Ca²⁺-induced fusion of large unilamellar phosphatidylserine/cholesterol vesicles

The effect of cholesterol on the Ca²⁺-induced aggregation and fusion of large unilamellar phosphatidylserine (PS) vesicles has been investigated. Mixing of aqueous vesicle contents was followed continuously with the Tb/dipicolinate assay, while the dissociation of pre-encapsulated Tb/dipicolinate complex was taken as a measure of the release of vesicle contents. Vesicles consisting of pure PS or PS/cholesterol mixtures at molar ratios of 4:1, 2:1 and 1:1 were employed at three different lipid concentrations, each at four different Ca²⁺ concentrations. The results could be well simulated in terms of a mass-action kinetic model, providing separately the rate constants of vesicle aggregation, c₁₁, and of the fusion reaction itself, f₁₁. In the analyses the possibility of deaggregation of aggregated vesicles was considered explicitly. Values of both c₁₁ and f₁₁ increase steeply with the Ca²⁺ concentration increasing from 2 to 5 mM. With increasing cholesterol content of the vesicles the value of c₁₁ decreases, while the rate of the actual fusion reaction, f₁₁, increases. Remarkably, the effect of cholesterol on both aggregation and fusion is quite moderate. The presence of cholesterol in the vesicle bilayer does not affect the leakage of vesicle contents during fusion.