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Paxilline (PAX) is a tremorgenic mycotoxin that is thought to be associated with various syndromes in animals. To facilitate the determination of this toxin, a hapten was chemically synthesized and then respectively conjugated to Bovine Thyroglobulin (BTG) and Bovine serum albumin (BSA) for antibody production. Monoclonal antibody (mAb) 4F8 was finally obtained and used to establish an accurate and sensitive indirect competitive ELISA (icELISA). Various parameters that could impact the performance of icELISA were optimized, including the ion concentration, pH value and organic solvent. Under optimal condition, this assay exhibits an IC50 value as low as 0.84 µg L−1. The limit of detection (LOD) of the established icELISA method for PAX was 0.03–0.3 µg L−1. In conclusion, this study provided a rapid, sensitive, and high-throughput immunoassay for the detection of PAX in grains. [ABSTRACT FROM AUTHOR]

An eleven-year-old Pit Bull Terrier was presented to the veterinary practice with an acute onset of whole-body seizures. The clinical signs developed in a garden where the dog was kept that morning. There was a suspicion of tremorgenic mycotoxin poisoning by compost as the dog had vomited parts of compost right before the onset of the seizures and there was a pile of compost located in the garden. The dog underwent immediate decontamination following supportive treatment and recovered fully within 24 h of intensive care. The samples of the vomit and parts of the compost were cultivated. In the sample of the vomit, Penicillium sp. was found. Subsequently, tremorgenic mycotoxins paxilline, penitrem A and roquefortine C were determined chromatographically at significant concentrations in the vomit and a growth medium with cultivated Penicillium sp. The aim of this work is to describe the complex therapeutic and diagnostic approach to the patient with a suspected tremorgenic mycotoxin poisoning where a combination of mycological and chromatographic analyses was used to confirm the diagnosis. To the best of our knowledge, this is the first confirmed case of canine tremorgenic mycotoxicosis in the Czech Republic and the first reported case of paxilline poisoning in a dog.

An eleven-year-old Pit Bull Terrier was presented to the veterinary practice with an acute onset of wholebody seizures. The clinical signs developed in a garden where the dog was kept that morning. There was a suspicion of tremorgenic mycotoxin poisoning by compost as the dog had vomited parts of compost right before the onset of the seizures and there was a pile of compost located in the garden. The dog underwent immediate decontamination following supportive treatment and recovered fully within 24 h of intensive care. The samples of the vomit and parts of the compost were cultivated. In the sample of the vomit, Penicillium sp. was found. Subsequently, tremorgenic mycotoxins paxilline, penitrem A and roquefortine C were determined chromatographically at significant concentrations in the vomit and a growth medium with cultivated Penicillium sp. The aim of this work is to describe the complex therapeutic and diagnostic approach to the patient with a suspected tremorgenic mycotoxin poisoning where a combination of mycological and chromatographic analyses was used to confirm the diagnosis. To the best of our knowledge, this is the first confirmed case of canine tremorgenic mycotoxicosis in the Czech Republic and the first reported case of paxilline poisoning in a dog. [ABSTRACT FROM AUTHOR]

Introduction: slowly adapting mechanoreceptors in the skin provide vital tactile information to animals. The ionic channels that underlie their functioning is the subject of intense research. Previous work suggests that potassium channels may play particular roles in the activation and firing of these mechanoreceptors. Objective: We used a range of potassium channel blockers and openers to observe their effects on different phases of mechanoreceptor responses. Methods: Extracellular recording of neural activity of slowly adapting mechanoreceptors was carried out in an in vitro preparation of the sinus hair follicles taken from rat whisker pads. A range of potassium (K+) channel modulators were tested on these mechanoreceptor responses. The channel blockers tested were: tetraethylammonium (TEA), barium chloride (BaCl2), dequalinium, 4-aminopyridine (4-AP), paxilline, XE 991, apamin, and charybdotoxin. Results: Except for charybdotoxin and apamin, these drugs increased the activity of both types of slowly adapting units, St I and St II. Generally, both spontaneous and evoked (dynamic and static) activities increased. The channel opener NS1619 was also tested. NS1619 clearly decreased evoked activity (both dynamic and static) while leaving spontaneous activity relatively unaffected, with no clear discrimination of effects on the two types of St receptor Conclusion: These findings are consistent with the targets of the drugs suggesting that K+ channels play an important role in the maintenance of spontaneous firing and in the production of and persistence of mechanoreceptor activity.

