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The formation of amphotericin B ion channels in lipid bilayers
Insights
Amphotericin B (AmB) forms ion channels in lipid bilayers at a critical concentration, requiring at least 16 molecules per liposome. Cholesterol
Area of Science:
- Biophysics
- Membrane Biology
- Antifungal Drug Research
Background:
- Amphotericin B (AmB) is a crucial antifungal agent.
- Its mechanism involves forming channels in fungal cell membranes.
- Cholesterol's role in AmB channel formation is not fully understood.
Purpose of the Study:
- To investigate the formation of ion-permeable channels by AmB in cholesterol-containing lipid bilayers.
- To determine the critical concentration of AmB for channel activity.
- To elucidate the influence of cholesterol on AmB-mediated channel formation.
Main Methods:
- UV/visible absorbance, circular dichroism, and fluorescence spectroscopy were employed.
- Liposomes with varying ratios of distearoylphosphatidylcholine, cholesterol, distearoylphosphatidylglycerol, and AmB were prepared.
- Singular value decomposition analysis was used to study AmB states in the bilayer.
Main Results:
- AmB exists in monomeric and aggregated states in lipid bilayers.
- Channel activity appears at a critical concentration of 1 AmB per 1000 lipids.
- Optimal AmB channel formation occurs when cholesterol forms tail-to-tail dimers.
Conclusions:
- AmB channel formation is dependent on reaching a critical drug concentration.
- Cholesterol's structural organization, specifically tail-to-tail dimers, facilitates AmB channel initiation.
- These findings support AmB's mechanism of action via ion-permeable channel formation in fungal cells.
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.