Gambierol, a marine dinoflagellate toxin, potently increases evoked quantal transmitter release and reverses pre- and post-synaptic neuromuscular block at vertebrate junctions

International audienceGambierol is a marine polycyclic ether toxin that was first isolated together with ciguatoxins from cultured Gambierdiscus toxicus dinoflagellates collected in French Polynesia. The chemical synthesis of gambierol permitted the analyses of its mode of action, which includes the selective inhibition of voltage-gated K+ (KV) channels in various cells and tissues expressing such channels. In the present study, we investigated the action of synthetic gambierol at vertebrate skeletal neuromuscular junctions using conventional techniques. Nanomolar concentrations of gambierol inhibited the fast K+ current and prolonged the duration of the presynaptic action potential in motor nerve terminals, as revealed by presynaptic focal current recordings, and increased stimulus-evoked quantal transmitter release in neuromuscular junctions blocked either by a high Mg2+-low Ca2+ medium, or by botulinum neurotoxin type-A. Also, gambierol reversed the postsynaptic block produced by d-tubocurarine. In motor nerve terminals loaded with fluo-3/AM, gambierol increased the transient Ca2+-signals in response to nerve-stimulation at 1-10 Hz. The results suggest that gambierol, which on equimolar basis is more potent than 3,4-diaminopyridine, can have potential application in pathologies in which it is necessary to antagonize pre- or post-synaptic neuromuscular block, or both

Gambierol blocks a K+^+ current fraction and affects action potential duration and the firing rate in rat fetal chromaffin cells

The dinoflagellate genus Gambierdiscus, particularly Gambierdiscus toxicus is known to produce numerous ladder polycyclic ether compounds, including gambierol, characterized by a transfused octacyclic polyether core, as well as the identified ciguatoxins responsible for ciguatera poisoning. Gambierol inhibits voltage-gated K$^+$ (K$_V$) channels in various mammalian excitable and nonexcitable cells, and motor nerve terminals of the neuromuscular junction. In the present study, we investigated the action of gambierol on outward K$^+$ currents of cultured rat fetal adrenal medulla chromaffin cells using the whole-cell configuration voltage-clamp method. The pharmacological dissection of the outward K$^+$current, using selective K$^+$ channel inhibitors, revealed that gambierol reduced just a small fraction of the total outward current without affecting calcium-activated K$^+$ currents that were sensitive to apamin and iberiotoxin, and ATP-sensitive K$^+$ currents sensitive to glibenclamide. Cultured fetal chromaffin cells were excitable and generated either evoked (upon direct electric stimulation) or spontaneous action potentials. These action potentials were sensitive to tetrodotoxin that blocks voltage-gated Na$^+$(Na$_V$) channels. Gambierol neither affected the rising phase nor the overshoot of action potentials but significantly prolonged their repolarizing phase and increased the firing rate of action potentials during sustained current depolarization, as determined using current-clamp recordings. Our results highlight the effects of gambierol on K$_V$ channels and on the electrical properties of rat fetal chromaffin cells. It is likely that gambierol may cross the placental barrier as ciguatoxins do. Further studies are needed to determine whether such actions may have deleterious effects on fetuses

Gambierol effects on K(+) currents and catecholamine release in single rat fetal adrenomedullary cultured chromaffin cells

International audienceThe purpose of this work was to study the action of the polycyclic ether gambierol on K+ currents and catecholamine secretion in single rat fetal adrenomedullary cultured chromaffin (AMC) cells using perforated whole-cell voltage-clamp recordings, and current-clamp and electrochemical recordings. From the several types of voltage-gated K+ channels (KV) contributing to the total outward current of rat fetal AMC cells, gambierol only partly inhibited the total K+ current, when added after or before KCa and KATP blockers, and affected neither KCa nor KATP channels. After blocking of Nav and KATP channels, and preventing the activation of KCa channels, gambierol blocked 50% of the maximal K+ current fraction with an inhibitory concentration (IC50) of 5.8 ± 1.56 nM (n = 9). In marked contrast to ciguatoxins, gambierol slowed the kinetics of K+-current activation by 75.4 ± 10.1% (n = 4) with respect to controls (p = 0.031). Hence, before and after gambierol the activation time constants of K+ current were 3.82 ± 0.39 ms (n = 4) and 6.80 ± 1.02 ms (n = 4), respectively. Simultaneous current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11-seconds depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent block of KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells respectively, highlighting the key modulatory role played by KCa channels in the control of exocytosis from rat fetal AMC cells

Gambierol blocks a K+^+ current fraction without affecting catecholamine release in rat fetal adrenomedullary cultured chromaffin cells

International audienceGambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19–F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of KV channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K$^+$ current and slowed the kinetics of K$^+$ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K$^+$ (KCa) and ATP-sensitive K$^+$ (KATP) channels. The gambierol concentration necessary to inhibit 50% of the K$^+$ current-component sensitive to the polyether (IC50) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by KCa channels in the control of exocytosis from fetal (F19–F20) adrenomedullary chromaffin cells

Gambierol blocks a K(+) current component affecting action potential duration in fetal adrenomedullary chromaffin cells. Is this action sufficient to modulate catecholamine release?

