The gene encoding the BK_Ca channel (slo1/KCNMA1) was first cloned from Drosophila (Atkinson et al., 1991; Adelman et al., 1992), and a mammalian gene was identified later (Butler et al., 1993). The BK_Ca channel is encoded by a single gene, and alternative splicing allows this channel to respond to a variety of regulatory inputs in a tissue-specific manner. To date, over 30 exons have been reported in the human KCNMA1 gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=KCNMA1), leading to a large number of potential isoforms of the channel. Early studies demonstrated that splice variants of the BK_Ca channel have altered Ca²⁺ and voltage sensitivities (Tseng-Crank et al., 1994), and key phosphorylation sites are created by the inclusion of certain exons (Tian et al., 2001). In mouse myometrium, the expression of BK_Ca channel isoforms with low sensitivity to Ca²⁺ increases at mid-pregnancy (Benkusky et al., 2000). In human myometrium, expression of specific spliced isoforms can be altered during pregnancy and at the juncture between non-laboring and laboring states (Curley et al., 2004), allowing the uterus to attain a more excitable state during labor. For example, although the overall levels of BK_Ca channel transcript and protein decrease as term approaches (Matharoo-Ball et al., 2003; Gao et al., 2009), the proportion of the mK44 isoform transcript increases at this time (Curley et al., 2004). This isoform bears a unique 44 amino-acid insertion and undergoes endoproteolytic cleavage, with membrane localization of the N terminus variant and intracellular retention of the remaining cleaved pore-forming C terminus (Korovkina et al., 2006). Additionally, mK44 is less sensitive to Ca²⁺ and voltage than the canonical (lacking the insert) channel (Korovkina et al., 2001), suggesting that this isoform may modulate uterine activity near the time of labor (Curley et al., 2004).

Other splice variants that are widely expressed could play an important role in myometrial excitability during gestation, such as the stress axis regulated exon (STREX) isoform, which introduces 59 amino acids into the linker between cytosolic domains S8 and S9 (Saito et al., 1997). This idea is supported by studies showing that the STREX variant is regulated during pregnancy (Benkusky et al., 2000) in mice and rats by adrenocorticotropic hormone, estrogen, and progesterone (Xie and McCobb, 1998; Zhu et al., 2005). Additionally, STREX harbors a consensus PKA phosphorylation motif, whose phosphorylation inhibits channel activity (Tian et al., 2001). STREX expression decreases in rat myometrium during pregnancy, likely due to an estrogenic effect (Zhu et al., 2005) (see Section Hormonal regulation). Although this isoform does not appear to play a dominant role in human myometrium, it may affect myometrial excitability in other species.

Alternative splicing is usually considered a mechanism to derive variability from single gene products, but it may also regulate protein trafficking, as suggested by the existence of yet another splice variant termed SV1. In this protein, 33 amino acids that include an endoplasmic reticulum (ER) retention motif (CVLF) are inserted within the S1 transmembrane domain. Thus, this isoform is retained in the ER, where it acts as a naturally occurring dominant negative (Zarei et al., 2001). Although the role of this isoform in controlling myometrial excitability has not been fully explored, its expression could provide an important mechanism for BK_Ca channel modulation and regulation of uterine contraction. Table 1 presents a summary of the known myometrial splice variants and their modified functions.

Membrane trafficking of the BK_Ca channel regulates a wide variety of physiological processes including pregnancy (Song et al., 1999), aging (Marijic et al., 2001), and aldosterone-induced K⁺ secretion from the gut (Sorensen et al., 2008). Two regions that control BK_Ca channel surface localization are the intracellular C-terminal linker between the RCK1 and RCK2 domains (Lee et al., 2009; Chen et al., 2010) and an actin-binding domain in the C terminus (Zou et al., 2008). In addition, isoforms containing different C-terminal sequences have distinct trafficking to the cell surface (Kim et al., 2007a; Ma et al., 2007).

Variation of the α-subunit by alternative splicing can add or delete signal sequences that modify channel localization by facilitating its retention in or targeting to intracellular organelles, including the ER (Zarei et al., 2001; Chen et al., 2010) and mitochondria (Singh et al., 2013). In rat myometrium, a splice variant containing the SV1 exon is retained in the ER, thereby preventing surface localization and affecting cell excitability (Zarei et al., 2001, 2004). In addition to splicing, co-expression with the auxiliary β1-subunit enhances internalization of the BK_Ca α-subunit into endosomes, thus controlling its membrane localization (Toro et al., 2006). Likewise, a related β4-subunit has an ER retention signal at its C terminus and prevents the α-subunit from exiting the ER (Shruti et al., 2012). As noted above, ER retention mechanisms have been explored in the myometrium, but their physiological relevance in modulating uterine contractility during pregnancy is still unknown.

