand, Heinen et al. (2007a,b) demonstrated that in the absence of rotenone, under substrate conditions that allow reverse electron flow, mitoBK activation reduces H₂O₂ production by 73% by accelerating forward electron flow.

We note here that hemin, a by-product of hemoglobin with oxidative properties, can inhibit the electrical activity of BK channels. Therefore, the mitoBK channel can be considered a redox sensor. (Augustynek et al., 2014). Moreover, mitochondria of ventricular muscle fibers lacking mitoBK channels (by knockout) showed an increase in the production of postanoxic ROS, indicating that these channels regulate the oxidative state in hypoxia and reoxygenation (Soltysinska et al., 2014).

MitoBK channel regulation of mitochondrial Ca²⁺ retention capacity could be observed pharmacologically activating the channel with NS1619, increasing the number of Ca²⁺ pulses necessary to cause a massive release of Ca²⁺ from the mitochondria (Singh et al., 2013). Ca²⁺ retention capacity in mitochondria is closely related to mPTP activation, which mediates Ca²⁺ release from mitochondria to the cytosol (Singh et al., 2013). In fact, in a study with rat liver, mitoplasts, and astrocytes, hypoxia inhibited mPTP but substantially increased mitoBK activity, with an increase in Ca²⁺ retention capacity, which was reduced using iberiotoxin (Cheng et al., 2008). This finding may suggest a functional link between mitoBK and mPTP, where the reduction of the activity of the mitoBK channel by mitochondrial substrates can support the activation of mPTP, leading to cell death by apoptosis (Laskowski et al., 2016).

Possibly by the opening of mPTP, apoptosis results from the complex interaction between Ca²⁺ and ROS. The activation of mitoBK is involved in both processes, linking this channel to a delay in the formation and/or closure of mPTP (Goswami et al., 2019). In single-channel recordings in rat astrocyte mitoplasts and hepatic mitochondria, inhibition of mitoBK channels by the pro-apoptotic protein BAX (B-cell lymphoma (Blc) -2-associated X) was observed, which in turn activated mPTP and induced cytochrome C release (an effect like that obtained using iberiotoxin (Cheng et al., 2008)). Conversely, BCL-Xl (an anti-apoptotic protein) inhibited the impact of BAX on mitoBK and mPTP blockade. mitoBK channel is related to apoptotic mechanisms mediated by BAX, which exerts its pro-apoptotic effect by inhibiting mitoBK and thus promotes the opening of mPTP (Cheng et al., 2008, 2011).

In the mitochondria of the human glioblastoma cell line U-87 MG, the substrates of the ETC (NADH, succinate, and malate or glutamate) and artificial donors of electrons (tetramethyl-p-phenylenediamine TMPD/ascorbate) inhibited the mitoBK channel (Bednarczyk et al., 2013b). These results suggest that the mitoBK channel is regulated by the cytochrome C oxidase and that a redox signal is “transferred” from ETC to mitoBK through CCO (Ohya et al., 2005). Together, these observations suggest a structural and functional coupling of the respiratory chain and mitoBK channels, although the underlying molecular mechanisms are still unknown (Laskowski et al., 2016).

Cytoprotection induced by mitoBK activators may also be mediated by inhibiting the mitochondrial respiratory chain (Kicinska and Szewczyk, 2004). Activating the mitoBK channel sing NS1619 in IMM of isolated rat brain mitochondria inhibited ROS production of the respiratory chain using the complex I (Kulawiak et al., 2008). In cultures of hippocampal sections exposed to glutamate, preincubation with NS1619 showed an increase in basal respiration (Piwońska et al., 2016). Activating cardiac mitoBK channels produced an improvement in mitochondrial respiration due to a decrease in state 4 respiration (characterized as a state without any ATP usage/production), while state 3 of respiration (described as a state with saturating ATP usage/production) was unchanged (Aon et al., 2010). These findings suggest a probable mitoBK-dependent mechanism for both cardiac and neuronal cytoprotection (Testai et al., 2015; Wawrzkiewicz-Jałowiecka et al., 2020).

To date, a possible role for mitoBK in cancer development has not been reported; despite that, it has been related to the survival and motility of glioma cells after irradiation (Steinle et al., 2011). Irradiation and hypoxia (Gu et al., 2014) have been found to increase the P_o of mitoBK, which in turn activates Calmodulin kinase II (CaMKII), leading to increased migration of glioblastoma cells (Steinle et al., 2011; Peruzzo et al., 2016), as well as resistance to hypoxic conditions (Gu et al., 2014). MitoBK in gliomas may also regulate the respiratory chain and confer cytoprotection, which may be one reason that makes this type of cancer incurable (Wawrzkiewicz-Jałowiecka et al., 2020).

Shrum et al. (2019) demonstrated that mitoBK channels might represent a therapeutic target to prevent cold storage (CS) preservation and rewarming (RW)-induced kidney injury that is very common in kidneys routinely subjected to transplant. To do this, they added NS11021 to the CS solution and evaluated the effect on normal rat kidney proximal tubular epithelial cells. The addition of this activator of mitoBK prevented the deterioration induced by CS+RW in the uptake of K⁺ mediated by mitoBK, as well as a reduction in cell death and mitochondrial damage. In addition, they observed mitigation in respiratory dysfunction, depolarization, and superoxide production (Shrum et al., 2019).

Even though the presence of BK channels has been reported in the NE of many cell types such as pancreatic cells, brain endothelial cells and macrophages (Maruyama et al., 1995; Gobeil et al., 2002; Selezneva et al., 2021), little is known about its functional role.

In pancreatic acinar cell nuclei, nBK channels only localize in ONM, and their activation is sensitive to Ca²⁺ lumen levels (Maruyama et al., 1995). Whether nBK can regulate the nuclear transmembrane potential was proven in mice hippocampal neurons using a potentiometric probe. This experiment showed that the perinuclear lumen got more negative when nBK was blocked by paxilline. Usage of paxilline also indicated an increase in nuclear Ca²⁺ through RyR, mainly due to intracellular BK inhibition without pmBK being involved (Li et al., 2014). We recall here that RyR is sensitive to changes in nuclear transmembrane potential (Zahradníková and Mészáros, 1998). This increase in nuclear Ca²⁺ due to nBK inhibition showed to activate CREB through phosphorylation in a nuclear Ca^2+/CaMKIV-dependent manner, which also causes changes in neuronal dendritic arborization (Li et al., 2014).

Not only nBK but also pmBK are found in macrophages from the nervous system (microglia). These channels are involved in pro-inflammatory mechanisms induced by Toll-like receptors 4 (TLR4) activated by lipopolysaccharides (LPS). pmBK is activated through TLR4, which induces translocation of NF-kB (nuclear factor kappa B) to the nucleus, where it prompts gene expression regarding cytokine production. Treatment with paxilline at different times after LPS application showed that after 6h, paxilline did not affect cytokine production, indicating the existence of BK modulation on gene expression NF-kB-independent. This result becomes clear by considering that the nBK expression is induced by LPS long-lasting activation on TLR4 (Yang et al., 2019). Another signaling mechanism in macrophages regarding nBK was described using the RAW264.7 cell line. The blockage of nBK using paxilline in preparations of the cell line and isolated nuclei showed an increase in CREB phosphorylation due to CaMKII Calmodulin kinase II) and CaMIV (Calmodulin kinase IV) activity (see Figure 1B; Selezneva et al., 2021). It is important to note that CREB is related to many roles for macrophages, particularly preventing apoptosis (Park et al., 2005).