BK channels, also called MaxiK/Slo1/K_Ca1.1 channels, are a class of K⁺ ion channels that undergo extensive pre- and post-translational modification. BK channel α subunits are encoded by the KCNMA1 gene, also known as SLO, and are ubiquitously expressed throughout mammalian tissues (e.g., neurons, smooth and skeletal muscles, exocrine cells). BK channels are assembled and strategically positioned on membrane surfaces, including the plasma membrane (Latorre et al., 1989), mitochondria and nucleus (Singh et al., 2012). Functional BK channels are multimeric structures composed of four similar pore-forming α subunits (Shen et al., 1994) and up to four regulatory β subunits can co-assemble with the tetrameric α subunit complex. The synergistic activation of BK channels by Ca²⁺ ions and depolarization causes a substantial K⁺ current that exhibits a large or “big” single channel conductance (i.e., up to 250 pS under symmetric K⁺ conditions). Activation of this formidable ionic current serves to drive membrane potential in the negative direction.

The transmembrane portion of the BK channel α subunit structure is thought to largely resemble that of voltage-gated K⁺ (K_v) channel subunits in terms of voltage-sensing and pore-forming domains. Notably, BKα subunits contain an additional transmembrane segment, termed S0, resulting in an extracellular N-terminus. Specialized charged residues are present within the transmembrane segments S2–S4 of the BKα subunit that contribute to its voltage-sensing properties. While topologically similar to their K_v channel counterparts, BK channels display weaker or less sensitive voltage-dependent activation (i.e., the ionic conductance-voltage relation is less steep), due to an altered distribution of voltage-sensing residues within the S2–S4 segments (Ma et al., 2006). Mechanistically, membrane depolarization drives conformational re-arrangements in the voltage sensor domains, resulting in an upward twisting of the S4 segment relative to the pore domain; these conformational movements are reversed upon repolarization (Hoshi et al., 2013).

The C-terminal domain of the BKα subunit contains a considerable range of specialized structures that regulate channel function. These include several binding sites for divalent cations (i.e., Ca²⁺ and Mg²⁺) and regions that undergo dynamic post-translational modification such as phosphorylation. Each mammalian BKα subunit contains two “regulators of K⁺ conductance” (RCK) domains, arranged in tandem along the C-terminus; in the tetrameric channel complex, these RCK domains co-assemble to form an octomeric gating ring structure in the cytosol (Yuan et al., 2010). The RCK domains also have Ca²⁺-binding regions and are crucial in conferring the channel's Ca²⁺ ion sensing properties (Cui et al., 2009). Ca²⁺ ions bind to these specialized regions within the BKα C-terminus, leading to a structural expansion of the intracellular region of the ion conduction pathway that facilitates gating and K⁺ efflux (Yuan et al., 2012; Hoshi et al., 2013).

Unlike the K_v channel superfamily, which uses different genes to increase its genetic diversity, BK channels derive functional diversity through the alternative post-transcriptional splicing of mRNA derived from the single KCNMA1 gene encoding the BKα subunit (Shipston, 2001). Up to ten distinct splice sites have been described in KCNMA1 (Poulsen et al., 2009), leading to the generation of BKα subunits with different phenotypes and various functional roles, including altered sensitivity to Ca²⁺ and/or voltage (Shipston, 2001; Johnson et al., 2011), responses to phosphorylation (Tian et al., 2001), signaling cascades (Schubert and Nelson, 2001; Tian et al., 2001, 2004), membrane expression regulation (Alioua et al., 2008; Ahrendt et al., 2014), trafficking and lipidation (Toro et al., 2006; Zarei et al., 2007; Shipston, 2014). The impressive range of phenotypic products that can result from differential splicing of the KCNMA1 gene product contributes to diversity of BK channel function between tissues, cells and intracellular compartments.

