Differential expression of BK auxiliary subunits confers to the channel functional diversity (Figure 1). In the smooth muscle, the α subunit is co-expressed with the β1 subunit, which prompts membrane hyperpolarization and vasorelaxation, decreasing the risk of pathologies associated with vascular tone regulation (Amberg and Santana, 2003). The β1-induced effects are associated with the improvement of apparent sensitivity to Ca²⁺ and slowing down the activation and deactivation kinetics. These effects are elicited by changes of the allosteric coupling between gating and the Ca²⁺ binding sites along with the reduction of the voltage dependence of the voltage sensor activation process (Nimigean and Magleby, 2000; Orio and Latorre, 2005; Contreras et al., 2012). β2 subunits also increase the apparent Ca²⁺ sensitivity and slowed the gating kinetics, associated with an increase in the allosteric coupling factors (Orio and Latorre, 2005). The β3 subunit has four members (β3a, β3b, β3c, and β3d). The most studied members are β3a and β3b, which induce BK channel inactivation (Brenner et al., 2000a; Uebele et al., 2000; Gonzalez-Perez and Lingle, 2019). It has been reported that β4 subunit slows the gating kinetics, reduces the apparent voltage-sensitive of the channel activation, and modifies the apparent Ca²⁺ sensitivity in two ways: by the inhibition of the channel activity in low [Ca²⁺]_i and by the increase of the channel activity in high [Ca²⁺]_i. These effects are associated with the stabilization of the active conformation of the voltage sensor, and a reduction in the number of the gating charges per sensor (Brenner et al., 2000a; Wang B. et al., 2006; Contreras et al., 2012).

γ1 (LRRC26), γ2 (LRRC52), γ3 (LRRC55), and γ4 (LRRC38) subunits display tissue-specific expression and function (Yan and Aldrich, 2012; Evanson et al., 2014). They produce a significant modification in the voltage dependence of BK channel activation, recognized as a shift to negative potentials of ∼140, 100, 50, and 20 mV in the presence of γ1 (LRRC26), γ2 (LRRC52), γ3 (LRRC55), and γ4 (LRRC38), respectively (Yan and Aldrich, 2012; Zhang and Yan, 2014). The increase in voltage sensitivity induced by γ1 has been associated with vasodilation observed in arterial smooth muscle cells (Evanson et al., 2014). Moreover, knocking down the expression of the γ1 subunit contributes to vasoconstriction (Yan and Aldrich, 2012). γ1 subunit also induces the acceleration of the activation kinetics, while the deactivation kinetics become slower. It has been proposed that γ2, γ3, and γ4 subunits can modify the apparent Ca²⁺ sensitivity (Latorre et al., 2017). LINGO1 is an accessory subunit of BK channels that induces a rapid inactivation of the channel, slowing down the deactivation process and shifting their activation to negative potentials (Dudem et al., 2020).

Palmitoylation is a post-translational modification associated with regulation of the BK channel plasma membrane localization without variations in single-channel conductance or the Ca²⁺ and voltage sensitivity. BK channel palmitoylation occurs in the STREX insert and in the residues C53, C54, and C56, located in the intracellular loop S0-S1 (Jeffries et al., 2010; Zhou et al., 2012).

On the other hand, BK α or β subunits undergo reversible posttranslational modifications as protein phosphorylation. These control BK channel activity. Protein kinase A (PKA), Ca²⁺—and diacylglycerol-activated protein kinase C (PKC), cyclic guanosine monophosphate (cGMP) -activated protein kinase G (PKG), and adenosine monophosphate (AMP)-activated protein kinase (AMPK) have been reported to phosphorylate BK channel α subunit in serine, threonine, and tyrosine residues. The phosphorylation in the BK channel activity is depending on the channel variant. For instance, PKA stimulates ZERO variant activation but induces STREX splice variant inhibition. The final effects of phosphorylation are tissue-depending, like inducing BK channel activation in smooth muscle but inhibit the channel activity in pituitary cells (Contreras et al., 2013; Kyle and Braun, 2014; Shipston and Tian, 2016).

