Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes.
The interaction of Actinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.
Belmonte
G
G
Dipartimento di Fisica, Universitá di Trento, Povo, Italy.
Pederzolli
C
C
Macek
P
P
Menestrina
G
G
D000818:Animals; D002463:Cell Membrane Permeability / Q000187:drug effects; D003064:Cnidarian Venoms / Q000633:toxicity*; D004910:Erythrocyte Membrane / Q000187:drug effects*; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000187:drug effects; D008051:Lipid Bilayers / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008567:Membranes, Artificial
D003064:Cnidarian Venoms; D007473:Ion Channels; D008051:Lipid Bilayers; D008563:Membrane Lipids; D008567:Membranes, Artificial; C012307:equinatoxin