The Formation of Amphotericin B Ion Channels in Lipid Bilayers

Abstract

The ability of amphotericin B (AmB) to form ion-permeable channels in cholesterol containing lipid bilayers was studied by UV/visible absorbance, circular dichroism, and fluorescence spectroscopy. Stable liposomes composed of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylglycerol, and AmB were prepared so that a wide range of AmB concentrations in the bilayer could be studied. Singular value decomposition analysis (Henry & Hofrichter, 1992) of the circular dichroism spectra of AmB at different AmB/lipid ratios suggests that AmB exists primarily in only two states in the bilayer, a "monomeric" state and an "aggregated" state. The transition from the "monomeric" to the "aggregated" state begins to occur at a critical concentration of 1 AmB per 1000 lipids in the membrane and coincides with the appearance of channel activity. The data support the recent theoretical conclusions of Weakliem et al. (1995) which predict that pore formation in the lipid bilayer will occur when the drug molecule concentration exceeds a critical value. At this critical concentration, it is calculated that a minimum number of 16 AmB molecules per liposome are required to observe channel activity. The results are consistent with the sterol-dependent AmB channel models proposed by de Kruijff and Demel (1974), Andreoli (1974), and Khutorsky (1992). To further elucidate the effects of sterol on AmB-mediated channel formation, liposomes were prepared with varying ratios of cholesterol and AmB. At cholesterol mole percentages greater than 1, channel activity was observed to occur at AmB concentrations just above the critical value. Previous reports show that cholesterol forms "tail-to-tail" dimers at mole percentages greater than 2 (Harris et al., 1995). This suggests that formation of the bilayer-spanning channels by AmB is initiated most efficiently when the tail-to-tail dimer of cholesterol is present. Although the structural nature of the AmB channel could not be unambiguously determined, these experiments provide further evidence in support of the widely held view that AmB's primary mechanism of killing fungal cells occurs by forming ion-permeable channels.

Structural insights on biologically relevant cationic membranes by ESR spectroscopy

“…1) is a polyene antifungal drug widely used for the treatment of systemic fungal infections (Hartsel and Bolard 1996;Denning and Hope 2010;Gallis et al 1990). Its mechanism of action is supposed to be based on changes in membrane permeability (Bolard 1986;Fujii et al 1997;Cohen 2010), although a mechanism based on the production of free radicals has also been suggested (Lamy-Freund et al 1985;Sokol-Anderson et al 1988;Sangalli-Leite et al 2011). The use of AmB is limited by several side effects, including nephrotoxicity and hemolytic activity; therefore much effort has been made to develop formulations able to reduce its toxicity and increase its therapeutic effect (Brajtburg and Bolard 1996;Torrado et al 2008).…”

Molecular organization of antifungal antibiotic amphotericin B in lipid monolayers studied by means of Fluorescence Lifetime Imaging Microscopy

“…According to a popular conviction AmB present in biomembranes associates into molecular aggregates in the form of transmembrane pores that affect physiological ion transport [4][5][6][7][8]. The concept that such structures are formed more efficiently in the presence of ergosterol, the sterol present in the membranes of fungi, than in the presence of cholesterol is a key paradigm of selectivity of AmB.…”

The Design of Molecular Switches for Biosensors

“…[5][6][7][8][9] The presence of the cholesterol molecules in the AmB channel is critical for the channel to be open. 10 The removal of the cholesterol from the channel shuts the channel off.…”