BK channels can co-assemble with modulatory auxiliary subunits BKβ 1-4 (Knaus et al., 1994a; Tanaka et al., 1997; Brenner et al., 2000a; Uebele et al., 2000), as well as a newly defined family of leucine-rich repeat containing subunits (LRRCs), referred to as γ subunits (Yan and Aldrich, 2010, 2012). Both BKβ and γ subunits contain sizeable extracellular regions and it is thought that these regions physically interact with the membrane-spanning domains of the BKα subunit. In particular, BKβ subunits appear to interact mainly with the N-terminal S0–S2 segments of the pore-forming BKα subunit (Morrow et al., 2006; Liu et al., 2008; Morera et al., 2012), thereby regulating channel opening through allosteric effects on the intramolecular processes underlying Ca²⁺ and/or voltage-dependent activation. As these auxiliary subunits are expressed in a tissue-specific manner, they confer distinct functional consequences by impacting BK channel kinetics and gating behavior. For instance, BKβ 1 subunits are typically expressed in smooth muscle, whereas BKβ 4 are expressed in neural tissue. BKβ subunits 1, 2 and 4 are reported to stabilize the channel's voltage sensor domains in the active conformation (Contreras et al., 2012), thereby enhancing channel activity, In contrast, BKβ 2 and β 3 subunits confer BK channel inactivation via an N-terminal “inactivation ball” (Wallner et al., 1999; Brenner et al., 2000a; Uebele et al., 2000) (Figure 1), which will limit K⁺ efflux and membrane hyperpolarization. To date, two functionally-distinct BKβ 2 splice variants (BKβ 2_a−b) have been described in mammals, although BKβ 2_b does not appear to inactivate the channel complex (Ohya et al., 2010). Similarly, four functionally-distinct BKβ 3 splice variants (BKβ 3_a−d) are known, with splice variants A-C conferring partial inactivation of BK channel current (Uebele et al., 2000). BKβ 4 subunits are the most distantly-related of the β subunits in terms of sequence similarity and produce mixed effects on BK channel gating, depending on the local Ca²⁺ concentration. At low Ca²⁺ concentrations, BKβ 4 appears to decrease channel activation, but at high Ca²⁺ concentrations, activation is enhanced (Brenner et al., 2000a; Wang et al., 2006).

The molecular mechanisms by which γ-subunits interact with and influence BK channel gating and kinetics are currently an area of active investigation. All four known LRRC proteins (i.e., LRRC26, 38, 52, and 55) have been reported to enhance voltage-dependent activation of BK channels (Yan and Aldrich, 2010, 2012), with LRRC26 producing an impressive shift of up to −150 mV.

Phasic smooth muscles, such as those lining the urinary bladder, urethra and ureters, undergo action potential (AP) events, with rapid depolarization-repolarization fluctuations. APs cause a significant global increase in intracellular [Ca²⁺] and BK channels are largely responsible for the rapid down-stroke (repolarization) phase (Burdyga and Wray, 2005; Thorneloe and Nelson, 2005; Kyle et al., 2013b). In contrast, tonic smooth muscles, such as those found throughout vascular tissue and much of the gastrointestinal tract and airways, regulate lower magnitude changes in membrane potential by principally responding to localized elevations in intracellular [Ca²⁺] mediated by ryanodine receptors (RyRs) (Figure 2). The dynamic post-translational “tuning” of BK channels permits considerable diversity in the biophysical properties of the current.

In common with many other tetrameric K⁺ channels in smooth muscles, the amplitude of K⁺ current carried through BK channels in smooth muscles can be dynamically regulated by post-translational modifications to the channel complex, including the reversible phosphorylation of the pore-forming BKα subunit by a number of protein kinases, as described below. Almost all phosphorylation sites are conserved in mammalian BK channel splice variants.

Many tissues have distinct macromolecular signaling complexes underlying the function of ion channels. Smooth muscles, for instance, generally have closely-associated RyRs, which periodically release Ca²⁺ and cause local elevations in [Ca²⁺]_i (i.e., 10–20 μM) (Pérez et al., 1999; ZhuGe et al., 2002) near BK channels positioned on the plasma membrane, which is sufficient to significantly raise the P_o and efflux K⁺ (Figure 2). The RyRs themselves are often close to Ca²⁺ influx pathways, for instance voltage-gated Ca²⁺ channels, or in proximity to IP₃ receptors (Ohi et al., 2001).

The primary role of BK channels in vascular smooth muscle (VSM) is to repolarize/hyperpolarize the cell membrane potential in the face of chronic depolarizing stimuli, thereby reducing contractile activity. It is now well-recognized that enhancement of BK channel current in VSM via phosphorylation is principally-regulated by nitric oxide (NO)/cGMP/PKG signaling (Feil et al., 2003) (see Section BK Channel Modulation via Protein Phosphorylation below). NO is a gaseous second messenger synthesized mainly by the adjacent endothelial cell layer lining the lumen of all blood vessels (Fleming and Busse, 2003). Therefore, BK channel activity is considered to be closely linked with endothelial cell activity. Therapeutically, NO and synthetic NO donors are used to treat a range of vascular disorders, including angina pectoris and hypertension (Wimalawansa, 2008).