Diverse endogenous molecules, including heme, carbon monoxide (CO), and reactive oxygen/nitrogen species, have been reported to activate BK channels (Hou et al., 2009; Kyle and Braun, 2014). The free intracellular heme binds to a region in the RCK1 and RCK2 segments and decrease or increase the BK channel Po at positive or negative voltages, respectively. These effects are modulated by the action of heme oxygenase enzyme (HO) by regulating the heme group degradation (Horrigan et al., 2005; Muñoz-Sánchez and Chánez-Cárdenas, 2014). Electrophysiological assays demonstrated that CO increase the Po of the channel in cell-free membrane patches, suggesting channel modulation by CO direct binding in RCK1 domain (Jaggar et al., 2002; Hou et al., 2008; Williams et al., 2008).

Calcium and voltage-activated potassium (BK) channels redox modulation has been widely described (Hou et al., 2009). H₂O₂ decreases the Po through amino acid modifications. These are reversed by the addition of reducing agents as glutathione (GSH) or dithiothreitol (DTT). C433 and C911, located in the RCK1 and COOH terminus, respectively, have been reported to contribute to the sensitivity of the BK channel to reactive species (DiChiara and Reinhart, 1997; Tang et al., 2001, 2004; Soto et al., 2002; Brakemeier et al., 2003). STREX variant has additional cysteine residues that increase the inhibitory effect of oxidation in the channel (Erxleben et al., 2002). O₂^–, NO, and peroxynitrite are reactive molecules also affecting the BK channel activity (Hou et al., 2009). Finally, fatty acids, phospholipids, steroids, and other lipid metabolites can also modulate BK channel properties (Hou et al., 2009). In another way, it has been reported that decreases in intracellular pH increase BK channel activation by inducing a shift to the left in the voltage-dependence (Avdonin et al., 2003; Hou et al., 2009; Kyle and Braun, 2014).

As was described, the structural properties of the BK channels play different important physiological roles. In addition, both BK channel expression and its activity are regulated by various mechanisms which confer functional diversity to the channel. In the next section, we will describe the reported mechanism involved in the modulation of the BK channel by hypoxia and the possible physiological impact of that modulation.

It has been previously reported that hypoxia promotes a rise in the BK channel activity through membrane depolarization, and increases in [Ca²⁺]_i. Hypoxia promotes membrane depolarization by different mechanisms including the inhibition of K⁺ channels as K_v1.2 and K_v1.5 (Seta et al., 2004). Blocking K_v channels with the antagonist tetraethylammonium (TEA) evoked a similar response as hypoxia in glomus cells (Pardal et al., 2000; López-Barneo et al., 2001; Wang and Kim, 2018). TASK channels expressed in glomus cells are also hypoxic-inhibited channels. It has been reported that a decrease in cytosolic Mg-ATP or phosphorylation via AMP-activated kinases, among others, are events that promote TASK inhibition leading to membrane depolarization, consequent activation of L-type voltage-dependent Ca²⁺ channels, and rising in [Ca²⁺]_i, and consequently increasing BK channel Po (Buckler, 2015; O’Donohoe et al., 2018).

Hippocampal neurons exposed to hypoxia for 6 h exhibited a significant rise in BK channel Po and unitary conductance after 6 h of reoxygenation. The normal parameters in channel activity were recovered after 24 h of reoxygenation. These results were associated with an increase in [Ca²⁺]_i without changes in the BK α subunit expression (Chen et al., 2013; Wang and Kim, 2018). Some authors have proposed that BK channels are active at rest, and that hypoxia inhibits their activity (Peers and Wyatt, 2007). On the contrary, some reports suggest that BK channels are closed in normoxia while hypoxia induces the channel activation to diminish the hypoxia-induced cell damage (Pardal et al., 2000; Gomez-Niño et al., 2009). The overall effect of hypoxia-induced BK channel activity on cell survival depends on specific factors present in a given cell type or tissue. For instance, the activation of the BK channel after hypoxia/reoxygenation induces hippocampal neuronal apoptosis. Meanwhile the hyperpolarization induced in glomus cells by BK channel activation constraints the rise in [Ca²⁺]_i, and, therefore, limits the hypoxic response (Chen et al., 2013; Wang and Kim, 2018).

