Vascular smooth muscle BK channels limit ouabain-induced vasocontraction: Dual role of the Na/K-ATPase as a hub for Src-kinase and the Na/Ca-exchanger.

Large-conductance, calcium-activated potassium channels (BK channels) and the Na/K-ATPase are expressed universally in vascular smooth muscle. The Na/K-ATPase may act via changes in the intracellular Ca2+ concentration mediated by the Na/Ca exchanger (NCX) and via Src kinase. Both pathways are known to regulate BK channels. Whether BK channels functionally interact in vascular smooth muscle cells with the Na/K-ATPase remains to be elucidated. Thus, this study addressed the hypothesis that BK channels limit ouabain-induced vasocontraction. Rat mesenteric arteries were studied using isometric myography, FURA-2 fluorimetry and proximity ligation assay. The BK channel blocker iberiotoxin potentiated methoxamine-induced contractions. The cardiotonic steroid, ouabain (10-5 M), induced a contractile effect of IBTX at basal tension prior to methoxamine administration and enhanced the pro-contractile effect of IBTX on methoxamine-induced contractions. These facilitating effects of ouabain were prevented by the inhibition of either NCX or Src kinase. Furthermore, inhibition of NCX or Src kinase reduced the BK channel-mediated negative feedback regulation of arterial contraction. The effects of NCX and Src kinase inhibition were independent of each other. Co-localization of the Na/K-ATPase and the BK channel was evident. Our data suggest that BK channels limit ouabain-induced vasocontraction by a dual mechanism involving the NCX and Src kinase signaling. The data propose that the NCX and the Src kinase pathways, mediating the ouabain-induced activation of the BK channel, act in an independent manner.

PTHrP attenuates spontaneous contractions in detrusor smooth muscle of the rat bladder by activating spontaneous transient outward potassium currents.

Parathyroid hormone-related protein (PTHrP) released from detrusor smooth muscle (DSM) cells upon bladder distension attenuates spontaneous phasic contractions (SPCs) in DSM and associated afferent firing to facilitate urine storage. Here, we investigate the mechanisms underlying PTHrP-induced inhibition of SPCs, focusing on large-conductance Ca2+-activated K+ channels (BK channels) that play a central role in stabilizing DSM excitability. Perforated patch-clamp techniques were applied to DSM cells of the rat bladder dispersed using collagenase. Isometric tension changes were recorded from DSM strips, while intracellular Ca2+ dynamics were visualized using Cal520 AM -loaded DSM bundles. DSM cells developed spontaneous transient outward potassium currents (STOCs) arising from the opening of BK channels. PTHrP (10 nM) increased the frequency of STOCs without affecting their amplitude at a holding potential of - 30 mV but not - 40 mV. PTHrP enlarged depolarization-induced, BK-mediated outward currents at membrane potentials positive to + 20 mV in a manner sensitive to iberiotoxin (100 nM), the BK channel blocker. The PTHrP-induced increases in BK currents were also prevented by inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) (CPA 10 µM), L-type voltage-dependent Ca2+ channel (LVDCC) (nifedipine 3 µM) or adenylyl cyclase (SQ22536 100 µM). PTHrP had no effect on depolarization-induced LVDCC currents. PTHrP suppressed and slowed SPCs in an iberiotoxin (100 nM)-sensitive manner. PTHrP also reduced the number of Ca2+ spikes during each burst of spontaneous Ca2+ transients. In conclusion, PTHrP accelerates STOCs discharge presumably by facilitating SR Ca2+ release which prematurely terminates Ca2+ transient bursts resulting in the attenuation of SPCs.

Targeting Large-Conductance Calcium-Activated Potassium Channels to Ameliorate Lipopolysaccharide-Induced Depressive-Like Behavior in Mice.

