Targeting BK (big potassium) Channels in Epilepsy

Abstract

Introduction

Epilepsies are disorders of neuronal excitability characterized by spontaneous and recurrent seizures. Ion channels are critical for regulating neuronal excitability and, therefore, can contribute significantly to epilepsy pathophysiology. In particular, large conductance, Ca²⁺-activated K⁺ (BK_Ca) channels play an important role in seizure etiology. These channels are activated by both membrane depolarization and increased intracellular Ca²⁺. This unique coupling of Ca²⁺ signaling to membrane depolarization is important in controlling neuronal hyperexcitability, as outward K⁺ current through BK_Ca channels hyperpolarizes neurons.

Areas covered

This review focuses on BK_Ca channel structure-function and discusses the role of these channels in epilepsy pathophysiology.

Expert opinion

Loss-of-function BK_Ca channels contribute neuronal hyperexcitability that can lead to temporal lobe epilepsy, tonic-clonic seizures and alcohol withdrawal seizures. Similarly, BK_Ca channel blockade can trigger seizures and status epilepticus. Paradoxically, some mutations in BK_Ca channel subunit can give rise to the channel gain-of-function that leads to development of idiopathic epilepsy (primarily absence epilepsy). Seizures themselves also enhance BK_Ca channel currents associated with neuronal hyperexcitability, and blocking BK_Ca channels suppresses generalized tonic-clonic seizures. Thus, both loss-of-function and gain-of-function BK_Ca channels might serve as molecular targets for drugs to suppress certain seizure phenotypes including temporal lobe seizures and absence seizures, respectively.

1. Introduction

Epilepsies are disorders of neuronal hyperexcitability that are characterized by spontaneous and recurrent seizures. Seizure disorder etiologies are usually divided into idiopathic, cryptogenic and symptomatic forms. Idiopathic epilepsies are characterized by focal or generalized seizures that do not result from brain lesion or metabolic abnormality. Seizure predisposition is common in idiopathic epilepsies, and genetic defects including mutations in genes encoding for Ca²⁺-dependent ion channels are important in the etiology of this neurological disease. Dysregulation of Ca²⁺ homeostasis and Ca²⁺ signaling are also important factors that contribute to epileptogenesis in acquired epilepsy including temporal lobe epilepsy (TLE).

Potassium channels control the resting membrane potential and therefore play a critical role in regulating neuronal excitability. Based on their structure, biophysical characteristics, pharmacological sensitivities and physiology, K⁺ channels are classified as voltage-gated (K_v), inwardly rectifying, Ca²⁺-activated and tandem-pore K⁺ channels. Potassium channels contribute to the pathophysiology of a broad range of neurological disorders. In particular, inherited dysfunctions of K_v channels, KQT-like subfamily, member 2 and 3, caused by gene mutations are known to contribute to the development of common idiopathic epilepsy syndromes in humans [1]. Also important to epilepsy causation is the large conductance, voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels, which are gated by membrane depolarization and rise in intracellular Ca²⁺ ([Ca²⁺]_i) levels. This unique feature of BK_Ca channels signaling to membrane depolarization is important for controlling neuronal hyperexcitability as the resulting outward K⁺ current through these channels serves to repolarise or hyperpolarize the membrane. In addition, the exquisite sensitivity of BK_Ca channels to Ca²⁺ provides an important negative-feedback system for controlling Ca²⁺ entry into neurons, either by deactivating voltage-gated Ca²⁺ (Ca_v) channels or increasing the activity of the Na⁺/Ca²⁺ exchangers, thus regulating Ca²⁺-dependent processes.

Under physiological conditions, activation of neuronal BK_Ca channels contributes to action potential (AP) repolarization, gives rise to the fast afterhyperpolarization (fAHP) that follows the AP, shapes dendritic Ca²⁺ spikes and influences neurotransmitter release [2, for review]. Thus, activation of BK_Ca channels is an intrinsic inhibitory mechanism to counter membrane depolarization and the excessive accumulation of cytosolic Ca²⁺ that occurs during seizures. Accordingly, putative loss-of-function mechanisms of BK_Ca channels have been associated with TLE, as fAHP conductances (mainly mediated by BK_Ca channels) were reduced in epileptic patients [3]. Paradoxically, gain-of-function mutations in BK_Ca channel subunit genes actually contribute to the development of certain forms of idiopathic generalized epilepsy in humans [4]. Thus, both gain-of-function and loss-of-function BK_Ca channels are potentially important molecular targets for developing drugs to prevent epileptogenesis and suppress both temporal lobe seizures (TLS) and absence seizures.

This review summarizes our current knowledge of BK_Ca channel structure-function and discusses the contribution of BK_Ca channels gain-of-function and loss-of-function mechanisms to epileptogenic neuronal hyperexcitability, and their potential as therapeutic targets for epilepsy.

2. BK_Ca channels

2.1 Structure and biophysical characteristics of BK_Ca channels

When unitary conductance is determined from current-voltage plots in symmetrical [K⁺] (>100 mM), three groups of Ca²⁺-activated K⁺ channels can be distinguished: large conductance (100-300 pS; BK_Ca), intermediate conductance (25-100 pS; IK_Ca), and small conductance (2-25 pS; SK_Ca) channels [5, for review]. Unlike SK_Ca and IK_Ca channels, BK_Ca (also known as BK, Slo1, Big K⁺, MaxiK or KCNMA1) channels are activated by both elevated [Ca²⁺]_i and membrane depolarization. The mechanisms of Ca²⁺ and voltage dependency of BK_Ca channels are independent, with each mechanism being able to increase the open-channel probability [6]. Under physiological conditions, BK_Ca channels have a low affinity for Ca²⁺ such that >10 μM [Ca²⁺]i is required for channel activation [7]. This sensitivity of BK_Ca channels to Ca²⁺ is an important negative feedback mechanism for controlling Ca²⁺ entry and subsequent Ca²⁺-dependent processes. Structurally, a minimal functional BK_Ca channel consists of four identical pore-forming α subunits, which determine the basic properties of unitary conductance, voltage- and Ca²⁺-sensitivity, and channel opening probability. In most tissues, however, the BK_Ca channel α subunits are associated with up to three regulatory β subunits, whose expression are rather tissue-specific.