It is known that BK β subunits expression contributes to the molecular diversity of BK channels (Torres et al., 2014). An important role for BK β1 subunit in regulating vascular tone was demonstrated by systemic hypertension in mice carrying a deletion of the Kcnmb1, the gene encoding for β1 subunit (Brenner et al., 2000b; Chang et al., 2006). Additionally, the association between severity of asthma and a loss-of-function polymorphism in Kcnmb1, suggests a role of this subunit in modulating smooth muscle cells tone of airway in humans (Seibold et al., 2008). Different reports show that hypoxic response can also be modulated by differential expression of β subunits. In ovine pulmonary artery smooth muscle cells (PASMC), primary cultures, and ovine cerebral artery smooth muscle, hypoxia induced a rise in mRNA and protein expression level of both BKα and β1 subunits. Similar results were observed in rats that have been maintained in hypobaric hypoxic chambers for 3 weeks where mRNA levels of BKα and β1 subunits increased approximately threefold and twofold, respectively (Resnik et al., 2006; Tao et al., 2015).

The rise in the β1 subunit expression enhances the BK channel apparent sensitivity to [Ca²⁺]_i and the current density, leading to hyperpolarization, subsequent closing of voltage-gated Ca²⁺ channels, and decreasing the [Ca²⁺]_i (Torres et al., 2007; Castillo et al., 2015). The increase in BK channel activity may offer an adequate brain O₂ level when there is a lower arterial O₂ concentration, as it was reported in ovine artery smooth muscle (Tao et al., 2015).

Reported changes in the BK subunits expression are suggested to occur via post-translational modification by phosphorylation/dephosphorylation and by transcriptional regulation through the interaction with HIF-1α (Resnik et al., 2006; Ahn et al., 2012; Tao et al., 2015). After hypoxic treatment of hPASMC, it was observed an increase in the expression of both HIF-1α and KCNMB1 and knocking down HIF-1α avoided the hypoxia-induced KCNMB1 expression. In addition, it was demonstrated that human KCNMB1 promoter has HREs that are critical for HIF-1α binding and hypoxic-modulation of KCNMB1 expression (Ahn et al., 2012). Kcnmb1^–/– mice showed higher right ventricular systolic pressure after hypoxic stimulus, compared with WT mice. These experiments demonstrated the importance of the BKβ1 subunit in the modulation of the pulmonary vascular response to chronic and acute hypoxia, and suggests a connection between BKβ1 expression and HIF-1α activity in the regulation of the tone in the microcirculation (Barnes et al., 2018).

Besides the increase in the BKβ subunit expression, it was reported that chronic hypoxia enhanced α/β colocalization at the plasma membrane without changes in mRNA expression, suggesting post-transcriptional regulation of the BK β subunit (Hartness et al., 2003). From these results, it has been proposed that hypoxic events originated from diverse cardiorespiratory diseases, conduct to adaptative cellular responses, including changes in the BK subunit expression or increase in functional α/β complex at the plasma membrane (Hartness et al., 2003).

In some cells/tissues a decrease in β subunit expression has also been reported after hypoxic stimuli. Rat myocytes exposed to prenatal hypoxia showed a reduction in the channel voltage and Ca²⁺ -sensitivity, associated with a drop in the BK β1 subunit mRNA and protein expression. These changes were without variations in the BKα subunit expression (Liu et al., 2018a,b). All these findings suggest that BK channel modulation induced by hypoxia is cell specific. BK channels expressed in rat vascular smooth muscle cells are functionally less active, decreasing the vasorelaxant effect of the channel and leading to high blood pressure, vascular dysfunction, and cardiovascular alterations (Lewis et al., 2002; Navarro-Antolín et al., 2005; Liu et al., 2018a). However, in ovine PASMC primary cultures and ovine cerebral smooth muscle, hypoxia induced a rise in mRNA and protein expression level of both BKα and β1 subunits.

