Eva Cuypers

Jellyfish and other cnidarian envenomations cause pain by affecting TRPV1 channels

Cnidarian envenomations cause a burning‐pain sensation of which the underlying mechanisms are unknown. Activation of TRPV1, a non‐selective cation channel expressed in nociceptive neurons, leads to cell depolarisation and pain. Here, we show in vitro and in vivo evidence for desensitization‐dependent TRPV1 activation in cnidarian envenomations. Cnidarian venom induced a nociceptive reactivity, comparable to capsaicin, in laboratory rats, which could be reduced by the selective TRPV1 antagonist, BCTC. These findings are the first to explain at least part of the symptomology of cnidarian envenomations and provide insights into the design of more effective treatments for this global public health problem.

Gambierol, a toxin produced by the dinoflagellate Gambierdiscus toxicus, is a potent blocker of voltage-gated potassium channels

In this study, we pharmacologically characterized gambierol, a marine polycyclic ether toxin which is produced by the dinoflagellate Gambierdiscus toxicus. Besides several other polycyclic ether toxins like ciguatoxins, this scarcely studied toxin is one of the compounds that may be responsible for ciguatera fish poisoning (CFP). Unfortunately, the biological target(s) that underlies CFP is still partly unknown. Today, ciguatoxins are described to specifically activate voltage-gated sodium channels by interacting with their receptor site 5. But some dispute about the role of gambierol in the CFP story shows up: some describe voltage-gated sodium channels as the target, while others pinpoint voltage-gated potassium channels as targets. Since gambierol was never tested on isolated ion channels before, it was subjected in this work to extensive screening on a panel of 17 ion channels: nine cloned voltage-gated ion channels (mammalian Na(v)1.1-Na(v)1.8 and insect Para) and eight cloned voltage-gated potassium channels (mammalian K(v)1.1-K(v)1.6, hERG and insect ShakerIR) expressed in Xenopus laevis oocytes using two-electrode voltage-clamp technique. All tested sodium channel subtypes are insensitive to gambierol concentrations up to 10 microM. In contrast, K(v)1.2 is the most sensitive voltage-gated potassium channel subtype with almost full block (>97%) and an half maximal inhibitory concentration (IC(50)) of 34.5 nM. To the best of our knowledge, this is the first study where the selectivity of gambierol is tested on isolated voltage-gated ion channels. Therefore, these results lead to a better understanding of gambierol and its possible role in CFP and they may also be useful in the development of more effective treatments.

Potent modulation of the voltage-gated sodium channel Nav1.7 by OD1, a toxin from the scorpion Odonthobuthus doriae.

Voltage-gated sodium channels are essential for the propagation of action potentials in nociceptive neurons. Nav1.7 is found in peripheral sensory and sympathetic neurons and involved in short-term and inflammatory pain. Nav1.8 and Nav1.3 are major players in nociception and neuropathic pain, respectively. In our effort to identify isoform-specific and high-affinity ligands for these channels, we investigated the effects of OD1, a scorpion toxin isolated from the venom of the scorpion Odonthobuthus doriae. Nav1.3, Nav1.7, and Nav1.8 channels were coexpressed with β1-subunits in Xenopus laevis oocytes. Na+ currents were recorded with the two-electrode voltage-clamp technique. OD1 modulates Nav1.7 at low nanomolar concentrations: 1) fast inactivation is dramatically impaired, with an EC50 value of 4.5 nM; 2) OD1 substantially increases the peak current at all voltages; and 3) OD1 induces a substantial persistent current. Nav1.8 was not affected by concentrations up to 2 μM, whereas Nav1.3 was sensitive only to concentrations higher than 100 nM. OD1 impairs the inactivation process of Nav1.3 with an EC50 value of 1127 nM. Finally, the effects of OD1 were compared with a classic α-toxin, AahII from Androctonus australis Hector and a classic α-like toxin, BmK M1 from Buthus martensii Karsch. At a concentration of 50 nM, both toxins affected Nav1.7. Nav1.3 was sensitive to AahII but not to BmK M1, whereas Nav1.8 was affected by neither toxin. In conclusion, the present study shows that the scorpion toxin OD1 is a potent modulator of Nav1.7, with a unique selectivity pattern.

