Probing the Dynamic Pore Formation of Antimicrobial Peptide in Nanoscale Lipid Membranes

Antimicrobial peptides (AMPs), key components of innate immunity, kill bacteria by forming structured pores on their lipid membranes. Understanding how AMPs form pores is of fundamental interest and holds significant promise for various biomedical and biotechnological applications. However, the underlying molecular mechanism of pore formation remains elusive, and directly observing the dynamic pore formation process poses significant measurement challenges. Here, we report a method to probe the dynamic pore formation process driven by AMPs. This approach involves forming a stable suspended artificial planar phospholipid bilayer at the orifice of a glass nanopipette and monitoring the leakage ionic current across the membrane. By using nanoscale-sized membranes, we enhanced their stability, enabling longer time-resolved ionic current measurements with a high signal-to-noise ratio. We also added and optimized the cholesterol content in the diphytanoylphosphatidyl choline (DPhPC) bilayer to 20 mol %, which improved both the yield and stability of the membrane. We first validated this method by investigating voltage-controlled electroporation and subsequently applied it to study the pore-forming process of a model AMP, alamethicin. The interaction between alamethicin and the membrane, as well as its pore formation process, could be effectively controlled by the applied voltage and repeatedly probed. Analysis of the rich single-channel conductance changes revealed the alamethicin pore formation process in detail. The pore formation dynamics and the resulting pore structures were found to be sensitive to the applied voltage, alamethicin concentration, lipid composition, ionic strength, and cation size.