Studies on the mechanism of membrane fusion: evidence for an intermembrane calcium(2+) ion-phospholipid complex, synergism with magnesium(2+) ion, and inhibition by spectrin

Abstract

The interaction of Ca2+ and Mg2+ with phosphatidylserine (PS) vesicles in 0.1 M NaCl aqueous solution was studied by equilibrium dialysis binding, X-ray diffraction, batch microcalorimetry, kinetics of cation-induced vesicle aggregation, release of vesicle contents and fusion. Addition of either cation causes aggregation of PS vesicles and produces complexes with similar stoichiometry (1:2 cation/PS) at saturating concentrations, although the details of the interactions and the resulting complexes are quite different. Addition of Ca2+ to PS vesicles at T (temperature) .gtoreq. 25.degree. C induces the formation of an anhydrous complex of closely apposed membranes with highly ordered crystalline acyl chains and a very high transition temperature (Tc (transition temperature) > 100.degree. C). The formation of this complex is accompanied by a release of heat (5.5 kcal/mol), rapid release of vesicle contents and fusion of the vesicles into larger membranous structures. Addition of Mg2+ produces a complex with PS which is much more hydrated, has no crystallization of the acyl chains at T .gtoreq. 20.degree. C, and has comparatively little fusion. Studies with both Ca2+ and Mg2+ added simultaneously indicate that there is a synergistic effect between the 2 cations, which results in an enhancement of the ability of Ca2+ to form its specific complex with PS at lower concentrations. The presence of the [human] erythrocyte protein spectrin inhibits this synergism and interferes with the formation of the specific PS/Ca complex. It also inhibits the fusion of PS vesicles. The unique PS/Ca complex, which involves close apposition of vesicle membranes, may be an intermembrane trans complex. Such a complex may be a key step for the resultant phase transition and fusion of PS vesicles. By contrasts, the PS/Mg complex is proposed to be a cis complex with respect to each membrane. The results are discussed in terms of the mechanism of membrane fusion.