SUMMARY At a synapse, fast synchronous neurotransmitter release requires localization of Ca2+-channels to presynaptic active zones. How Ca2+-channels are recruited to active zones, however, remains unknown. Using unbiased yeast two-hybrid screens, we here identify a direct interaction of the central PDZ-domain of the active-zone protein RIM with the C-termini of presynaptic N- and P/Q-type Ca2+-channels, but not L-type Ca2+-channels. To test the physiological significance of this interaction, we generated conditional knockout mice lacking all presynaptic RIM isoforms. Deletion of all RIMs ablated most neurotransmitter release by simultaneously impairing the priming of synaptic vesicles and by decreasing the presynaptic localization of Ca2+-channels. Strikingly, rescue of the decreased Ca2+-channel localization required the RIM PDZ-domain, whereas rescue of vesicle priming required the RIM N-terminus. We propose that RIMs tether N- and P/Q-type Ca2+-channels to presynaptic active zones via a direct PDZ-domain mediated interaction, thereby enabling fast, synchronous triggering of neurotransmitter release at a synapse.

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H abc domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.exocytosis ͉ membrane fusion ͉ neurotransmitter release ͉ Sec1/Munc18-like proteins ͉ synapse E very eukaryotic cell relies on the precise and regulated trafficking of proteins, membranes, and other types of cargo between cellular compartments. The molecular machinery responsible for membrane traffic is partly conserved to provide for basic membrane fusion and fission reactions and is partly cell type-and compartment-specific to meet the unique requirements of a particular cellular locale (1). Neurons have arguably the most complex membrane trafficking of all cells, primarily because synaptic transmission requires continuous (but at the same time tightly regulated) synaptic membrane traffic. At a synapse, neurotransmitter release is effected by fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptic vesicle fusion requires two conserved protein families that are universally involved in membrane fusion reactions: SNARE proteins, which are thought to pull membranes together by forming tight ''SNARE complexes,'' and Sec1/Munc18-like proteins (SM proteins), which perform an unknown but essential role in fusion and interact with SNARE proteins (reviewed in refs. 2-4).Synaptic vesicle exocytosis involves one SM protein (Munc18-1) and three SNARE proteins (synaptobrevin/VAMP on synaptic vesicles, and SNAP-25 and syntaxin-1A/1B on the plasma membrane; syntaxin-1A and -1B are highly homologous and thought to have identical functions). All SNARE proteins contain a conserved 60-to 70-residue sequence, the SNARE motif, that assembles into a four-helical bundle during SNARE complex formation (5). Synaptobrevin/VAMP and SNAP-25 are ''minimal SNARE proteins'' that are composed of one (synaptobrevin) or two (SNAP-25) SNARE motifs and a membraneanchor sequence. In syntaxin-1, however, the SNARE motif and transmembrane region occupy less than half of the sequence, with the...

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.