During synaptic vesicle fusion, the SNARE-protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a ‘closed’ conformation outside the SNARE-complex, and an ‘open’ conformation in the SNARE-complex. Whereas SNARE-complexes containing ‘open’ syntaxin-1 and Munc18-1 are essential for exocytosis, the significance of ‘closed’ syntaxin-1 is unknown. Here, we generated knockin/knockout mice that expressed only ‘open’ syntaxin-1B. Syntaxin-1BOpen mice were viable, but succumbed to generalized seizures at 2-3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1BOpen synapses, and the size of the readily-releasable vesicle pool was decreased, whereas the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies.

Ca 2؉-dependent phospholipid binding to the C 2 A and C 2 B domains of synaptotagmin 1 is thought to trigger fast neurotransmitter release, but only Ca 2؉ binding to the C 2 B domain is essential for release. To investigate the underlying mechanism, we have compared the role of basic residues in Ca The synaptic vesicle protein synaptotagmin 1 acts as a major Ca 2ϩ sensor in neurotransmitter release at excitatory and inhibitory synapses (1, 2). This function can be attributed to Ca 2ϩ binding to the two C 2 domains of synaptotagmin 1 (referred to as the C 2 A and C 2 B domain; Ref.3). The C 2 A and C 2 B domains bind three and two Ca 2ϩ ions, respectively, through loops located at the tips of similar ␤-sandwich structures (4 -7). Both C 2 domains bind to negatively charged phospholipids, including phosphoinositides, as a function of Ca 2ϩ , and exhibit comparable apparent Ca 2ϩ affinities (7-9). Furthermore, in the absence of Ca 2ϩ , the C 2 B domain, but not the C 2 A domain, of synaptotagmin 1 avidly binds to inositolpolyphosphates (such as inositol 1,3,4,5-tetrakisphosphate) and to phosphoinositides (such as phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 2 ) via a polybasic sequence that is located in a ␤-strand on the side of the domain (10, 11). Moreover, the C 2 domains interact Ca 2ϩ -dependently and -independently with individual SNARE proteins such as syntaxin1 and SNAP-25 and with SNARE complexes (12-17). Finally, the synaptotagmin C 2 domains engage in additional interactions in vitro, including binding of the clathrin adaptor protein complex and Ca 2ϩ channels (21-23). Although the biochemical properties of synaptotagmin 1 have been studied in detail, the functional importance of individual properties has remained unclear. Ca 2ϩ -dependent phospholipid binding by synaptotagmin 1 in vitro correlates with its functional role in Ca 2ϩ triggering of release in vivo, as demonstrated with both loss-of-function and gain-offunction mutations (1,24). This correlation suggests that Ca 2ϩ -dependent phospholipid binding represents a crucial step in synaptotagmin 1 function. However, mutational studies revealed that although both C 2 domains of synaptotagmin 1 are involved in Ca 2ϩ -triggered release, Ca 2ϩ binding to the C 2 A domain only boosts release, whereas Ca 2ϩ binding to the C 2 B domain is essential for synchronous release (1, 25-29). Thus, it is puzzling that the two C 2 domains of synaptotagmin 1 appear to exhibit similar Ca 2ϩ -dependent phospholipid binding properties in vitro but a striking functional asymmetry in vivo. The differential requirements of the C 2 A versus C 2 B domain for the Ca 2ϩ triggering of release could potentially arise from the unique ability of the C 2 B domain (but not the C 2 A domain) to bind to phosphoinositides in a Ca 2ϩ -independent manner (10, 11). Indeed, consistent with this idea, microinjection of soluble inositol polyphosphates into nerve terminals potently inhibits release (30). Two observations, however, argue against this interpretation. First, the C 2 B domai...