The skin secretion of tree frogs contains a vast array of bioactive chemicals for repelling predators, but their structural and functional diversity is not fully understood. Paxilline (PAX), a compound synthesized by Penicillium paxilli, has been known as a specific antagonist of large conductance Ca2+-activated K+ Channels (BKCa). Here, we report the presence of PAX in the secretions of tree frogs (Hyla japonica) and that this compound has a novel function of inhibiting the potassium channel subfamily K member 18 (KCNK18) channels of their predators. The PAX-induced KCNK18 inhibition is sufficient to evoke Ca2+ influx in charybdotoxin-insensitive DRG neurons of rats. By forming π-π stacking interactions, four phenylalanines located in the central pore of KCNK18 stabilize PAX to block the ion permeation. For PAX-mediated toxicity, our results from animal assays suggest that the inhibition of KCNK18 likely acts synergistically with that of BKCa to elicit tingling and buzzing sensations in predators or competitors. These results not only show the molecular mechanism of PAX-KCNK18 interaction, but also provide insights into the defensive effects of the enriched PAX. [ABSTRACT FROM AUTHOR]

• Quercetin potently activates the mitoBK Ca channel, and this effect is not inhibited by the canonical blocker of this channel, paxilline. • Analog of quercetin, isorhamnetin, has no effect on the mitoBK Ca channel activity. • Due to steric hindrance, the quercetin is large enough to block paxilline from reaching its binding site. Large-conductance calcium-regulated potassium channel (BK Ca) is known to play an important role in physiological and pathological processes. Despite the BK Ca channel being encoded by one gene, this channel has been found to be located not only in the cell membrane but also in the membranes of intracellular compartments, such as in the inner mitochondrial membrane. With some differences, the mitochondrial BK Ca (mitoBK Ca) channel has been shown to be activated or inhibited by both synthetic and natural compounds. One of them, paxilline, has been considered to be a canonical blocker of this channel. In the previous study, we showed that the natural origin substance quercetin activates the mitoBK Ca channel at ten times lower the concentration compared to channel present in the plasma membrane. Here, using the patch-clamp technique, we report that after inhibition of mitoBK Ca channels by paxilline, quercetin activates these channels, indicating a paxilline and quercetin binding competition in the regulation of the mitoBK Ca channel. To support our hypothesis, we used an analog of quercetin – isorhamnetin, a substance with one substituent changed. Isorhamnetin has no effect on the mitoBK Ca channel activity, and after its application, paxilline fully inhibits the channel. Additionally, the molecular modeling studies were used. The results of docking quercetin and paxilline to the BK Ca channel suggest that paxilline cannot bind after activation of the channel with quercetin. It seems that the likely mechanism of this phenomenon is the formation of spatial hindrance by quercetin. The results obtained shed a completely new, groundbreaking in the paxilline context, light on the current knowledge about mitochondrial potassium channel regulation. [ABSTRACT FROM AUTHOR]

• The expression of MaxiK was elevated significantly in cerebral cortex of blast-induced traumatic brain injury (bTBI) rats. • MaxiK might be involved in the process of neuroinflammation of bTBI. • Paxilline could alleviate bTBI by blocking MaxiK channel and subsequently inhibition of NLRP3 inflammasome activation. Abundant findings including our previous work proved that the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome exerts a key role in the process of neuroinflammation following blast-induced traumatic brain injury (bTBI). The opening of potassium channels leads to low K+ environment in cells, which appears to be an essential requirement for NLRP3 inflammasome activation. Notably, MaxiK (BK) channel is significant for K+ transport. The present study is aim to investigate the potential role of MaxiK in the activation of NLRP3 and to evaluate whether MaxiK channel blocker paxilline could confer beneficial effects on attenuating the severity of bTBI in rats. Rats were randomly assigned into five groups (n = 8). MaxiK channel expression was measured in bTBI rats. The effect of paxilline on the expression of NLRP3 inflammasome, the level of inflammatory cytokines, brain injury biomarkers in serum and brain edema were also evaluated in bTBI rats. The results showed that the expression of MaxiK was elevated significantly in the cerebral cortex of bTBI rats. The treatment of MaxiK channel blocker paxilline suppressed the NLRP3 inflammasome expression substantially. In addition, paxilline could also decrease the level of pro-inflammatory cytokines and the biomarkers of brain injury and alleviate brain edema of bTBI rats. Our findings have revealed that MaxiK channel might be involved in the process of neuroinflammation of bTBI. Paxilline could depress neuro-inflammation response and alleviate brain injury by blocking MaxiK channel and subsequently inhibition of NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Caactivated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (~100 ls) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing. [ABSTRACT FROM AUTHOR]

Reduced Cl− conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK−/−) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK−/− mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC).