Neuroinflammation plays crucial role in the development and progression of depression. Large conductance calcium- and voltage-dependent potassium (BK) channels mediate the activation of microglia. Herein, we investigated whether BK channels could serve as a target for the treatment of inflammation-associated depression. Lipopolysaccharide (LPS, 0.83 mg/kg) was injected intraperitoneally (i.p.) to induce neuroinflammation and depressive-like behavior in 6-8 week ICR mice. Adeno-associated virus (AAV) constructs (AAV9-Iba1p-BK shRNA-EGFP (BK shRNA-AAV) or AAV9-Iba1p-NC shRNA-EGFP (NC shRNA-AAV)) were unilaterally injected intracerebroventricularly to selectively knock down BK channels in microglia. The tail suspension test (TST) and forced-swim test (FST) were used to evaluate depressive-like behavior in mice 24 h after LPS challenge. The morphology of microglia, expression of BK channels, levels of cytokines, and expression and activity of indoleamine 2,3-dioxygenase (IDO) were measured by immunohistochemistry, western blot, quantitative real time PCR, and enzyme-linked immunosorbent assay (ELISA), respectively. Either paxilline (i.p.), a specific BK channel blocker, or BK shRNA-AAV effectively inhibited the activation of microglia, reduced the production of IL-1ß in the hippocampus and suppressed the expression and activity of IDO in the hippocampus and prefrontal cortex, resulting in the amelioration of depressive-like behavior in mice. These data suggest for the first time that BK channels are involved in LPS-induced depressive-like behaviors. Thus, microglia BK channels may be a potential drug target for the depression treatment.

The BK Channel Limits the Pro-Inflammatory Activity of Macrophages.

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1ß production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1ß production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions.

Hypoxia, Ion Channels and Glioblastoma Malignancy.

The malignancy of glioblastoma (GBM), the most aggressive type of human brain tumor, strongly correlates with the presence of hypoxic areas within the tumor mass. Oxygen levels have been shown to control several critical aspects of tumor aggressiveness, such as migration/invasion and cell death resistance, but the underlying mechanisms are still unclear. GBM cells express abundant K+ and Cl- channels, whose activity supports cell volume and membrane potential changes, critical for cell proliferation, migration and death. Volume-regulated anion channels (VRAC), which mediate the swelling-activated Cl- current, and the large-conductance Ca2+-activated K+ channels (BK) are both functionally upregulated in GBM cells, where they control different aspects underlying GBM malignancy/aggressiveness. The functional expression/activity of both VRAC and BK channels are under the control of the oxygen levels, and these regulations are involved in the hypoxia-induced GBM cell aggressiveness. The present review will provide a comprehensive overview of the literature supporting the role of these two channels in the hypoxia-mediated GBM malignancy, suggesting them as potential therapeutic targets in the treatment of GBM.

Role of Na+/Ca2+ Exchanger (NCX) in Glioblastoma Cell Migration (In Vitro).

Glioblastoma (GBM) is the most malignant form of primary brain tumor. It is characterized by the presence of highly invasive cancer cells infiltrating the brain by hijacking neuronal mechanisms and interacting with non-neuronal cell types, such as astrocytes and endothelial cells. To enter the interstitial space of the brain parenchyma, GBM cells significantly shrink their volume and extend the invadopodia and lamellipodia by modulating their membrane conductance repertoire. However, the changes in the compartment-specific ionic dynamics involved in this process are still not fully understood. Here, using noninvasive perforated patch-clamp and live imaging approaches on various GBM cell lines during a wound-healing assay, we demonstrate that the sodium-calcium exchanger (NCX) is highly expressed in the lamellipodia compartment, is functionally active during GBM cell migration, and correlates with the overexpression of large conductance K+ channel (BK) potassium channels. Furthermore, a NCX blockade impairs lamellipodia formation and maintenance, as well as GBM cell migration. In conclusion, the functional expression of the NCX in the lamellipodia of GBM cells at the migrating front is a conditio sine qua non for the invasion strategy of these malignant cells and thus represents a potential target for brain tumor treatment.