The BKβ4 subunit expression is also modulated by hypoxia. In podocytes, an increase in BK β4 mRNA and protein levels were observed after chronic hypoxia without modifications in the expression of the BK α subunit. The higher expression of the β4 subunit decreases the BK channel activity by a shift in the voltage-dependent activation toward depolarized voltages, and a significant increase in the time constant for channel activation. In conclusion, Zhang et al. (2012), suggested that reducing BK channel activity promotes podocyte depolarization, leading to a decrease in Ca²⁺ influx through TRPC6 channels. The consequent change in [Ca²⁺]_i modifies Ca²⁺-dependent signaling pathways associated with hypoxic response (Zhang et al., 2012).

In addition to increases in BK β subunit expression, a rise in mRNA from BK channel α subunit, and changes in the protein distribution were positively associated with the extent of the hypoxic response in pulmonary arteria. That response correlates with structural changes as alterations in the intimal thickening, suggesting an adaptative response in patients with the COPD to attenuate hypoxic pulmonary vasoconstriction (Peinado et al., 2008).

Calcium and voltage-activated potassium (BK) channel activity is modulated by additional mechanisms, including heme oxygenase-2 (HO-2), ROS regulation, and STREX-associated interaction (Hoshi and Lahiri, 2004; Navarro-Antolín et al., 2005; Figure 1). In mammalian cells, the enzyme HO-2 is implicated in the degradation of the heme group to produce biliverdin, iron, and carbon monoxide (CO). HO-2 function has been associated to O₂ sensing and hypoxic response through the regulation of the BK channel activity (Hoshi and Lahiri, 2004; Ortega-Sáenz et al., 2006; Muñoz-Sánchez and Chánez-Cárdenas, 2014). The mechanism involves the BK channel activation by CO and the physical interaction of HO-2 with the channel. It has been reported that knocking down of HO-2 induces a decrease in the expression of BK α subunit. Hypoxia can induce a redox dysregulation that prompts the HO-2 deficiency, decreases the CO levels, and reduces the channel activity (Naik and Walker, 2003; Williams et al., 2004; Ortega-Sáenz et al., 2006). Co-immunoprecipitation with HO-2 in carotid body cells was only observed when channels were expressing both α and β subunit (Riesco-Fagundo et al., 2001; Williams et al., 2004; Ortega-Sáenz et al., 2006). On the contrary, a weak interaction between HO-2 and BK channel was observed in pulmonary arterial smooth muscle. Furthermore, in mouse lines deficient in either HO-2 or BKα subunit it was not observed any role of these proteins in the hypoxic effects (Roth et al., 2009), suggesting that hypoxic-modulated interaction and function of HO-2 and BK channels could be tissue-specific and the interaction HO-2/BK channel could not be considered as a universal O₂ sensor. Currently, there are not reports related to the role of γ or LINGO1 subunits in the modulation of BK channel function by hypoxia.

Another mechanism involved in the BK channel modulation is associated with redox regulation of BK channel binding of the heme group. In normoxia, the heme group has a low affinity to the channel in contrast to the high affinity that shows for HO-2. That event induces heme degradation and an increase in CO, promoting channel activation. On the contrary, in hypoxia, HO-2 has a lower affinity for the heme group, generating an increase in heme concentration and decreasing CO production. Both stimuli reduce the channel activity (Tang et al., 2003; Yi and Ragsdale, 2007; Ragsdale and Yi, 2011) (Figure 1).

BK channel is also modulated through cAMP and GMP-dependent protein kinase (PKG) by specific phosphorylation of multiples Ser residues in the C terminus of the α subunit. PKC-dependent phosphorylation shifts the voltage-dependence to more negative potentials, increasing BK channel voltage-sensitivity (Zhou et al., 2001; Kyle et al., 2013). In middle cerebral arteries, long-term hypoxia affected the PKG-modulation of the BK channel activity. It was found that hypoxia reduces the BKα subunit expression and decreases the association of PKG with the BK channel, inhibiting the channel activation and the consequent vasorelaxation (Thorpe et al., 2017).

