The Journal of Membrane Biology

, Volume 118, Issue 3, pp 243–249

Cytotoxicity of equinatoxin II from the sea anemoneActinia equina involves ion channel formation and an increase in intracellular calcium activity

  • R. Zorec
  • M. Tester
  • P. Maček
  • W. T. Mason


Equinatoxin Il is a 20-kDa basic protein isolated from the sea anemoneActinia equina. The aim of our work was to investigate the primary molecular basis for the cytotoxic effects of equinatoxin II in two model systems: single bovine lactotrophs and planar lipid bilayers. Previous work has shown that equinatoxin II produces rapid changes in cell morphology, which are dependent on external calcium. It has also been reported that addition of equinatoxin II increases membrane electrical conductance, which suggests that the cytotoxic action of equinatoxin II involves an increase in the permeability of membranes to Ca2+. Extensive changes in cytosolic Ca2+ activity are thought to invoke irreversible changes in cell physiology and morphology. In this paper, we show that morphological changes brought about by equinatoxin II in bovine lactotrophs are associated with a rapid rise in cytosolic Ca2+ activity, monitored with a fura-2 video imaging apparatus. Moreover, incorporation of equinatoxin II into planar lipid bilayers produces Ca2+ permeable ion channels. This suggests that the mode of equinatoxin II cytotoxicity involves the formation of cation (Ca2+) permeable channels in cell membranes.

Key Words

fura-2 imaging planar lipid bilayers Ca channels bovine lactotrophs equinatoxin sea anemone Aclinia equina 