We recently established a role for the stretch-activated two-poredomain K1 (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-a-induced acute lung injury. We have now discovered the expression of large conductance, Ca21-activated K1 (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or theEmin either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca21 concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca21 concentrations. [ABSTRACT FROM AUTHOR]

Reduced Cl− conductance causes inhibited muscle relaxation after forceful voluntary contraction due to muscle membrane hyperexcitability. This represents the pathomechanism of myotonia congenita. Due to the prevailing data suggesting that an increased potassium level is a main contributor, we studied the effect of a modulator of a big conductance Ca2+- and voltage-activated K+ channels (BK) modulator on contraction and relaxation of slow- and high-twitch muscle specimen before and after the pharmacological induction of myotonia. Human and murine muscle specimens (wild-type and BK−/−) were exposed to anthracene-9-carboxylic acid (9-AC) to inhibit CLC-1 chloride channels and to induce myotonia in-vitro. Functional effects of BK-channel activation and blockade were investigated by exposing slow-twitch (soleus) and fast-twitch (extensor digitorum longus) murine muscle specimens or human musculus vastus lateralis to an activator (NS1608) and a blocker (Paxilline), respectively. Muscle-twitch force and relaxation times (T90/10) were monitored. Compared to wild type, fast-twitch muscle specimen of BK−/− mice resulted in a significantly decreased T90/10 in presence of 9-AC. Paxilline significantly shortened T90/10 of murine slow- and fast-twitch muscles as well as human vastus lateralis muscle. Moreover, twitch force was significantly reduced after application of Paxilline in myotonic muscle. NS1608 had opposite effects to Paxilline and aggravated the onset of myotonic activity by prolongation of T90/10. The currently used standard therapy for myotonia is, in some individuals, not very effective. This in vitro study demonstrated that a BK channel blocker lowers myotonic stiffness and thus highlights its potential therapeutic option in myotonia congenital (MC). [ABSTRACT FROM AUTHOR]

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca2+-activated K+ (BK Ca) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BK Ca channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BK Ca channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and β2-4 subunits of the BK Ca channel, the novel regulatory γ1 subunit (BK Ca γ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BK Ca γ1. Store-operated Ca2+ entry was smaller in BK Ca γ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BK Ca γ1. These results strongly suggest that BK Ca γ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BK Ca γ1 may be a novel therapeutic target for somatostatinoma. • Somatostatinoma is a pancreatic neuroendocrine tumor with high malignancy. • Somatostatinoma QGP-1 cells have large-conductance Ca2+-activated K+ (BK Ca) currents. • The γ1 subunit of BK Ca channel (BK Ca γ1) was functionally expressed in QGP-1 cells. • The proliferation of QGP-1 cells was facilitated by the presence of BK Ca γ1. • BK Ca γ1 may be a novel therapeutic target for somatostatinoma. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The indole diterpenoid toxin lolitrem B is a tremorgenic agent found in the common grass species, perennial ryegrass (Lolium perenne). The toxin is produced by a symbiotic fungus Epichloë festucae (var. lolii) and ingestion of infested grass with sufficient toxin levels causes a movement disorder in grazing herbivores known as 'ryegrass staggers'. Beside ataxia, lolitrem B intoxicated animals frequently show indicators of cognitive dysfunction or exhibition of erratic and unpredictable behaviours during handling. Evidence from field cases in livestock and controlled feeding studies in horses have indicated that intoxication with lolitrem B may affect higher cortical or subcortical functioning. In order to define the role of lolitrem B in voluntary motor control, spatial learning and memory under controlled conditions, mice were exposed to a known dose of purified lolitrem B toxin and tremor, coordination, voluntary motor activity and spatial learning and memory assessed. Motor activity, coordination and spatial memory were compared to tremor intensity using a novel quantitative piezo-electronic tremor analysis. Peak tremor was observed as frequencies between 15 and 25Hz compared to normal movement at approximately 1.4–10Hz. A single exposure to a known tremorgenic dose of lolitrem B (2 mg/kg IP) induced measureable tremor for up to 72 h in some animals. Initially, intoxication with lolitrem B significantly decreased voluntary movement. By 25 h post exposure a return to normal voluntary movement was observed in this group, despite continuing evidence of tremor. This effect was not observed in animals exposed to the short-acting tremorgenic toxin paxilline. Lolitrem B intoxicated mice demonstrated a random search pattern and delayed latency to escape a 3 h post intoxication, however by 27 h post exposure latency to escape matched controls and mice had returned to normal searching behavior indicating normal spatial learning and memory. Together these data indicate that the tremor exhibited by lolitrem B intoxicated mice does not directly impair spatial learning and memory but that exposure does reduce voluntary motor activity in intoxicated animals. Management of acutely affected livestock suffering toxicosis should be considered in the context of their ability to spatially orientate with severe toxicity. Image 1 • Single exposure to lolitrem B toxin (2 mg/kg) causes tremor lasting up to 72 hours in mice. • Tremor quantitation is accurately measured mice exposed to tremorgenic toxins using piezoelectric pressure sensor analysis. • Lolitrem B exerts a negative, but resolving, effect on voluntary movement despite continued presence of tremor. • Lolitrem B intoxication does not impede spatial learning and memory in mice. • Acute effects on spatial cognition likely contribute to mortality and morbidity cases of perennial ryegrass toxicosis. [ABSTRACT FROM AUTHOR]

