MECHANISM OF MEMBRANE PERMEABILIZATION BY STICHOLYSIN-I, A CYTOLYSIN ISOLATED FROM THE VENOM OF THE SEA-ANEMONE STICHODACTYLA-HELIANTHUS

Actinaria cytolysins are very potent basic toxins isolated from the ve nom of sea anemones, which are supposed to exert their toxic activity through formation of oligomeric pores in the host plasma membrane. To gain insight into their mechanism of action, the interaction of Sticho dactyla helianthus sticholysin I (St-I) with lipid bilayers was studie d. St-I increased the permeability of calcein-loaded lipid vesicles co mposed of different phospholipids. The rate of permeabilization improv ed when sphingomyelin (SM) was introduced into phosphatidylcholine (PC ) vesicles, reaching an optimum value at equimolar concentrations of t hese two phospholipids. It was also a function of the pH showing a loc al maximum of activity between pH 8 and 9 and a marked decrease at pH 10 and 11. Under optimal conditions (e.g., PC:SM 1:1, pH 8, toxin to v esicle ratio < 200), most of the toxin is bound to the lipid phase. Th e reduced toxin effect at low and high SM content, or at high pH, is p rincipally due to a decreased toxin binding. From the dose dependence of the permeabilization, at constant lipid concentration, it was infer red that St-I increases membrane permeability by forming oligomeric po res comprising at least three cytolysin monomers. The involvement of o ligomers was also suggested by the dependence of calcein release on th e vesicle concentration at constant toxin dose. In fact, the time cour se of dye release was well described under all circumstances by a kine tic model which assumes that trimerization leads to a conductive pore. All the relevant equilibrium and rate constants were derived. Additio n of St-I to one side of a planar lipid membrane increased the conduct ivity of the film in discrete steps of defined amplitude, indicating t he formation of ion channels. The dose dependence of this effect was t he same as with LUV. The channel was cation-selective and its conducta nce suggested a functional radius of about 1.0 nm, consistent with the size of the lesion previously observed in red blood cells. Pores exhi bited rectification and voltage-dependent gating.