Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.

The Bi-Functional Paxilline Enriched in Skin Secretion of Tree Frogs (Hyla japonica) Targets the KCNK18 and BKCa Channels.

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.

Tremorgenic mycotoxin poisoning in a dog: A case report.

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.

Effects of potassium channel modulators on the responses of mammalian slowly adapting mechanoreceptors.

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.

Flavonoid quercetin abolish paxilline inhibition of the mitochondrial BKCa channel.

Large-conductance calcium-regulated potassium channel (BKCa) is known to play an important role in physiological and pathological processes. Despite the BKCa 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 BKCa (mitoBKCa) 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 mitoBKCa 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 mitoBKCa channels by paxilline, quercetin activates these channels, indicating a paxilline and quercetin binding competition in the regulation of the mitoBKCa channel. To support our hypothesis, we used an analog of quercetin - isorhamnetin, a substance with one substituent changed. Isorhamnetin has no effect on the mitoBKCa 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 BKCa 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.

The Effect of MaxiK Channel on Regulating the Activation of NLRP3 Inflammasome in Rats of Blast-induced Traumatic Brain Injury.

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.

Identification of the Large-Conductance Ca2+-Regulated Potassium Channel in Mitochondria of Human Bronchial Epithelial Cells.

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 ß3-ß4 subunits of BKCa channel in HBE cells. Western blot analysis of cellular fractions confirmed the mitochondrial localization of α pore-forming and predominately ß3 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.

Inhibition of Potassium Channels Affects the Ability of Pig Spermatozoa to Elicit Capacitation and Trigger the Acrosome Exocytosis Induced by Progesterone.

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.

BK Channel Regulation of Afterpotentials and Burst Firing in Cerebellar Purkinje Neurons.

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 Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 µs) 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.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.

Paxilline Prevents the Onset of Myotonic Stiffness in Pharmacologically Induced Myotonia: A Preclinical Investigation.

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).

Mitigating effect of paxilline against injury produced by Cd2+ in rat pheochromocytoma PC12 and ascites hepatoma AS-30D cells.

On two rat cell lines, pheochromocytoma PC12 and ascites hepatoma AS-30D, and on rat liver mitochondria we studied action of paxilline (lipophilic mycotoxin from fungus Penicillium paxilli which is blocker of large-conductance potassium channels) against harmful effects of Cd(II) - one of the most dangerous toxic metals and environmental pollutants. We investigated an influence of paxilline on cell viability and mitochondrial function in the presence and in the absence of Cd2+. As found, paxilline protected partially from the Cd2+-induced cytotoxicity, namely taken in concentration of 1 µM it decreased the Cd2+-induced cell necrosis in average by 10-14 or 13-23% for AS-30D and PC12 cells, respectively. Nevertheless, paxilline did not affect the Cd2+-induced apoptosis of AS-30D cells. The alleviating concentration of paxilline reduced an intracellular production of reactive oxygen species (ROS) in PC12 cells intoxicated by Cd2+ and enhanced the ROS production in control AS-30D cells; however, it weakly affected mitochondrial membrane potential of the cells in the absence and in the presence of Cd2+. The ameliorative concentration of paxilline decreased the maximal respiration rates of control cells of both types after short-term (3-5 h) treatment with it while the rates reached their control levels after long-term (24-48 h) incubation with the drug. Paxilline was not protective against the Cd2+-induced membrane permeability and respiration rate changes in isolated rat liver mitochondria. As result, the mitochondrial electron transport chain was concluded to contribute in the mitigating effect of paxilline against the Cd2+-produced cell injury.

Involvement of the γ1 subunit of the large-conductance Ca2+-activated K+ channel in the proliferation of human somatostatinoma cells.

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+ (BKCa) 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 BKCa 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 BKCa 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 ß2-4 subunits of the BKCa channel, the novel regulatory γ1 subunit (BKCaγ1) 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 BKCaγ1. Store-operated Ca2+ entry was smaller in BKCaγ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BKCaγ1. These results strongly suggest that BKCaγ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BKCaγ1 may be a novel therapeutic target for somatostatinoma.