2.1.1 Pore-forming α subunit

The BK_Ca channel pore-forming α-subunit is encoded by the KCNMA1 (human) or Kcnma1 (mouse) gene [8]. Like Ca_v channels, voltage-gated Na⁺ (Na_v) channels and other K_v channels, BK_Ca channel α subunits have six transmembrane segments (S1-S6) at the short extracellular N-terminus [5]. In addition, BK_Ca channels have a unique hydrophobic segment (S0) that leads to an exoplasmic N-terminus and additional four hydrophobic segments (S7-S10) in the large intracellular C-terminus [9]. The K⁺-selective pore is located at the center of four α subunits and is surrounded by the voltage-sensing domain (S1-S4 segments) [10]. The acidic residues in S2 and S3 segments, together with basic (positively charged) residues in the S4 segment, confer voltage sensitivity to BK_Ca channels [10,11], and membrane depolarization causes movement of these charged residues, resulting in the opening (gating) of the K⁺-selective pore [6,9,11]. The pore-forming motif is a loop between S5 and S6 segments that controls channel permeation and the S6 segment is believed to serve as the major structural determinant for the channel gate [12].

The intracellular C-terminal domain contains two regulators of conductance for K⁺ (RCK1 and RCK2) domains and contributes to the physiological regulation of BK_Ca channels by Ca²⁺ and other intracellular stimuli [13,14,15]. The distal region (S9-S10 segments) or tail domain of the C-terminus contains the Ca²⁺ bowl, an aspartate-rich region that is a structural element of the RCK2 domain [13,15]. Each BK_Ca channel α-subunit contains two tandems RCK1-RCK2 domains [14], and the channel sensitivity to Ca²⁺ is determined by a “gating ring” of eight RCK domains from four assembled α subunits [13,15,16]. The BK_Ca gating ring has a wide central hole that allows ions to move freely between the conduction pore and the intracellular side [15]. Thus, BK_Ca channel gating occurs via two independent sensing mechanisms, one that is voltage-dependent in the N-terminus and the other one that is Ca²⁺-dependent in the C-terminus.

BK_Ca channels have at least one low-affinity and two high-affinity Ca²⁺-recognition sites. The high-affinity Ca²⁺ recognition sites are located in RCK1 domain [17,18] and in the Ca²⁺ bowl of RCK2 domain [13,15,19]. The low-affinity Ca²⁺ recognition site is located in the RCK1 domain, but its function has not been clearly established [17]; it is known, however, that under physiological conditions (in which [Ca²⁺]_i is in the range of a few hundred nanomolar to tens of micromolar), the low-affinity Ca²⁺-recognition site(s) is not occupied by Ca²⁺ [20]. This observation suggests that this site could exert only a small effect (if any) in increasing the open-channel probability following an increase in [Ca²⁺]_i.

In addition to membrane depolarization and increased [Ca²⁺]_i, BK_Ca channels are also activated by intracellular Mg²⁺ [20,21] and the low-affinity Ca²⁺-recognition site in RCK1 also mediates the channel's sensitivity to Mg²⁺ [20,21,22]. In addition, the Ca²⁺ bowl in RCK2 also binds Mg²⁺, albeit with low-affinity [23]. BK_Ca channels are further regulated by intracellular H⁺. Although some reports have conflicted, evidence shows that unlike other K_v channels, lowering [24] or increasing [25] intracellular pH, under defined conditions, enhances or decreases BK_Ca channel conductances, respectively. The RCK1 domain of BK_Ca channel also serves as the proton sensor [26], suggesting a common sensing domain for Ca²⁺ and protons. Other regulatory domains sites located in the C-terminus include phosphorylation sites for c-AMP-dependent protein kinase (PKA) and protein kinase C (PKC). Phosphorylation by PKA and PKC activates and inhibits BK_Ca channels, respectively [27]. Thus, metabolic factors can play an important role in controlling BK_Ca channel activity.

The BK_Ca channel KCNMA1gene is subjected to alternative splicing that results in number of transcription variants. Alternatively spliced BK_Ca channel variants exhibit both Ca²⁺ and voltage sensitivity [28]. Multiple sites of alternative splicing in the KCNMA1 gene have been identified, with the majority being located within the C-terminus. Interestingly, two splice isoforms of the KCNMA1 gene are expressed in the brain including the so-called stress axis hormone-regulated exon (STREX) and ZERO variant [28,29]. Thus, STREX-containing BK_Ca channel splice variant has a cysteine-rich insertion that the ZERO variant lacks [28,29]. Compared to the ZERO variant, the STREX variant accelerates and slows the channel's activation and deactivation kinetics, respectively, resulting in a dramatic enhancement of BK_Ca channel open time [29]. Thus, STREX-containing BK_Ca channel splice variant exhibits biophysical characteristics that may have functional relevance, as KCNMA1 mutation that enhances BK_Ca conductances (gain-of-function) give rise to idiopathic generalized epilepsy [4].

