Distinct effects of α-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis

Abstract

We have used stage-specific assays for MgATP-dependent priming and for Ca2+-activated triggering in the absence of free MgATP to examine the effects of α-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca2+- dependent increase in catecholamine secretion. Both α-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of α- SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca2+-dependency, time course and extent of stimulation and their effects were additive. α-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that α-SNAP acts in an early MgATP-requiring stage and not in the late Ca2+- triggered steps immediately prior to membrane fusion as previously suggested.