INSECT SPECIFIC NEUROTOXINS FROM SCORPION-VENOM THAT AFFECT SODIUM CURRENT INACTIVATION

A new toxin, Lqh alpha IT, 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, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT 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, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions, a degree of homology comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT 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 Lqh alpha IT is an a toxin which acts on insect sodium channels. Binding studies with the radioiodinated Lqh alpha IT toxin reveal that Lqh alpha IT 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: (I) distinct from binding sites of other sodium channel neurotoxins; (2) inhibited by sea anemone toxin 2; (3) cooperatively interacting with veratridine. The binding of Lqh alpha IT is not dependent on membrane potential, in contrast to the binding of alpha 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 alpha scorpion toxins. The limited sequence variation between Lqh alpha IT and typical ex, 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 Lqh alpha IT 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 Lqh alpha IT-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.