Gilles Ouanounou

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16‐membered macrocycle. The toxic potential and the mechanism of action of GYM‐A, highly purified from contaminated clams, have been assessed. GYM‐A in isolated mouse phrenic hemidiaphragm preparations produced a concentration‐ and time‐dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM‐A paralyzed frog and mouse isolated neuromuscular preparations. Patch‐clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM‐A in a reversible manner. GYM‐A also blocked, in a voltage‐independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes. Competition‐binding assays confirmed that GYM‐A is a powerful ligand interacting with muscle‐type nAChR, heteropentameric α3β2, α4β2, and chimeric α7‐5HT3 neuronal nAChRs. Our data show for the first time that GYM‐A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM‐A detection in shellfish samples.

Ca2+ and Na+ contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.

The marine polyether gambierol enhances muscle contraction and blocks a transient K(+) current in skeletal muscle cells.

Gambierol is a complex marine toxin first isolated with ciguatoxins from cell cultures of the toxic dinoflagellate Gambierdiscus toxicus. Despite the chemical complexity of the polycyclic ether toxin, the total successful synthesis of gambierol has been achieved by different chemical strategies. In the present work the effects of synthetic gambierol on mouse and frog skeletal neuromuscular preparations and Xenopus skeletal myocytes have been studied. Gambierol (0.1-5 muM) significantly increased isometric twitch tension in neuromuscular preparations stimulated through the motor nerve. Less twitch augmentation was observed in directly stimulated muscles when comparing twitch tension-time integrals obtained by nerve stimulation. Also, gambierol induced small spontaneous muscle contraction originating from presynaptic activity that was completely inhibited by d-tubocurarine. Gambierol slowed the rate of muscle action potential repolarization, triggered spontaneous and/or repetitive action potentials, and neither affected action potential amplitude nor overshoot in skeletal muscle fibers. These results suggest that gambierol through an action on voltage-gated K(+) channels prolongs the duration of action potentials, enhances the extent and time course of Ca(2+) release from the sarcoplasmic reticulum, and increases twitch tension generation. Further evidence is provided that gambierol at sub-micromolar concentrations blocks a fast inactivating outward K(+) current that is responsible for action potential prolongation in Xenopus skeletal myocytes.

The Boltzmann equation in molecular biology

In the 1870s, Ludwig Boltzmann proposed a simple equation that was based on the notion of atoms and molecules and that defined the probability of finding a molecule in a given state. Several years later, the Boltzmann equation was developed and used to calculate the equilibrium potential of an ion species that is permeant through membrane channels and to describe conformational changes of biological molecules involved in different mechanisms including: open probability of ion channels, effect of molecular crowding on protein conformation, biochemical reactions and cell proliferation. The aim of this review is to trace the history of the developments of the Boltzmann equation that account for the behaviour of proteins involved in molecular biology and physiology.

A novel synaptic plasticity rule explains homeostasis of neuromuscular transmission.

Excitability differs among muscle fibers and undergoes continuous changes during development and growth, yet the neuromuscular synapse maintains a remarkable fidelity of execution. Here we show in two evolutionarily distant vertebrates (Xenopus laevis cell culture and mouse nerve-muscle ex-vivo) that the skeletal muscle cell constantly senses, through two identified calcium signals, synaptic events and their efficacy in eliciting spikes. These sensors trigger retrograde signal(s) that control presynaptic neurotransmitter release, resulting in synaptic potentiation or depression. In the absence of spikes, synaptic events trigger potentiation. Once the synapse is sufficiently strong to initiate spiking, the occurrence of these spikes activates a negative retrograde feedback. These opposing signals dynamically balance the synapse in order to continuously adjust neurotransmitter release to a level matching current muscle cell excitability. DOI: http://dx.doi.org/10.7554/eLife.12190.001

Sodium-dependent activity of aquaporin-1 in rat glioma cells: a new mechanism of cell volume regulation

