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The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K⁺ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.

Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.

Inactivation is an intrinsic property of several voltage-dependent ion channels, closing the conduction pathway during membrane depolarization and dynamically regulating neuronal activity. BK K + channels undergo N-type inactivation via their β2 subunit, but the physiological significance is not clear. Here, we report that inactivating BK currents predominate during the day in the suprachiasmatic nucleus, the brain’s intrinsic clock circuit, reducing steady-state current levels. At night inactivation is diminished, resulting in larger BK currents. Loss of β2 eliminates inactivation, abolishing the diurnal variation in both BK current magnitude and SCN firing, and disrupting behavioural rhythmicity. Selective restoration of inactivation via the β2 N-terminal ‘ball-and-chain’ domain rescues BK current levels and firing rate, unexpectedly contributing to the subthreshold membrane properties that shift SCN neurons into the daytime ‘upstate’. Our study reveals the clock employs inactivation gating as a biophysical switch to set the diurnal variation in suprachiasmatic nucleus excitability that underlies circadian rhythm. BK potassium channels have been previously shown to mediate SCN circadian firing, although the precise mechanisms are unclear. Here, using knockout and rescue approaches, the authors find that the ß2 ‘ball-and-chain’ confers BK channel inactivation during the day, promoting SCN electrical upstate.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BK Ca , K Ca 1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K + secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BK Ca antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BK Ca activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BK Ca in the lung yet may lead to adverse events through BK Ca activation elsewhere.

Extracellular vesicles (EVs) are associated with intercellular communications, immune responses, viral pathogenicity, cardiovascular diseases, neurological disorders, and cancer progression. EVs deliver proteins, metabolites, and nucleic acids into recipient cells to effectively alter their physiological and biological response. During their transportation from the donor to the recipient cell EVs face differential ionic concentrations, which can be detrimental to their integrity and impact their cargo content. EVs are known to possess ion channels and transporters in their membrane but neither the function nor the role of these channels in EVs is known. In this study, we discover a functional calcium-activated large-conductance potassium channel (BK Ca ) in the membrane of EVs. Furthermore, we establish that BK Ca is essential for the structural and functional integrity of EVs. Together, these findings establish the critical role of ion channels such as BK Ca in functioning as gatekeepers and maintaining EV-mediated signaling. Extracellular vesicles secreted by cells play a crucial role in cellular communication. Here, the authors identify a functional BK channel in extracellular vesicle membranes and establish its role in preserving vesicle integrity, K+ homeostasis, and physiological function.

Mitochondrial potassium channels have been implicated in myocardial protection mediated through pre-/postconditioning. Compounds that open the Ca2+- and voltage-activated potassium channel of big-conductance (BK) have a pre-conditioning-like effect on survival of cardiomyocytes after ischemia/reperfusion injury. Recently, mitochondrial BK channels (mitoBKs) in cardiomyocytes were implicated as infarct-limiting factors that derive directly from the KCNMA1 gene encoding for canonical BKs usually present at the plasma membrane of cells. However, some studies challenged these cardio-protective roles of mitoBKs. Herein, we present electrophysiological evidence for paxilline- and NS11021-sensitive BK-mediated currents of 190 pS conductance in mitoplasts from wild-type but not BK-/- cardiomyocytes. Transmission electron microscopy of BK-/- ventricular muscles fibres showed normal ultra-structures and matrix dimension, but oxidative phosphorylation capacities at normoxia and upon re-oxygenation after anoxia were significantly attenuated in BK-/- permeabilized cardiomyocytes. In the absence of BK, post-anoxic reactive oxygen species (ROS) production from cardiomyocyte mitochondria was elevated indicating that mitoBK fine-tune the oxidative state at hypoxia and re-oxygenation. Because ROS and the capacity of the myocardium for oxidative metabolism are important determinants of cellular survival, we tested BK-/- hearts for their response in an ex-vivo model of ischemia/reperfusion (I/R) injury. Infarct areas, coronary flow and heart rates were not different between wild-type and BK-/- hearts upon I/R injury in the absence of ischemic pre-conditioning (IP), but differed upon IP. While the area of infarction comprised 28±3% of the area at risk in wild-type, it was increased to 58±5% in BK-/- hearts suggesting that BK mediates the beneficial effects of IP. These findings suggest that cardiac BK channels are important for proper oxidative energy supply of cardiomyocytes at normoxia and upon re-oxygenation after prolonged anoxia and that IP might indeed favor survival of the myocardium upon I/R injury in a BK-dependent mode stemming from both mitochondrial post-anoxic ROS modulation and non-mitochondrial localizations.

