Pores formed by Baxα5 relax to a smaller size and keep at equilibrium.

Pores made by amphipathic cationic peptides (e.g., antimicrobials and fragments of pore-forming proteins) are typically studied by examining the kinetics of vesicle leakage after peptide addition or obtaining structural measurements in reconstituted peptide-lipid systems. In the first case, the pores have been considered transient phenomena that allow the relaxation of the peptide-membrane system. In the second, they correspond to equilibrium structures at minimum free energy. Here we reconcile both approaches by investigating the pore activity of the α5 fragment from the proapoptotic protein Bax (Baxα5) before and after equilibrium of peptide/vesicle complexes. Quenching assays on suspensions of large unilamellar vesicles suggest that in the presence of Baxα5, the vesicles maintain a leaky state for hours under equilibrium conditions. We proved and analyzed stable pores on single giant unilamellar vesicles (GUVs) in detail by monitoring the entrance of dyes added at different times after incubation with the peptide. When the GUVs came in contact with Baxα5, leakage started stochastically, was delayed for various periods of time, and in the majority of cases proceeded rapidly to completion. After hours in the presence of the peptide, the same individual GUVs that refilled completely at first instance maintained a porated state, which could be observed in subsequent leak-in events for serially added dyes. However, these long-term pores were smaller in size than the initial equilibration pores. Stable pores were also detected in GUVs made in the presence of Baxα5. The latter pores can be considered equilibrium states and may correspond to structures measured previously in bilayer stacks. Although pore formation may occur as a kinetic process, equilibrium pores may also be functionally relevant structures, especially in highly regulated systems such as the apoptotic mitochondrial pores induced by Bax.