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Title: Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity

Abstract

In general, scorpion {beta}-toxins have been well examined. However, few in-depth studies have been devoted to species selectivity and affinity comparisons on the different voltage-activated Na{sup +} channels since they have become available as cloned channels that can be studied in heterologous expression systems. As a result, their classification is largely historical and dates from early in vivo experiments on mice and cockroach and fly larvae. In this study, we aimed to provide an updated overview of selectivity and affinity of scorpion {beta}-toxins towards voltage-activated Na{sup +} channels of vertebrates or invertebrates. As pharmacological tools, we used the classic {beta}-toxins AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the neuronal vertebrate voltage-activated Na{sup +} channel, rNa{sub v}1.2a. For comparison, its invertebrate counterpart, DmNav1, was also tested. Both these channels were expressed in Xenopus laevis oocytes and the currents measured with the two-electrode voltage-clamp technique. We supplemented this data with several binding displacement studies on rat brain synaptosomes. The results lead us to propose a general classification and a novel nomenclature of scorpion {beta}-toxins based on pharmacological activity.

Authors:
 [1];  [2];  [3]
  1. Laboratory of Toxicology, University of Leuven, O and N 2, Postbus 922, Herestraat 49, 3000 Leuven (Belgium)
  2. CNRS FRE 2738, Biologie des Interactions Moleculaires et Cellulaires, Faculte de Medecine secteur Nord, Institut Jean Roche, Universite de la Mediterranee, Bd Pierre Dramard, 13916, Marseille, Cedex 20 (France)
  3. Laboratory of Toxicology, University of Leuven, O and N 2, Postbus 922, Herestraat 49, 3000 Leuven (Belgium). E-mail: Jan.Tytgat@pharm.kuleuven.be
Publication Date:
OSTI Identifier:
20976837
Resource Type:
Journal Article
Resource Relation:
Journal Name: Toxicology and Applied Pharmacology; Journal Volume: 218; Journal Issue: 1; Other Information: DOI: 10.1016/j.taap.2006.10.009; PII: S0041-008X(06)00370-X; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; BRAIN; CLASSIFICATION; COCKROACHES; ELECTRIC POTENTIAL; ELECTRODES; IN VIVO; LARVAE; MICE; OOCYTES; RATS; SCORPIONS; SODIUM IONS; TOXINS

