Activation mechanism and novel binding sites of the BKCa channel activator CTIBD.

The large-conductance calcium-activated potassium (BKCa) channel, which is crucial for urinary bladder smooth muscle relaxation, is a potential target for overactive bladder treatment. Our prior work unveiled CTIBD as a promising BKCa channel activator, altering V 1/2 and G max This study investigates CTIBD's activation mechanism, revealing its independence from the Ca2+ and membrane voltage sensing of the BKCa channel. Cryo-electron microscopy disclosed that two CTIBD molecules bind to hydrophobic regions on the extracellular side of the lipid bilayer. Key residues (W22, W203, and F266) are important for CTIBD binding, and their replacement with alanine reduces CTIBD-mediated channel activation. The triple-mutant (W22A/W203A/F266A) channel showed the smallest V 1/2 shift with a minimal impact on activation and deactivation kinetics by CTIBD. At the single-channel level, CTIBD treatment was much less effective at increasing P o in the triple mutant, mainly because of a drastically increased dissociation rate compared with the WT. These findings highlight CTIBD's mechanism, offering crucial insights for developing small-molecule treatments for BKCa-related pathophysiological conditions.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Evaluation of Progesterone Receptor Antagonist and Maxi-K Channel Agonist as Neuroprotective in Feeney's Weight Drop Model of TBI.

Background: Neuroprotection in traumatic brain injury (TBI) is an unmet medical need. Objective: We evaluated two agents, aglepristone (progesterone receptor antagonist) and N-salicyloyltryptamine (STP) (activator of Maxi-K channel in GH3 cells), for neuroprotection in Feeney's weight drop model of TBI. Material and Methods: Forty-eight male Wistar rats were divided into six groups (n = 8 per group). A battery of six neurobehavioral tests was evaluated at the end of the first week (EO1W), second week (EO2W), and third week (EO3W). In addition, histopathological and immunohistochemistry (BAX, Bcl-2, and M30 Cytodeath) tests were performed at EO3W. Results: Aglepristone at 10 mg/kg showed significant neuroprotection compared to control as assessed by Rota-rod test at EO1W, VEFP right paw and 28-point neurobehavioral test at EO2W, MWM test at EO3W, and positive histopathological and IHC findings. Aglepristone at 20 mg/kg showed negative results as assessed by BAX expression, downregulation of Bcl-2, and positive M30 Cytodeath, thereby suggesting toxicity at higher doses. STP 100 mg/kg showed modest neuroprotective activity but failed to show a dose-response relationship at a dose of 50 mg/kg. Conclusion: The study shows that progesterone receptor antagonists have neuroprotection at lower doses and toxicity at higher doses.

BKCa Channel Activation Attenuates the Pathophysiological Progression of Monocrotaline-Induced Pulmonary Arterial Hypertension in Wistar Rats.

PURPOSE: In the present study, the therapeutic efficacy of a selective BKCa channel opener (compound X) in the treatment of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) was investigated. METHODS: PAH was induced in male Wistar rats by a single injection of MCT. After two weeks, the MCT-treated group was divided into two groups that were either treated with compound X or vehicle. Compound X was administered daily at 28 mg/kg. Electrocardiographic, echocardiographic, and haemodynamic analyses were performed; ex vivo evaluations of pulmonary artery reactivity, right ventricle (RV) and lung histology as well as expression levels of α and ß myosin heavy chain, brain natriuretic peptide, and cytokines (TNFα and IL10) in heart tissue were performed. RESULTS: Pulmonary artery rings of the PAH group showed a lower vasodilatation response to acetylcholine, suggesting endothelial dysfunction. Compound X promoted strong vasodilation in pulmonary artery rings of both control and MCT-induced PAH rats. The untreated hypertensive rats presented remodelling of pulmonary arterioles associated with increased resistance to pulmonary flow; increased systolic pressure, hypertrophy and fibrosis of the RV; prolongation of the QT and Tpeak-Tend intervals (evaluated during electrocardiogram); increased lung and liver weights; and autonomic imbalance with predominance of sympathetic activity. On the other hand, treatment with compound X reduced pulmonary vascular remodelling, pulmonary flow resistance and RV hypertrophy and afterload. CONCLUSION: The use of a selective and potent opener to activate the BKCa channels promoted improvement of haemodynamic parameters and consequent prevention of RV maladaptive remodelling in rats with MCT-induced PAH.

Preclinical pharmacological in vitro investigations on low chloride conductance myotonia: effects of potassium regulation.