On the other hand, hypoxia and endogenous H₂S induce similar inhibition of the BK channels in the carotid body. The inhibition mediated by H₂S endogenous production demonstrated the involvement of that molecule in the hypoxic-modulated BK channel activity, and suggest it as sensor for hypoxia in vasculature (Li Q. et al., 2010). A contrary effect was reported in vascular smooth muscle, where H₂S has a regulating function of myogenic tone through BK channel activation (Jackson-Weaver et al., 2011). These results demonstrated that BK channel modulation depends on the cell type, however, more studies are necessary to establish the effect of H₂S in hypoxic modulated BK channel activity.

Calcium and voltage-activated potassium (BK) channel inhibition, induced by low O₂ in neocortical neurons, is mediated by variations in the cellular redox potential, and depends on cytosolic factors, demonstrating the channel regulation by oxidative stress (Liu et al., 1999). Similarly, in rat CA1 hippocampal neurons, hypoxia induced a decrease in the BK channel Po due to a reduced mean channel open time and an increased closed time. Channel activity was restored after treatment with oxidizing agents, suggesting that hypoxia decreases cellular oxidation potential in CA1 neurons (Gao and Fung, 2002). Furthermore, hypoxia stimulates the production of reactive species ROS and reactive nitrogen species (RNS), and increases oxidative stress (Fresquet et al., 2006; Hu et al., 2016). In smooth muscle from sheep, uterine artery was reported a significant effect of stress oxidative in the inhibition of BK channel activity by hypoxia. These changes were showed to be mediated by downregulation of the BK β1 subunit (Zhu et al., 2014; Hu et al., 2016). The authors reported that the treatment with the antioxidant N-acetylcysteine (NAC) eliminates the hypoxic-mediated inhibition of the BK channel’s current density, reestablished β1 expression, and restored the arterial tone. After NAC treatment, there were no differences in relaxation induced by the BK channel agonist NS1619 in normoxic and hypoxic animals. In addition, the antioxidant treatment also rescues the hormonal steroid effect induced on the BK channel activity. The proposed mechanism involves post-translational modifications induced by ROS and KCNMB1 gene repression (Zhu et al., 2014; Hu et al., 2016).

Reactive oxygen species (ROS) effect has been reported in cardiovascular diseases. Moreover, it has been proposed that oxidative stress induced by ROS impairs the BK channel activation by changing the cysteine 911 (C911), located in RCK2 near to the Ca²⁺ bowl. It was shown that adding H₂O₂ to the intracellular side induced the BKα + β1 channel inhibition through a decrease in Po and Ca²⁺ sensitivity without modification of the unitary conductance. Authors suggest that observed functional properties of the BK channel after oxidative stress are comparable to the found in absence of β1 subunit (Soto et al., 2002; Tang et al., 2004; Tano and Gollasch, 2014).

Hypoxia promotes a decrease in the modulatory effect that tamoxifen and steroid hormone induces on the BK channel activity, which was associated with the diminished expression of the BK β1 subunit (Navarro-Antolín et al., 2005; Liu et al., 2018a). Recently, we reported that changes in cholesterol concentration reduces the effects of 17β-Estradiol (E2) in the BK channel activity. However, that effect was not induced by changes in the expression of the BK β1 subunit (Granados et al., 2021). Considering that hypoxia reduces the membrane cholesterol concentration, we suggested that reported changes in the modulation of Tamoxifen and E2 after hypoxia could also be related to changes in membrane cholesterol concentration. However, it is necessary to carry on more experiments to unveil the complete mechanisms associated with that effect (Zhang et al., 2018).

McCartney et al. (2005) reported that BK channel sensitivity to hypoxia is splice-variant-specific, conferred by the stress-regulated exon (STREX) (Figure 1). The hypoxic inhibition induced in the STREX variant is Ca²⁺-sensitive with no effect on single-channel conductance nor the voltage sensitivity. The hypoxic inhibition required Serine S24 and Cysteines C23 and C24, suggesting an important role of these residues in the hypoxic response. The ZERO variant was non-sensitive to hypoxia (McCartney et al., 2005; Tano and Gollasch, 2014).

