Mechanism of Membrane Permeabilization by Sticholysin I, a Cytolysin Isolated from the Venom of the Sea Anemone Stichodactyla helianthus ² Mayra Tejuca, ‡,§ Mauro Dalla Serra, ‡ Mercedes Ferreras, ‡ Maria E. Lanio, § and Gianfranco Menestrina* ,‡ Centro di Fisica degli Stati Aggregati, Consiglio Nazionale delle Richerche-Istituto Trentino di Cultura, I-38050 PoVo (Trento), Italy, and Departamento de Bioquimica, Facultad de Biologia, UniVersidad de la Habana, La Habana, Cuba ReceiVed April 2, 1996; ReVised Manuscript ReceiVed September 16, 1996 X ABSTRACT: Actinaria cytolysins are very potent basic toxins isolated from the venom 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 Stichodactyla helianthus sticholysin I (St-I) with lipid bilayers was studied. St-I increased the permeability of calcein-loaded lipid vesicles composed of different phospholipids. The rate of permeabilization improved when sphingomyelin (SM) was introduced into phosphatidylcholine (PC) vesicles, reaching an optimum value at equimolar concentrations of these two phospholipids. It was also a function of the pH, showing a local 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 vesicle ratio < 200), most of the toxin is bound to the lipid phase. The reduced toxin effect at low and high SM content, or at high pH, is principally due to a decreased toxin binding. From the dose dependence of the permeabilization, at constant lipid concentration, it was inferred that St-I increases membrane permeability by forming oligomeric pores comprising at least three cytolysin monomers. The involvement of oligomers was also suggested by the dependence of calcein release on the vesicle concentration at constant toxin dose. In fact, the time course of dye release was well described under all circumstances by a kinetic model which assumes that trimerization leads to a conductive pore. All the relevant equilibrium and rate constants were derived. Addition of St-I to one side of a planar lipid membrane increased the conductivity of the film in discrete steps of defined amplitude, indicating the formation of ion channels. The dose dependence of this effect was the same as with LUV. The channel was cation-selective and its conductance suggested a functional radius of about 1.0 nm, consistent with the size of the lesion previously observed in red blood cells. Pores exhibited rectification and voltage- dependent gating. Potent cytolysins have been extracted from the venom of at least 16 species of sea anemones [for recent reviews see Kem (1988), Bernheimer (1990), Harvey (1990), Turk (1991), and Macek (1992)]. Many such toxins (at least 27) have been isolated and characterized. They share a number of common properties: they are basic proteins, with pI between 8 and 12; they comprise a single polypeptide chain of molecular mass ranging from 16 to 20 kDa; they contain no cysteines or cystines; and they are strongly inhibited by sphingomyelin. A few of them have been sequenced (Blumenthal & Kem, 1983; Simpson et al., 1990; Mebs et al., 1992; Belmonte et al., 1994). At subnanomolar con- centrations they cause erythrocyte lysis by opening pores, which are probably oligomeric. With some of them, the formation of discrete channels permeable to small ions and solutes has been demonstrated also in model lipid membranes (Michaels, 1979; Varanda & Finkelstein, 1980; Chanturya et al., 1990; Zorec et al., 1990; Belmonte et al., 1993). For their common ability to form pores in lipid membranes they have received the family name of actinoporins (Kem, 1988). Due to their extraordinary potency and chemical stability, these toxins can be used to construct chimeric molecules targeted at killing tumor cells (Avila et al., 1989; Pederzolli et al., 1995). The sea anemone Stichodactyla helianthus, widely diffused in the Caribbean area, produces a venom that contains several active proteinaceous factors, among which are a neurotoxin acting on potassium channels (Castan ˜eda et al., 1995), a protease inhibitor (Delfin et al., 1994), a phospholipase (Pazos et al., 1996), and at least two cytolysins belonging to the above-mentioned group (Alvarez et al., 1994, 1996). The isolation, purification to homogeneity, and primary sequence of one of these cytolysins, designated sticholysin I, has been described recently (Alvarez et al., 1996). This toxin is similar to the cytolysin C III 1 described earlier (Blumenthal & Kem, 1983); however, it contains a 22-residue ² This work was financially supported by the Italian Consiglio Nazionale delle Ricerche, by a grant from the Istituto Trentino di Cultura to M.T., and by a short-term fellowship from the European Molecular Biology Organization to M.F. (EMBO ASTF 7745) . * Address correspondence to this author. ‡ Centro di Fisica degli Stati Aggregati, CNR-ITC. § Universidad de la Habana. X Abstract published in AdVance ACS Abstracts, November 1, 1996. 1 Abbreviations: C III, Stichodactyla helianthus cytolysin III; St-I, Stichodactyla helianthus sticholysin I; EqT-II, Actinia equina equinatox- in II; BSA, bovine serum albumin; SM, sphingomyelin; PC, phos- phatidylcholine; PE, phosphatidylethanolamine; POPC, palmitoylo- leoylphosphatidylcholine; SUV, small unilamellar vesicles; LUV, large unilamellar vesicles; PLM, planar lipid membranes; TLC, thin-layer chromatography; SDS, sodium dodecyl sulfate; Triton X-100, octylphe- noxy(polyethoxy)ethanol; PAGE, polyacrylamide gel electrophoresis; RBC, red blood cells. 14947 Biochemistry 1996, 35, 14947-14957 S0006-2960(96)00787-8 CCC: $12.00 © 1996 American Chemical Society