  We investigated the phase separation of dioleoylphosphatidylserine (DOPS) and
dipalmitoylphosphatidylcholine (DPPC) in giant unilamellar vesicles in
hypotonic solution using fluorescence and confocal laser scanning microscopy.
Although phase separation in charged lipid membranes is generally suppressed by
the electrostatic repulsion between the charged headgroups, osmotic stress can
promote the formation of charged lipid domains. Interestingly, we observed
three-phase coexistence even in DOPS/DPPC binary lipid mixtures. The three
phases were DPPC-rich, dissociated DOPS-rich, and nondissociated DOPS-rich
phases. The two forms of DOPS were found to coexist owing to the ionization of
the DOPS headgroup, such that the system could be regarded as quasi-ternary.
The three formed phases with differently ionized DOPS domains were successfully
identified experimentally by monitoring the adsorption of positively charged
particles. In addition, coarse-grained molecular dynamics simulations confirmed
the stability of the three-phase coexistence. Attraction mediated by hydrogen
bonding between protonated DOPS molecules and reduction of the electrostatic
interactions at the domain boundaries stabilized the three-phase coexistence.