In myotonia, reduced Cl- conductance of the mutated ClC-1 channels causes hindered muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. Repetitive contraction temporarily decreases myotonia, a phenomena called "warm up." The underlying mechanism for the reduction of hyperexcitability in warm-up is currently unknown. Since potassium displacement is known to reduce excitability in, for example, muscle fatigue, we characterized the role of potassium in native myotonia congenita (MC) muscle. Muscle specimens of ADR mice (an animal model for low gCl- conductance myotonia) were exposed to increasing K+ concentrations. To characterize functional effects of potassium ion current, the muscle of ADR mice was exposed to agonists and antagonists of the big conductance Ca2+-activated K+ channel (BK) and the voltage-gated Kv7 channel. Effects were monitored by functional force and membrane potential measurements. By increasing [K+]0 to 5 mM, the warm-up phenomena started earlier and at [K+]0 7 mM only weak myotonia was detected. The increase of [K+]0 caused a sustained membrane depolarization accompanied with a reduction of myotonic bursts in ADR mice. Retigabine, a Kv7.2-Kv7.5 activator, dose-dependently reduced relaxation deficit of ADR myotonic muscle contraction and promoted the warm-up phenomena. In vitro results of this study suggest that increasing potassium conductivity via activation of voltage-gated potassium channels enhanced the warm-up phenomena, thereby offering a potential therapeutic treatment option for myotonia congenita.

Fe2+-Mediated Activation of BKCa Channels by Rapid Photolysis of CORM-S1 Releasing CO and Fe2.

Heme catabolism by heme oxygenase (HO) with a decrease in intracellular heme concentration and a concomitant local release of CO and Fe2+ has the potential to regulate BKCa channels. Here, we show that the iron-based photolabile CO-releasing molecule CORM-S1 [dicarbonyl-bis(cysteamine)iron(II)] coreleases CO and Fe2+, making it a suitable light-triggered source of these downstream products of HO activity. To investigate the impact of CO, iron, and cysteamine on BKCa channel activation, human Slo1 (hSlo1) was expressed in HEK293T cells and studied with electrophysiological methods. Whereas hSlo1 channels are activated by CO and even more strongly by Fe2+, Fe3+ and cysteamine possess only marginal activating potency. Investigation of hSlo1 mutants revealed that Fe2+ modulates the channels mainly through the Mg2+-dependent activation mechanism. Flash photolysis of CORM-S1 suits for rapid and precise delivery of Fe2+ and CO in biological settings.

Modulation of TRPV4 and BKCa for treatment of brain diseases.

As a member of transient receptor potential family, the transient receptor potential vanilloid 4 (TRPV4) is a kind of nonselective calcium-permeable cation channel, which belongs to non-voltage gated Ca2+ channel. Large-conductance Ca2+-activated K+ channel (BKCa) represents a unique superfamily of Ca2+-activated K+ channel (KCa) that is both voltage and intracellular Ca2+ dependent. Not surprisingly, aberrant function of either TRPV4 or BKCa in neurons has been associated with brain disorders, such as Alzheimer's disease, cerebral ischemia, brain tumor, epilepsy, as well as headache. In these diseases, vascular dysfunction is a common characteristic. Notably, endothelial and smooth muscle TRPV4 can mediate BKCa to regulate cerebral blood flow and pressure. Therefore, in this review, we not only discuss the diverse functions of TRPV4 and BKCa in neurons to integrate relative signaling pathways in the context of cerebral physiological and pathological situations respectively, but also reveal the relationship between TRPV4 and BKCa in regulation of cerebral vascular tone as an etiologic factor. Based on these analyses, this review demonstrates the effective mechanisms of compounds targeting these two channels, which may be potential therapeutic strategies for diseases in the brain.

The BK activator NS11021 partially protects rat kidneys from cold storage and transplantation-induced mitochondrial and renal injury.

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS induces mitochondrial and cellular injury, which exacerbates renal graft dysfunction, highlighting the need for therapeutic interventions. Using an in vitro model of renal CS, we recently reported that pharmacological activation of the mitochondrial BK channel (mitoBK) during CS protected against CS-induced mitochondrial injury and cell death. Here, we used an in vivo syngeneic rat model of renal CS (18 h) followed by transplantation (24 h reperfusion) (CS + Tx) to similarly evaluate whether addition of a mitoBK activator to the CS solution can alleviate CS + Tx-induced renal injury. Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from rat kidneys, and mitoBK protein level was reduced after CS + Tx compared to sham surgery. The addition of the BK activator NS11021 (3 µM) to the CS solution partially protected against CS + Tx-induced mitochondrial respiratory dysfunction, oxidative protein nitration, and cell death, but not acute renal dysfunction (SCr and BUN). In summary, the current preclinical study shows that pharmacologically targeting mitoBK channels during CS may be a promising therapeutic intervention to prevent CS + Tx-induced mitochondrial and renal injury.