Key points: Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa) by BK‐β subunits is not established.MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK‐β1 (left‐shifted voltage dependence of activation, V1/2 = −55 mV, 12 µm matrix Ca2+).In BK‐β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+)Co‐expression of BKCa with the BK‐β1 subunit in HeLa cells doubled the density of BKCa in mitochondria.The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. Association of the plasma membrane BKCa channel with auxiliary BK‐β1–4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK‐β1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium‐ and voltage‐activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po) and voltage half‐activation (V1/2 = −55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK‐β1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa‐α and its BK‐β1 subunit. Mitochondria from the BK‐β1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK‐β1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK‐β1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two‐fold. Importantly, both paxilline‐treated and BK‐β1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK‐β1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis. Key points: Association of plasma membrane BKCa channels with BK‐β subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa) by BK‐β subunits is not established.MitoBKCa‐α and the regulatory BK‐β1 subunit associate in mouse cardiac mitochondria.A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK‐β1 (left‐shifted voltage dependence of activation, V1/2 = −55 mV, 12 µm matrix Ca2+).In BK‐β1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+)Co‐expression of BKCa with the BK‐β1 subunit in HeLa cells doubled the density of BKCa in mitochondria.The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK‐β1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. [ABSTRACT FROM AUTHOR]

Mitochondria play a key role in energy metabolism within the cell. Potassium channels such as ATP-sensitive, voltage-gated or large-conductance Ca2+-regulated channels have been described in the inner mitochondrial membrane. Several hypotheses have been proposed to describe the important roles of mitochondrial potassium channels in cell survival and death pathways. In the current study, we identified two populations of mitochondrial large-conductance Ca2+-regulated potassium (mitoBKCa) channels in human bronchial epithelial (HBE) cells. The biophysical properties of the channels were characterized using the patch-clamp technique. We observed the activity of the channel with a mean conductance close to 285 pS in symmetric 150/150 mM KCl solution. Channel activity was increased upon application of the potassium channel opener NS11021 in the micromolar concentration range. The channel activity was completely inhibited by 1 µM paxilline and 300 nM iberiotoxin, selective inhibitors of the BKCa channels. Based on calcium and iberiotoxin modulation, we suggest that the C-terminus of the protein is localized to the mitochondrial matrix. Additionally, using RT-PCR, we confirmed the presence of α pore-forming (Slo1) and auxiliary β3-β4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately β3 subunits. Additionally, the regulation of oxygen consumption and membrane potential of human bronchial epithelial mitochondria in the presence of the potassium channel opener NS11021 and inhibitor paxilline were also studied. In summary, for the first time, the electrophysiological and functional properties of the mitoBKCa channel in a bronchial epithelial cell line were described.