The evaluation of the applicability of a high pH mobile phase in ultrahigh performance liquid chromatography tandem mass spectrometry analysis of benzodiazepines and benzodiazepine-like hypnotics in urine and blood

A sensitive liquid chromatography tandem mass spectrometry method was developed and validated for simultaneous detection of benzodiazepines, benzodiazepine-like hypnotics and some metabolites (7-aminoflunitrazepam, alprazolam, bromazepam, brotizolam, chlordiazepoxide, chlornordiazepam, clobazam, clonazepam, clotiazepam, cloxazolam, diazepam, ethylloflazepate, flunitrazepam, flurazepam, loprazolam, lorazepam, lormetazepam, midazolam, N-desmethylflunitrazepam, nitrazepam, N-methylclonazepam (internal standard), nordiazepam, oxazepam, prazepam, temazepam, tetrazepam, triazolam, zaleplon, zolpidem, zopiclone) in urine and whole blood. Sample preparation was performed on a mixed-mode cation exchange solid phase extraction cartridge. Electrospray ionization was found to be more efficient than atmospheric pressure chemical ionization. The use of a mobile phase of high pH resulted in higher retention and higher electrospray ionization signals than the conventional low pH mobile phases. Considering the benefits of a high pH mobile phase on both chromatography and mass spectrometry, its use should be encouraged. In the final method, gradient elution with 10 mM ammonium bicarbonate (pH 9) and methanol was performed on a small particle column (Acquity C18, 1.7 μm, 2.1 mm × 50 mm). The optimized method was fully validated.

A polyether biotoxin binding site on the lipid-exposed face of the pore domain of Kv channels revealed by the marine toxin gambierol.

Gambierol is a marine polycyclic ether toxin belonging to the group of ciguatera toxins. It does not activate voltage-gated sodium channels (VGSCs) but inhibits Kv1 potassium channels by an unknown mechanism. While testing whether Kv2, Kv3, and Kv4 channels also serve as targets, we found that Kv3.1 was inhibited with an IC50 of 1.2 ± 0.2 nM, whereas Kv2 and Kv4 channels were insensitive to 1 μM gambierol. Onset of block was similar from either side of the membrane, and gambierol did not compete with internal cavity blockers. The inhibition did not require channel opening and could not be reversed by strong depolarization. Using chimeric Kv3.1–Kv2.1 constructs, the toxin sensitivity was traced to S6, in which T427 was identified as a key determinant. In Kv3.1 homology models, T427 and other molecular determinants (L348, F351) reside in a space between S5 and S6 outside the permeation pathway. In conclusion, we propose that gambierol acts as a gating modifier that binds to the lipid-exposed surface of the pore domain, thereby stabilizing the closed state. This site may be the topological equivalent of the neurotoxin site 5 of VGSCs. Further elucidation of this previously undescribed binding site may explain why most ciguatoxins activate VGSCs, whereas others inhibit voltage-dependent potassium (Kv) channels. This previously undescribed Kv neurotoxin site may have wide implications not only for our understanding of channel function at the molecular level but for future development of drugs to alleviate ciguatera poisoning or to modulate electrical excitability in general.

OD1, the first toxin isolated from the venom of the scorpion Odonthobuthus doriae active on voltage-gated Na+ channels

In this study, we isolated and pharmacologically characterized the first α‐like toxin from the venom of the scarcely studied Iranian scorpion Odonthobuthus doriae. The toxin was termed OD1 and its primary sequence was determined: GVRDAYIADDKNCVYTCASNGYCNTECTKNGAESGYCQWIGRYGNACWCIKLPDEVPIRIPGKCR. Using the two‐electrode voltage clamp technique, the pharmacological effects of OD1 were studied on three cloned voltage‐gated Na+ channels expressed in Xenopus laevis oocytes (Nav1.2/β1, Nav1.5/β1, para/tipE). The inactivation process of the insect channel, para/tipE, was severely hampered by 200 nM of OD1 (EC50 = 80 ± 14 nM) while Nav1.2/β1 still was not affected at concentrations up to 5 μM. Nav1.5/β1 was influenced at micromolar concentrations.

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms.