The functionally relevant site for paxilline inhibition of BK channels.

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.

MitoBKCa channel is functionally associated with its regulatory ß1 subunit in cardiac mitochondria.

KEY POINTS: Association of plasma membrane BKCa channels with BK-ß subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-ß subunits is not established. MitoBKCa -α and the regulatory BK-ß1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-ß1 (left-shifted voltage dependence of activation, V1/2  = -55 mV, 12 µm matrix Ca2+ ). In BK-ß1 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-ß1 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-ß1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT: Association of the plasma membrane BKCa channel with auxiliary BK-ß1-4 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-ß1 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-ß1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -α and its BK-ß1 subunit. Mitochondria from the BK-ß1 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-ß1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-ß1subunit 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-ß1 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-ß1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.

A single exposure to the tremorgenic mycotoxin lolitrem B inhibits voluntary motor activity and spatial orientation but not spatial learning or memory in mice.

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.

Large-conductance Ca2+-activated potassium channels are potently involved in the inverse neurovascular response to spreading depolarization.

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.

Inhibition of BKCa negatively alters cardiovascular function.

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.

The Role of Ca2+ and BK Channels of Locus Coeruleus (LC) Neurons as a Brake to the CO2 Chemosensitivity Response of Rats.

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 CO2-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 Ca2+ 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 Ca2+ 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.

Immunochemical Analysis of Paxilline and Ergot Alkaloid Mycotoxins in Grass Seeds and Plants.

Limited availability of toxin standards for lolitrem B and ergovaline impedes routine control of grasses for endophyte toxins. This study aimed at assessing the applicability of an enzyme immunoassay (EIA) for the indole-diterpene mycotoxin paxilline, in combination with a generic EIA for ergot alkaloids, as alternative parameters for screening purposes. Analysis of grass seeds and model pastures of four different grass species showed that both EIAs yielded highly positive results for paxilline and ergot alkaloids in perennial ryegrass seeds. Furthermore, evidence for natural occurrence of paxilline in grass in Germany was obtained. High performance liquid chromatography-tandem mass spectrometry analysis qualitatively confirmed the paxilline EIA results but showed that paxilline analogues 1'-O-acetylpaxilline and 13-desoxypaxilline were the predominant compounds in seeds and grass. In the absence of easily accessible reference standards for specific analysis of some major endophyte toxins, analysis of paxilline and ergot alkaloids by EIA may be suitable substitute parameters. The major advantage of this approach is its ease of use and speed, providing an analytical tool which could enhance routine screening for endophyte toxins in pasture.

Nongenomic modulation of the large conductance voltage- and Ca2+-activated K+ channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17ß-estradiol regulates large conductance voltage- and Ca2+-activated K+ (BK) channels, the most prominent K+ channels in human DSM We employed single BK channel recordings on inside-out excised membrane patches, perforated whole-cell patch-clamp on freshly isolated DSM cells, and isometric tension recordings on DSM-isolated strips to investigate the mechanism by which 17ß-estradiol activates BK channels. 17ß-Estradiol (100 nmol/L) rapidly increased depolarization-induced whole-cell K+ currents in DSM cells. The 17ß-estradiol stimulatory effects on whole-cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 µmol/L), clearly indicating that 17ß-estradiol specifically activates BK channels. 17ß-Estradiol also increased the frequency of ryanodine receptor-mediated transient BK currents. Single BK channel recordings showed that 17ß-estradiol (100 nmol/L) significantly increased the BK channel open probability of inside-out excised membrane patches, revealing that 17ß-estradiol activates BK channels directly. 17ß-Estradiol reduced spontaneous phasic contractions of human DSM-isolated strips in a concentration-dependent manner (100 nmol/L-1 µmol/L), and this effect was blocked by paxilline (1 µmol/L). 17ß-Estradiol (100 nmol/L) also reduced nerve-evoked contractions of human DSM-isolated strips. Collectively, our results reveal that 17ß-estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.