Citation Formats

Bosmans, Frank, Martin-Eauclaire, Marie-France, and Tytgat, Jan. Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity. United States: N. p., 2007. Web. doi:10.1016/j.taap.2006.10.009.
Bosmans, Frank, Martin-Eauclaire, Marie-France, & Tytgat, Jan. Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity. United States. doi:10.1016/j.taap.2006.10.009.
Bosmans, Frank, Martin-Eauclaire, Marie-France, and Tytgat, Jan. Mon . "Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity". United States. doi:10.1016/j.taap.2006.10.009.
@article{osti_20976837,
title = {Differential effects of five 'classical' scorpion {beta}-toxins on rNa{sub v}1.2a and DmNav1 provide clues on species-selectivity},
author = {Bosmans, Frank and Martin-Eauclaire, Marie-France and Tytgat, Jan},
abstractNote = {In general, scorpion {beta}-toxins have been well examined. However, few in-depth studies have been devoted to species selectivity and affinity comparisons on the different voltage-activated Na{sup +} channels since they have become available as cloned channels that can be studied in heterologous expression systems. As a result, their classification is largely historical and dates from early in vivo experiments on mice and cockroach and fly larvae. In this study, we aimed to provide an updated overview of selectivity and affinity of scorpion {beta}-toxins towards voltage-activated Na{sup +} channels of vertebrates or invertebrates. As pharmacological tools, we used the classic {beta}-toxins AaHIT, Css II, Css IV, Css VI and Ts VII and tested them on the neuronal vertebrate voltage-activated Na{sup +} channel, rNa{sub v}1.2a. For comparison, its invertebrate counterpart, DmNav1, was also tested. Both these channels were expressed in Xenopus laevis oocytes and the currents measured with the two-electrode voltage-clamp technique. We supplemented this data with several binding displacement studies on rat brain synaptosomes. The results lead us to propose a general classification and a novel nomenclature of scorpion {beta}-toxins based on pharmacological activity.},
doi = {10.1016/j.taap.2006.10.009},
journal = {Toxicology and Applied Pharmacology},
number = 1,
volume = 218,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Scorpion neurotoxins targeting the Na{sub v} channel can be classified into two classes: {alpha}- and {beta}-neurotoxins and are reported as highly active in mammalian brain. In this work, we evaluate the effects of Tityus serrulatus venom (Ts venom) and its {alpha}-neurotoxin TsTX-V on {gamma}-aminobutyric acid (GABA), dopamine (DA) and glutamate (Glu) uptake in isolated rat brain synaptosomes. TsTX-V was isolated from Ts venom by ion exchange chromatography followed by reverse-phase (C18) high-performance liquid chromatography. Neither Ts venom nor TsTX-V was able to affect {sup 3}H-Glu uptake. On the other hand, Ts venom (0.13 {mu}g/mg) significantly inhibited both {sup 3}H-GABA andmore » {sup 3}H-DA uptake ({approx} 50%). TsTX-V showed IC{sub 5} values of 9.37 {mu}M and 22.2 {mu}M for the inhibition of {sup 3}H-GABA and {sup 3}H-DA uptake, respectively. These effects were abolished by pre-treatment with tetrodotoxin (TTX, 1 {mu}M), indicating the involvement of voltage-gated Na{sup +} channels in this process. In the absence of Ca{sup 2+}, and at low Ts venom concentrations, the reduction of {sup 3}H-GABA uptake was not as marked as in the presence of Ca{sup 2+}. TsTX-V did not reduce {sup 3}H-GABA uptake in COS-7 cells expressing the GABA transporters GAT-1 and GAT-3, suggesting that this toxin indirectly reduces the transport. The reduced {sup 3}H-GABA uptake by synaptosomes might be due to rapid cell depolarization as revealed by confocal microscopy of C6 glioma cells. Thus, TsTX-V causes a reduction of {sup 3}H-GABA and {sup 3}H-DA uptake in a Ca{sup 2+}-dependent manner, not directly affecting GABA transporters, but, in consequence of depolarization, involving voltage-gated Na{sup +} channels.« less
  • Several scorpion and snake toxins were radioiodinated using the lactoperoxydase method of (/sup 125/I)iodide oxidation. Two techniques of labeling were set up: Using carrier-free Na/sup 125/I and 5 ..mu..g of toxin, about one iodine atom was incorporated per mole of protein without loss of toxicity. Specific radioactivities about 2,000 Ci/mmol (280 ..mu..Ci/..mu..g) were obtained. The modified toxin, purified by immunoprecipitation with an antiserum prepared against the native toxin, was obtained in a short time (4 hr), with a good yield (50 to 80%), and in a small volume (1 ml). Using Na/sup 127/I traced with Na /sup 125/I and largermore » amounts (200 ..mu..g) of toxin, more than one iodine atom was incorporated per mole of protein without loss of activity. Lower specific radioactivities (1 to 1.5 Ci/mmol) were obtained. The iodinated toxins were purified by gel filtration of the radioiodination mixtures on a column made of two layers of Sephadex (G-15 and G-50). The modified proteins were extensively analyzed by paper electrophoresis and polyacrylamide gel electrophoresis. Their content of monoiodotyrosine and diiodotyrosine was estimated and, in the case of toxin I of Androctonus australis Hector, it was possible to follow the iodination rate of its three tyrosine residues by automatic Edman degradation. The mode of purification of the iodinated scorpion toxins affects their behavior on molecular sieving on Sephadex G-50 and on electrophoresis on polyacrylamide gel. The results are discussed.« less
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