Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA

Abstract

The synthetic, amphipathic peptide GALA undergoes a pH-dependent conformational change and induces leakage of contents from large unilamellar phosphatidylcholine vesicles when in a helical conformation. The kinetics of this process have been investigated over a wide range of pH and lipid and peptide concentrations. Leakage from lipid vesicles is rapidly initiated (within 2 s) when the pH is lowered below 6 and is rapidly terminated when the pH is raised to 7.5. The leakage shows a selectivity to the size of the entrapped molecules and occurs by an all or none mechanism; vesicles either leak or retain all of their contents. Using this experimental data, we have developed a mathematical description of the kinetics of leakage induced by GALA. This model assumes that GALA becomes incorporated into the vesicle bilayer and aggregates to form a pore. Leakage occurs when a critical number of peptides assemble into a supramolecular aggregate in the bilayer. Leakage curves generated at lipid/peptide ratios ranging from 500/1 to 30000/1 can be well described by this formalism. On the basis of the results and the model, we suggest that GALA forms a transbilayer channel composed of 8-12 monomers. The channel diameter ranges from 5 to 10 A. To the best of our knowledge, this is the first model that can predict the leakage kinetics of solutes entrapped in lipid vesicles induced by a pore-forming peptide. The analysis may be of general use in defining the kinetics and state of aggregation of similarly acting peptides and proteins which form multimeric assemblies in membranes.

Interactions between human defensins and lipid bilayers: Evidence for formation of multimeric pores

“…Fujii et al (1993) have also observed that reduced human and rabbit defensins are more active than the native forms. The gen- era1 features of defensin-induced leakage described here have also been observed for melittin (Schwarz et al, 1992) and the channel-forming peptide GALA (Parente et al, 1990). …”

“…Even after more than 80% of the total entrapped ANTS has been released by native HNP-2, the encapsulated remainder is quenched by an amount indistinguishable from the initial value. All-or-none leakage of vesicle contents, which has been observed for melittin (Schwarz et al, 1992), magainin (Grant et al, 1992), GALA (Parente et al, 1990), and a-hemolysin (Ostolaza et al, 1993), is consistent with Fraction Released (f0J Fig. 11.…”

“…It could be a graded process, in which all of the vesicles release portions of their contents, or it could be all-or-none, in which some fraction of the vesicles lose either all of their contents or none. These 2 possibilities are usually distinguished by determining the level of quenching in vesicle fractions chromatographically separated from external fluorophor after peptide-induced leakage has occurred (Parente et al, 1990;Grant et al, 1992;Ostolazaet al, 1993). Alternately, thequenching can be determined more easily and accurately without separating free and entrapped fluorophor by measuring the overall quenching factor Qroral following the incremental addition of DPX quencher to the vesicle/peptide preparation after the fluorescence plateau (Fig.…”

“…The bacterial toxin a-hemolysin (Ostolaza et al, 1993) and the amphipathic peptide GALA (Parente et al, 1990) have also Table 1). …”

“…However, the a-hemolysin-induced leakage of FITC-dextrans was found to be independent of their molecular weight up to 17,200 Da, leading Ostolaza et al (1993) to conclude that ahemolysin probably causes all-or-none leakage through a detergent-like disruption of vesicular structure. The GALA peptide, on the other hand, was shown only to release molecules smaller than approximately 800 Da, leading Parente et al (1990) to conclude that GALA forms pores that are 10-20 A in diameter. Fujii et al (1993) reported that the human defensin HNP-I induced 20-50% fusion of sonicated vesicles of 3: 1 DPPC:DOPS.…”