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Isolation and characterization of the genes encoding gamma toxin of the Brazilian scorpions Tityus serrulatus, Tityus bahiensis and Tityus stigmurus
Abstracts
305
mechanisms. The most striking changes appear in the a-scorpion toxin (toxin IV-5) which, in contrast to the /?-scorpion toxins (I and III-8), act by a Ca 2+-independent mechanism. We will present our findings in recent studies of these mechanisms in the context of molecular structural characteristics. lsofation and characterization qf the genes encoding gamma toxin of the Brazilian scorpions Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. B. Becerril, M. Corona and L. D. Possani (Department of Molecular Recognition and Structural Biology, Instituto de Biotechnologia, Universidad National Autonoma de Mexico. Apartado Postal 510-3, Cuernavaca-62271, Mexico). The genes encoding toxin gamma from the scorpions Tiryus serrulutus, T. stigmurus and T. bahiensis were amplified from genomic DNA by means of PCR using synthetic oligonucleotides designed from the reported cDNA sequence of gamma toxin from 7’. serrulatus. The analysis of the nucleotide sequence of these genes revealed the presence of introns of 475, 474 and 464 base pairs in the genes of T. serrulaius, T. stigrnurus and ‘Z. bnhiensis, respectively, which interrupt the region that encodes the signal peptide of the respective precursor toxin. A distinctive feature of gamma toxin from 7: stigmurus concerns the presence of an additional glycine residue at the amino end of the mature toxin compared with the other two gamma toxins mentioned above. The interpretation of the mechanism for the processing at the amino terminus of this peptide will be discussed. Finally, a comparison of the intron boundary sequences of the gamma toxin genes with those from other arachnid genes will also be presented. This work was supported in part by grants of the Howard National University of Mexico (DGAPA-205893) and Consejo LDP.
Hughes Medical Institute (No. 75191-527104), National de Ciencia y Tecnologia of Mexico to
Selective rnod$cation of sodium channel inactivation by versutoxin, an Australian funnel-web spider toxin. G. M. Nicholson’ and T. Narahashi*(’ Department of Biochemistry and Physiology, University of Technology, Sydney, NSW 2007, Australia; and 2Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611, U.S.A.). Venom from the Australian funnel-web spiders Hadronyche versuta and Atrax robustus has previously been shown to produce repetitive action potentials in nerve fibres leading to spontaneous transmitter release at motor and autonomic nerve endings, This action may involve an alteration to voltage-gated sodium channel function. To assess this possibility the effects of versutoxin, a novel 42 amino acid peptide from the venom of the Australian Blue Mountains funnel-web spider H. rersura, was investigated on ionic currents in acutely dissociated rat dorsal root ganglion cells. Whole-cell patch clamp experiments revealed that versutoxin (VTX), at concentrations up to 2 FM, had no effect on potassium currents or tetrodotoxin-resistant sodium currents. In contrast, VTX produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive sodium current (TTX-S INil) inactivation when applied via the external but not the internal bathing solution. The prolonged steady-state ZNa seen in the presence of VTX was maintained during prolonged depolarizing pulses greater than 200 msec. At higher test potentials, however, there was a reduction in the amplitude of the steady-state Z,, which may reflect a voltage-dependent dissociation of the toxin from the extracellular binding site. Unlike fi-scorpion toxins, VTX did not significantly alter the time-course of TTX-S 6, tail currents. Examination of the steady-state inactivation curve (IT,) of the sodium current revealed that 30 nM VTX produced a significant 7 mV hyperpolarizing shift in the voltage dependence of h, and that there was a significant non-inactivatable Z,,. This non-inactivating component (14 + 2% of maximal ZNa) was present at prepulse potentials more depolarized than -4OmV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an increase in the rate of recovery from channel inactivation in the presence of VTX. In conclusion, these selective actions of VTX on sodium channel gating, particularly the voltage-dependent slowing of channel inactivation and the faster rate of recovery from inactivation, are similar to those of x-scorpion toxins. They also support the model of a two-state inactivation process proposed by Strichartz and Wang (1986) to explain the modification of sodium channel gating by cw-scorpion toxins. Strichartz,
G. R. and Wang,
G. K. (1986) J. gen. Physiol. 88, 413435.
Ciguatera @sh poisoning): progress andperspectives. R. J. Lewis (Queensland Department QABC, Gehrmann Laboratories, The University of Queensland, Qld 4072, Australia).
of Primary
Industries,
Ciguatera, a pleomorphic syndrome consisting of a range of gastrointestinal, neurological and cardiovascular signs and symptoms, follows the consumption of warm-water marine fish that have become contaminated with the ciguatoxin class of polyether toxins. The disease is rarely fatal, and the severity and duration of illness can be markedly reduced with intravenous mannitol. The ciguatoxins (CTX) arise from the oxidative biotransformation of gambiertoxin(s) produced by certain strains of the benthic dinoflagellate, Gambierdiscus toxicus.