International audienceThe genera Gambierdiscus and Fukuyoa produce numerous ladder polycyclic ether compoundsincluding gambierol, characterized by a transfused octacyclic polyether core, and the identified ciguatoxins, responsible for ciguatera poisoning. Gambierol inhibits voltage-gated K+ (Kv) channels in various cells, and motor nerve terminals and enhances acetylcholine release. The purpose of this work was to study the effects of gambierol on cultured rat fetal adrenomedullary chromaffin cells. These excitable cells have different types of Kv channels and release catecholamines. Perforated patch-clamp current-recordings revealed that gambierol (100 nM) only blocked a small component of the total outward K+ current, and did not affect calciumâactivated K+ channels (KCa) and ATP-sensitive K+ (KATP) channels. When KATP and KCa channel activation were blocked, the gambierol concentration inhibiting 50 % of the K+ current-component (IC50) was 7.6 ± 1.10 nM (n = 9). Gambierol also slowed the kinetics of K+ current activation. The recording of all-or-none action potentials in chromaffin cells disclosed that gambierol (50 nM) prolonged by 33 % their repolarizing phase. Gambierol did not modify the membrane potential following 15-second depolarizing current-steps, and did not increase the number of exocytotic catecholamine release events, as determined by simultaneous patchclamp and single-cell amperometry recordings. Under, the same conditions gambierol enhanced catecholamine secretion provided KCa and KATP channels were blocked. In conclusion, the specific inhibition of Kv channels by gambierol is not enough to modulate catecholaminesecretion, emphasizing the key role played by KCa and KATP channels

Gambierol action on K(+) currents and catecholamine release in cultured single chromaffin cells from fetal rat adrenal medulla

International audienceGambierol, characterized by a transfused octacyclic polyether core, was first isolated and chemically typified from cultured Gambierdiscus toxicus dinoflagellates collected in French Polynesia. Subsequently, distinct groups in Japan and the USA, using various strategies, achieved its total chemical synthesis. This allowed detailed studies on its mode of action. Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells, as well as in motor nerve terminals of the skeletal neuromuscular junction. In the present study, we have investigated first, the effects of nanomolar concentrations of gambierol on K+ current of cultured chromaffin cells from fetal rat adrenal medulla using perforated patch-clamp current-recordings. Our results show that gambierol only blocked a small component of the total K+ current, and affected neither calcium‐activated K+ (KCa) nor ATP-sensitive K+ (KATP) channels, as revealed using apamin and iberiotoxin (selective KCa channel blockers) and glibenclamid (KATP channel blocker). After inhibiting KATP and KCa channel activation, the gambierol concentration blocking 50% the K+ current-component (IC50) was 7.6 ± 1.1 nM. The repolarizing phase of all-or-none elicited action potentials, recorded under current-clamp conditions (triggered by 1-ms depolarizing pulses), was sensitive to the action of gambierol but insensitive to the action of apamin and iberiotoxin, indicating that KCa channels do not participate in the modulation of action potential duration triggered by short depolarizing pulses. The use of simultaneous patch-clamp and single-cell amperometry allowed controlling the membrane potential and detecting exocytosis events (with a carbon electrode polarized to +650 mV to allow the oxidation of released catecholamines). Such recordings revealed that gambierol did not modify the membrane potential following 14-second depolarizing current-steps, and did not increase significantly the number of exocytotic catecholamine release events with respect to controls. The addition of KCa channel blockers (in the continuous presence of gambierol) enhanced the membrane depolarization by about 15 mV (during the 14-second current step), and at the same time increased significantly the number of exocytotic events related to catecholamine secretion. Such enhanced depolarization induced by the KCa channel blockers probably brings the membrane potential above the activation threshold of high-voltage activated CaV channels triggering both Ca2+ influx and subsequent catecholamine secretion. These results emphasize the diversity of KV channels in chromaffin cells from fetal rat adrenal medulla and highlight the modulatory role played by KCa channels in the control of exocytosis in the absence of splanchnic innervation

Purification, Toxicity and Functional Characterization of a New Proteinaceous Mussel Biotoxin from Bizerte Lagoon

International audienceThe marine environment is known to be occupied by microorganisms. The potential toxicity of some of these marine microorganisms, that are capable of producing unknown biotoxins, has always been underestimated. Indeed, these biotoxins may be a threat to human health through the consumption of contaminated seafood and fish. For more than ten years, recurrent but atypical toxicity has been detected in mussels from Bizerte lagoon (North of Tunisia) during routine tests. In this study, we have isolated and characterized a new proteinaceous marine biotoxin, named Mussel Toxic Peptide (MTP). Using HPLC, electrophoresis and LC/MS studies, we showed that MTP has a protein characteristic UV-spectrum, can be visualized by protein specific reagents such as Coomassie, and has a molecular mass of 6.4 kDa. Patch-clamp experiments performed on cultured N18 neuroblastoma cells revealed that MTP (0.9-18 µM) markedly inhibited voltage-gated Na current, but was about 23 times less active in blocking voltage-gated K current at equimolar concentrations. To the best of our knowledge, this is the first time that a proteinaceous marine biotoxin with relatively high molecular mass is isolated and involved in the contamination of mussels harvested from shellfish farming areas