A mitochondrial BK_Ca (mitoBK_Ca) channel was first identified by patch clamp studies performed on mitoplasts prepared from human glioma cells (Siemen et al., 1999). The structure of mitoBK_Ca is similar to the plasmalemmal BK_Ca except for the inclusion of a mitochondrial-targeting sequence, DEC, in the C-terminal region (Singh et al., 2013). Located in the inner mitochondrial membrane, mitoBK_Ca channels appear to be structurally and functionally coupled to the respiratory chain (Bednarczyk et al., 2013). In cardiac myocytes, activation of mitoBK_Ca channels attenuates mitochondrial Ca²⁺ overload (Sato et al., 2005). A similar effect is observed after activation of mitochondrial ATP-sensitive K⁺ channels, but these effects seem to be independent (Sato et al., 2005). The link between the mitoBK_Ca channel and myometrial function has not been explored. However, disruption of mitochondrial function decreases the amplitude and frequency of spontaneous contractions in non-pregnant mouse uterus, and some data suggest that this effect is, at least in part, mediated by Ca²⁺-activated K⁺ channels, such as the BK_Ca channel (Gravina et al., 2010). Notably, the effect occurs through modulation of Ca²⁺ influx and membrane potential. The idea that mitoBK_Ca functions in the myometrium is appealing. For example, activation of mitoBK_Ca improves mitochondrial respiratory function and thus protects the heart from ischemic injury (Xu et al., 2002). Moreover, mitoBK_Ca channels are more sensitive to hypoxia than plasma membrane BK_Ca channels in glioma cells (Gu et al., 2014), suggesting functional differences between these forms. Therefore, further work is required to determine (i) whether the mitochondria-dependent modulation of Ca²⁺ levels and uterine contractility changes during pregnancy, and (ii) whether mitoBK_Ca function affects mitochondria to accommodate changes in Ca²⁺ dynamics in the myometrium.

Localization of proteins in cholesterol- and sphingolipid-rich membrane microdomains has been proposed as a mechanism to modulate membrane excitability and intracellular signaling (Razani et al., 2002). Several lines of evidence indicate that such microdomains play important roles in controlling myometrial excitability. First, the number of a specific type of microdomain, caveolae, increases in myometrial cells toward the end of pregnancy (Turi et al., 2001). Second, two isoforms of the scaffolding proteins that form caveolae, caveolin-1, and caveolin-2, are down regulated by estrogen (Turi et al., 2001) and labor (Chan et al., 2014). Third, depletion of membrane cholesterol and consequent disruption of membrane microdomains, induces an increase in uterine contractions and Ca²⁺ transients (Smith et al., 2005). Finally, multiple studies have shown that BK_Ca channels localize to membrane microdomains in both cells used for heterologous expression and smooth muscle cells (Bravo-Zehnder et al., 2000; Babiychuk et al., 2004). For example, co-localization of BK_Ca channels with downstream effectors and other receptors in caveolae alters channel function in vascular smooth muscle cells (Lu et al., 2010).

The discrete membrane localization of the BK_Ca channel with its effectors and regulators might be an important mechanism to modulate BK_Ca function in myometrium. In support of this idea, a sub-population of BK_Ca channels in MSMCs localizes to caveolae where they associate with both structural components of caveolae, caveolin-1, and caveolin-2, and cytoskeletal proteins, α- and γ-actin (Brainard et al., 2005). Specific down-regulation of caveolin-1 decreases BK_Ca currents and alters localization of BK_Ca channels from detergent-resistant to detergent-soluble membrane microdomains (Brainard et al., 2009). This effect is also observed by deleting the entire caveolin-binding motif in the C terminus of the BK_Ca channel (Alioua et al., 2008) or by mutating key amino acids in this region (Brainard et al., 2009). Moreover, disruption of caveolae by depletion of membrane cholesterol or depolymerization of the actin cytoskeleton increases BK_Ca activity in human MSMCs (Brainard et al., 2005). Conversely, cholesterol depletion decreases BK_Ca activity in rat MSMCs (Shmygol et al., 2007b). These contradictory observations might be explained if the cholesterol-depleting agent used in both studies differentially affected other membrane-bound proteins such as Ca²⁺ or K⁺ channels (Levitan et al., 2010). Nonetheless, it is tempting to speculate that differential localization of BK_Ca isoforms within caveolar domains of the plasma membrane partially explains the Ca²⁺-insensitive BK_Ca currents that are observed in laboring myometrium (Khan et al., 1993).

The BK_Ca channel possesses numerous phosphorylation sites, and the phosphorylation state of these residues can regulate channel activity (Toro et al., 1998; Schubert and Nelson, 2001; Kyle et al., 2013). Below, we discuss three potential kinase modulators of BK_Ca channel activity in the myometrium: protein kinase A (PKA), protein kinase C (PKC), and protein kinase G (PKG).

