Analysis of the interactions of amphotericin B with the Leishmania plasma membrane using EPR spectroscopy

Abstract

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to study the interactions of amphotericin B (AmB) with the plasma membrane of Leishmania amazonensis promastigotes, erythrocytes, and J774 macrophages. Spin labels embedded into the cell membranes detected strong interactions with putative AmB/sterol complexes that resulted in pronounced changes in the EPR spectra, which can be interpreted as a reduction in membrane fluidity or an increase in the polarity assessed by the spin probe. The EPR spectra of spin-labeled lipids corroborated the findings that AmB does not enter phospholipid membrane-sterol models and probably forms extramembranous aggregates, as predicted by the sterol sponge model. Furthermore, these aggregates were shown to extract the spin probe androstanol from the lipid bilayer. However, in contrast to the results for the model membrane, EPR spectroscopy suggested that AmB easily enters the membranes of the studied cells, implying that the entry process is dependent on interactions with the membrane proteins.