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 cal led actinoporins. This family comprises at least 27 members, which are all hemolytic at rather low concentrations. Red blood cell (RBC) hemo lysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced pores. Using osmotic protectants of differen t size the functional radius of the lesion was estimated to be approxi mately 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 electrophore sis (PAGE) after covalent cross-linking. EqT-II is active also against a variety of mammalian cells including leukocytes, platelets and card iomiocytes. An increased permeability of the plasma membrane after Eq- II attack is compatible with the notion that the toxin forms pores als o on these cells. Eq-II permeabilises even purely lipidic model membra nes, suggesting a protein receptor is not necessary. Using calcein-loa ded unilamellar vesicles (UVs) comprised of phosphatydylcholine (PC) m ixed with other lipids we observed that the rate and extent of permeab ilization 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 incre ased level of toxin binding to such vesicles. The formation of cation- selective channels by EqT-II was directly demonstrated using planar li pid membranes where the toxin induced discrete increases of the film c onductivity. The conductance of the channel was consistent with the es timated size of the lesion formed in RBC. Several factors can affect t oxin activity: serum, low pH, low ionic strength and multivalent catio ns are potent inhibitors. pH Dependence is bell shaped, optimum activi ty being between pH 8 and 9. Similarly the action of Ca2+ is also biva lent: up to a concentration of approximately 2 mM it stimulates hemoly sis, but above this concentration it inhibits (with 50% inhibition occ urring at approximately 10 mM). When the known amino acid sequences of actinoporins are examined a common trait emerges: the presence of a w ell conserved, amphiphilic, putative alpha-helix at the N-terminus, wh ich might be involved in the insertion of EqT-II in lipid membranes.