Co-Authors:
Zlotkin, E., Cell and Animal Biology, Life Science Institute, Hebrew University, 91904, Jerusalem, Israel
Eitan, M., Cell and Animal Biology, Life Science Institute, Hebrew University, 91904, Jerusalem, Israel
Pelhate, M., Neurophysiologie, Faculte de Medecine, Universite d'Angers, F-49045, Angers Cedex, France
Chejanovsky, N., Entomology Volcani Center for Agriculture, 50250, Bet Dagan, Israel
Gurevitz, M., Botany Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978, Tel Aviv, Israel
Gordon, D., Cell and Animal Biology, Life Science Institute, Hebrew University, 91904, Jerusalem, Israel
Abstract:
A new toxin, LqhαlT, which causes a unique mode of paralysis in blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, LqhαlT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) LqhαlT lacked strict selectivity for insects; was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin from its binding sites in insect neuronal membrane. However, in its primary structure and its effect on excitable tissues, LqhαlT strongly resembled the well-characterized α scorpion toxins, which affect mammals. The amino acid sequence was identical with α toxin sequences in 55%75% of positions, a degree of homology comparable to that seen among the α toxins themselves. Voltage-and current-clamp studies showed that LqhαlT caused an extreme prolongation of the action potential in cockroach giant axon preparation as a result of slowing of the inactivation of sodium currents. These observations indicate that LqhαlT is an α toxin which acts on insect sodium channels. Binding studies with the radioiodinated LqhαlT toxin reveal that LqhαlT does not bind to rat brain membrane and possesses in locust neuronal membranes a single class of high affinity (Kd= 1.06 + 0.15 nM) and low capacity (Bmax = 0.7 + 0.19 pmol/mg protein) binding sites. The latter are: (1) distinct from binding sites of other sodium channel neurotoxins; (2) inhibited by sea anemone toxin 2; (3) cooperatively interacting with veratridine. The binding of LqhαlT is not dependent on membrane potential, in contrast to the binding of α toxins in vertebrate systems. These data suggest the occurrence of (a) conformational-structural differences between insect and mammal sodium channels and (b) the animal group specificity and pharmacological importance of the α scorpion toxins. The limited sequence variation between LqhαlT and typical α mammal toxins provide a convenient model for clarifying the structural basis for animal group specificity of scorpion neurotoxins by a genetic approach. Therefore (1) the cDNA encoding the LqhαlT was cloned and sequenced. (2) The clone was successfully expressed in a transformed E. coli culture resulting in a recombinant toxin which is chemically and pharmacologically indistinguishable from the native toxin. (3) A recombinant baculovirus armed with the LqhαT-cDNA was constructed and it demonstrates the feasibility of implementation of insect selective neurotoxins for insect pest control. The unusual mode of animal group specificity exhibited by scorpion alpha toxins provides a unique lead for (a) the study of structural elements related to the gating of sodium channels and (b) the choice of new targets and models for the design of selective insecticides. © 1994 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.
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