2.1.2 Regulatory β subunits

Four different types of BK_Ca channel β subunit (called β1 trough β4) have been identified thus far, each encoded by a specific gene KCNMB1-4 (human) or Kcnmb1-4 (mouse) [30]. In non-excitable cells, a novel BK_Ca channel auxiliary subunit, leucine-rich repeat containing protein (LRRC26) that is unrelated to the neuronal β1–4 subunits has been identified and characterized [31]. Structurally, the BK_Ca channel β subunit has two membrane-spanning domains and each subunit is interposed between two adjacent α subunits [32]. Functionally, β subunits alter the gating and pharmacological sensitivity of BK_Ca channels, as well as their regulation by phosphorylation [33,34]. In particular, the regulatory β1 subunit enhances Ca²⁺ sensitivity of BK_Ca channels and slows the rate of channel opening, whereas β2 subunit confers fast inactivation kinetics [35,36]. The β3 subunit increases BK_Ca channel activity [7,36] and alternative splicing of this gene's RNA produces four distinct isoforms (β3a-d), each of which has four sequence variants (V1-V4) [37,38]. The (β3a through (β3c subunits cause partial inactivation of BK_Ca channels [38], whereas the β3d subunit has no obvious effect on channel gating [38]. Of the four variants in the β3 subunit family, only the V4 variant of the β3 subunit markedly alters the channel's gating properties, including its fast inactivation kinetics [37]. Finally, the brain-specific BK_Ca channel regulatory β4 subunit increases and decreases channel activity in high and low [Ca²⁺]_i, respectively [7]. The β4 subunit also slows the activation and deactivation kinetics of BK_Ca channels [7] and decreases the channel's sensitivity to pore blockers such as charybdotoxin and iberiotoxin [34,39]. Moreover, the BK_Ca channel β4 subunit inhibits STREX variant channels via a Ca²⁺-dependent mechanism [40]. Thus, at low [Ca²⁺]_i, the β4 subunit reduces the open probability of the STREX variant channel by slowing its activation. At intermediate and high [Ca²⁺]_i (i.e., >5.3 μM), the β4 subunit reduces BK_Ca channel open probability by opposing the slow deactivation conferred by the STREX exon. In contrast to its effect on STREX variant channels, β4 subunit slows both the activation and deactivation kinetics of ZERO variant channels. Thus, it has been suggested that local Ca²⁺ environment in combination with ZERO or STREX variant BK_Ca channels will determine whether β4 subunit containing BK_Ca channels can contribute to shaping the AP or regulating neurotransmitter release [40].

2.2 Localization of BK_Ca channels in the central nervous system

2.2.1 BK_Ca channel α subunit

BK_Ca channel α subunits are ubiquitously expressed in the central nervous system (CNS), yet they are preferentially located at the terminal areas of primary projection tracts [41], which is consistent with the role of these channels in regulating neurotransmitter release. Accordingly, in the cortex and hippocampus, BK_Ca channel α subunits are more abundantly expressed in glutamatergic nerve terminals than in GABAergic nerve terminals [42]. BK_Ca channel α subunits are also present in neuronal post-synaptic compartments [43], where they contribute to AP repolarization at the soma and generate the fAHP [44]. High expression levels of α subunit protein and mRNA are present in the neocortex, olfactory system, piriform cortex and hippocampus [41,45]. BK_Ca channel α subunit proteins are also found in mossy fibers, the axons of dentate gyrus (DG) granule cells that innervate CA3 pyramidal neurons, and within the perforant pathway, which is the principal excitatory projection innervating the hippocampus [41]. The presence of BK_Ca channel α subunit proteins in CA3 and CA1 pyramidal neurons and in hippocampal projections [46] may be of pathophysiological relevance because these limbic networks contribute to TLE epileptogenesis. The BK_Ca channel α subunit is also moderately expressed in the cortical and basolateral nuclei of the amygdala [45,46], a limbic structure that is also implicated in the pathogenesis of TLE.

In the basal ganglia, BK_Ca channel α subunit proteins are highly concentrated in globus pallidus, substantia nigra pars reticulata and entopeduncular nucleus [41,45,46], where this channel may play a role in the pathophysiology of ataxia and tremors. The BK_Ca channels α subunit proteins are also expressed in the thalamus, hypothalamus and habenula [45,46]. Interestingly, the thalamus plays a critical role in the pathophysiology of absence epilepsy in humans. Mesencephalic nuclei, including the periaqueductal gray, which plays an important role in the networks for pain and seizures, express BK_Ca channel α subunit proteins, at low to moderate levels [46]. The inferior colliculus (IC), which is critical in initiating acoustically evoked generalized tonic-clonic seizures, has moderate expression of BK_Ca channel α subunit proteins in the central nucleus and external cortex and high expression in the dorsal cortex [46]. Finally, in the cerebellum, the Purkinje cells, molecular layers and deep nuclei as well as the interpeduncular nucleus, all express BK_Ca channel α subunit mRNA at high levels [46].

ZERO variant BK_Ca channels are abundant in the brain, whereas STREX variant channels are only weakly expressed [28]. STREX-containing BK channels are primarily present in the pituitary gland, neocortex, hippocampus and cerebellum [40]. In the hippocampus, STREX variant channels are present in the DG granule cells and in CA3 pyramidal neurons, whereas these channels exhibit a weak expression in CA1 neurons [40]. Thus, STREX variant channels may play an important role in controlling excitability of DG granule cells and CA3 pyramidal neurons.

In CNS neurons, BK_Ca channel α subunits are also present in the mitochondria [47]. These channels are localized in the inner membrane and found in a limited subset of mitochondria in several brain structures, including the cerebral cortex, hippocampus and cerebellum [47]. The mitochondrial BK_Ca (called mitoBK_Ca) channel is also selective for K⁺ and activated by both Ca²⁺ and voltage, and has a conductance of 260-320 pS [48]. The structure of brain mitoBK_Ca channels remains unknown. Nevertheless, it is possible that both neuronal membrane and mitoBKCa channels are splice variants of the same KCNMA1 gene, as this gene may undergo extensive pre-mRNA splicing [49].

2.2.2 BK_Ca channel β subunits

In contrast to the widely distributed BK_Ca channel α subunit, the four regulatory β subunits display a restricted pattern of expression [37,38]. BK_Ca channel β1 and β4 subunit expression seems focused to non-CNS and CNS tissues, respectively [50]. In the CNS, the expression of BK_Ca channel β2, β3, and β4 subunit mRNA ranges from very weak to high in various structures including the neocortex, hippocampus, amygdala, substantia nigra, thalamus, and spinal cord [7,50,51].

Co-expression of BK_Ca channel α and β1 subunit mRNA has been clearly demonstrated in Purkinje cells, in some brainstem nuclei and in the hypothalamus [45]. Surprisingly, no co-expression of BK_Ca channel α and β subunits was found in the hippocampal formation (DG, CA1-3 areas), the amygdala, and all thalamic areas except zona incerta [45]. This apparent lack of BK_Ca channel β subunit expression in these areas might be due to weak expression that could not be detected using in situ hybridization methods. Interestingly, both the STREX variant and the β4 subunit are present in CA3 pyramidal cells and in DG granule cells [40], and this may be of relevance in TLE pathophysiology. Of the four regulatory BK_Ca channel β-subunits, only β4-subunit are expressed in the inner membrane of neuronal mitochondria [51], which suggests an important role for this subunit in the mechanisms of neuroprotection or neurodegeneration.