305
mechanisms. The most striking changes appear in the a-scorpion toxin (toxin IV-5) which, in contrast to the /?-scorpion toxins (I and III-8), act by a Ca 2+-independent mechanism. We will present our findings in recent studies of these mechanisms in the context of molecular structural characteristics. lsofation and characterization qf the genes encoding gamma toxin of the Brazilian scorpions Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. B. Becerril, M. Corona and L. D. Possani (Department of Molecular Recognition and Structural Biology, Instituto de Biotechnologia, Universidad National Autonoma de Mexico. Apartado Postal 510-3, Cuernavaca-62271, Mexico). The genes encoding toxin gamma from the scorpions Tiryus serrulutus, T. stigmurus and T. bahiensis were amplified from genomic DNA by means of PCR using synthetic oligonucleotides designed from the reported cDNA sequence of gamma toxin from 7’. serrulatus. The analysis of the nucleotide sequence of these genes revealed the presence of introns of 475, 474 and 464 base pairs in the genes of T. serrulaius, T. stigrnurus and ‘Z. bnhiensis, respectively, which interrupt the region that encodes the signal peptide of the respective precursor toxin. A distinctive feature of gamma toxin from 7: stigmurus concerns the presence of an additional glycine residue at the amino end of the mature toxin compared with the other two gamma toxins mentioned above. The interpretation of the mechanism for the processing at the amino terminus of this peptide will be discussed. Finally, a comparison of the intron boundary sequences of the gamma toxin genes with those from other arachnid genes will also be presented. This work was supported in part by grants of the Howard National University of Mexico (DGAPA-205893) and Consejo LDP.
Hughes Medical Institute (No. 75191-527104), National de Ciencia y Tecnologia of Mexico to
Selective rnod$cation of sodium channel inactivation by versutoxin, an Australian funnel-web spider toxin. G. M. Nicholson’ and T. Narahashi*(’ Department of Biochemistry and Physiology, University of Technology, Sydney, NSW 2007, Australia; and 2Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611, U.S.A.). Venom from the Australian funnel-web spiders Hadronyche versuta and Atrax robustus has previously been shown to produce repetitive action potentials in nerve fibres leading to spontaneous transmitter release at motor and autonomic nerve endings, This action may involve an alteration to voltage-gated sodium channel function. To assess this possibility the effects of versutoxin, a novel 42 amino acid peptide from the venom of the Australian Blue Mountains funnel-web spider H. rersura, was investigated on ionic currents in acutely dissociated rat dorsal root ganglion cells. Whole-cell patch clamp experiments revealed that versutoxin (VTX), at concentrations up to 2 FM, had no effect on potassium currents or tetrodotoxin-resistant sodium currents. In contrast, VTX produced a concentration-dependent slowing or removal of tetrodotoxin-sensitive sodium current (TTX-S INil) inactivation when applied via the external but not the internal bathing solution. The prolonged steady-state ZNa seen in the presence of VTX was maintained during prolonged depolarizing pulses greater than 200 msec. At higher test potentials, however, there was a reduction in the amplitude of the steady-state Z,, which may reflect a voltage-dependent dissociation of the toxin from the extracellular binding site. Unlike fi-scorpion toxins, VTX did not significantly alter the time-course of TTX-S 6, tail currents. Examination of the steady-state inactivation curve (IT,) of the sodium current revealed that 30 nM VTX produced a significant 7 mV hyperpolarizing shift in the voltage dependence of h, and that there was a significant non-inactivatable Z,,. This non-inactivating component (14 + 2% of maximal ZNa) was present at prepulse potentials more depolarized than -4OmV, potentials which normally inactivate all TTX-S sodium channels. Finally, there was an increase in the rate of recovery from channel inactivation in the presence of VTX. In conclusion, these selective actions of VTX on sodium channel gating, particularly the voltage-dependent slowing of channel inactivation and the faster rate of recovery from inactivation, are similar to those of x-scorpion toxins. They also support the model of a two-state inactivation process proposed by Strichartz and Wang (1986) to explain the modification of sodium channel gating by cw-scorpion toxins. Strichartz,
G. R. and Wang,
G. K. (1986) J. gen. Physiol. 88, 413435.
Ciguatera @sh poisoning): progress andperspectives. R. J. Lewis (Queensland Department QABC, Gehrmann Laboratories, The University of Queensland, Qld 4072, Australia).
of Primary
Industries,
Ciguatera, a pleomorphic syndrome consisting of a range of gastrointestinal, neurological and cardiovascular signs and symptoms, follows the consumption of warm-water marine fish that have become contaminated with the ciguatoxin class of polyether toxins. The disease is rarely fatal, and the severity and duration of illness can be markedly reduced with intravenous mannitol. The ciguatoxins (CTX) arise from the oxidative biotransformation of gambiertoxin(s) produced by certain strains of the benthic dinoflagellate, Gambierdiscus toxicus.