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. 1980 Feb;299:507–520. doi: 10.1113/jphysiol.1980.sp013139

Binding of scorpion toxin to sodium channels in vitro and its modification by β-bungarotoxin

Harumasa Okamoto 1
PMCID: PMC1279239  PMID: 6247482

Abstract

1. Binding of a purified scorpion toxin to membrane fragments isolated from electroplaque of an electric eel Electrophorus electricus was studied using a radio-iodinated toxin.

2. A scorpion toxin was purified from the venom of Leiurus quinquestriatus and iodinated with 125I in a lactoperoxidase-catalysed reaction. Monoiodinated toxin, isolated by an ion exchange chromatography, retarded the inactivation kinetics of Na current to a similar extent as the native toxin, indicating that radioiodination did not appreciably affect physiological and binding properties of the native toxin.

3. Analyses of binding properties by Scatchard plots showed the presence of two classes of binding sites (with low and high affinities) in the membrane preparation from eel electroplaque; similar preparation from an electric skate, of which the electroplaque is known to be devoid of Na channels, possessed only the low affinity sites.

4. The number of high affinity sites in the eel preparation was 41·8 ± 10·5 p-mole/g tissue; the value was within the range reported for tetrodotoxin binding to similar preparations (15-148 p-mole/g tissue).

5. A variety of cations (Na+, Mn2+ and La3+) inhibited the high affinity scorpion toxin binding, as indicated for the toxin binding to Na channels by a previous electrophysiological study. KD value in the presence of 120 mM-Na+ (approx. 8 nM) agreed reasonably with that (approx. 10nM) reported for the scorpion toxin binding to excitable neuroblastoma cells or synaptic nerve ending particles under conditions where membrane potential was depolarized by the addition of 135 mM-KCl.

6. Pretreatment of the eel membrane preparation with β-bungarotoxin (7-44 ng/ml.) in the presence of Ca ions (10-200 μM) resulted in a substantial loss of high affinity binding of scorpion toxin. When phospholipase A2 activity of the β-toxin was inactivated by a chemical modification with p-bromophenacyl bromide, the inhibitory action of the β-bungarotoxin was abolished.