Er-xian decoction drug-containing serum promotes Mc3t3-e1 cell proliferation and osteogenic differentiation via regulating BK channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Er-xian Decoction (EXD) is a well-known prescription widely used to prevent and treat climacteric syndrome and osteoporosis in China. BK channel positively affects osteoblast bone formation in vitro. However, it is still unclear whether the effect of EXD on promoting osteoblasts osteogenic differentiation is related to BK channel. AIM OF THE STUDY: The study is aimed at determining whether the EXD-containing serum promotes the proliferation of osteoblasts and their differentiation through BK channel. MATERIALS AND METHODS: The chemical compounds of EXD were analyzed by UPLC-Q-TOF/MS. An osteogenic induction medium (OM) was used to induce MC3T3-E1 cells' osteogenic differentiation. The effects of EXD-containing serum and tetraethylammonium (TEA) on the proliferation activity of Mc3t3-e1 cells were detected by CCK-8 assay. ALP activity was determined by an alkaline phosphatase kit. The protein expression (BMP2, OPG, and COL1) was analyzed by Western blot, and the mRNA expression (Runx2, OPG, and BMP2) was assessed by real-time PCR. Alizarin red was used to stain the mineralized region of osteoblasts. In addition, we analyzed the relationship between BK channel and its downstream PTEN/Akt/Foxo1 signaling pathway. RESULTS: 72 compounds were identified by UPLC-Q-TOF/MS analysis in EXD. Mangiferin, ferulic acid, berberine, and icariin were main active components of EXD. EXD-containing serum could enhance the cell viability of MC3T3-E1 cells by decreasing the expression of BKα protein. EXD-containing serum increased ALP activity and calcium nodule formation of Mc3t3-e1 cells, promoted the protein expression of BKα, COL1, BMP2, OPG, and the mRNA expression of RUNX2, OPG, and BMP2, however, these effects can be reversed after adding TEA. In addition, EXD-containing serum could upregulate phosphorylation of Akt and Foxo1 in osteogenic-induced Mc3t3-e1 cells, and lower the expression of PTEN. And these effects of EXD-containing serum could be reduced by TEA. CONCLUSIONS: The effect of EXD-containing serum on promoting cell proliferation and osteogenic differentiation of Mc3t3-e1 cells might be linked to the regulation of BK channel.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate.

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

BK Channel in the Physiology and in the Cancer of Pancreatic Duct: Impact and Reliability of BK Openers.

BK (KCa 1.1, Slo-1) is a K+ channel characterized by an allosteric regulation of the gating mechanism by Ca2+ binding and voltage, and a high unitary conductance. The channel is expressed in many different tissues, where it is involved in the regulation or the fine-tuning of many physiological processes. Among other organs, BK is expressed in the pancreatic duct, a part of the gland important for the correct ionic composition of the pancreatic juice. Unfortunately, the pancreatic duct is also the site where one of the deadliest cancer types, the pancreatic duct adenocarcinoma (PDAC), develops. In the past years, it has been reported that continuous exposure of cancer cells to BK openers can have a significant impact on cell viability as well as on the ability to proliferate and migrate. Here, we first summarize the main BK channel properties and its roles in pancreatic duct physiology. Then we focus on the potential role of BK as a pharmacological target in PDAC. Moreover, we discuss how results obtained when employing BK activators on cancer cells can, in some cases, be misleading.

Chronic stress facilitates bursting electrical activity in pituitary corticotrophs.