Significant splicing of the α subunit-encoding KCNMA1 (see 2.1.1) or β subunit-encoding KCNMB3 (see 2.1.2) gene, differential tissue-specific expression of the various BK_Ca channel β subunits, and post-translational modification of channel subunits, all contribute to the particular BK_Ca channel phenotype that is expressed in a given tissue or brain region.

2.3 Regulation of BK_Ca channel activity

Given their role in controlling neuronal excitability, modulating BK_Ca channels may have clinical relevance. Many factors important to epilepsy pathophysiology, including Ca²⁺ and pH, regulate BK_Ca channel activity [52,53]. For instance, BK_Ca channels are activated by micromolar increases in [Ca²⁺]_i levels. Such increases in [Ca²⁺]_i levels are restricted to “Ca²⁺-nano/microdomains” in the immediate vicinity of the Ca²⁺ source. In the CNS, the Ca²⁺ that activates BK_Ca channels is provided primarily by Ca_v channels and N-methyl-D-aspartate (NMDA) receptors [45,54,55]. Indeed, BK_Ca channels co-localize with Ca_v channels, such that Ca²⁺ diffuse only ∼13 nanometers after entering the neuron before binding to BK_Ca channels [56]. The specific Ca_v type that interacts with BK_Ca channels varies from neuron to neuron and includes Ca_v1.2 (L-type), Ca_v2.1 (P/Q-type), Ca_v2.2 (N-type) and Ca_v3 (T-type), but not Ca_v2.3 (R-type) channels [55]. In some CNS neurons, BK_Ca channels that contain β2 or β4 subunits can associate with Ca_v1.2, Ca_v2.1 and Ca_v2.2 channels [55]. Co-localization of BK_Ca channels with NMDA receptors in CNS neurons [54] suggests that BK_Ca channels may an important role in shaping the action of glutamate and controlling its release in various brain regions. In addition to depolarization-dependent Ca²⁺ influx, BK_Ca channels also are activated by Ca²⁺ that is released from intracellular ryanodine-gated Ca²⁺ pools [57]. Whether Ca²⁺ released from inositol triphosphate-gated Ca²⁺ pools can also activate neuronal BK_Ca channels remains unknown. Based on their distribution, BK_Ca channels in the plasma membrane belong to two distinct pools, namely clustered and scattered channels, which are activated by Ca²⁺ release from intracellular Ca²⁺-gated stores and Ca²⁺ entry through Ca_v channels, respectively [58].

Membrane depolarization often leads to intracellular acidification, and many types of ion channels, including BK_Ca channels are sensitive to H⁺ exposure. Interestingly, acidification reduces the activity of BK_Ca channels, under high [Ca²⁺]_i conditions [26,59,60]. This sensitivity of BK_Ca channels to protons may play an important role in the pathophysiology of epilepsy, as intracellular acidification in some CNS neurons is tightly coupled to seizure termination [61]. Zinc (Zn²⁺) is an important intracellular molecule that may play an essential role in the control of neuronal excitability, as intracellular Zn²⁺ ([Zn²⁺]_i) activates BK_Ca channels [62]. Conversely, reduced [Zn²⁺]_i can contribute to neuronal hyperexcitability. In TLE, recurrent mossy fibers (which project from the DG granule cells to CA3 pyramidal neurons) synapse the DG, thus facilitating the synchronous firing of the granule cells. Interestingly, vesicular Zn²⁺ is abundant in mossy fibers. In a model of TLE, Zn²⁺ released from recurrent mossy fibers is thought to contribute to DG granule cells bursting [63]. Such epileptiform activity may be due to altered sensitivity of BK_Ca channels to [Zn²⁺]_i.

Numerous BK_Ca channel blockers and activators (i.e., openers) are used to identify these channels and study their functions. Several potent and commonly used BK_Ca channel blockers include tetraethylamonium (TEA), paxilline, penitrem A, charybdotoxin, iberiotoxin and quinoline. TEA has poor selectivity for BK_Ca channels, as it also blocks other K_v channels, including K_v7.2 and K_v7.4 channels [64]. Charybdotoxin, a peptide isolated from the venom of the scorpion Leiurus quinquestriatus, is a nonselective BK_Ca blocker that also blocks K_v1.2 and IK_Ca channels [65]. Iberiotoxin, a peptide isolated from the venon of the scorpion Buthus tamulus, the tremorgenic fungal toxins paxilline and penitrem A, and the quinoline alkaloid tetrandrine are all fairly selective for BK_Ca channels, but also suppress Ca²⁺-dependent chloride channels [66]. Paxilline blocks BK_Ca channels-containing β4 subunits whereas iberiotoxin exhibits low activity for these channels [34]. Interestingly, mitoBK_Ca channels are sensitive to both charybdotoxin and iberiotoxin [67]. Another BK_Ca channel blocker of interest include Slotoxin (αKTX 1.11), that was isolated from the venom of scorpion Centruroides noxius Hoffman. Slotoxin appears to selectively block the BK_Ca channels and can pharmacologically distinguish BK_Ca channel complexes containing α, α/β1 and α/β4 subunits [68]. Finally, cholesterol, an endogenous compound, inhibits BK_Ca channels activity by decreasing both the mean open and closed times [69].

BK_Ca channel activators are predicted to stabilize the neuron's resting membrane potential by increasing K⁺ efflux, which lead to membrane hyperpolarization and subsequently dampens neuronal excitability. Many compounds activate BK_Ca channels including synthetic benzimidazolone derivatives such has NS11021, biaryl amines (mefenamic and flufenamide acid), diaryl ureas (NS 1609), pyridyl amines, 3-aryloxin-doles, benzopyrans, dihydropyridines, and natural modulators such as dehydrosoyasaponin-1 and flavonoids. Among the commonly used BK_Ca channel activators, NS11021 is a compound of interest, as it increases the open probability of BK_Ca channels but does not affect Ca_v, K_v, or Na_v channels [70].