In addition to the urinary tract and VSM, BK channels are also important regulators in mediating the proper function of various other smooth muscles, including those found in the gastrointestinal tract, airway, and uterus. Their function, however, varies between cell types and layers, and generally is dependent on the associated macromolecular signaling complex. In the colon, for instance, BK channels contribute to setting the resting membrane potential in longitudinal smooth muscle, whereas in the circular layer, they limit excitatory responses (Sanders, 2008).

In VSM, a single amino acid polymorphism in the BKβ 1 subunit (i.e., E65K) is reported to have a gain-of-function effect on BKs channel activation and has been associated with lower systolic and diastolic blood pressures and a decreased prevalence of diabetic hypertension in humans (Fernández-Fernández et al., 2004; Nielsen et al., 2008). In contrast, BKβ 1 subunit expression is decreased in some forms of genetic hypertension (Amberg and Santana, 2003). Moreover, a point mutation (R140W) in the BKβ 1 subunit that modestly impairs channel opening has been linked with asthma severity in African-American males (Seibold et al., 2008). Provocative data from Jaggar and colleagues further suggest that the majority of BKβ 1 subunits reside within the cell interior and assemble with α subunits at the cell surface in a dynamic fashion (Leo et al., 2014). NO signaling appears to promote the forward trafficking of internal BKβ 1 subunits to the cell membrane, where they co-associate with BKα subunits to enhance channel activation. The authors suggest that auxiliary BKβ 1 subunits undergo selective endocytosis from the plasma membrane, followed by re-insertion in response to a vasodilatory stimulus, such as NO. These data imply that native BK channels in VSM may not always contain a full complement of β 1 subunits (i.e., the ratio of β 1 to α subunits in a single channel complex is <1), as described in rat cremaster artery (Yang et al., 2009), and that the subunit stoichiometry of these channels is not permanent. Dynamic regulation of BK channel subunit co-assembly and interaction at the plasma membrane may thus represent a novel paradigm for the modulation of ion channel activity.

Many research groups have reported that BK channel activity is upregulated during hypertension, and its contribution is apparently enhanced compared to normotensive animals (for review, see Joseph et al., 2013). It should be noted, however, that downregulation of BK channel activity has also been reported during hypertension (Amberg et al., 2003; Amberg and Santana, 2003; Nieves-Cintrón et al., 2007; Yang et al., 2013). Investigators have speculated that this decrease may be due to reduced BKβ 1 subunit expression/coupling, which would dampen the Ca²⁺ sensitivity of BK channel activation. Several research groups have reported that BK current density is positively-correlated to blood pressure in hypertensive animals (Rusch et al., 1992; England et al., 1993; Rusch and Runnells, 1994; Liu et al., 1998). Aortic smooth muscle isolated from rats with renal hypertension, spontaneously-hypertensive rats (SHR) and stroke-prone SHR (Rusch et al., 1992; England et al., 1993; Liu et al., 1998) exhibits significantly-upregulated BK channel activity, likely as a compensatory response. Collectively, these studies indicate that the expression and function of BK channels in the vasculature involves complex expression and signaling pathways, and may vary between cells, tissues, vascular beds and pathophysiological profiles.

BK channels are densely-expressed in mammalian bladder tissues (~20 channels per square micrometer) (Ohi et al., 2001) with BKβ 1 auxiliary subunits. BKα subunit knockout mice have demonstrated bladder dysfunction and exhibit a depolarized resting membrane potential in isolated bladder smooth muscle cells and intact tissues, indicating a role for BK channels in setting the membrane potential (Sprossmann et al., 2009). Inhibition of BK channel current with iberiotoxin in the bladders of healthy mice led to similar effects (Heppner et al., 1997; Hristov et al., 2011). BKβ 1-knockout mice similarly display overactive bladder symptoms, and a significant decrease in BK channel activity (Petkov et al., 2001). Intriguingly, bladder smooth muscle tissue taken from patients with neurogenic bladder over-activity exhibit little to no response to BK channel inhibition by iberiotoxin, or the channel agonist NS1619, indicating severe BK channel dysfunction (Oger et al., 2010). Macroscopic current recordings from these tissues demonstrated a significantly lower BK channel current density that mirrors that reported for experimentally-induced partial urethral obstruction in rats (Aydin et al., 2012). Patients with benign prostatic hyperplasia experiencing overactive bladder symptoms also demonstrate a parallel reduction in BK channel expression (Chang et al., 2010). Overexpression of BK channel protein in rats with experimentally-induced partial urethral obstruction proved to be an effective treatment for the existing overactive bladder activity (Christ and Hodges, 2006). These data collectively indicate that BK channels are important regulators of bladder smooth muscle excitability, and a potential target for therapeutic intervention for overactive bladder conditions.