In the myometrium, the association of PKA with the plasma membrane is regulated by progesterone and labor (Ku and Sanborn, 2002; Ku et al., 2005). Activation of the PKA pathway by cyclic AMP contributes to uterine quiescence during pregnancy through phosphorylation of various proteins (Lopez Bernal, 2007; Tyson et al., 2008). The BK_Ca channel is one such target; in non-pregnant myometrium, PKA inhibits BK_Ca channels, whereas in pregnant myometrium, phosphorylation by PKA activates the channel (Perez and Toro, 1994). This disparity may be explained by the fact that, as mentioned in section Splice variants, different splice variants of the BK_Ca channel respond in distinctive ways to PKA modulation (Tian et al., 2001; Zhou et al., 2001).

PKC is a serine/threonine kinase activated by increasing intracellular levels of diacylglycerol or Ca²⁺. In vascular SMCs, PKC directly phosphorylates the BK_Ca channel α-subunit, reducing its activity (Schubert and Nelson, 2001; Zhou et al., 2010). In these cells, PKC can also reduce BK_Ca channel activity indirectly by decreasing the release of Ca²⁺ sparks from the sarcoplasmic reticulum (Bonev et al., 1997; Hristov et al., 2014). Although the PKC modulation of agonist-dependent myometrial contractions has been explored (Phillippe, 1994; Breuiller-Fouche et al., 1998; Eude et al., 2000), the role of BK_Ca channels in this process remains elusive.

PKG, a serine/threonine-specific protein kinase that is activated by intracellular cyclic GMP, enhances BK_Ca activity by direct phosphorylation of serine residues (Alioua et al., 1998; Kyle et al., 2013). In SMCs, PKG has been shown to activate BK_Ca channels (Robertson et al., 1993; Archer et al., 1994; Zhou et al., 1996). Likewise, PKG enhances the activity of BK_Ca channels originally cloned from myometrium and subsequently expressed in a heterologous system (Zhou et al., 1998). Furthermore, PKG activation increases the activity of BK_Ca channels in myometrium (Zhou et al., 2000b), suggesting a role for PKG in maintaining uterine quiescence by modulation of BK_Ca channel activity. Functional contraction studies aimed at dissecting the effects of PKG on BK_Ca currents in non-pregnant and pregnant myometrium are required to elucidate whether this interaction has a role in the myometrium during pregnancy or labor.

The pore-forming BK_Ca channel α-subunits can associate with and be regulated by auxiliary β- and γ-subunits (Knaus et al., 1994b; Tanaka et al., 1997; Yan and Aldrich, 2012). Four distinct β-subunits proteins (β1-4) have been found to regulate the function and localization of the BK_Ca channel α-subunit (Knaus et al., 1994a; Wallner et al., 1999; Behrens et al., 2000; Brenner et al., 2000; Uebele et al., 2000). We will focus on the β1- and β2-subunits as these are expressed in MSMCs (Behrens et al., 2000; Chan et al., 2014). In addition, four members of a γ-subunit family, also known as leucine-rich repeat-containing (LRRC) proteins, that associate with the BK_Ca channel α-subunits: LRRC26 (γ1), LRRC52 (γ2), LRRC55 (γ3), and LRRC38 (γ4) (Yan and Aldrich, 2012) will be examined.

Numerous hormones regulate BK_Ca channel expression and activity in different tissues. Two relevant steroid hormones in the uterus, estrogens and progesterone, are key regulators for both maintaining uterine quiescence during pregnancy and for inducing labor at term. Although the levels of both hormones increase during pregnancy in humans (Boroditsky et al., 1978; Buster et al., 1979; Montelongo et al., 1992), changes in responsiveness of the target cells are key for their function. Here, we discuss ways in which BK_Ca might contribute to myometrial cell responsiveness to estrogens, progesterone, and also the hormone human chorionic gonadotropin.

The steroid hormone 17β-estradiol (E₂) helps maintain pregnancy. As such, circulating E₂ levels rise throughout pregnancy (Boroditsky et al., 1978; Buster et al., 1979; Montelongo et al., 1992), and the activity of the estrogen receptor α (ERα) is increased in myometrium near term (Mesiano and Welsh, 2007; Welsh et al., 2012). E₂ regulates expression of the BK_Ca channel by species-specific mechanisms. For example, expression of the mouse BK_Ca gene (mSlo1) is up-regulated by E₂ through activation of ERα and binding to estrogen response elements in the mSlo1 promoter (Kundu et al., 2007). Expression of the human homolog (KCNMA1 or hSlo1) is also up-regulated by E₂ interaction with ERα, but through the phosphatidylinositol 3-kinase pathway (Danesh et al., 2011). Furthermore, E₂ activation of ER decreases expression of the STREX variant in rat myometrium, mimicking the effect of pregnancy on this variant (Zhu et al., 2005). In addition, E₂ augments the expression of the BK_Ca auxiliary β1-subunit in mouse uterus (Benkusky et al., 2002). Although less studied, the estrogen receptor β (ERβ) has also been suggested to play a role in myometrial quiescence and labor (Wu et al., 2000). Furthermore, ERβ is necessary for the E₂-induced increase in BK_Ca currents in a neuronal cell line (Nishimura et al., 2008), but whether ERβ modulates myometrial BK_Ca currents has not been studied.