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  1. Alsen, C. 1983. Biological significance of peptides fromAnemonia sulcata.Fed. Proc. 42:101–108PubMedGoogle Scholar
  2. Barrett, J.N., Magleby, K.L., Pallotta, B.S. 1982. Properties of single calcium-activated potassium channels in cultured rat muscle.J. Physiol. (London) 331:211–230Google Scholar
  3. Batista, U., Jezernik, K., Maček, P., Sedmak, B. 1987. Morphological evidence of cytotoxic and cytolytic activity of equinatoxin II.Period. Biol. 89:347–348Google Scholar
  4. Batista, U., Maček, P., Sedmak, B. 1986. The influence of equinatoxin II on V-79-379 A cell line.Period. Biol. 88:97–98Google Scholar
  5. Bernheimer, A.W., Avigad, L.S. 1976. Properties of a toxin from the sea anemoneStoichactis helianthus, including specific binding to sphingomyelin.Proc. Natl. Acad. Sci. USA 73:467–471PubMedGoogle Scholar
  6. Bernheimer, A.W., Rudy, B. 1986. Interactions between membranes and cytolytic peptides.Biochim. Biophys. Acta 864:123–141PubMedGoogle Scholar
  7. Choi, D.W. 1988. Calcium-mediated neurotoxicity: Relationship to specific channel types and role in ischemic damage.Trends Neurosci. 11: 465–469PubMedGoogle Scholar
  8. Doyle, J.W., Kem, W.R., Vilallonga, F.A. 1989. Interfacial activity of an ion channel-generating protein cytolysin from the sea anemoneStichodactyla helianthus.Toxicon 27:465–471PubMedGoogle Scholar
  9. Ferlan, I., Lebez, D. 1974. Equinatoxin, a lethal protein fromActinia equina-I: Purification and characterization.Toxicon 12:57–61PubMedGoogle Scholar
  10. Geletyuk, V.I., Kazachenko, V.N. 1985. Single Cl channels in molluscan neurons: Multiplicity of the conductance states.J. Membrane Biol. 86:9–15Google Scholar
  11. Giraldi, T., Ferlan, I., Romeo, D. 1976. Antitumor activity of equinatoxin.Chem. Biol. Interact. 13:199–203PubMedGoogle Scholar
  12. Goldman, D.E. 1943. Potential, impedance and rectification in membranes.J. Gen. Physiol. 27:37–60Google Scholar
  13. Grynkiewicz, G.M., Poenie, M., Tsien, R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties.J. Biol. Chem. 260:3440–3450PubMedGoogle Scholar
  14. Hamill, O.P., Sakmann, B. 1981. Multiple conductance states of single acetylcholine receptor channels in embryonic cells.Nature (London) 194:462–464Google Scholar
  15. Ho, C.L., Ko, J.L., Lue, H.M., Lee, C.Y., Ferlan, I. 1987. Effects of equinatoxin on the guinea-pig atrium.Toxicon 25:659–664PubMedGoogle Scholar
  16. Hodgkin, A.L., Katz, B. 1949. The effect of sodium ions on the electrical activity of the giant axon of the squid.J. Physiol. (London) 108:37–77Google Scholar
  17. Hughes, D., McBurney, R.N., Smith, S.M., Zorec, R. 1987. Caesium ions activate chloride channels in rat cultured spinal cord neurones.J. Physiol. (London) 392:231–251Google Scholar
  18. Ingram, C.D., Keefe, P.D., Wooding, F.B.P., Bicknell, R.J. 1988. Morphological characterisation of lactotrophs separated from the bovine pituitary by rapid enrichment technique.Cell Tissue Res. 252:655–659PubMedGoogle Scholar
  19. Kem, W.R. 1988. Sea anemone toxins: Structure and action.In: Biology of Nematocysts. D. Hessinger and H. Lenhoff, editors. Academic, LondonGoogle Scholar
  20. Knowles, B.H., Blatt, M.R., Tester, M., Horsnell, J.M., Carroll, J., Menestrina, G., Ellar, D.J. 1989. A cytolytic δ-endotoxin fromBacillus thurinigiensis var.israelensis forms cation-selective channels in planar lipid bilayers.FEBS Lett. 244:259–262PubMedGoogle Scholar
  21. Krouse, M.E., Schneider, G.T., Gage, P.W. 1986. A large anionselective channel has seven conductance levels.Nature (London) 319:58–60Google Scholar
  22. Lee, C.Y., Lin, W.W., Chen, Y.M., Lee, S.Y. 1988. On the causes of acute death produced by animal venoms and toxins.In: Progress in Venom and Toxin Research. P. Gopalakrishnakone, and C.K. Tan, editors. pp. 3–14. University of Medicine, National University of Singapore, SingaporeGoogle Scholar
  23. Linder, R., Bernheimer, A.W., Kim, K.-S. 1977. Interaction between sphingomyelin and a cytolysin from the sea anemoneStoichactis helianthus.Biochim. Biophys. Acta 467:290–300PubMedGoogle Scholar
  24. Maček, P., Lebez, D. 1981. Kinetics of hemolysis induced by equinatoxin, a cytolytic toxin from the sea anemoneActinia equina. Effect of some ions and pH.Toxicon 19:233–240PubMedGoogle Scholar
  25. Maček, P., Lebez, D. 1988. Isolation and characterization of three lethal and hemolytic toxins from the sea anemoneActinia equina L.Toxicon 26:441–451PubMedGoogle Scholar
  26. Malgaroli, A., Milani, D., Meldolesi, J., Pozzan, T. 1987. Fura-2 measurements of cytosolic free Ca++ in monolayers and suspensions of various types of animal cells.J. Cell Biol. 105:2145–2155PubMedGoogle Scholar
  27. Mason, W.T., Rawlings, S.R., Cobbett, P., Sikdar, S.K., Zorec, R., Akerman, S.N., Benham, C.D., Berridge, M.J., Cheek, T., Moreton, R.B. 1988. Control of secretion in anterior pituitary cells—linking ion channels, messengers and exocytosis.J. Exp. Biol. 139:287–316PubMedGoogle Scholar
  28. McBurney, R.N., Neering, I.R. 1987. Neuronal calcium homeostasis.Trends Neurosci. 10:164–169Google Scholar
  29. Michaels, D.W. 1979. Membrane damage by a toxin from the sea anemoneStoichactis helianthus. 1. Formation of transmembrane channels in lipid bilayers.Biochim. Biophys. Acta 555:67–78PubMedGoogle Scholar
  30. Miller, C. 1982. Open-state substructure of single chloride channels fromTorpedo electroplax.Phil. Trans. R. Soc. London B 299:401–411Google Scholar
  31. Miller, R.J., 1988. Calcium signalling in neurons.Trends Neurosci. 11:415–419PubMedGoogle Scholar
  32. Nagy, K. 1987. Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells.Eur. Biophys. J. 15:129–132PubMedGoogle Scholar
  33. Robinson, R.A., Stokes, R.H. 1959. Electrolyte Solutions. Butterworths, LondonGoogle Scholar
  34. Shin, M.L., Michaels, D.W., Mayer, M.M. 1979. Membrane damage by a toxin from the sea anemoneStoichactis helianthus: II. Effect of membrane lipid composition in a liposome system.Biochim. Biophys. Acta 555:79–88PubMedGoogle Scholar
  35. Sket, D., Drašlar, K., Ferlan, I., Lebez, D. 1974. Equinatoxin, a lethal protein fromActinia equina: II. Pathophysiological action.Toxicon 12:63–68PubMedGoogle Scholar
  36. Smith, S.J., Augustine, G.J. 1988. Calcium ions, active zones and synaptic transmitter release.Trends Neurosci. 11:458–464PubMedGoogle Scholar
  37. Smith, S.M., Zorec, R., McBurney, R.N. 1989. Conductance states activated by glycine and GABA in rat cultured spinal neurones.J. Membrane Biol. 108:45–52Google Scholar
  38. Šuput, D. 1986. Effect of equinatoxin on the membrane of skeletal muscle fiber.Period. Biol. 88:210–211Google Scholar
  39. Šuput, D., Rubly, N., Meves, H. 1988. Effects of equinatoxins on single myelinated nerve fibres.In: Progress in Venom and Toxin Research. pp. 467–470. P. Gopalakrishnakone and C.K. Tan, editors. National University of Singapore, SingaporeGoogle Scholar
  40. Teng, C.-M., Lee, L.-G., Lee, C.-Y., Ferlan, I. 1988. Platelet aggregation induced by equinatoxin.Thromb. Res. 52:401–411PubMedGoogle Scholar
  41. Turk, T., Maček, P. 1986. Effect of different membrane lipids on the hemolytic activity of equinatoxin II fromActinia equina.Period. Biol. 88:216–217Google Scholar
  42. Turk, T., Maček, P., Gubenšek, F. 1989. Chemical modification of equinatoxin II, a lethal and cytolytic toxin from the sea anemoneActinia equina L.Toxicon 27:357–384Google Scholar
  43. Varanda, W., Finkelstein, A. 1980. Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone,Stoichactis helianthus.J. Membrane Biol. 55:203–211Google Scholar

Copyright information

© Springer-Verlag New York Inc 1990

Authors and Affiliations

  • R. Zorec
    • 1
  • M. Tester
    • 2
  • P. Maček
    • 3
  • W. T. Mason
    • 4
  1. 1.Institute of PathophysiologyUniversity of LjubljanaLjubljanaYugoslavia
  2. 2.Botany SchoolUniversity of CambridgeCambridgeUK
  3. 3.Department of BiologyBiotechnical FacultyLjubljanaYugoslavia
  4. 4.A.F.R.C.Institute of Animal PhysiologyCambridgeUK
  5. 5.Department of BotanyUniversity of AdelaideAdelaideAustralia

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