BK channels are abundantly expressed in both central and peripheral neurons, with prominent expression reported in both the cell body and pre-synaptic terminals (Faber and Sah, 2003). Functionally, these channels are key regulators of neuronal excitability, as channel opening will reduce action potential (AP) amplitude and duration, increase the magnitude of the fast after-hyperpolarization (fAHP) immediately following repolarization and limit the frequency of AP burst firing (Bielefeldt and Jackson, 1993; Faber and Sah, 2003; Gu et al., 2007; Haghdoost-Yazdi et al., 2008). At the pre-synaptic nerve terminal, localized BK channel activity can modulate both the amplitude and duration of depolarization-evoked Ca²⁺ entry as a result of the rapid repolarization and deactivation of voltage-gated Cav 2.1 (i.e., P/Q-type) and 2.2 (N-type) Ca²⁺ channels (Robitaille and Charlton, 1992; Issa and Hudspeth, 1994; Marrion and Tavalin, 1998; Fakler and Adelman, 2008). Reduced Ca²⁺ influx will limit vesicle fusion at active zones, leading to decreased neurotransmitter release (Roberts et al., 1990; Hu et al., 2001; Raffaelli et al., 2004).

Dissecting the functional roles of BK channels in the nervous system has been greatly aided by the availability of highly selective toxins (i.e., iberiotoxin) (Kaczorowski and Garcia, 1999) and small molecule inhibitors (e.g., penitrem A, paxilline, lolitrem B) (Knaus et al., 1994b; Imlach et al., 2008; Nardi and Olesen, 2008), along with the generation of genetically-engineered mice lacking either BKα or β subunits (Brenner et al., 2000b, 2005; Plüger et al., 2001; Meredith et al., 2004; Sausbier et al., 2004). Such strategies have revealed that the loss of neuronal BK current, either acutely or chronically, increases membrane excitability by decreasing the magnitude of the fAHP. Reducing the fAHP facilitates more rapid membrane depolarization in response to a tonic stimulus, leading to higher frequency AP firing. Such alterations in neuronal activity are typically associated with neurological disorders in the CNS, including tremor and ataxia (Sausbier et al., 2004; Brenner et al., 2005; Imlach et al., 2008). Interestingly, a point mutation in the RCK1 domain of the BKα subunit (i.e., D434G) identified in a subset of epileptic patients has been shown to increase neuronal BK channel activity by enhancing Ca²⁺-dependent channel gating (Du et al., 2005; Wang et al., 2009; Yang et al., 2010). Functionally, increasing BK activity and the associated fAHP may augment membrane excitability in the soma by enhancing the recovery rate of fast Na⁺ currents from voltage-dependent inactivation and reducing the absolute refractory period of neuronal firing.

In the CNS of mice and humans, genetic knockout or mutational disruption of the molecular chaperone cysteine string protein (CSPα) is linked with early onset neurodegeneration (Fernandez-Chacon et al., 2004; Donnelier and Braun, 2014), and interestingly, these conditions are associated with a significant up-regulation of BK channel expression in mouse brain and cultured neurons (Kyle et al., 2013a; Ahrendt et al., 2014). Although the mechanistic link between increased BK expression/activity and neurodegeneration remains undefined, it is hypothesized that increased BK current density in pre-synaptic terminals and/or the soma may lead to disrupted synaptic membrane excitability and neurotransmitter release. As described below, elevated BK channel expression in the CNS is closely linked with epilepsy, strongly suggesting that increased BK current density can lead to neurological disorders and possibly synaptic dysfunction/degeneration.

Heteromeric BK channel complexes are the subject of extensive post-translational modifications, which can significantly alter channel behavior. Some modifications are highly-complex and require prior upstream modification(s) to the channel subunits.

BK channel activity is regulated both directly and indirectly through a diverse range of modulatory pathways involving covalent modifications, metabolic factors, trafficking events and transcriptional processes (see Figure 3). Given the formidable effect that BK channels can exert on membrane excitability, as a result of their large single channel conduction and dual activation by membrane depolarization/cytosolic free Ca²⁺, such “fine-tuning” affords cells the ability to precisely control the impact of these channels on their function and responsiveness to both acute and chronic stimuli. As reinforced by the accompanying articles in this thematic issue, BK channels represent powerful effectors in tissue health and dysfunction and that understanding their modes of regulation may lead to novel therapeutic strategies in disease treatment.