Mechanism of action of equinatoxin II, a cytolysin from the sea anemone Actinia equina L. belonging to the family of actinoporins.

Actinia equina equinatoxin II (EqT-II) is a representative of a family of pore-forming, basic, polypeptide toxins from sea anemones, now called actinoporins. This family comprises at least 27 members, which are all hemolytic at rather low concentrations. Red blood cell (RBC) hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced pores. Using osmotic protectants of different size the functional radius of the lesion was estimated to be approximately 1.1 nm. These pores are most probably constituted by oligomeric aggregates of cytolysin molecules, whose presence on the membrane of lysed RBC was directly demonstrated by polyacrylamide gel electrophoresis (PAGE) after covalent cross-linking. EqT-II is active also against a variety of mammalian cells including leukocytes, platelets and cardiomiocytes. An increased permeability of the plasma membrane after Eq-II attack is compatible with the notion that the toxin forms pores also on these cells. Eq-II permeabilises even purely lipidic model membranes, suggesting a protein receptor is not necessary. Using calcein-loaded unilamellar vesicles (UVs) comprised of phosphatydylcholine (PC) mixed with other lipids we observed that the rate and extent of permeabilization greatly increases when sphingomyelin (SM) or the ganglioside GM1 were introduced, particularly in the case of large UVs (which are more sensitive to the toxin than small UVs). PAGE indicated that the increased effect of Eq-II on SM containing vesicles is due to an increased level of toxin binding to such vesicles. The formation of cation-selective channels by EqT-II was directly demonstrated using planar lipid membranes where the toxin induced discrete increases of the film conductivity. The conductance of the channel was consistent with the estimated size of the lesion formed in RBC. Several factors can affect toxin activity: serum, low pH, low ionic strength and multivalent cations are potent inhibitors. pH Dependence is bell shaped, optimum activity being between pH 8 and 9. Similarly the action of Ca2+ is also bivalent: up to a concentration of approximately 2 mM it stimulates hemolysis, but above this concentration it inhibits (with 50% inhibition occurring at approximately 10 mM). When the known amino acid sequences of actinoporins are examined a common trait emerges; the presence of a well conserved, amphiphilic, putative alpha-helix at the N-terminus, which might be involved in the insertion of EqT-II in lipid membranes.