The interaction of Staphylococcus aureus alpha-toxin with planar lipid membranes results in the formation of ionic channels whose conductance can be directly measured in voltage-clamp experiments. Single-channel conductance depends linearly on the solution conductivity suggesting that the pores are filled with aqueous solution; a rough diameter of 11.4 +/- 0.4 A can be estimated for the pore. The conductance depends asymmetrically on voltage and it is slightly anion selective at pH 7.0, which implies that the channels are asymmetrically oriented into the bilayer and that ion motion is restricted at least in a region of the pore. The pores are usually open in a KCl solution but undergo a dose- and voltage-dependent inactivation in the presence of di- and trivalent cations, which is mediated by open-closed fluctuations at the single-channel level. Hill plots indicate that each channel can bind two to three inactivating cations. The inhibiting efficiency follows the sequence Zn2+ greater than Tb3+ greater than Ca2+ greater than Mg2+ greater than Ba2+, suggesting that carboxyl groups of the protein may be involved in the binding step. A voltage-gated inactivation mechanism is proposed which involves the binding of two polyvalent cations to the channel, one in the open and one in the closed configuration, and which can explain voltage, dose and time dependence of the inactivation.

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.

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.

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X-ray absorption spectroscopy data show different metal binding site structures in beta-amyloid peptides according to whether they are complexed with Cu(2+) or Zn(2+) ions. While the geometry around copper is stably consistent with an intra-peptide binding with three metal-coordinated Histidine residues, the zinc coordination mode depends on specific solution conditions. In particular, different sample preparations are seen to lead to different geometries around the absorber that are compatible with either an intra- or an inter-peptide coordination mode. This result reinforces the hypothesis that assigns different physiological roles to the two metals, with zinc favoring peptide aggregation and, as a consequence, plaque formation.