Coordination of an appropriate stress response is dependent upon anterior pituitary corticotroph excitability in response to hypothalamic secretagogues and glucocorticoid negative feedback. A key determinant of corticotroph excitability is large conductance calcium- and voltage-activated (BK) potassium channels that are critical for promoting corticotrophin-releasing hormone (CRH)-induced bursting that enhances adrenocorticotrophic hormone secretion. Previous studies revealed hypothalamic-pituitary-adrenal axis hyperexcitability following chronic stress (CS) is partly a function of increased corticotroph output. Thus, we hypothesise that chronic stress promotes corticotroph excitability through a BK-dependent mechanism. Corticotrophs from CS mice displayed significant increase in spontaneous bursting, which was suppressed by the BK blocker paxilline. Mathematical modelling reveals that the time constant of BK channel activation, plus properties and proportion of BK channels functionally coupled to L-type Ca2+ channels determines bursting activity. Surprisingly, CS corticotrophs (but not unstressed) display CRH-induced bursting even when the majority of BK channels are inhibited by paxilline, which modelling suggests is a consequence of the stochastic behaviour of a small number of BK channels coupled to L-type Ca2+ channels. Our data reveal that changes in the stochastic behaviour of a small number of BK channels can finely tune corticotroph excitability through stress-induced changes in BK channel properties. Importantly, regulation of BK channel function is highly context dependent allowing dynamic control of corticotroph excitability over a large range of time domains and physiological challenges in health and disease. This is likely to occur in other BK-expressing endocrine cells, with important implications for the physiological processes they regulate and the potential for therapy. KEY POINTS: Chronic stress (CS) is predicted to modify the electrical excitability of anterior pituitary corticotrophs. Electrophysiological recordings from isolated corticotrophs from CS male mice display spontaneous electrical bursting behaviour compared to the tonic spiking behaviour of unstressed corticotrophs. The increased spontaneous bursting from CS corticotrophs is BK-dependent and mathematical modelling reveals that the time constant of activation, properties and proportion of BK channels functionally coupled to L-type calcium channels determines the promotion of bursting activity. CS (but not unstressed) corticotrophs display corticotrophin-releasing hormone-induced bursting even when the majority of BK channels are pharmacologically inhibited, which can be explained by the stochastic behaviour of a small number of BK channels with distinct properties. Corticotroph excitability can be finely tuned by the stochastic behaviour of a small number of BK channels dependent on their properties and functional co-localisation with L-type calcium channels to control corticotroph excitability over diverse time domains and physiological challenges.

Blocking the BKCa channel induces NF-κB nuclear translocation by increasing nuclear calcium concentration†.

Nuclear factor kappa B (NF-κB) transcriptionally regulates several genes involved in initiating uterine contractions. A key factor controlling NF-κB activity is its translocation to the nucleus. In myometrial smooth muscle cells (MSMCs), this translocation can be stimulated by the inflammatory molecule lipopolysaccharide (LPS) or by blocking the potassium calcium-activated channel subfamily M alpha 1 (KCNMA1 or BKCa) with paxilline (PAX). Here, we sought to determine the mechanism by which blocking BKCa causes NF-κB-p65 translocation to the nucleus in MSMCs. We show that LPS- and PAX-induced NF-κB-p65 translocation are similar in that neither depends on several mitogen-activated protein kinase pathways, but both require increased intracellular calcium (Ca2+). However, the nuclear transport inhibitor wheat germ agglutinin prevented NF-κB-p65 nuclear translocation in response to LPS but not in response to PAX. Blocking BKCa located on the plasma membrane resulted in a transient NF-κB-p65 nuclear translocation that was not sufficient to induce expression of its transcriptional target, suggesting a role for intracellular BKCa. We report that BKCa also localizes to the nucleus and that blocking nuclear BKCa results in an increase in nuclear Ca2+ in MSMCs. Together, these data suggest that BKCa localized on the nuclear membrane plays a key role in regulating nuclear Ca2+ and NF-κB-p65 nuclear translocation in MSMCs.

NS-11021 Modulates Cancer-Associated Processes Independently of BK Channels in Melanoma and Pancreatic Duct Adenocarcinoma Cell Lines.