Several endogenous factors that have been implicated in the pathophysiology of epilepsy including nitric oxide, arachidonic acid metabolites, and estradiol also modulate BK_Ca channel activity through G-proteins, phosphorylation or dephosphorylation. Both arachidonic acid and nitric oxide potentiate BK channel activity by slowing inactivation kinetics and enhancing channel opening, respectively [71,72]. Similarly, 17β-estradiol increases BK_Ca channel currents by enhancing both channel opening and the expression of both α and β4 subunits [73]. Finally, ethanol, an exogenous compound that contributes to the etiology of alcohol withdrawal seizures, activates BK_Ca channels by increasing their mean open time and promoting Ca²⁺-driven channel gating [74].

2.4 The physiological role of BK_Ca channels

The primarily function of BK_Ca channels is to generate a fAHP after contributing to AP repolarization. The BK_Ca channel-mediated fAHP that follows an AP controls the shape and duration of that AP, thus determining the amount of Ca²⁺ that enters the neuron. Following a fAHP, many neurons exhibit a prolonged AHP that is comprised of medium (mAHP) and slow (sAHP) both of which are generated by SK_Ca channels; moreover, the sAHP is thought to play an important role in controlling spike frequency [75]. Based on their biophysical characteristics, two functionally groups of BK_Ca channels have been identified, namely inactivating and non-inactivating BK_Ca channels. The non-inactivating BK_Ca channels are further classified into two types including type I and type II channels. The type I non-inactivating BK_Ca channels contribute to AP repolarization and the induction of the fAHP and are sensitive to charybdotoxin and iberiotoxin, whereas the type II non-inactivating BK_Ca channels are slowly gated, iberiotoxin-resistant channels [34,76]. The molecular correlates of type I and type II non-inactivating BK_Ca channels are poorly understood. Nevertheless, BK_Ca channels that contain β4 subunits contribute to type II non-inactivating channels [34].

BK_Ca channels also play an important role in regulating neurotransmitter release at CNS nerve terminals. Calcium influx into presynaptic terminal via Ca_v channels initiates neurotransmitter release and activates BK_Ca channels, which in turn inhibit the release of neurotransmitter. It has been suggested that presynaptic BK_Ca channels are recruited only following conditions associated with a massive [Ca²⁺]_i accumulation in presynaptic terminals, for example during seizures [77]. Under such conditions, BK_Ca channels would limit depolarization-induced bursting activity by hyperpolarizing the presynaptic membrane, leading to deactivation of Na_v and Ca_v channels, and thereby inhibiting subsequent APs. This mechanism provides a rapid inhibitory response at the presynaptic terminals that is consistent with their role as an “emergency brake” that protects against neuronal hyperexcitability and prevents seizure-related neurodegeneration [78]. Another important role for BK_Ca channels is to prevent excessive [Ca²⁺]_i increases by repolarizing the membrane, deactivating Ca_v channels and increasing activity of the Na⁺/Ca²⁺ exchanger.

Neuronal mitochondria are an important system for sequestering [Ca²⁺]_i. Physiologically, mitoBKCa channels are important for the K⁺ transport. Unlike BK_Ca channels in the plasma membrane, opening of mitoBK_Ca channels results in depolarization of the inner mitochondrial membrane, which in turn modulates the mitochondria function. MitoBK_Ca channels may constitute a link between cytosolic and intramitochondrial Ca²⁺ and mitochondrial membrane potential dependent reactions. The functional role of mitoBK_Ca channels is poorly understood. Nevertheless, these channels may play an important role in both buffering [Ca²⁺]_i and neuroprotection or neurodegeneration.

In general, the activation of BK_Ca channel reduces neuronal excitability, an action that should dampen epileptiform bursts. However, in some neurons BK_Ca channel activation enhances neuronal excitability. For instance, in hippocampal CA1 pyramidal cells, BK_Ca channels-containing β2 subunits promote frequency-dependent AP broadening during repetitive firing, an effect that is reduced by BK_Ca channel blockers [76,78,79]. This effect is mediated through rapid spike repolarization and the fAHP, which may limit the inactivation of Na_v channels and the activation of other slower K_v channels (such as delayed rectifier K⁺ channels) during interspike interval [80]. Similarly, in DG granule cells, β4 subunits are believed to prevent BK_Ca channels from contributing to membrane repolarization, as genetically deleting of these subunits promotes both high frequency firing and AP sharpening (or narrowing), thus reducing AHP duration [81].

2.5 The role of BK_Ca channels in epilepsy pathophysiology

Complex spike bursts caused by enhanced inward currents through Ca_v channels constitute a critical epileptogenic mechanism [52]. BK_Ca channel currents, which are Ca²⁺ dependent, oppose neuronal activity and reduce neuronal hyperexcitability that could otherwise lead to seizures. Surprisingly, blocking BK_Ca channels inhibits epileptiform bursting in models of acute seizures and inherited epilepsy [82]. BK_Ca channel blockade also inhibits bursting activity induced by elevated [Ca²⁺]_i following Ca²⁺ release from intracellular Ca²⁺-gated pools [82]. Because BK_Ca channels are important in controlling neuronal excitability under normal physiological conditions, it may seem counterintuitive to postulate that BK_Ca channel activation might contribute to neuronal hyperexcitability. Nevertheless, such effects can occur in the presence of BK_Ca channels that carry a gain-of-function mutation in their a-subunits [4].

2.5.1 Clinical studies

BK_Ca channels were first implicated in seizure-inducing neuronal hyperexcitability when it was reported that fAHP conductances were reduced in DG granule cells obtained from surgical samples from TLE patients [3]. At the time, the mechanism(s) by which altered BK_Ca channels contributed to seizures include downregulation of channel expression, altered channel trafficking and insertion to the plasma membrane or a loss-of-function mutation that gives rise to nonfunctional or hypo-functional channel. However, recent evidence indicates that some mutations in the genes encoding BK_Ca channels give rise to a gain-of-function mechanism that is associated with human idiopathic generalized epilepsy and absence epilepsy in particular [4].