In the last decades, Ts1 has not only been the subject of many studies, it has also been considered as a very useful tool to investigate NaV channels and to explore the exact role of NaV channels in channelopathies. Ts1 is believed to modulate the activation process of NaV upon interaction at the neurotoxin binding site 4. Our aim was to carry out an in depth functional characterization of Ts1 on a wide array of Nav channels, in order to investigate its mechanism of action and to verify if Ts1 can indeed be considered as a prototype site 4 selective toxin, valid for all the Nav isoforms we know currently. Ts1 has been subjected to an in-depth functional investigation on 9 NaV isoforms expressed in Xenopus laevis oocytes. Ts1 does not only interfere with the activation process but also modulates the inactivation in a bell-shaped voltage-dependent matter. Furthermore, Ts1 altered the ion selectivity through insect NaV. without influencing the tetrodotoxin selectivity of the channels. Finally, Ts1 was also found to inhibit the sodium current through the cardiac Nav1.5 isoform. On the basis of the totally unexpected plethora of Nav modulations as induced by Ts1, we demonstrate that caution is required in interpretation the in vivo experiments when using Ts1. The electrophysiological characterization of Ts1 indeed shows that the general accepted contours of NaV binding sites are much more obscure than believed and that interpretation of NaV pharmacology upon toxin binding is more complex than believed thus far.

Consequences of Decontamination Procedures in Forensic Hair Analysis Using Metal-Assisted Secondary Ion Mass Spectrometry Analysis

Today, hair testing is considered to be the standard method for the detection of chronic drug abuse. Nevertheless, the differentiation between systemic exposure and external contamination remains a major challenge in the forensic interpretation of hair analysis. Nowadays, it is still impossible to directly show the difference between external contamination and use-related incorporation. Although the effects of washing procedures on the distribution of (incorporated) drugs in hair remain unknown, these decontamination procedures prior to hair analysis are considered to be indispensable in order to exclude external contamination. However, insights into the effect of decontamination protocols on levels and distribution of drugs incorporated in hair are essential to draw the correct forensic conclusions from hair analysis; we studied the consequences of these procedures on the spatial distribution of cocaine in hair using imaging mass spectrometry. Additionally, using metal-assisted secondary ion mass spectrometry, we are the first to directly show the difference between cocaine-contaminated and user hair without any prior washing procedure.

Development and validation of a new TD-GC/MS method and its applicability in the search for human and animal decomposition products.

Differentiation between human and animal remains by means of analysis of volatile compounds released during decomposition is impossible since no volatile marker(s) specific for human decomposition has been established today. Hence, the identification of such a marker for human decomposition would represent great progression for the discovery of buried cadavers by analytical techniques. Cadaver dogs can be trained more efficiently, the understanding of forensic entomology can be enhanced, and the development of a portable detection device may be within reach. This study describes the development and validation of a new analytical method that can be applied in the search of such (a) specific marker(s). Sampling of the volatile compounds released by decomposing animal and human remains was performed both in a laboratory environment and outdoors by adsorption on sorbent tubes. Different coatings and several sampling parameters were investigated. Next, the volatile compounds were analyzed and identified by a thermal desorber combined with gas chromatography coupled to mass spectrometry (TD-GC/MS). Different GC columns were tested. Finally, the analytical method was validated using a standard mixture of nine representative compounds.

Steviol glycosides enhance pancreatic beta-cell function and taste sensation by potentiation of TRPM5 channel activity

Steviol glycosides (SGs), such as stevioside and rebaudioside A, are natural, non-caloric sweet-tasting organic molecules, present in extracts of the scrub plant Stevia rebaudiana, which are widely used as sweeteners in consumer foods and beverages. TRPM5 is a Ca2+-activated cation channel expressed in type II taste receptor cells and pancreatic β-cells. Here we show that stevioside, rebaudioside A and their aglycon steviol potentiate the activity of TRPM5. We find that SGs potentiate perception of bitter, sweet and umami taste, and enhance glucose-induced insulin secretion in a Trpm5-dependent manner. Daily consumption of stevioside prevents development of high-fat-diet-induced diabetic hyperglycaemia in wild-type mice, but not in Trpm5−/− mice. These results elucidate a molecular mechanism of action of SGs and identify TRPM5 as a potential target to prevent and treat type 2 diabetes.