The phase separation in lipid bilayers that include negatively charged lipids is examined experimentally. We observed phase-separated structures and determined the membrane miscibility temperatures in several binary and ternary lipid mixtures of unsaturated neutral lipid, dioleoylphosphatidylcholine (DOPC), saturated neutral lipid, dipalmitoylphosphatidylcholine (DPPC), unsaturated charged lipid, dioleoylphosphatidylglycerol (DOPG (-) ), saturated charged lipid, dipalmitoylphosphatidylglycerol (DPPG (-) ), and cholesterol. In binary mixtures of saturated and unsaturated charged lipids, the combination of the charged head with the saturation of hydrocarbon tail is a dominant factor for the stability of membrane phase separation. DPPG (-) enhances phase separation, while DOPG (-) suppresses it. Furthermore, the addition of DPPG (-) to a binary mixture of DPPC/cholesterol induces phase separation between DPPG (-) -rich and cholesterol-rich phases. This indicates that cholesterol localization depends strongly on the electric charge on the hydrophilic head group rather than on the ordering of the hydrocarbon tails. Finally, when DPPG (-) was added to a neutral ternary system of DOPC/DPPC/Cholesterol (a conventional model of membrane rafts), a three-phase coexistence was produced. We conclude by discussing some qualitative features of the phase behaviour in charged membranes using a free energy approach.

We investigated the effect of charge on the membrane morphology of giant unilamellar vesicles (GUVs) composed of various mixtures containing charged lipids. We observed the membrane morphologies by fluorescent and confocal laser microscopy in lipid mixtures consisting of a neutral unsaturated lipid [dioleoylphosphatidylcholine (DOPC)], a neutral saturated lipid [dipalmitoylphosphatidylcholine (DPPC)], a charged unsaturated lipid [dioleoylphosphatidylglycerol (DOPG (-) )], a charged saturated lipid[dipalmitoylphosphatidylglycerol (DPPG (-) )], and cholesterol (Chol). In binary mixtures of neutral DOPC/DPPC and charged DOPC/DPPG (-) , spherical vesicles were formed. On the other hand, pore formation was often observed with GUVs consisting of DOPG (-) and DPPC.In a DPPC/DPPG (-) /Chol ternary mixture, pore-formed vesicles were also frequently observed. The percentage of pore-formed vesicles increased with the DPPG (-) concentration.Moreover, when the head group charges of charged lipids were screened by the addition of salt, pore-formed vesicles were suppressed in both the binary and ternary charged lipid mixtures. We discuss the mechanisms of pore formation in charged lipid mixtures and the relationship between phase separation and the membrane morphology. Finally, we reproduce the results seen in experimental systems by using coarse-grained molecular dynamics simulations. Ⅰ. INTRODUCTIONThe basic structure of a biomembrane is a lipid bilayer that is composed of various types of phospholipids. Biomembranes not only separate the inner and outer environments of living cells, but also play a role in a wide range of life-related phenomena through dynamic structural changes. In biomembranes, the components are not uniformly dispersed, and it is believed that such compositional heterogeneity emerges spontaneously. This heterogeneous structure is known as a "lipid raft" [1][2][3]. Lipid rafts, which are enriched with saturated lipids, cholesterol, or various membrane proteins, are expected to function as platforms to which proteins are attached during signal transduction and membrane trafficking [4,5].Synthetic lipid vesicles consisting of several lipid molecules are commonly used as models of biomembranes to investigate the physicochemical properties of lipid membranes. In particular, ternary lipid mixtures consisting of a saturated lipid, unsaturated lipid, and cholesterol exhibit phase separation between the saturated lipid and the cholesterol-rich phase (the liquid-ordered (Lo) phase) and the unsaturated lipid-rich phase (the liquid-disordered (Ld) phase) [6,7]. The spontaneous domain formation that results from this phase separation has attracted great attention in connection to raft formation in biomembranes.Most previous studies have investigated the primary physical properties of lipid membranes composed of electrically neutral lipids [6,8]. However, biomembranes also contain negatively charged lipids. For instance, the membranes of prokaryotes such asStaphylococcus aureus and Escherichia co...

We propose a model describing the phase behavior of two-component membranes consisting of binary mixtures of electrically charged and neutral lipids. We take into account the structural phase transition (main-transition) of the hydrocarbon chains, and investigate the interplay between this phase transition and the lateral phase separation. The presence of charged lipids significantly affects the phase behavior of the multicomponent membrane. Due to the conservation of lipid molecular volume, the main-transition temperature of charged lipids is lower than that of neutral ones. Furthermore, as compared with binary mixtures of neutral lipids, the membrane phase separation in binary mixtures of charged lipids is suppressed, in accord with recent experiments. We distinguish between two types of charged membranes: mixtures of charged saturated lipid/neutral unsaturated lipid and a second case of mixtures of neutral saturated lipid/charged unsaturated lipid. The corresponding phase behavior is calculated and shown to be very different. Finally, we discuss the effect of added salt on the phase separation and the temperature dependence of the lipid molecular area.

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