Nuclear calcium levels affect gene expression, but little is known about how they are regulated. The authors show that large-conductance calcium-activated potassium (BK) channels are present on the nuclear envelope in rodent hippocampal neurons. Blockade of nuclear BK channels revealed that they regulate nucleoplasmic Ca 2+ , gene expression and dendritic arborization. Ion channels are essential for the regulation of neuronal functions. The significance of plasma membrane, mitochondrial, endoplasmic reticulum and lysosomal ion channels in the regulation of Ca 2+ is well established. In contrast, surprisingly little is known about the function of ion channels on the nuclear envelope (NE). Here we demonstrate the presence of functional large-conductance, calcium-activated potassium channels (BK channels) on the NE of rodent hippocampal neurons. Functionally, blockade of nuclear BK channels (nBK channels) induces NE-derived Ca 2+ release, nucleoplasmic Ca 2+ elevation and cyclic AMP response element binding protein (CREB)-dependent transcription. More importantly, blockade of nBK channels regulates nuclear Ca 2+ –sensitive gene expression and promotes dendritic arborization in a nuclear Ca 2+ –dependent manner. These results suggest that the nBK channel functions as a molecular link between neuronal activity and nuclear Ca 2+ to convey signals from synapse to nucleus and is a new modulator, operating at the NE, of synaptic activity–dependent neuronal functions.

Calcium- and voltage-gated potassium channels of large conductance (BK channels) in smooth muscle (SM) act as part of a negative feedback mechanism on SM contraction and associated decrease in cerebral artery diameter. Functional BK channels result from tetrameric association of α subunits encoded by KCNMA1 (Slo1). Ionic current from slo1 channels is inhibited by cholesterol in artificial lipid bilayers, an effect significantly reduced by the slo1 Y450F substitution. Whether such substitution affects cholesterol action on cerebral artery SM BK channel function and diameter remains unknown. Using the KCNMA1Y450F knock-in (K/I) mouse, we determined the effect of cholesterol enrichment on BK currents in native SM cells from middle cerebral artery using patch-clamp electrophysiology and the artery diameter ex vivo response to cholesterol. Results show that the KCNMA1Y450F K/I mutation modifies both basal function and the channel’s response to cholesterol enrichment. Such modifications are detectable solely in SM cells from males, demonstrating sexual dimorphism. Unexpectedly, the modifications introduced by the Y450F substitution do not translate into observable changes in middle cerebral artery diameter ex vivo, suggesting that mechanisms at the SM level compensate for changes driven by the KCNMA1 point mutation under study.

Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol’s direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary β subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.

Key Points Identification of the Slo gene family came hot on the heels of new advances in molecular cloning, and involved Drosophila melanogaster neurogenetics as well as numerous physiological and biophysical studies. These biophysical studies had identified Ca 2+ -dependent K + currents in many systems, as well as unusually large (maxi-K) Ca 2+ - and Na + -dependent single-channel currents. The key to identifying the genes underlying these phenomena turned out to be the D rosophila slowpoke (slo) mutant. The structures of the α-subunits of SLO family channels resemble those of voltage-gated K + channels. However, they differ from those of voltage-gated ion channels in that they have an extensive carboxyl extension — the 'tail' — which is thought to confer distinctive properties, such as calcium-sensing, to SLO1 channels, whereas the 'core' domain containing the membrane-spanning segments confers voltage sensitivity. Voltage-dependent channels are gated (opened and closed) in response to changes in transmembrane voltage. In ligand-gated channels, the binding of a ligand, such as a neurotransmitter, causes a conformational change of a 'ligand-binding domain' that is physically coupled to the pore of the channel. For SLO1 and SLO3 channels, this distinction breaks down because both voltage-gating and ligand-gating domains are present, indicating two independent sensing mechanisms that converge near the gates of the pore. A complicated system of cooperativity, involving several Ca 2+ -sensing sites both on the same subunit and on different subunits, could underlie the responsiveness of SLO1 channels to a broad range of Ca 2+ concentrations. This provides versatility and allows these channels to serve a wide variety of physiological roles in different cell types and cellular microdomains, where Ca 2+ concentrations can vary extensively. SLO1 channels have the largest single-channel conductance of all K + - selective channels. Several studies have indicated the presence of at least two salient underlying structural mechanisms: two rings of negative charges in the inner and outer pore of the channel's ionic conduction pathway and the size of the SLO1 inner pore region. The functional diversity of SLO1 channels comes from many sources, such as alternative RNA splicing, post-translational modifications and β-subunits. β-subunits alone account for a great deal of diversity, such as enhanced sensitivity to Ca 2+ and rapid channel inactivation. The sensitivity of SLO1 to Ca 2+ makes it an important negative feedback system for Ca 2+ entry in many cell types. SLO1 channels are ubiquitously expressed in most tissues and have roles in neurons, smooth muscles, secretory endocrine cells and specialized sensory receptors. SLO channels comprise a family of high-conductance potassium channels with several distinctive features that give them the versatility to function in various cellular contexts. The authors describe the key properties and physiological roles of these channels in the nervous system. High-conductance, 'big' potassium (BK) channels encoded by the Slo gene family are among the largest and most complex of the extended family of potassium channels. The family of SLO channels apparently evolved from voltage-dependent potassium channels, but acquired a large conserved carboxyl extension, which allows channel gating to be altered in response to the direct sensing of several different intracellular ions, and by other second-messenger systems, such as those activated following neurotransmitter binding to G-protein-coupled receptors (GPCRs). This versatility has been exploited to serve many cellular roles, both within and outside the nervous system.