Ca2+ stop-induced phase separation in the membrane of palmitate-containing liposomes and its possible relation to membrane permeabilization

A Ca2+-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca2+, fluorescence decreased, which indicated separation of PA/Ca2+ complexes into distinct membrane domains. The Ca2+-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca2+ in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca2+-induced membrane permeabilization relates to the initial stage of Ca2+-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.