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrations of PAX required to inhibit signal development by 50% (IC50s) ranged from 1.2 to 2.5 ng/mL. One mAb (2-9) was applied to the detection of PAX in maize silage. The assay was sensitive to the effects of solvents, with 5% acetonitrile or 20% methanol causing a two-fold or greater increase in IC50. For analysis of silage samples, extracts were cleaned up by adsorbing potential matrix interferences onto a solid phase extraction column. The non-retained extract was then diluted with buffer to reduce solvent content prior to assay. Using this method, the limit of detection for PAX in dried silage was 15 µg/kg and the limit of quantification was 90 µg/kg. Recovery from samples spiked over the range of 100 to 1000 µg/kg averaged 106% ± 18%. The assay was applied to 86 maize silage samples, with many having detectable, but none having quantifiable, levels of PAX. The results suggest the CI-ELISA can be applied as a sensitive technique for the screening of PAX in maize silage.

Abstract Recurrent spreading depolarizations occur in the cerebral cortex from minutes up to weeks following acute brain injury. Clinical evidence suggests that the immediate reduction of cerebral blood flow in response to spreading depolarization importantly contributes to lesion progression as the wave propagates over vulnerable tissue zones, characterized by potassium concentration already elevated prior to the passage of spreading depolarization. Here we demonstrate with two-photon microscopy in anesthetized mice that initial vasoconstriction in response to SD triggered experimentally with 1 M KCl is coincident in space and time with the large extracellular accumulation of potassium, as shown with a potassium indicator fluorescent dye. Moreover, pharmacological manipulations in combination with the use of potassium-sensitive microelectrodes suggest that large-conductance Ca2+-activated potassium (BK) channels and L-type voltage-gated calcium channels play significant roles in the marked initial vasoconstriction under elevated baseline potassium. We propose that potassium efflux through BK channels is a central component in the devastating neurovascular effects of spreading depolarizations in tissue at risk. Graphical abstract Unlabelled Image Highlights • Potassium wave of spreading depolarization (SD) initiates arteriolar constriction. • BK channels are significantly implicated in SD-related vasoconstriction. • L-type voltage-gated Ca2+ channels partially mediate SD-related vasoconstriction. • K+ efflux during SD manifests through BK channels to a significant degree. [ABSTRACT FROM AUTHOR]

Physiological functions of type 3 ryanodine receptors (RyR3) in smooth muscle (SM) tissues are not well understood, in spite of their wide expression. However, the short isoform of RyR3 is known to be a dominant-negative variant (DN-RyR3), which may negatively regulate functions of both RyR2 and full-length (FL) RyR3 by forming hetero-tetramers. Here, functional roles of RyR3 in the regulation of Ca2+ signaling in mesenteric artery SM cells (MASMCs) were examined using RyR3 homozygous knockout mice (RyR3-/-). Quantitative PCR analyses suggested that the predominant RyR3 subtype in MASMs from wild-type mice (RyR3+/+) was DN-RyR3. In single MASMCs freshly isolated from RyR3-/-, the EC50 of caffeine to induce Ca2+ release was lower than that in RyR3+/+ myocytes. The amplitude and frequency of Ca2+ sparks and spontaneous transient outward currents in MASMCs from RyR3-/- were all larger than those from RyR3+/+. Importantly, mRNA and functional expressions of voltage-dependent Ca2+ channel and large-conductance Ca2+- activated K+ (BK) channel in MASMCs from RyR3-/- were identical to those from RyR3+/+. However, in the presence of BK channel inhibitor, paxilline, the pressure rises induced by BayK8644 in MA vascular beds of RyR3-/- were significantly larger than in those of RyR3+/+. This indicates that the negative feedback effects of BK channel activity on intracellular Ca2+ signaling was enhanced in RyR3-/-. Thus, RyR3, and, in fact, mainly DN-RyR3, via a complex with RyR2 suppresses Ca2+ release and indirectly regulated membrane potential by reducing BK channel activity in MASMCs and presumably can affect the regulation of intrinsic vascular tone. [ABSTRACT FROM AUTHOR]

