In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centfugation. The nonsoluble form was favored by a high concentration ofAmB of the stock solution.The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB.Amphotericin B (AmB) remains the drug of choice for treating most systemic fungal infections. The recent introduction of liposomal AmB formulations with lower toxicity to patients (see, for instance, reference 5), has increased the clinical interest in this drug. However, its mechanism of action is still imperfectly understood. Inducing permeability to K+ is thought to be the first event following the addition of AmB to cells and is therefore an indicator of its activity. In order to understand the mechanism of its selectivity between fungal (ergosterol-containing membranes) and mammalian (cholesterol-containing membranes) cells, several studies have been performed from the functional point of view with mammalian erythrocytes (see references 2 [and references therein], 6, 26, and 32) as well as from the molecular point of view (3,29,30,33). In a previous study (3), we were able to show that AmB induces permeability to K+ in cholesterol-containing membranes, particularly in erythrocytes only when AmB is in the self-associated form in the incubation medium. The threshold of concentration beyond which AmB self-association starts therefore appeared to be a determining factor in the activity of the drug. This point is very important for the design of new less toxic derivatives of AmB. It can also be a clue for the mechanism by which the selectivity of the drug is increased when it is incorporated into liposomes.However, a more detailed analysis of the toxicity of the self-associated form was necessary, because it is well-known (23) that two self-assoc...

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The polyene antibiotic amphotericin B (AmB) is known to form aqueous pores in lipid membranes and biological membranes. Here, membrane potential and ion permeability measurements were used to demonstrate that AmB can form two types of selective ion channels in human erythrocytes, differing in their interaction with cholesterol. We show that AmB induced a cation efflux (negative membrane polarization) across cholesterol-containing liposomes and erythrocytes at low concentrations (< or =1.0 x 10(-6) M), but a sharp reversal of such polarization was observed at concentrations greater than 1.0 x 10(-6) M AmB, an indication that aqueous pores are formed. Cation-selective AmB channels are also formed across sterol-free liposomes, but aqueous pores are only formed at AmB concentrations 10 times greater. The effect of temperature on the AmB-mediated K+ efflux across erythrocytes revealed that the energies of activation for channel formation are negative and positive at AmB concentrations that lead predominantly to the formation of cation-selective channels and aqueous pores, respectively. These findings support the conclusion that the two types of AmB channels formed in human erythrocytes differ in their interactions with cholesterol and other membrane components. In effect, a membrane lipid reorganization, as induced by incubation of erythrocytes with tetrathionate, a cross-linking agent of the lipid raft-associated protein spectrin, led to differential changes in the activation parameters for the formation of both types of channels, reflecting the different lipid environments in which such structures are formed.

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