Evidence indicates that the D434G missense mutation in the α subunit KCNMA1 gene may contribute to inherited generalized epilepsy (manifesting primarily as absence epilepsy, although generalized tonic-seizures occur in some patients), which is associated with paroxysmal dyskinesia [4]. This mutation is characterized by an increase in BK_Ca channel's sensitivity to Ca²⁺, enhanced voltage- and Ca²⁺-dependent channel activation, increased membrane currents, and an increased channel mean open time, all of which are consistent with BK_Ca channels gain-of-function effect [4,36,83]. This mutation specifically alters Ca²⁺-dependent activation initiated at the Ca²⁺ binding site within the RCK1 domain [84]. Several mechanisms for how D434G mutation causes BK_Ca channel gain-of-function effect (and the subsequent contribution to neuronal hyperexcitability and seizures) have been suggested and include rapid repolarization of APs and relief of Na_v channels inactivation, up-regulation of hyperpolarization-activated channels or inhibition of GABAergic interneurons with subsequent disinhibition of thalamocortical circuits [4]. The D434G mutation may also confer specific changes to the proprieties of the regulatory BK_Ca channel β subunits, thus contributing to the pathophysiology of idiopathic epilepsy. Indeed, this mutation enhances the availability of BK_Ca channels that contain β2 subunits, and increases the channel function (i.e., opening rate) of BK_Ca channels containing β1 and β4 subunits but slows rates of both channel opening and closing of BK_Ca channels that contain β3b subunits [35]. Interestingly, a polymorphism in the gene encoding the BK_Ca channel β4 subunit is associated with human TLE [85]. Thus, some BK_Ca channel variants that have a gain-of-function phenotype may contribute to certain forms of inherited generalized epilepsy in humans when the thalamus and thalamo-cortical circuits are involved in the pathophysiology of seizures, whereas a loss-of-function BK_Ca channels phenotype may be associated with TLE.

The gene encoding the regulatory BK_Ca β3 subunit (KCNMB3) has also been implicated in the pathophysiology of epilepsy. The KCNMB3 gene maps a region in the human chromosome 3 (3q26.3-q27) [86]. This segment is duplicated in the dup(3q) syndrome, which is characterized by congenital and neurological abnormalities, including seizures [86]. Because dup(3q) syndrome has early onset during development, the duplication of the KCNMB3 gene suggests that overexpression of this gene and the resulting increase in the β3 subunit protein levels may contribute to seizure etiology. Similarly, a susceptibility locus for idiopathic generalized epilepsy maps to the 3q26 chromosome region [87]. In humans, mutations in the KCNMB3 gene may also contribute to both neuronal hyperexcitability and seizures as a single nucleotide deletion in exon 4 (delA750) is associated with idiopathic generalized epilepsy (mostly in the form of typical absence epilepsy) [88]. Interestingly, BK_Ca channels containing the β3b-V4 (delA750 mutation) variant exhibit fast inactivation kinetics [38], suggesting a reduced ability of these channels to repolarize the membrane during an AP, and thus leading to neuronal hyperexcitability.

2.5.2 Experimental studies

In sharp contrast to clinical studies that suggest gain-of-function BK_Ca channels essentially contribute to the pathophysiology of absence epilepsy, experimental studies indicate BK_Ca channels gain-of-function mechanisms can also trigger TLE. Thus, BK_Ca channel β4 subunit knockout mice exhibit TLS associated with a gain-of-function phenotype in their BK_Ca channels that sharpens (narrows) APs and supports higher firing frequency in DG granule cells [81]. The mechanisms by which a gain-of-function of BK_Ca channels without β4 subunits triggers seizures may include i) enhanced Ca²⁺ sensitivity of BK_Ca channels, ii) reduced AHP size, iii) de novo generation of type I, fast-gated BK_Ca channels, or iv) limited recruitment of SK_Ca channels between spikes [81]. A loss of β4 subunits may also remove their inhibitory effect on STREX variant BK_Ca channels, leading to an increase in BK_Ca channel function. Interestingly, DG granule cells have the strongest expression of β4 subunits in the hippocampal formation, and upregulation of the STREX variant channels (and downregulation of ZERO variant channels) in DG granule cells are associated with TLE [89]. Thus, upregulation of STREX variant may contribute to gain-of-function BK_Ca channels that give rise to TLS, as this variant exhibit faster activation and slower deactivation kinetics. The DG has a very high threshold for seizure initiation and is resistant to seizure-induced neurodegeneration. Thus, neuronal BK_Ca channel β4 subunits may provide a molecular basis for both seizure resistance and neuroprotection.

The BK_Ca channel regulatory β4 subunit in the inner mitochondrial membrane may also contribute to neurprotection, as the functional role of mitoBK_Ca channel is in part associated with this subunit. Mitochondrial dysfunction is associated with several multiple epileptic phenotypes, including TLE [90]. The mechanisms of how neuronal mitochondrial dysfunction contributes to epileptogenesis are not fully understood. Evidence indicates that seizures can elicit both significant increases in mitochondrial Ca²⁺ levels and changes in mitochondrial membrane potential [91]. Neuronal mitoBK_Ca channels would then, be activated due to rising [Ca²⁺] in the matrix in response to an increase in cytosolic Ca²⁺, and the consequence of these channels opening is K⁺ efflux into mitochondria matrix, resulting in depolarization of mitochondrial inner membrane, which would then stimulate respiration. Mitochondria are a major source of reactive oxygen species (ROS), and the mitochondria-mediated catalytic removal of ROS prevents status epilepticus-induced neuronal cell loss [92]. Thus, activation of mitoBK_Ca channels would contribute to seizure suppression and confer neuroprotection via inhibition of ROS synthesis. Alternatively, downregulation of mitoBK_Ca channels would contribute to (i.e., facilitate) seizure-induced neuronal cell loss.

Seizures themselves also can confer a gain-of-function effect to BK_Ca channels. Accordingly, picrotoxin-induced generalized tonic-clonic seizures give rise to a BK_Ca channel gain-of-function that is characterized by enhanced BK_Ca currents and increased neuronal firing in the somatosensory (barrel) cortex [93]. Interestingly, BK_Ca channel blockers suppressed generalized tonic-clonic seizures in pre-sensitized animals and reversed the elevated neuronal firing that follows tonic-clonic seizures [93,94]. These findings suggest that blocking gain-of-function BK_Ca channels may be a potential mechanism for suppressing seizures. The extent to which seizure-induced gain-of-function BK_Ca channels contribute to seizure generation and the development of chronic epilepsy remains unknown.