The cellular mechanisms by which LC neurons respond to hypercapnia are usually attributed to an “accelerator” whereby hypercapnic acidosis causes an inhibition of K + channels or activation of Na + and Ca +2 channels to depolarize CO 2 -sensitive neurons. Nevertheless, it is still unknown if this “accelerator” mechanism could be controlled by a brake phenomenon. Whole-cell patch clamping, fluorescence imaging microscopy and plethysmography were used to study the chemosensitive response of the LC neurons. Hypercapnic acidosis activates L-type Ca 2+ channels and large conductance Ca-activated K + (BK) channels, which function as a “brake” on the chemosensitive response of LC neurons. Our findings indicate that both Ca 2+ and BK currents develop over the first 2 weeks of postnatal life in rat LC slices and that this brake pathway may cause the developmental decrease in the chemosensitive firing rate response of LC neurons to hypercapnic acidosis. Inhibition of this brake by paxilline (BK channel inhibitor) returns the magnitude of the chemosensitive firing rate response from LC neurons in rats older than P10 to high values similar to those in LC neurons from younger rats. Inhibition of BK channels in LC neurons by bilateral injections of paxilline into the LC results in a significant increase in the hypercapnic ventilatory response of adult rats. Our findings indicate that a BK channel-based braking system helps to determine the chemosensitive respiratory drive of LC neurons and contributes to the hypercapnic ventilatory response. Perhaps, abnormalities of this braking system could result in hypercapnia-induced respiratory disorders and panic responses. [ABSTRACT FROM AUTHOR]

Abstract: Large conductance calcium and voltage‐activated potassium channels (BKCa) are transmembrane proteins, ubiquitously expressed in the majority of organs, and play an active role in regulating cellular physiology. In the heart, BKCa channels are known to play a role in regulating the heart rate and protect it from ischemia–reperfusion injury. In vascular smooth muscle cells, the opening of BKCa channels results in membrane hyperpolarization which eventually results in vasodilation mediated by a reduction in Ca2+ influx due to the closure of voltage‐dependent Ca2+ channels. Ex vivo studies have shown that BKCa channels play an active role in the regulation of the function of the majority of blood vessels. However, in vivo role of BKCa channels in cardiovascular function is not completely deciphered. Here, we have evaluated the rapid in vivo role of BKCa channels in regulating the cardiovascular function by using two well‐established, rapid‐acting, potent blockers, paxilline and iberiotoxin. Our results show that BKCa channels are actively involved in regulating the heart rate, the function of the left and right heart as well as major vessels. We also found that the effect on BKCa channels by blockers is completely reversible, and hence, BKCa channels can be exploited as potential targets for clinical applications for modulating heart rate and cardiac contractility. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) impairs autoregulation of cerebral blood flow, which contributes to the development of secondary brain injury, increasing mortality of patients. Impairment of pressure-induced myogenic constriction of cerebral arteries plays a critical role in autoregulatory dysfunction; however, the underlying cellular and molecular mechanisms are not well understood. To determine the role of mitochondria-derived H2O2 and large-conductance calcium-activated potassium channels (BKCa) in myogenic autoregulatory dysfunction, middle cerebral arteries (MCAs) were isolated from rats with severe weight drop-impact acceleration brain injury. We found that 24 h post-TBI MCAs exhibited impaired myogenic constriction, which was restored by treatment with a mitochondria-targeted antioxidant (mitoTEMPO), by scavenging of H2O2 (polyethylene glycol [PEG]-catalase) and by blocking both BKCa channels (paxilline) and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels (HC 067047). Further, exogenous administration of H2O2 elicited significant dilation of MCAs, which was inhibited by blocking either BKCa or TRPV4 channels. Vasodilation induced by the TRPV4 agonist GSK1016790A was inhibited by paxilline. In cultured vascular smooth muscle cells H2O2 activated BKCa currents, which were inhibited by blockade of TRPV4 channels. Collectively, our results suggest that after TBI, excessive mitochondria-derived H2O2 activates BKCa channels via a TRPV4-dependent pathway in the vascular smooth muscle cells, which impairs pressure-induced constriction of cerebral arteries. Future studies should elucidate the therapeutic potential of pharmacological targeting of this pathway in TBI, to restore autoregulatory function in order to prevent secondary brain damage and decrease mortality. [ABSTRACT FROM AUTHOR]

The tremorgenic paxilline (PAX), an indolediterpene alkaloid mycotoxin, was recently detected as a natural contaminant of ergot of barley and rye. To check the possibility of a transfer of this mycotoxin into beer, a rapid and sensitive immunochemical method for the analysis of PAX in commercial bottled beer was developed. A straightforward sample preparation procedure could be established, including degassing, pH adjustment, optional filtering, and finally a 1:5 dilution with a methanolic phosphate-buffered saline solution. Analysis of PAX was performed by a competitive indirect enzyme immunoassay (EIA). The detection limit at a cut-off value of 80% B/B0 of the EIA standard curve was evaluated by analysis of spiked beer. PAX at levels of 3 μg/L and 5 μg/L yielded 69% and 100% positive results, therefore the detection limit in beer was at 5 μg/L. Recoveries of PAX at levels of 5-20 μg/L were 88-97%, coefficients of variation were 17-22%.With these characteristics, the EIAwas considered to be a suitable screening method for PAX in beer. A survey of bottled beer from the German market was conducted which included 38 samples of domestic and international brands, the latter containing various flavoring ingredients. [ABSTRACT FROM AUTHOR]

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the fungal communities found in dispensary based Cannabis flowers by ITS2 sequencing, and demonstrate the sensitive detection of several toxigenic Penicillium and Aspergillus species, including P. citrinum and P. paxilli, that were not detected by one or more culture-based methods currently in use for safety testing.