Impact of Ca2+-Sensitive Potassium Channels in Levosimendan-Induced Postconditioning.

PURPOSE: Small and big conductance Ca2+-sensitive potassium (KCa) channels are involved in cardioprotective measures aiming at reducing myocardial reperfusion injury. For levosimendan, infarct size-reducing effects were shown. Whether activation of these channels is involved in levosimendan-induced postconditioning is unknown. We hypothesized that levosimendan exerts a concentration-dependent cardioprotective effect and that both types of Ca2+-sensitive potassium channels are involved. METHODS: In a prospective blinded experimental laboratory investigation, hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. At the onset of reperfusion, hearts were perfused with various concentrations of levosimendan (0.03-1 µM) in order to determine a concentration-response relationship. To elucidate the involvement of KCa-channels for the observed cardioprotection, in the second set of experiments, 0.3 µM levosimendan was administered in combination with the subtype-specific KCa-channel inhibitors paxilline (1 µM, big KCa-channel) and NS8593 (0.1 µM, small KCa-channel) respectively. Infarct size was determined by tetrazolium chloride (TTC) staining. RESULTS: Infarct size in controls was 60 ± 7% and 59 ± 6% respectively. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 5% (P < 0.0001 vs. control). Higher concentrations of levosimendan did not induce a stronger effect. Paxilline but not NS8593 completely abolished levosimendan-induced cardioprotection while both substances alone had no effect on infarct size. CONCLUSIONS: Cardioprotection by levosimendan-induced postconditioning shows a binary phenomenon, either ineffective or with maximal effect. The cardioprotective effect requires activation of big but not small KCa channels.

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.

A computational model of large conductance voltage and calcium activated potassium channels: implications for calcium dynamics and electrophysiology in detrusor smooth muscle cells.

The large conductance voltage and calcium activated potassium (BK) channels play a crucial role in regulating the excitability of detrusor smooth muscle, which lines the wall of the urinary bladder. These channels have been widely characterized in terms of their molecular structure, pharmacology and electrophysiology. They control the repolarising and hyperpolarising phases of the action potential, thereby regulating the firing frequency and contraction profiles of the smooth muscle. Several groups have reported varied profiles of BK currents and I-V curves under similar experimental conditions. However, no single computational model has been able to reconcile these apparent discrepancies. In view of the channels' physiological importance, it is imperative to understand their mechanistic underpinnings so that a realistic model can be created. This paper presents a computational model of the BK channel, based on the Hodgkin-Huxley formalism, constructed by utilising three activation processes - membrane potential, calcium inflow from voltage-gated calcium channels on the membrane and calcium released from the ryanodine receptors present on the sarcoplasmic reticulum. In our model, we attribute the discrepant profiles to the underlying cytosolic calcium received by the channel during its activation. The model enables us to make heuristic predictions regarding the nature of the sub-membrane calcium dynamics underlying the BK channel's activation. We have employed the model to reproduce various physiological characteristics of the channel and found the simulated responses to be in accordance with the experimental findings. Additionally, we have used the model to investigate the role of this channel in electrophysiological signals, such as the action potential and spontaneous transient hyperpolarisations. Furthermore, the clinical effects of BK channel openers, mallotoxin and NS19504, were simulated for the detrusor smooth muscle cells. Our findings support the proposed application of these drugs for amelioration of the condition of overactive bladder. We thus propose a physiologically realistic BK channel model which can be integrated with other biophysical mechanisms such as ion channels, pumps and exchangers to further elucidate its micro-domain interaction with the intracellular calcium environment.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.

Preconditioning by Levosimendan is Mediated by Activation of Mitochondrial Ca2+-Sensitive Potassium (mBKCa) Channels.