In addition to plasma membrane BK channels, there are diverse reports about the involvement of mitochondrial BK channels (mitoBK_Ca) in the hypoxic response and protection against ischemic injury (Xu et al., 2002; Shi et al., 2007; Goswami et al., 2019). mitoBK_Ca is expressed in adult cardiomyocytes and it is encoded by the splice variant of the Kcnma1 gene DEC, which has an insert of 50 amino acids in the C-terminal region that is responsible for targeting the channel to mitochondria (Singh et al., 2013; Szteyn and Singh, 2020). mitoBK_Ca has been reported to have similar properties to BK channels expressed in the plasma membrane (Xu et al., 2002; Balderas et al., 2019). These channels induce a cardioprotective mechanism that involves the decrease of ROS production and the depolarization of the mitochondria matrix by K⁺ flux and the reduction of Ca²⁺ overloading during ischemia and reperfusion (Xu et al., 2002; Stowe et al., 2006; Shi et al., 2007; Borchert et al., 2011; Goswami et al., 2019). Interestingly, the reported effect was observed only in the normoxic hearts but not in chronic hypoxic hearts. Considering that BKα subunit expression was not changed, it is suggested that the effect was associated with a significant reduction in mitoBK_Ca channel activity, probably via decreasing in Ca²⁺ sensitivity (Riesco-Fagundo et al., 2001; Shi et al., 2007). In addition to BKα subunit, it has been demonstrated the mitochondrial expression of β1 subunit in cardiac tissue and myocytes, where it is critical to mediate cardioprotective response (Wang et al., 2008; Borchert et al., 2011; Balderas et al., 2019). These effects are not related to changes in the β1 subunit expression but are associated with activation of the channel possibly induced by modifications in glycosylation level of β1 subunit. However, more assays are necessary to prove the suggested hypothesis (Wang et al., 2008; Borchert et al., 2011).

Experiments using BK β1 KO mice demonstrated that mitoBK_Ca channel activity in mitochondria was negligible when BK β1 subunit was absent, suggesting a reduction in the number of channels in mitochondria. Moreover, it was proposed a role of β1 in targeting BK channel to mitochondria. Activity of mitoBK_Ca was associated with Ca²⁺ homeostasis in cardiac cells (Balderas et al., 2019). The presence of a second population of mitoBK_Ca that activates at more depolarizing potentials suggests that BK can be associated with other regulatory subunits to produce channels with different voltage-sensitivity (Balderas et al., 2019). In addition, Frankenreiter et al. (2017), reported a positive regulation of mitoBK_Ca by cGMP, which regulates ROS homeostasis in oxidatively stressed cardiomyocyte mitochondria and induces a significant increase in channel Po. That effect is associated with cardioprotective properties in models of ischemia/reperfusion injury possible through cGMP/cGKI (cGMP-dependent protein kinase type I) pathway (Frankenreiter et al., 2017).

Hypoxic modulation of the BK channel depends on cells or tissue. In some cells, hypoxic stimuli inhibit the BK channel activity. Meanwhile, in other cells, hypoxia promotes channel activation. Modulation of the channel activity has an important role in the hypoxic response, as it has been proposed to act as an “emergency brake.” BK channel response to hypoxia has been associated with the development of diverse pathologies such as preeclampsia (Hu et al., 2012; Zhu et al., 2013) and neuronal injury (Tjong et al., 2008). In addition, there are several reports about the role of BK channel in hypoxic-modulated cardiovascular diseases, like pulmonary artery hypertension derived from COPD, chronic inhalation of CO, or OSA (Bonnet et al., 2003; Dubuis et al., 2005; Peinado et al., 2008; Navarro-Antolín et al., 2009; Roth et al., 2009; Ahn et al., 2012; Barnes et al., 2018; Liu et al., 2018b; Li et al., 2019). Moreover, it has been proposed that BK channel openers might be used in stroke, epilepsy, asthma, and hypertension (Kirby et al., 2013).