VAMP‐2 and cellubrevin are expressed in pancreatic beta‐cells and are essential for Ca(2+)‐but not for GTP gamma S‐induced insulin secretion

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta‐cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP‐2 and cellubrevin, but not VAMP‐1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta‐cells contain secretory granules that store and secrete insulin as well as synaptic‐like microvesicles carrying gamma‐aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP‐2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP‐2 with insulin‐containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta‐cells. Pretreatment of streptolysin‐O permeabilized insulin‐secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP‐2 and cellubrevin and abolished Ca(2+)‐induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S‐stimulated insulin secretion. Taken together, our results show that pancreatic beta‐cells express VAMP‐2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)‐mediated insulin secretion.