Potassium channels have emerged as regulators of carcinogenesis, thus introducing possible new therapeutic strategies in the fight against cancer. In particular, the large-conductance Ca2+-activated K+ channel, often referred to as BK channel, is involved in several cancer-associated processes. Here, we investigated the effects of different BK activators, NS-11021, NS-19504, and BMS-191011, in IGR39 (primary melanoma cell line) and Panc-1 (primary pancreatic duct carcinoma cell line), highly expressing the channel, and in IGR37 (metastatic melanoma cell line) that barely express BK. Our data showed that NS-11021 and NS-19504 potently activated BK channels in IGR39 and Panc-1 cells, while no effect on channel activation was detected in IGR37 cells. On the contrary, BK channel activator BMS-191011 was less effective. However, only NS-11021 showed significant effects in cancer-associated processes, such as cell survival, migration, and proliferation in these cancer cell lines. Moreover, NS-11021 led to an increase of intracellular Ca2+ concentration, independent of BK channel activation, thus complicating any interpretation of its role in the regulation of cancer-associated mechanisms. Overall, we conclude that the activation of the BK channel by itself is not sufficient to produce beneficial anti-cancer effects in the melanoma and PDAC cell lines examined. Importantly, our results raise an alarm flag regarding the use of presumably specific BK channel openers as anti-cancer agents.

Secondhand Smoke Exposure Impairs Ion Channel Function and Contractility of Mesenteric Arteries.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

Nuclear BK channels regulate CREB phosphorylation in RAW264.7 macrophages.

BACKGROUND: Macrophages are important cells of the innate immune system and contribute to a variety of physiological and pathophysiological responses. Monovalent and divalent ion channels have been studied in macrophage function, and while much research is still required, a role for these channels is beginning to emerge in macrophages. In addition to the plasma membrane, ion channels are also found in intracellular membranes including mitochondrial, lysosomal and nuclear membranes. While studying the function of plasma membrane located large conductance voltage- and calcium-activated potassium channels (BK channels) in a macrophage cell line RAW264.7, we became aware of the expression of these ion channels in other cellular locations. METHODS: Immunofluorescence and Western blot analysis were used to identify the expression of BK channels. To demonstrate a functional role for the nuclear located channel, we investigated the effect of the lipid soluble BK channel inhibitor paxilline on CREB phosphorylation. RESULTS: Treatment of resting macrophages with paxilline resulted in increased CREB phosphorylation. To confirm a role for nuclear BK channels, these experiments were repeated in isolated nuclei and similar results were found. Ca2+ and calmodulin-dependent kinases (CaMK) have been demonstrated to regulate CREB phosphorylation. Inhibition of CaMKII and CaMKIV resulted in the reversal of paxilline-induced CREB phosphorylation. CONCLUSIONS: These results suggest that nuclear BK channels regulate CREB phosphorylation in macrophages. Nuclear located ion channels may therefore be part of novel signalling pathways in macrophages and should be taken into account when studying the role of ion channels in these and other cells.

Deeper and Deeper on the Role of BK and Kir4.1 Channels in Glioblastoma Invasiveness: A Novel Summative Mechanism?

In the last decades, increasing evidence has revealed that a large number of channel protein and ion pumps exhibit impaired expression in cancers. This dysregulation is responsible for high proliferative rates as well as migration and invasiveness, reflected in the recently coined term oncochannelopathies. In glioblastoma (GBM), the most invasive and aggressive primary brain tumor, GBM cells modify their ionic equilibrium in order to change their volume as a necessary step prior to migration. This mechanism involves increased expression of BK channels and downregulation of the normally widespread Kir4.1 channels, as noted in GBM biopsies from patients. Despite a large body of work implicating BK channels in migration in response to an artificial intracellular calcium rise, little is known about how this channel acts in GBM cells at resting membrane potential (RMP), as compared to other channels that are constitutively open, such as Kir4.1. In this review we propose that a residual fraction of functionally active Kir4.1 channels mediates a small, but continuous, efflux of potassium at the more depolarized RMP of GBM cells. In addition, coinciding with transient membrane deformation and the intracellular rise in calcium concentration, brief activity of BK channels can induce massive and rapid cytosolic water loss that reduces cell volume (cell shrinkage), a necessary step for migration within the brain parenchyma.

