Lung lamellar bodies are distinct organelles that are enclosed by a limiting membrane and maintain an ATP-dependent pH gradient (inside acidic). Their major physiologic function is the storage and secretion of alveolar surfactant. The importance of surfactant secretion is underscored by the fact that it is an integral part of the lung surfactant metabolism that operates to tightly regulate the alveolar pool of surfactant. Moreover, it is the only means to acutely upregulate the alveolar pool of functionally active surfactant. The mechanisms of membrane fusion between the lamellar bodies and plasma membrane that must occur during surfactant secretion has remained uninvestigated. Our studies show that lung synexin (annexin VII), a member of annexins family of proteins, may have a role in membrane fusion during surfactant secretion. First, synexin specifically binds to an approximate 76 kDa protein in the lamellar bodies and plasma membrane fractions and promotes in vitro fusion between these two fractions in a Ca2+-dependent manner, and increases surfactant secretion in permeabilized type II cells. Second, inhibitors of synexin activity inhibit surfactant secretion. Finally, secretagogue treatment of type II cells increase in vitro binding of synexin to membranes. We now propose to obtain further evidence for the role of synexin in surfactant secretion by following secretion in type II cells made synexin-deficient after cell-permeabilization, by introduction of synexin antibodies in intact type II cells, and by transfection of cells (in vitro or in vivo) with antisense oligodeoxynucleotides to synexin mRNA. We also propose that intracellular regulation of synexin function is achieved by two mechanisms. One, by release of lamellar body Ca2+ in response to changes in lamellar body pH, and two, by relocation of soluble synexin to the membranes that may occur with secretagogue-induced phosphorylation of membrane proteins. Finally, we will evaluate structural requirements of the amino terminus domain of synexin to understand the mechanisms of synexin action by comparing the properties of recombinant truncated and mutant synexins with the wild type annexin. This is now feasible as we have recently obtained complete cDNA sequence for rat synexin and because we can express recombinant synexin peptide. The proposed studies will establish a physiologic function for synexin in type II cells and provide insights in to the mechanism and regulation of membrane fusion during exocytosis of lung surfactant.
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