Loss-of-function or downregulated BK_Ca channels may also contribute to the pathophysiology of seizures in models of inherited generalized tonic-clonic epilepsy and TLE. For instance, the severe seizure strain of genetically epilepsy-prone rats (GEPR-9), which exhibit enhanced susceptibility to acoustically evoked seizures, have reduced fAHP conductances in hippocampal CA3 neurons [95]. In line with these studies, BK_Ca channel current density is significantly reduced in IC neurons of the moderate seizure strain (GEPR-3) [N'Gouemo, unpublished results]. However, the reduced BK_Ca current density was not associated with downegulation of BK_Ca channel α-subunit in IC neurons of GEPR-3s [96]; thus, other mechanisms (such as phosphorylation or dephosphorylation of the channel) may account for the reduced current density. In a model of TLE, however, a downregulation of the BK_Ca channel α subunits was reported in the cortex and hippocampus (specifically in the mossy fibers), during the active (or chronic) phase of epilepsy [97,98]. Interestingly, BK_Ca channels are abundantly expressed at glutamatergic presynaptic terminals including at the mossy fibers where they can control glutamate release under conditions of excessive neuronal activity. Thus, downregulation of presynaptic BK_Ca channels may contribute to mossy fibers hyperexcitability and aberrant glutamate release during epileptogenesis. Finally, penitrem A, a potent blocker of BK_Ca channels triggers seizures and status epilepticus [99]; thus providing direct evidence that these channels are critical for controlling neuronal hyperexcitability that leads to seizures.

Acute alcohol exposure potentiates BK_Ca channel activity [74] that should reduce neuronal excitability and provide a basis for the neurodepressive effect of alcohol. Interestingly, an abrupt reduction or cessation of chronic alcohol consumption leads to the development of alcohol withdrawal syndrome characterized by neuronal hyperexcitability and seizures (primarily generalized tonic-clonic seizures). Because of the role that BK_Ca channels play in controlling neuronal excitability, these channels may contribute to the etiology of alcohol withdrawal seizures. Consistent with this hypothesis, alcohol withdrawal seizures are associated with a reduction in both BK_Ca channel current density and BK_Ca channel α-subunit expression in IC neurons (N'Gouemo, unpublished data).

Thus, the aforementioned studies provide evidence to support the notion that both gain-of-function and loss-of-function BK_Ca channels play important roles in the pathophysiology of epilepsy. The specific role of these BK_Ca channels may ultimately depend, at least, in their cellular localization, anatomical distribution, variant channel expression and seizure type.

2.5.3 BK_Ca channel activators and blockers as antiepileptic drugs

In humans, mutations in the BK_Ca channels that result in a gain-of-function phenotype have been implicated in the pathophysiology of idiopathic generalized seizures [4]. Accordingly, blocking these BK_Ca channels should suppress seizures. In support of this hypothesis, ethosuximide, a drug of choice for human absence epilepsy inhibits neuronal BK_Ca channels, in addition to its primary effect of blocking Ca_v3 channels [100]. Furthermore, paxilline, a BK_Ca channel blocker, has anticonvulsant effects in models of picrotoxin- and pentylenetetrazole-induced generalized seizures in pre-sensitized animals [94]. Interestingly, at the tested dose, paxilline did not suppress acute picrotoxin- or pentlylenetetrazole-induced generalized tonic-clonic seizures in animals that were not pre-sensitized [94]. These findings suggest the convulsant pre-sensitization may contributes to the gain-of-function effect on BK_Ca channels, which may serve as molecular target for the anticonvulsant effect of paxilline. Loss-of-function of BK_Ca channels are also associated with inherited and acquired susceptibility to generalized tonic-clonic seizures and TLE, suggesting that activating BK_Ca channels can provide an elegant mechanism for seizure suppression. Accordingly, zonisamide, a clinically used antiepileptic drug, activates BK_Ca channels in addition to other mechanisms suggesting that this pharmacological activity may contribute to some extent to its anticonvulsant effect.

3. Concluding remarks

BK_Ca channels are important regulators of neuronal activity and represent an intrinsic inhibitory mechanism for critically restoring normal neuronal excitability. Thus, BK_Ca channel activators may serve as potential anticonvulsants. However, enhanced BK_Ca channel function may lead to increased excitability by modifying the refractory period of the associated conductances. Furthermore, mutations in the genes encoding the BK_Ca subunits can give rise to BK_Ca channel gain-of-function phenotype resulting in neuronal hyperexcitability and seizures. Seizures themselves also can induce a gain-of-function effect in BK_Ca channels, as a consequence of seizure activity. Blocking BK_Ca channels suppresses the channel gain-of-function effect and seizures, which strongly suggest these channels, are also of therapeutic interest as drug targets. Understanding the molecular basis of how both BK_Ca channels gain-of-function and loss-of-function BK_Ca channels contribute to epileptogenesis and seizure generation may lead to developing rational therapies for certain inherited and acquired epilepsy syndrome.

4. Expert opinion (1,000 words; actual:999)

The role of BK_Ca channels in epilepsy pathophysiology is complex as activation of these channels can lead to both anti- and pro-epileptic activities. This dual action of BK_Ca channels depends on many factors including the seizure type. Emerging evidence suggests that loss-of-function mechanisms of BK_Ca channels are associated with inherited seizure susceptibility, alcohol withdrawal seizures, and TLE, while gain-of-function effects on BK_Ca channels are thought to contribute to the etiology of absence epilepsy.

Despite significant progress in managing epilepsy syndromes, approximately one-third of patients with epilepsy live with uncontrolled seizures, and unwanted side effects from antiepileptic drugs are common. One of the major challenges in the management of seizures is drug resistance, the causes of which are both multiple and complex and include a failure of the antiepileptic drugs to block inward Ca²⁺ and Na⁺ currents through Ca_v and Na_v channels, respectively, or to enhance GABA-mediated inhibition. There is an urgent need to develop novel antiepileptic drugs based on the underlying mechanisms of neuronal hyperexcitability that may lead to both epileptogenesis and seizure generation. Thus, there is intense and growing interest in the BK_Ca channel loss-of-function mechanisms associated with epilepsy and the novel BK_Ca channel gain-of-function mechanisms that give rise to seizures, both of which can result from mutations in the gene that encodes for the channel's pore-forming α subunit. Likewise, there is intense interest in gain-of-function effect on BK_Ca channels that is caused by seizures themselves, as this may serve as putative mechanism in epileptogenesis.