7. It is concluded that a high affinity binding of scorpion toxin to the eel electroplaque membrane fragments represents the binding to Na channels in vitro, and that phospholipase A2 activity of β-bungarotoxin interferes with the binding of scorpion toxin to Na channels.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abe T., Alemá S., Miledi R. Isolation and characterization of presynaptically acting neurotoxins from the venom of Bungarus snakes. Eur J Biochem. 1977 Oct 17;80(1):1–12. doi: 10.1111/j.1432-1033.1977.tb11849.x. [DOI] [PubMed] [Google Scholar]
  2. Agnew W. S., Levinson S. R., Brabson J. S., Raftery M. A. Purification of the tetrodotoxin-binding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2606–2610. doi: 10.1073/pnas.75.6.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Almers W., Levinson S. R. Tetrodotoxin binding to normal depolarized frog muscle and the conductance of a single sodium channel. J Physiol. 1975 May;247(2):483–509. doi: 10.1113/jphysiol.1975.sp010943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Armstrong C. M. Ionic pores, gates, and gating currents. Q Rev Biophys. 1974 May;7(2):179–210. doi: 10.1017/s0033583500001402. [DOI] [PubMed] [Google Scholar]
  5. CHANG C. C., LEE C. Y. ISOLATION OF NEUROTOXINS FROM THE VENOM OF BUNGARUS MULTICINCTUS AND THEIR MODES OF NEUROMUSCULAR BLOCKING ACTION. Arch Int Pharmacodyn Ther. 1963 Jul 1;144:241–257. [PubMed] [Google Scholar]
  6. Cahalan M. D. Modification of sodium channel gating in frog myelinated nerve fibres by Centruroides sculpturatus scorpion venom. J Physiol. 1975 Jan;244(2):511–534. doi: 10.1113/jphysiol.1975.sp010810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Catterall W. A. Membrane potential-dependent binding of scorpion toxin to the action potential Na+ ionophore. Studies with a toxin derivative prepared by lactoperoxidase-catalyzed iodination. J Biol Chem. 1977 Dec 10;252(23):8660–8668. [PubMed] [Google Scholar]
  8. Catterall W. A., Morrow C. S. Binding to saxitoxin to electrically excitable neuroblastoma cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):218–222. doi: 10.1073/pnas.75.1.218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Catterall W. A. Purification of a toxic protein from scorpion venom which activates the action potential Na+ ionophore. J Biol Chem. 1976 Sep 25;251(18):5528–5536. [PubMed] [Google Scholar]
  10. Grundfest H. Comparative electrobiology of excitable membranes. Adv Comp Physiol Biochem. 1966;2:1–116. doi: 10.1016/b978-0-12-395511-1.50006-8. [DOI] [PubMed] [Google Scholar]
  11. Hinzen D. H., Tauc L. Membrane properties of Aplysia neurones intracellularly injected with phospholipases A and C. J Physiol. 1977 Jun;268(1):21–34. doi: 10.1113/jphysiol.1977.sp011844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kopeyan C., Martinez G., Rochat H. Amino acid sequence of neurotoxin V from the scorpion Leiurus quinquestriatus quinquestriatus. FEBS Lett. 1978 May 1;89(1):54–58. doi: 10.1016/0014-5793(78)80521-3. [DOI] [PubMed] [Google Scholar]
  13. Koppenhöfer E., Schmidt H. Die Wirkung von Skorpiongift auf die Ionenströme des Ranvierschen Schnürrings. I. Die Permeabilitäten PNa und PK. Pflugers Arch. 1968;303(2):133–149. doi: 10.1007/BF00592631. [DOI] [PubMed] [Google Scholar]
  14. Koppenhöfer E., Schmidt H. Die Wirkung von Skorpiongift auf die Ionenströme des Ranvierschen Schnürrings. II. Unvollständiage Natrium-Inaktivierung. Pflugers Arch. 1968;303(2):150–161. doi: 10.1007/BF00592632. [DOI] [PubMed] [Google Scholar]
  15. Levinson S. R. The purity of tritiated tetrodotoxin as determined by bioassay. Philos Trans R Soc Lond B Biol Sci. 1975 Jun 10;270(908):337–348. doi: 10.1098/rstb.1975.0013. [DOI] [PubMed] [Google Scholar]
  16. Linden C. D., Raftery M. A. Isolation of a scorpion toxin for use as a probe of the electrically excitable sodium channel. Biochem Biophys Res Commun. 1976 Sep 20;72(2):646–653. doi: 10.1016/s0006-291x(76)80089-7. [DOI] [PubMed] [Google Scholar]
  17. MONOD J., WYMAN J., CHANGEUX J. P. ON THE NATURE OF ALLOSTERIC TRANSITIONS: A PLAUSIBLE MODEL. J Mol Biol. 1965 May;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
  18. Morrison M., Bayse G. S. Catalysis of iodination by lactoperoxidase. Biochemistry. 1970 Jul 21;9(15):2995–3000. doi: 10.1021/bi00817a010. [DOI] [PubMed] [Google Scholar]
  19. Narahashi T., Shapiro B. I., Deguchi T., Scuka M., Wang C. M. Effects of scorpion venom on squid axon membranes. Am J Physiol. 1972 Apr;222(4):850–857. doi: 10.1152/ajplegacy.1972.222.4.850. [DOI] [PubMed] [Google Scholar]
  20. Okamoto H., Takahashi K., Yamashita N. One-to-one binding of a purified scorpion toxin to Na channels. Nature. 1977 Mar 31;266(5601):465–468. doi: 10.1038/266465a0. [DOI] [PubMed] [Google Scholar]
  21. Okamoto H., Takahashi K., Yoshii M. Membrane currents of the tunicate egg under the voltage-clamp condition. J Physiol. 1976 Jan;254(3):607–638. doi: 10.1113/jphysiol.1976.sp011249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Okamoto H., Takahashi K., Yoshii M. Two components of the calcium current in the egg cell membrane of the tunicate. J Physiol. 1976 Feb;255(2):527–561. doi: 10.1113/jphysiol.1976.sp011294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ray R., Morrow C. S., Catterall W. A. Binding of scorpion toxin to receptor sites associated with voltage-sensitive sodium channels in synaptic nerve ending particles. J Biol Chem. 1978 Oct 25;253(20):7307–7313. [PubMed] [Google Scholar]
  24. Reed J. K., Raftery M. A. Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus. Biochemistry. 1976 Mar 9;15(5):944–953. doi: 10.1021/bi00650a002. [DOI] [PubMed] [Google Scholar]
  25. Romey G., Chicheportiche R., Lazdunski M., Rochat H., Miranda F., Lissitzky S. Scorpion neurotoxin - a presynaptic toxin which affects both Na+ and K+ channels in axons. Biochem Biophys Res Commun. 1975 May 5;64(1):115–121. doi: 10.1016/0006-291x(75)90226-0. [DOI] [PubMed] [Google Scholar]
  26. Romine W. O., Jr, Schoepfle G. M., Smythies J. R., Al-Zahid G., Bradley R. J. Pharmacological evidence related to the existence of two sodium channel gating mechanisms. Nature. 1974 Apr 26;248(5451):797–799. doi: 10.1038/248797a0. [DOI] [PubMed] [Google Scholar]
  27. Strong P. N., Heuser J. E., Kelly R. B. Selective enzymatic hydrolysis of nerve terminal phospholipids by beta-bungarotoxin: biochemical and morphological studies. Prog Clin Biol Res. 1977;15:227–249. [PubMed] [Google Scholar]

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