Cell volume controls many functions and is itself regulated. To study cell volume regulations, the mean volume of C6-BU-1 rat glioma cells was electronically measured under isotonic and anisotonic conditions. Two isotonic solutions were used containing either normal (solution 1) or low (solution 2) NaCl. Anisotonicity was induced by changing NaCl or sucrose concentrations in solutions 1 and 2, respectively. The cells behaved like perfect osmometers when the tonicity was increased. In contrast, just after hypotonic challenges, the cell volume was smaller than that predicted by a perfect osmometer. This deviation reveals a new mechanism, which we call the volume increase limitation (VIL). When hypotonicity was induced by decreasing NaCl, a classical slow regulatory volume decrease (RVD) was also observed in addition to VIL. The cells expressed aquaporin-1 sensitive to HgCl2 and decreased by siRNA, which both reduced fast volume changes. In this study, we show that: (1) RVD is proportional to the change in external Cl− concentration and is inhibited by Cl− channel and K+–Cl− cotransporter blockers; (2) cell swelling due to the influx of H2O through aquaporins shows rectification with decreasing osmolarity and is sensitive to the internal Na+ concentration; (3) VIL is linearly related with hypotonicity and is abolished in solutions 1 and 2 by the Na+ ionophore monensin and in solution 1 by the Na+–K+ ATPase inhibitor ouabain. These results suggest that VIL is triggered by the decrease in internal Na+ caused by hyponatrema and cell swelling. In addition to RVD, VIL should protect cells during hyposmotic stress.

Heterogeneous firing rate response of mouse layer V pyramidal neurons in the fluctuation‐driven regime

We recreated in vitro the fluctuation‐driven regime observed at the soma during asynchronous network activity in vivo and we studied the firing rate response as a function of the properties of the membrane potential fluctuations. We provide a simple analytical template that captures the firing response of both pyramidal neurons and various theoretical models. We found a strong heterogeneity in the firing rate response of layer V pyramidal neurons: in particular, individual neurons differ not only in their mean excitability level, but also in their sensitivity to fluctuations. Theoretical modelling suggest that this observed heterogeneity might arise from various expression levels of the following biophysical properties: sodium inactivation, density of sodium channels and spike frequency adaptation.

The Neurotoxic Effect of 13,19-Didesmethyl and 13-Desmethyl Spirolide C Phycotoxins Is Mainly Mediated by Nicotinic Rather Than Muscarinic Acetylcholine Receptors

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4β2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes.

Local recording of biological magnetic fields using Giant Magneto Resistance-based micro-probes

The electrical activity of brain, heart and skeletal muscles generates magnetic fields but these are recordable only macroscopically, such as in magnetoencephalography, which is used to map neuronal activity at the brain scale. At the local scale, magnetic fields recordings are still pending because of the lack of tools that can come in contact with living tissues. Here we present bio-compatible sensors based on Giant Magneto-Resistance (GMR) spin electronics. We show on a mouse muscle in vitro, using electrophysiology and computational modeling, that this technology permits simultaneous local recordings of the magnetic fields from action potentials. The sensitivity of this type of sensor is almost size independent, allowing the miniaturization and shaping required for in vivo/vitro magnetophysiology. GMR-based technology can constitute the magnetic counterpart of microelectrodes in electrophysiology, and might represent a new fundamental tool to investigate the local sources of neuronal magnetic activity.

Secretagogue Activity of Trachynilysin, a Neurotoxic Protein Isolated from Stonefish (Synanceia trachynis) Venom

Approximately 400 to 500 species of marine fish may be poisonous to humans after ingestion. Most poisonous fish are nonmigratory reef fish and can be either herbivores or carnivores. Some of them have tissues that are toxic at all times, others are poisonous during certain periods of the year or in certain geographical areas, and still others have only specific organs that are toxic, and their toxicity may vary with time, location, and habitat (reviewed in ref. 1).