BK potassium currents contribute differently to action potential waveform and firing rate as rat hippocampal neurons mature in the first postnatal week.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator of neuronal action potential firing and follows two distinct trends in early postnatal development: an increase in total expression and a shift from the faster activating STREX isoform to the slower ZERO isoform. We analyzed the functional consequences of developmental trends in BK channel expression in hippocampal neurons isolated from neonatal rats aged 1 to 7 days. Following overnight cultures, action potentials and currents were recorded using whole cell patch-clamp electrophysiology. These neurons undergo a steady increase in excitability during this time, and the effect of blockade of BK channel activity with 100 nM iberiotoxin changes as the neurons mature. BK currents contribute significantly more to total potassium current and single action potentials in neurons of 1-day old rats (with BK blockade extending action potential duration by 0.46 ± 0.12 ms) than in those of 7-day old rats (with BK blockade extending action potential duration by 0.17 ± 0.05 ms). BK currents contribute consistently to maintain firing rates in neurons of 1-day old rats throughout extended action potential firing; BK blockade evenly depresses firing frequency across action potential trains. In neurons from 7-day old rats, BK blockade initially increases firing frequency and then progressively decreases frequency as firing continues, ultimately depressing neuronal firing rates to a greater extent than in the neurons from 1-day-old animals. These results are consistent with a transition from low expression of a fast-activating BK isoform (STREX) to high expression of a slower activating isoform (ZERO).NEW & NOTEWORTHY This work describes the early developmental trends of large-conductance calcium-activated potassium (BK) channel activity. Early developmental trends in expression of BK channels, both total expression and relative isoform expression, have been previously reported, but little work describes the effect of these changes in expression patterns on excitability. Here, we show that early changes in BK channel expression patterns lead to changes in the role of BK channels in determining the action potential waveform and neuronal excitability.

Neuronal Inactivity Co-opts LTP Machinery to Drive Potassium Channel Splicing and Homeostatic Spike Widening.

Homeostasis of neural firing properties is important in stabilizing neuronal circuitry, but how such plasticity might depend on alternative splicing is not known. Here we report that chronic inactivity homeostatically increases action potential duration by changing alternative splicing of BK channels; this requires nuclear export of the splicing factor Nova-2. Inactivity and Nova-2 relocation were connected by a novel synapto-nuclear signaling pathway that surprisingly invoked mechanisms akin to Hebbian plasticity: Ca2+-permeable AMPA receptor upregulation, L-type Ca2+ channel activation, enhanced spine Ca2+ transients, nuclear translocation of a CaM shuttle, and nuclear CaMKIV activation. These findings not only uncover commonalities between homeostatic and Hebbian plasticity but also connect homeostatic regulation of synaptic transmission and neuronal excitability. The signaling cascade provides a full-loop mechanism for a classic autoregulatory feedback loop proposed ∼25 years ago. Each element of the loop has been implicated previously in neuropsychiatric disease.

Evaluating the safety and potential activity of URO-902 (hMaxi-K) gene transfer by intravesical instillation or direct injection into the bladder wall in female participants with idiopathic (non-neurogenic) overactive bladder syndrome and detrusor overactivity from two double-blind, imbalanced, placebo-controlled randomized phase 1 trials.

