MODULATION OF MEMBRANE-FUSION BY ASYMMETRIC TRANSBILAYER DISTRIBUTIONS OF AMINO LIPIDS

The fusion of model lipid bilayers containing synthetic amino lipids a nd the regulation of this fusion by inducing transbilayer asymmetry of these amino lipids via imposed pH gradients are demonstrated. Liposom es of 100 nm diameter consisting of 5 mol % 1,2-dioleoyl-3-(N,N-dimeth ylamino)propane (AL1) in a mixture of egg phosphatidylcholine (EPC), d ioleoylphosphatidylethanolamine (DOPE), and cholesterol in a ratio of 35:20:45 do not fuse at pH 4.0. Fusion also is not observed upon incre asing the external pH of these vesicles to 7.5, which results in the r apid transport of All to the inner monolayer, as measured by a fluores cent probe sensitive to surface charge. However, dissipation of the im posed pH gradient leads to redistribution of AL1 to the outer monolaye r at pH 7.5 and causes liposomal fusion, as detected by fluorescent li pid-mixing assay and freeze-fracture electron microscopy. The effect o f varying the hydrocarbon structure of AL1 on the rate of fusion is de monstrated with five synthetic analogues, AL2-AL6. Higher rates of fus ion occur with lipids containing longer unsaturated acyl chains and wi th lower values of pK(a) for the membrane-bound amino lipids. Fusion i s also associated with destabilization of the bilayer at pH 7.5, as in dicated by the formation of the hexagonal H-II phase.