Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting ␤-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca 2؉ -induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary ␤-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca 2؉ -dependent insulin secretion from primary ␤-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.Exocytosis of insulin from pancreatic ␤-cells has been suggested to be mediated by the same core fusion machinery that controls all membrane fusion events in organisms ranging from yeast to human (1-3). The soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) 1 proteins are essential components of this machinery. Proteins localized in the membrane of the transported vesicle (v-SNAREs) specifically interact with proteins in the target membrane (t-SNAREs). In neurotransmitter release from synaptic vesicles, the plasma membrane-associated proteins syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) interact with the vesicular component synaptobrevin/vesicular-associated membrane protein (VAMP) (reviewed in Refs. 4 -6). Several synaptic proteins regulating neuronal exocytosis including syntaxin, SNAP-25, VAMP, and Munc18-1 have also been identified in pancreatic ␤-cells, supporting the idea that the molecular machinery regulating insulin secretion is similar to that of neurotransmitter release from synaptic vesicles (7-11).There are a series of discrete biochemical steps leading to trans-SNARE complex formation and vesicular fusion. The vesicles need to be transported and targeted to the cell surface where they are docked, primed, and finally fused with the plasma membrane. Munc18-1, a member of the sec1/Munc18 protein family has emerged as a critical regulator of exocytosis (12)(13)(14). Indeed, Munc18-1 is shown to be important for vesicle trafficking and essential for synaptic transmission since both synaptic transmission and spontaneous neurotransmitter release is abolished in neocortical neurons from Munc18-1-null mouse mutants (15). Albeit essential for regulated exocytosis, it has been demonstrated both in pancreatic ␤-cells and in neuronal systems, that Munc18-1 may also serve as a negative regulator of secretion (11,16,17). Munc18-1 binds to syntaxin 1 and might thus negatively regulate syntaxin 1 function if the expressi...

E xocytosis is delicately regulated via dynamic protein-protein interactions between different protein components localized to the plasma membrane, the secretory vesicle membrane, and the cytoplasm. According to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) hypothesis (1,2), the vesicular-SNARE vesicleassociated membrane protein (also called synaptobrevin) interacts with the cognate target-SNAREs syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) to form a core complex (also called SNARE complex) (1). The assembly of SNARE proteins between two opposing membranes and the formation of a core complex have been shown to be the key events that initiate membrane fusion and predict the specificity of vesicle fusion (1,2). That the compartmental specificity of cellular membrane fusion is encoded in SNARE proteins is further provided by the observation that these proteins have distinct localization in a cell (3). However, almost any combination of several members of vesicular-and target-SNARE proteins can form a SDS-resistant protein complex (4,5), suggesting that the interactions between SNARE proteins cannot provide all information for vesicle targeting. Additional specificity may be provided by other molecules that interact with SNARE proteins. An example of such a protein is the well-conserved syntaxin-binding protein Sec1/mammalian homolog of the Caenorhabditis elegans unc-18 gene (Munc-18). There are several Munc-18 isoforms in mammals, which are believed to support different vesicular trafficking events (rev. in 6). Munc-18-1 holds syntaxin in a closed conformation, thereby preventing the binding of SNAP-25 and vesicle-associated membrane protein to syntaxin (7). Moreover, each Munc-18 protein interacts more or less exclusively with one or two syntaxin isoforms, thereby providing further vesicle-targeting specificity (8 -13).The existence of another syntaxin-binding protein, designated tomosyn (tomo ϭ friend in Japanese, syn ϭ syntaxin), has been reported (14). Besides the original tomosyn protein, which has been named m-tomosyn, two further splice variants of tomosyn, designated big (b) and small (s) tomosyn, have been identified (15). The m-and s-tomosyn variants are mainly expressed in the brain, whereas b-tomosyn is found ubiquitously (15). More recently, two distinct genes that drive the expression of seven tomosyn isoforms in the mammalian brain have been described (16). Tomosyn is capable of dissociating Munc-18 from syntaxin 1 and thereby forming a novel complex with syntaxin 1, and synaptotagmin (14). The COOH-terminal domain of tomosyn spans a SNARE motif that allows tomosyn to form a stable complex with syntaxin 1A and SNAP-25 (17,18). Endogenous expression or overexpression of tomosyn has been shown to cause a reduction of Ca 2ϩ -dependent exocytosis (14,19 -23). The structural basis for the inhibitory role of tomosyn in exocytosis has recently been presented (24).In this study, we have investigated isoform expression and cellular localization of tomo...