Kinetics of a Ca-2+-triggered membrane aggregation reaction of phospholipid membranes

Ca-2+ and other divalent cations can trigger aggregation of phospholipid vesicles containing phosphatidic acid or phosphatidylserine. The reaction, which can be detected by an increase in light scattering, has a critical dependence on the Ca-2+ concentration, with a threshold near 4 mM Ca-2+. This is the concentration for half-saturation of the polar head groups and for full neutralization of the membrane surface charge. The aggregation proceeds as a "polymerization" reaction, eventually forming such large aggregates that the vesicles precipitate. The stopped-flow rapid mixing technique was used to study the vesicle dimerization reaction which is the first step in the overall aggregation process. Vesicle dimerization resulted in a doubling of light scattering and had a vesicle concentration-dependent time constant (t1/2) which varied between 0.4 and 2.0s under the conditions of the study. Analysis of the dependence of the reaction amplitude and l/t 1/2 on the concentrations of vesicles and Ca-2+ showed that the Ca-2+ binding is fast, and that the dimerization proceeds by a mechanism in which the vesicles first collide to form an encounter complex followed by a slower conversion of the encounter complex to a stable complex. For phosphatidic acid vesicles, about 200-700 collisions are necessary to achieve a stable dimer. The rate-limiting step in the overall reaction is thus the transformation of the encounter complex into a stable complex, requiring 0.5 and 1.0 ms. The above-mentioned results are relatively insensitive insensitive to the type of divalent cation or to the choice of negatively charged lipid (phosphatidic acid or phosphatidylserine). Evidence is given that the stable complex is effected by Ca-2+-mediated salt bridges between the two membranes and that the rate constant of the transformation step derives from the statistics of the distribution and the rate of redistribution of Ca-2+-occupied polar head groups on the membrane surfaces. The relevance of these results to the problem of Ca-2+-induced fusion of biological membranes is discussed.