Direct visualization of alamethicin ion pores formed in a floating phospholipid membrane supported on a gold electrode surface

Unilamellar DMPC/DMPG vesicles in the absence and presence of alamethicin were fused onto the surface of a gold electrode modified with a 1-thio-β-d-glucose self-assembled monolayer. The resulting floating bilayer lipid membranes (fBLMs) were investigated using atomic force microscopy (AFM) and electrochemical impedance spectroscopy (EIS). A corrugated film structure was observed for the pure DMPC/DMPG fBLMs due to surface stress between the tightly packed lipids. These corrugations are removed by the addition of alamethicin suggesting the lipid-peptide interactions alleviate the overall surface stress creating a more uniform bilayer. Both DMPC/DMPG films in the absence and presence of alamethicin had thickness of 5.5 ± 0.9 nm demonstrating that alamethicin has a minimal effect on the overall bilayer thickness. However, a significant decrease in membrane resistivity was observed when alamethicin was inserted into the fBLM indicating that the peptides are forming ion conducting pores. A direct visualization of the alamethicin pores was obtained by molecular resolution AFM images revealing that the pores are not randomly dispersed throughout the bilayer, but instead form hexagonal aggregates. The diameter of an individual pore within the aggregates is equal to 2.3 ± 0.3 nm, which is consistent with the size of a hexameric pore predicted by molecular dynamics simulations. Additionally, the image revealed a broad size distribution of alamethicin aggregates, which explains the origin of multiple conductivity states observed for the incorporation of alamethicin into free standing bilayer lipid membranes.