AIMS: Two phase 1 trials were performed in healthy women with the overactive bladder (OAB) syndrome and urodynamically demonstrated detrusor overactivity (DO), with the aim to demonstrate the safety and potential efficacy of URO-902, which comprises a gene therapy plasmid vector expressing the human big potassium channel α subunit. METHODS: ION-02 (intravesical instillation) and ION-03 (direct injection) were double-blind, placebo-controlled, multicenter studies without overlap in enrollment between studies. Active doses were administered and evaluated sequentially (lowest dose first) for safety. ION-02 participants received either 5000 µg or 10 000 µg URO-902, or placebo. ION-03 participants received either 16 000 or 24 000 µg URO-902, or placebo, injected directly into the bladder wall using cystoscopy. Primary outcome variables were safety parameters occurring subsequent to URO-902 administration; secondary efficacy variables also were evaluated. RESULTS: Among the safety outcomes, there were no dose-limiting toxicities or significant adverse events (AEs) preventing dose escalation during either trial, and no participants withdrew due to AEs. For efficacy, in ION-02 (N = 21), involuntary detrusor contractions on urodynamics at 24 weeks in patients receiving URO-902 (P < .0508 vs placebo) and mean urgency incontinence episodes in the 5000 µg group (P = .0812 vs placebo) each showed a downward trend. In ION-03 (N = 13), significant reduction versus placebo in urgency episodes (16 000 µg, P = .036; 24 000 µg, P = .046) and number of voids (16 000 µg, -2.16, P = .044; 24 000 µg, -2.73, P = .047) were observed 1 week after injection. CONCLUSION: Promising safety and efficacy results in these preliminary phase 1 studies suggest gene transfer may be a promising therapy for OAB/DO, warranting further investigation.

Roles of LRRC26 as an auxiliary γ1-subunit of large-conductance Ca2+-activated K+ channels in bronchial smooth muscle cells.

In visceral smooth muscle cells (SMCs), the large-conductance Ca2+-activated K+ (BK) channel is one of the key elements underlying a negative feedback mechanism that is essential for the regulation of intracellular Ca2+ concentration. Although leucine-rich repeat-containing (LRRC) proteins have been identified as novel auxiliary γ-subunits of the BK channel (BKγ) in several cell types, its physiological roles in SMCs are unclear. The BKγ expression patterns in selected SM tissues were examined using real-time PCR analyses and Western blotting. The functional contribution of BKγ1 to BK channel activity was examined by whole cell patch-clamp in SMCs and heterologous expression systems. BKγ1 expression in mouse bronchial SMCs (mBSMCs) was higher than in other several SMC types. Coimmunoprecipitation and total internal reflection fluorescence imaging analyses revealed molecular interaction between BKα and BKγ1 in mBSMCs. Under voltage-clamp, steady-state activation of BK channel currents at pCa 8.0 in mBSMCs occurred in a voltage range comparable to that of reconstituted BKα/BKγ1 complex. However, this range was much more negative than in mouse aortic SMCs (mASMCs) or in HEK293 cells expressing BKα alone and ß-subunit (BKß1). Mallotoxin, a selective activator of BK channel that lacks BKγ1, dose-dependently activated BK currents in mASMCs but not in mBSMCs. The abundant expression of BKγ1 in mBSMCs extensively facilitates BK channel activity to keep the resting membrane potential at negative values and prevents contraction under physiological conditions.

Zileuton, a 5-Lipoxygenase Inhibitor, Exerts Anti-Angiogenic Effect by Inducing Apoptosis of HUVEC via BK Channel Activation.

The arachidonic acid metabolism through 5-lipoxygenase (5-LO) pathways is involved in modulating both tumorigenesis and angiogenesis. Although anti-carcinogenic activities of certain 5-LO inhibitors have been reported, the role of zileuton, a well known 5-LO inhibitor, on the endothelial cell proliferation and angiogenesis has not been fully elucidated. Here, we report that zileuton has an anti-angiogenic effect, and the underlying mechanisms involved activation of the large-conductance Ca2+-activated K+ (BK) channel. Our results show that zileuton significantly prevented vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as in vivo. However, such anti-angiogenic effect of zileuton was abolished by iberiotoxin (IBTX), a BK channel blocker, suggesting zileuton-induced activation of BK channel was critical for the observed anti-angiogenic effect of zileuton. Furthermore, the anti-angiogenic effect of zileuton was, at least, due to the activation of pro-apoptotic signaling cascades which was also abolished by IBTX. Additionally, zileuton suppressed the expression of VCAM-1, ICAM-1, ETS related gene (Erg) and the production of nitric oxide (NO). Taken together, our results show that zileuton prevents angiogenesis by activating the BK channel dependent-apoptotic pathway, thus highlighting its therapeutic capacity in angiogenesis-related diseases, such as cancer.