CALCIUM-DEPENDENCE OF SYNEXIN BINDING MAY DETERMINE AGGREGATION AND FUSION OF LAMELLAR BODIES

Synexin (annexin VII) is a member of the annexin family of calcium and phospholipid binding proteins that promote calcium-dependent aggregat ion and fusion of Lipid vesicles or secretory granules. We have previo usly suggested that synexin may be involved in membrane fusion process es during exocytosis of lung surfactant since it promotes fusion in vi tro of lamellar bodies with plasma membranes. In this study, we charac terized calcium-dependency of synexin binding to lamellar bodies and p lasma membranes, since such binding is the initial, and, therefore, ma y be the rate-limiting step in membrane aggregation and fusion. The bi nding of biotinylated synexin to lamellar bodies and plasma membranes increased in a calcium-dependent manner reaching a maximum at approx. 200 mu M Ca2+. Binding to lamellar bodies was completely inhibited by unlabelled synexin. Gel-overlay analysis showed that synexin bound to an approx. 76 kDa protein in the lamellar body and plasma membrane fra ctions. The calcium kinetics were noticeably similar for synexin bindi ng to lamellar bodies and plasma membranes, aggregation of lamellar bo dies, and fusion of lamellar bodies with lipid vesicles. At low calciu m concentrations, aggregation of lamellar bodies could be increased wi th increasing synexin concentration, and arachidonic acid increased al l three parameters (binding, aggregation, and fusion) in a similar man ner. The effects of calcium and arachidonic acid on these three parame ters suggest that synexin binding to lamellar bodies may be a rate-det ermining step for fusion during surfactant secretion. Furthermore, at near physiological calcium levels, the membrane fusion may be enhanced by elevated concentrations of synexin and polyunsaturated fatty acids .