Although not yet fully explored, it is feasible that, at the onset of labor, E₂ triggers activation of BK_Ca channel activity directly rather than by activation of ERα and up-regulation of BK_Ca gene expression in MSMCs. This is a strong possibility because BK_Ca channel expression is reduced at the end of pregnancy (Matharoo-Ball et al., 2003; Gao et al., 2009; Chan et al., 2014). Additionally, E₂ can increase BK_Ca channel activity both in the presence (Valverde et al., 1999; De Wet et al., 2006) or absence (Wong et al., 2008) of the auxiliary β1-subunit by directly binding to the channel. An E₂-dependent increase in BK_Ca channel activity has also been observed in uterine vascular SMCs (Hu et al., 2011). However, a lower concentration of E₂ reduces BK_Ca currents and induces proteosomal degradation of the BK_Ca α-subunit (Korovkina et al., 2004). Hence, further studies are necessary to address the physiological significance of the E₂-BK_Ca channel interaction in the myometrium.

Myometrial quiescence during pregnancy is, in part, attributable to high plasma levels of the steroid hormone progesterone. Progesterone acts through its receptor PR to inhibit expression of contraction-associated proteins such as OTR, connexin 43, and cyclooxygenase-2, a key enzyme in the biosynthesis of prostaglandins (Renthal et al., 2010; Williams et al., 2012). Progesterone has been shown to inhibit BK_Ca channel currents in human sperm (Mannowetz et al., 2013) as well as in heterologous expression systems (Wong et al., 2008), suggesting a direct interaction between PR and the BK_Ca α-subunit. However, other evidence indicates that progesterone regulates expression of BK_Ca. For example, longer progesterone treatment increases mRNA and protein expression of the BK_Ca α-subunit in human immortalized MSMCs. Likewise, progesterone treatment decreases the expression of the β2-subunit (Soloff et al., 2011) without changing the expression of β1-subunit in mouse uterus (Xu et al., 2011). Although the effects of progesterone are wide and complex in the myometrium, elucidation of its effects on BK_Ca channel activity and expression will help to inform our understanding of the regulation of myometrial function by this hormone.

The human chorionic gonadotropin (hCG) is a glycoprotein produced mainly by the placenta. In addition to its role in sustaining early pregnancy, hCG may also participate in maintaining uterine quiescence during pregnancy. One study reported that hCG induces a potent relaxation of human myometrium in vitro, an effect partially attributable to an hCG-dependent increase in BK_Ca currents in MSMCs (Doheny et al., 2003). Simultaneously, another study found that certain unidentified chorionic-derived factors reduce oxytocin-mediated contraction in guinea pig myometrium in a paracrine manner, an effect that involves the activation of myometrial BK_Ca channels (Carvajal et al., 2003). Thus, BK_Ca channel seems to be a predominant effector of the uterorelaxant effects of chorionic-derived factors, including hCG.

Other modulators of vascular smooth muscle such as nitric oxide (NO) and certain eicosanoids have been reported to change BK_Ca channel activity in the myometrium. NO is a gaseous molecule that acts as a potent vasodilator mainly via activation of soluble guanylyl cyclase and production of cGMP in smooth muscle. NO production increases during pregnancy (Choi et al., 2002), and decreases toward labor, suggesting a role in regulating uterine contractility. NO has been shown to increase the open probability of the BK_Ca channel in human MSMCs (Shimano et al., 2000), but whether this occurs by a direct interaction or by cGMP-dependent pathways is unknown.

Another modulator of BK_Ca channels in the myometrium is the non-prostanoid eicosanoid, 5,6-epoxyeicosatrienoic acid (5,6-EET), a metabolite of arachidonic acid. The 5,6-EET isomer, the most abundant eicosanoid isomer in myometrial tissue (Zhang et al., 2007), reduces oxytocin-induced contractions in human pregnant myometrium by increasing BK_Ca currents (Pearson et al., 2009). Additional studies should elucidate the nature of this interaction and its physiological significance in the myometrium, as well as in other tissues.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.