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Gambierol enhances evoked quantal transmitter release and blocks a potassium current in motor nerve terminals of the mouse neuromuscular junction

In recent years, a great interest was developed to synthetize biologically-active natural products of marine origin. This is due to both their complex molecular structures and chemical diversity, and also to their unique biological activities. Among ladder-shaped toxins, gambierol, originally isolated from cultured Gambierdiscus toxicus dinoflagellate cells, together with ciguatoxins, has been successfully synthesized permitting detailed analyses of its mode and mechanism of action. Gambierol and analogs are known to inhibit some voltage-gated K+ (Kv) channel subtypes in various cell types. The aim of the present study was (i) to investigate whether gambierol has an action on quantal transmitter release evoked by nerve impulses and (ii) to determine whether Kv channels in motor nerve terminals of the mammalian neuromuscular junction are sensitive to the toxin action. Using electrophysiological techniques, the results obtained show that gambierol (2-20 nM) had no significant action on the resting membrane potential of mouse hemidiaphragm muscle fibers. In addition, spontaneous quantal transmitter release, measured by recording spontaneous miniature endplate potential frequency, remained unaffected by gambierol in resting neuromuscular junction. Gambierol (2 nM) increased about eight-fold the mean quantal content of evoked endplate potentials, as determined at individual junctions of the phrenic-hemidiaphragm preparation equilibrated in a low-Ca2+ and high-Mg2+ medium. The ability of gambierol to enhance quantal transmitter release was related to the reduction of a fast K+ current in nerve terminals. Overall, the present results show for the first time that gambierol enhances evoked quantal transmitter release in response to nerve stimuli, suggesting that it can be used to reverse pre- or post-synaptic neuromuscular blockade

Gambierol and analogs reverse paralysis caused by d-tubocurarine and botulinum type-A neurotoxin at vertebrate neuromuscular junctions

International audienceGambierol is a marine polycyclic ether toxin, first isolated with ciguatoxins by Satake, Murata, and Yasumoto (1993) from cultured Gambierdiscus toxicus dinoflagellates, collected in French Polynesia. The chemical synthesis of gambierol and truncated analogues has been achieved successfully allowing detailed analyses of their mode of action. Interestingly, gambierol and analogs inhibit voltage-gated K+ (Kv) channels. It is well known that Kv channels play important roles in modulating acetylcholine (ACh) release from motor nerve terminals of vertebrate neuromuscular junctions. Therefore, the aim of our study was to investigate the action of synthetic gambierol and analogues on K+ currents in nerve cells and nerve terminals, using electrophysiological techniques. In addition, gambierol was studied on neuromuscular junctions in which muscle nicotinic ACh receptors have been blocked with d-tubocurarine (postsynaptic block), or in junctions in which quantal ACh release has been greatly reduced (presynaptic block) by botulinum neurotoxin type-A (BoNT/A). This neurotoxin induces a selective proteolytic cleavage of SNAP-25 an important nerve terminal protein for the neurotransmitter release process. Our results showed that gambierol and analogues inhibited Kv channels in neuronal cells in a concentration-dependent manner. Also, nanomolar concentrations of gambierol prolonged the duration of the presynaptic action potential in motor nerve terminals, as revealed by focal current recording. This indicates that Kv channels in nerve terminals were also blocked by the polyether. The loading of the calcium-sensitive dye Fluo-3/AM to frog motor nerve terminals and the use of spectrometric imaging techniques showed that gambierol increased the transient calcium fluorescence signals in response to 1-10 Hz nerve-stimulation, indicating that blockade of Kv channels enhanced calcium entry into nerve terminals. At the neuromuscular junction, such actions of gambierol are essential for increasing nerve-evoked quantal ACh release and can explain the observed reversal of the neuromuscular block produced by d-tubocurarine, a competitive inhibitor of the muscle nicotinic ACh receptor. Interestingly, gambierol also increased the quantal content of endplate potentials in BoNT/A-intoxicated mouse neuromuscular junctions. These results indicate that blockade of Kv channels and enhanced calcium entry into terminals by gambierol increased the probability of quantal ACh release, supporting the view that SNAP-25 cleaved by BoNT/A can enter into the protein-complex required for calciumdependent neurotransmitter release. In conclusion, our results suggest that gambierol and analogues can have potential medical application in neuromuscular pathologies in which it is necessary to antagonize pre- or post-synaptic neuromuscular blockade, or both