PURPOSE: Activation of mitochondrial large-conductance Ca2+-sensitive potassium (mBKCa)-channels is a crucial step for cardioprotection by preconditioning. Whether activation of these channels is involved in levosimendan-induced preconditioning is unknown. We investigated if cardioprotection by levosimendan requires activation of mBKCa-channels in the rat heart in vitro. METHODS: In a prospective blinded experimental laboratory investigation, hearts of male Wistar rats were randomized and placed on a Langendorff system, perfused with Krebs-Henseleit buffer at a constant pressure of 80 mmHg. All hearts were subjected to 33 min of global ischemia and 60 min of reperfusion. Before ischemia, hearts were perfused with different concentrations of levosimendan (0.03-1 µM) for determination of a dose-effect curve. In a second set of experiments, 0.3 µM levosimendan was administered in combination with the mBKCa-channel inhibitor paxilline (1 µM). Infarct size was determined by TTC staining. RESULTS: In control, animal's infarct size was 58 ± 7%. Levosimendan at a concentration of 0.3 µM reduced infarct size to 30 ± 7% (P < 0.05 vs. control). Higher concentrations with 1 µM levosimendan did not confer stronger protection. Paxilline completely blocked levosimendan-induced cardioprotection while paxilline alone had no effect on infarct size. CONCLUSIONS: This study shows that activation of mBKCa-channels plays a pivotal role in levosimendan-induced preconditioning.

Inhibition of ZERO-BK by PKC is involved in carbachol-induced enhancement of rat colon smooth muscle motility.

BACKGROUND: Muscarinic acetylcholine receptor (mAChR) activation is an important factor to enhance the motility of gastrointestinal (GI) smooth muscle. Large conductance Ca2+ -activated potassium (BK) channels are widely expressed in GI smooth muscle. Roles of BK in carbachol (a mAChR agonist) induced enhancement of GI motility and the molecular mechanisms remains unknown and were investigated in this study. METHODS: Colonic smooth muscle (CSM) strip was perfused to record motility in vitro. The patch-clamp technique was used to record BK currents. RT-PCR was used to detect the expression of BK channels in rat CSM tissues. Two different types BK channels were constructed in HEK293 cells to investigate the regulation mechanism. Paired t tests were set with a P < .05 regarded as significant. KEY RESULTS: Carbachol enhanced CSM contraction through M3 receptor (M3 R) were attenuated by IbTX, an inhibitor of BK. Carbachol inhibited BK currents in CSM cells and Go6983, an inhibitor of protein kinase C (PKC), reversed the effect. PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited BK currents. Two types of BK channels (ZERO-BK and STREX-BK) were detected in CSM. ZERO- but not STREX-BK channels expressed in HEK293 cells were inhibited by PMA. CONCLUSION: Our results provide strong evidence that inhibition of ZERO-BK but not STREX-BK channels via PKC pathway is involved in the enhancement of CSM motility by mAChR activation. Besides the activation of BK by an increase in intracellular calcium, inhibition of BK played an important role in GI motility regulation during mAChR activation.

Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties.

Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models.

Early Alterations of Hippocampal Neuronal Firing Induced by Abeta42.

We studied the effect of Amyloid ß 1-42 oligomers (Abeta42) on Ca2+ dependent excitability profile of hippocampal neurons. Abeta42 is one of the Amyloid beta peptides produced by the proteolytic processing of the amyloid precursor protein and participates in the initiating event triggering the progressive dismantling of synapses and neuronal circuits. Our experiments on cultured hippocampal network reveal that Abeta42 increases intracellular Ca2+ concentration by 46% and inhibits firing discharge by 19%. More precisely, Abeta42 differently regulates ryanodine (RyRs), NMDA receptors (NMDARs), and voltage gated calcium channels (VGCCs) by increasing Ca2+ release through RyRs and inhibiting Ca2+ influx through NMDARs and VGCCs. The overall increased intracellular Ca2+ concentration causes stimulation of K+ current carried by big conductance Ca2+ activated potassium (BK) channels and hippocampal network firing inhibition. We conclude that Abeta42 alters neuronal function by means of at least 4 main targets: RyRs, NMDARs, VGCCs, and BK channels. The development of selective modulators of these channels may in turn be useful for developing effective therapies that could enhance the quality of life of AD patients during the early onset of the pathology.

Preconditioning with the BKCa channel activator NS-1619 prevents ischemia-reperfusion-induced inflammation and mucosal barrier dysfunction: roles for ROS and heme oxygenase-1.