Summary: Aims: We investigated the vasorelaxant effect of nateglinide and its related mechanisms using phenylephrine (Phe)‐induced precontracted aortic rings. Methods: Arterial tone measurement was performed in aortic smooth muscle. Results: The application of nateglinide induced vasorelaxation in a concentration‐dependent manner. Pretreatment with the large‐conductance Ca2+‐activated K+ (BKCa) channel inhibitor paxilline, the inwardly rectifying K+ (Kir) channel inhibitor Ba2+, and ATP‐sensitive K+ (KATP) channel inhibitor glibenclamide did not affect the vasorelaxant effect of nateglinide. However, pretreatment with the voltage‐dependent K+ (Kv) channel inhibitor 4‐aminopyridine (4‐AP) effectively reduced the vasorelaxant effect of nateglinide. Pretreatment with the Ca2+ inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase inhibitor thapsigargin did not change the vasorelaxant effect of nateglinide. Additionally, the vasorelaxant effect of nateglinide was not altered in the presence of an adenylyl cyclase, a protein kinase A, a guanylyl cyclase, or a protein kinase G inhibitor. The vasorelaxant effect of nateglinide was not affected by the elimination of the endothelium. In addition, pretreatment with a nitric oxide synthase inhibitor, L‐NAME, and a small‐conductance Ca2+‐activated K+ (SKCa) channel inhibitor, apamin, did not change the vasorelaxant effect of nateglinide. Conclusion: Nateglinide induced vasorelaxation via the activation of the Kv channel independent of other K+ channels, Ca2+ channels, intracellular Ca2+ ([Ca2+]i), and the endothelium. [ABSTRACT FROM AUTHOR]

The present study was designed to investigate whether large conductance Ca2+-activated K+ ( BK) channels were regulated by epidermal growth factor ( EGF) receptor ( EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK- HEK 293 cells expressing both functional α-subunits and the auxiliary β1-subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK- HEK 293 cells was increased by the broad spectrum protein tyrosine kinase ( PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase ( PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α- and β1-subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre-contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries. [ABSTRACT FROM AUTHOR]

A newly developed enzyme immunoassay (EIA) for the detection of the tremorgenic indole-diterpene alkaloid paxilline (PAX) and closely related analogs was used to analyze ergot sclerotia collected from rye and barley fields. The mean EIA standard curve detection limit was 0.47 ± 0.14 ng/mL; relative cross-reactivity of toxin standard solutions was found for 11-hydroxy-paspaline (terpendole E, 1.1%) but not for lolitrem B or ergot alkaloids. Sclerotia from all fields were positive in the PAX-EIA at concentration levels of 620 ± 200 and 160 ± 37 μg/kg in ergot of rye and 130 ± 47 μg/kg in ergot of barley. Confirmatory analyses of sclerotia by liquid chromatography-tandem mass spectrometric detection identified PAX and its analog 13-desoxypaxilline. To the best of our knowledge, this is the first report on the natural occurrence of tremorgenic indole-diterpene alkaloid mycotoxins in ergot sclerotia from rye and barley. Along with details on the analytical methodology developed in this study, particularly PAX-antibody production, the relevance and implications of these findings for food and feed safety are discussed. Presence or absence of elevated levels of tremorgenic mycotoxins, along with the ergot alkaloids, would help in explaining the difference between the two distinct manifestations of historic ergotism, the convulsive and the gangrenous form. Further method development for paxilline and other tremorgenic mycotoxins in cereals used for food and feed is a prerequisite for a comprehensive risk assessment, which seems to be necessary in light of the findings reported here. [ABSTRACT FROM AUTHOR]