Although the phenotype of human epilepsy that is caused by gain-of function BK_Ca channels seems limited to absence epilepsy, there is evidence implicating a polymorphism β4 subunit polymorphism in TLE (likely with secondary generalization), which is the most common form of refractory epilepsy in adults. All of this suggests that inhibiting gain-of-function BK_Ca channels is a promising means to treat some epilepsy syndromes. Interestingly, BK_Ca channel blockers suppress both gene mutation- and seizure-induced gain-of-function effect and these blockers exert anticonvulsant effects in models of picrotoxin- and pentylenetetrazole-induced seizures in pre-sensitized young animals. The BK_Ca channel gain-of-function mechanisms that lead to seizures may also include elevated extracellular [K⁺], which is tightly associated with neuronal hyperexcitability and seizures. Blocking BK_Ca channels may therefore stabilize extracellular [K⁺] at physiological levels and suppress seizures. Thus, gain-of-function BK_Ca channels along with the GABAergic system and T-type Ca²⁺ channels are important molecular targets underlying epileptogenesis in absence epilepsy.

Because gain-of-function BK_Ca channels can contribute to the pathogenesis of absence epilepsy, this raises the concern that clinically effective antiepileptic drugs which enhance BK_Ca channel conductances, for example zonisamide, may inappropriately affects thalamic networks activity, thereby leading to absence seizures (despite suppressing other seizure phenotypes). Thus, there is an urgent need for studies to investigate the extent to which i) selective activation of BK_Ca channels can suppress seizures and ii) activating BK_Ca channels worsens absence seizures and/or trigger additional seizure phenotypes in absence epilepsy.

There is also interest in BK_Ca channel loss-of-function mechanisms as downregulated BK_Ca channels and/or decreased channel activity are associated with neuronal hyperexcitability that can lead to seizures and epileptogenesis. To date, downregulation of BK_Ca channels has been associated with inherited seizure susceptibility (tonic-clonic seizures), alcohol withdrawal seizures (primarily tonic-clonic seizures) and TLE (likely with secondarily generalization). Thus, compounds that open and activate BK_Ca channels can be used to prevent and treat these seizures. Furthermore, presynaptic BK_Ca channels openers, by suppressing glutamate release and contributing neuroprotection, may have a promising in role in the management of seizure-induced lesions underlying the development of pharmacoresistant TLE.

To date, only the model of picrotoxin- and pentylenetetrazole-induced seizures in pre-sensitized young animals has sufficiently demonstrated that blocking BK_Ca channels suppresses seizures. Other models that would be useful for confirming this result include: animal models of acute generalized seizures (maximal electroshock) and epileptogenesis (chemical or electrical kindling; post-status epilepticus models of TLE). Future studies in relevant animal models of absence epilepsy (e.i., Genetic Absence Epilepsy Rat from Strasbourg, the Wistar Absence Glaxo from Rijwik, and the γ-hydroxybutyrate spike-and-wave model) and suitable clinical populations are needed to validate the anticonvulsant profile and potential clinical application of BK_Ca channel blockers. The BK_Ca channel β4 subunit knockout mouse is a useful model to determine the role of this subunit (and its dysfunction) in TLE. However, caution should be exercised when interpreting the relevance of this model, as knocking down the channel subunit results in a protein loss but not a production of dysfunctional protein that is the case for BK_Ca channel mutations that lead to inherited epilepsy in humans.

Our understanding of how BK_Ca channels activators and inhibitors can be used therapeutically to treat or prevent seizures and epilepsy is still in the early stages. A comprehensive structure-function analysis of gain-of-function and loss-of-function BK_Ca channels that leads to epileptogenesis and seizures may be helpful in designing new therapeutic strategies. Developing BK_Ca channel blockers (and activators) that are selective for channels containing β3 and β4 subunits would certainly help determine the extent to which these subunits might mediate antiepileptic/antiepileptogenic effects. In addition, it also would be interesting to determine whether certain BK_Ca channel openers that were originally developed to treat various diseases such as erectile dysfunction (3-thio-quinolines, 4-aryl-3-aminoquinolones), ischemic stroke (fluoro-oxindole,4-aryl-3-aminoquinolones) exhibit antiepileptic/antiepileptogenic effects.

This review raises the hypothesis that both gain-of-function and loss-of-function BK_Ca channels may contribute to epileptogenesis and seizure generation. Identification of altered BK_Ca channel mechanisms that lead to neuronal hyperexcitability will contribute to a better understanding of the molecular substrates for epileptogenesis and seizure generation and eventually lead to more effective strategies for treating seizures and epilepsy. Targeting BK_Ca channels to suppress seizures and treat epilepsy can be challenging because of both anti- and pro-epileptic activity following activation of these channels. Nevertheless, identification of diverse BK_Ca channel α and β subunit variants may enhance the possibility of targeting selective brain areas with selective BK_Ca channel openers and blockers, and therefore leading to better strategies for epileptogenesis prevention and treatment of certain seizure types and epilepsy syndromes.

Highlights Box.

• BK_Ca channels are activated by both membrane depolarization and increased intracellular Ca²⁺.

• Outward K⁺ currents through BK_Ca channels hyperpolarize neurons.

• Blockage of BK_Ca channels can trigger seizures and status epilepticus.

• Genetic deletion of the regulatory BK_Ca channel β4 subunit triggers temporal lobe seizures.

• Mutation in the KCNMB3 gene encoding for the regulatory BK_Ca channels β3 subunit is associated with idiopathic generalized epilepsy.

• Loss-of-function mechanisms of BK_Ca channels have been associated with temporal lobe seizures, tonic-clonic seizures and alcohol withdrawal seizures.

• Gain-of-function mutations in BK_Ca channel pore-forming α subunit contribute to the development of absence epilepsy.

• Generalized tonic-clonic seizures can confer a gain-of-function effect to BK_Ca channels

• Both loss-of-function and gain-of-function BK_Ca channels may be potential therapeutic targets for certain epilepsy syndromes.