Activation of large-conductance Ca2+-activated K+ (BKCa) channels evokes cell survival programs that mitigate intestinal ischemia and reperfusion (I/R) inflammation and injury 24 h later. The goal of the present study was to determine the roles of reactive oxygen species (ROS) and heme oxygenase (HO)-1 in delayed acquisition of tolerance to I/R induced by pretreatment with the BKCa channel opener NS-1619. Superior mesentery arteries were occluded for 45 min followed by reperfusion for 70 min in wild-type (WT) or HO-1-null (HO-1-/-) mice that were pretreated with NS-1619 or saline vehicle 24 h earlier. Intravital microscopy was used to quantify the numbers of rolling and adherent leukocytes. Mucosal permeability, tumor necrosis factor-α (TNF-α) levels, and HO-1 activity and expression in jejunum were also determined. I/R induced leukocyte rolling and adhesion, increased intestinal TNF-α levels, and enhanced mucosal permeability in WT mice, effects that were largely abolished by pretreatment with NS-1619. The anti-inflammatory and mucosal permeability-sparing effects of NS-1619 were prevented by coincident treatment with the HO-1 inhibitor tin protoporphyrin-IX or a cell-permeant SOD mimetic, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), in WT mice. NS-1619 also increased jejunal HO-1 activity in WT animals, an effect that was attenuated by treatment with the BKCa channel antagonist paxilline or MnTBAP. I/R also increased postischemic leukocyte rolling and adhesion and intestinal TNF-α levels in HO-1-/- mice to levels comparable to those noted in WT animals. However, NS-1619 was ineffective in preventing these effects in HO-1-deficient mice. In summary, our data indicate that NS-1619 induces the development of an anti-inflammatory phenotype and mitigates postischemic mucosal barrier disruption in the small intestine by a mechanism that may involve ROS-dependent HO-1 activity.NEW & NOTEWORTHY Antecedent treatment with the large-conductance Ca2+-activated K+ channel opener NS-1619 24 h before ischemia-reperfusion limits postischemic tissue injury by an oxidant-dependent mechanism. The present study shows that NS-1619-induced oxidant production prevents ischemia-reperfusion-induced inflammation and mucosal barrier disruption in the small intestine by provoking increases in heme oxygenase-1 activity.

Developing Molecular Pharmacology of BK Channels for Therapeutic Benefit.

High conductance, calcium-activated potassium (BK) channels (KCa1.1) are important in regulating physiologic responses in many types of tissues and, as such, present opportunities for development of new therapeutic agents. Both channel agonists and inhibitors could have therapeutic utility, depending on medical application under consideration. However, characterization of molecular pharmacology of BK channels is incomplete and has been difficult to accomplish because of paucity of chemical leads that are acceptable templates for Medicinal Chemistry investigation. Only through continued prosecution of new high-throughput screening campaigns can this situation be rectified. Examples are presented of BK channel agonist and inhibitor discovery paradigms which will be useful for progressing BK channel future drug discovery strategies.

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKß3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p < 0.05). CNTF-ACM produced a significant increase in BKα1 and BKß3 expression (p < 0.05) but had no significant effect upon SK2 or SK3 expression (p > 0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p > 0.05) as well as BKα1 and BKß3 expression in CNTF-ACM-treated neurons (p > 0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

Effect of a novel BKCa opener on BKCa currents and contractility of the rabbit corpus cavernosum.

Large-conductance Ca(2+)-activated K(+) (BKCa) channels are thought to play a key role in the regulation of corpus cavernosum smooth muscle (CCSM) excitability. Few BKCa channel openers have been accepted for clinical development. The effect of the novel BKCa channel opener GoSlo-SR5-130 on electrical activity in isolated rabbit CCSM cells and mechanical activity in strips of rabbit CCSM was examined. Single-channel currents were observed in inside-out patches. These channels were sensitive to Ca(2+), blocked by penitrem A, and had a conductance of 291 ± 20 pS (n = 7). In the presence of GoSlo-SR5-130, the number of open BKCa channels increased. Using voltage-ramp protocols, GoSlo-SR5-130 caused currents to activate at more negative potentials in a concentration-dependent manner, shifting the half-maximal activation voltage potential to the left on the voltage axis. Therefore, BKCa channels were open within the physiological range of membrane potentials in the presence of GoSlo-SR5-130. GoSlo-SR5-130 also resulted in an increase in the activity of spontaneous transient outward currents in myocytes isolated from CCSM, and this effect was reversed by iberiotoxin. In current-clamp mode, GoSlo-SR5-130 hyperpolarized the cell membrane. Isometric tension recording of strips of rabbit corpus cavernosum showed that GoSlo-SR5-130 inhibited spontaneous contractions in a concentration-dependent manner. This effect was reversed in the presence of iberiotoxin, suggesting that GoSlo-SR5-130 exerts its effect through BKCa channels. These findings suggest that GoSlo-SR5-130 is an effective tool for the study of BKCa channels and that these channels can modulate CCSM activity and are possible targets for the treatment of erectile dysfunction.