Key points Large conductance, Ca2+-activated K+ (BKCa) channels play important roles in mammalian retinal neurons, including photoreceptors, bipolar cells, amacrine cells and ganglion cells, but they have not been identified in horizontal cells., BKCa channel blockers paxilline and iberiotoxin, as well as Ca2+ free solutions and divalent cation Cav channel blockers, eliminate the outwardly rectifying current, while NS1619 enhances it. In symmetrical 150 m m K+, single channels had a conductance close to 250 pS, within the range of all known BKCa channels., In current clamped horizontal cells, BKCa channels subdue depolarizing membrane potential excursions, reduce the average resting potential and decrease oscillations. The results show that BKCa channel activation puts a ceiling on horizontal cell depolarization and regulates the temporal responsivity of the cells., Abstract Large conductance, calcium-activated potassium (BKCa) channels have numerous roles in neurons including the regulation of membrane excitability, intracellular [Ca2+] regulation, and neurotransmitter release. In the retina, they have been identified in photoreceptors, bipolar cells, amacrine cells and ganglion cells, but have not been conclusively identified in mammalian horizontal cells. We found that outward current recorded between −30 and +60 mV is carried primarily in BKCa channels in isolated horizontal cells of rats and mice. Whole-cell outward currents were maximal at +50 mV and declined at membrane potentials positive to this value. This current was eliminated by the selective BKCa channel blocker paxilline (100 n m), iberiotoxin (10 μ m), Ca2+ free solutions and divalent cation Cav channel blockers. It was activated by the BKCa channel activator NS1619 (30 μ m). Single channel recordings revealed the conductance of the channels to be 244 ± 11 pS ( n = 17; symmetrical 150 m m K+) with open probability being both voltage- and Ca2+-dependent. The channels showed fast activation kinetics in response to Ca2+ influx and inactivation gating that could be modified by intracellular protease treatment, which suggests β subunit involvement. Under current clamp, block of BKCa current increased depolarizing membrane potential excursions, raising the average resting potential and producing oscillations. BKCa current activation with NS1619 inhibited oscillations and hyperpolarized the resting potential. These effects underscore the functional role of BKCa current in limiting depolarization of the horizontal cell membrane potential and suggest actions of these channels in regulating the temporal responsivity of the cells. [ABSTRACT FROM AUTHOR]

The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.

The Center for Disease Control estimates 128,000 people in the U.S. are hospitalized annually due to food borne illnesses. This has created a demand for food safety testing targeting the detection of pathogenic mold and bacteria on agricultural products. This risk extends to medical Cannabis and is of particular concern with inhaled, vaporized and even concentrated Cannabis products . As a result, third party microbial testing has become a regulatory requirement in the medical and recreational Cannabis markets, yet knowledge of the Cannabis microbiome is limited. Here we describe the first next generation sequencing survey of the microbial communities found in dispensary based Cannabis flowers and demonstrate the limitations in the culture-based regulations that are being superimposed from the food industry.

Paxilline indole diterpenes have been a subject of scientific inquiry for over 30 years due to their diverse biological activities and unique structural features. These efforts resulted in a multitude of synthetic approaches and a dozen completed synthesis. Here, we discuss our contributions to this area that culminated in an 11-step synthesis of (±)-emindole SB. [ABSTRACT FROM AUTHOR]

Memory acquisition and synaptic plasticity are accompanied by changes in the intrinsic excitability of CA1 pyramidal neurons. These activity-dependent changes in excitability are mediated by modulation of intrinsic currents which alters the responsiveness of the cell to synaptic inputs. The afterhyperpolarization, a major contributor to the regulation of neuronal excitability, is reduced in animals that have acquired several types of hippocampus-dependent memory tasks and also following synaptic potentiation by high frequency stimulation. BK channels underlie the fast afterhyperpolarization and contribute to spike repolarization, and this afterhyperpolarization is reduced in animals that successfully acquired trace-eyeblink conditioning. This suggests that BK channel function is activity-dependent, but the mechanisms are unknown. In this study, we found that blockade of BK channels with paxilline (10µM) increased spike half-width and instantaneous frequency in response to a +100pA depolarization. In addition, induction of LTP by theta burst stimulation (TBS) in CA1 pyramidal neurons reduced BK channel’s contribution to spike repolarization and instantaneous frequency. This result indicates that BK channel activity is decreased following synaptic potentiation. Interestingly, blockade of mammalian target of rapamycin (MTORC1) with rapamycin (400 nM) following synaptic potentiation restored BK channel function, suggesting a role for protein translation in signaling events which decreased postsynaptic BK channel activity following synaptic potentiation.