A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly

The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca(2+)-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.     

Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.     

Munc18-bound syntaxin readily forms SNARE complexes with synaptobrevin in native plasma membranes

Munc18–1, a protein essential for regulated exocytosis in neurons and neuroendocrine cells, belongs to the family of Sec1/Munc18-like (SM) proteins. In vitro, Munc18–1 forms a tight complex with the SNARE syntaxin 1, in which syntaxin is stabilized in a closed conformation. Since closed syntaxin is unable to interact with its partner SNAREs SNAP-25 and synaptobrevin as required for membrane fusion, it has hitherto not been possible to reconcile binding of Munc18–1 to syntaxin 1 with its biological function. We now show that in intact and exocytosis-competent lawns of plasma membrane, Munc18–1 forms a complex with syntaxin that allows formation of SNARE complexes. Munc18–1 associated with membrane-bound syntaxin 1 can be effectively displaced by adding recombinant synaptobrevin but not syntaxin 1 or SNAP-25. Displacement requires the presence of endogenous SNAP-25 since no displacement is observed when chromaffin cell membranes from SNAP-25–deficient mice are used. We conclude that Munc18–1 allows for the formation of a complex between syntaxin and SNAP-25 that serves as an acceptor for vesicle-bound synaptobrevin and that thus represents an intermediate in the pathway towards exocytosis.     

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.     

High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.     

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.     

Proteolysis of SNAP-25 Isoforms by Botulinum Neurotoxin Types A, C, and E

Abstract : Tetanus toxin and the seven serologically distinct botulinal neurotoxins (BoNT/A to BoNT/G) abrogate synaptic transmission at nerve endings through the action of their light chains (L chains), which proteolytically cleave VAMP (vesicle-associated membrane protein)/synaptobrevin, SNAP-25 (synaptosome-associated protein of 25 kDa), or syntaxin. BoNT/C was reported to proteolyze both syntaxin and SNAP-25. Here, we demonstrate that cleavage of SNAP-25 occurs between Arg¹⁹⁸ and Ala¹⁹⁹, depends on the presence of regions Asn⁹³ to Glu¹⁴⁵ and Ile¹⁵⁶ to Met²⁰², and requires about 1,000-fold higher L chain concentrations in comparison with BoNT/A and BoNT/E. Analyses of the BoNT/A and BoNT/E cleavage sites revealed that changes in the carboxyl-terminal residues, in contrast with changes in the amino-terminal residues, drastically impair proteolysis. A proteolytically inactive BoNT/A L chain mutant failed to bind to VAMP/synaptobrevin and syntaxin, but formed a stable complex ( K _D = 1.9 × 10^-7 M ) with SNAP-25. The minimal essential domain of SNAP-25 required for cleavage by BoNT/A involves the segment Met¹⁴⁶-Gln¹⁹⁷, and binding was optimal only with full-length SNAP-25. Proteolysis by BoNT/E required the presence of the domain Ile¹⁵⁶-Asp¹⁸⁶. Murine SNAP-23 was cleaved by BoNT/E and, to a reduced extent, by BoNT/A, whereas human SNAP-23 was resistant to all clostridial L chains. Lys¹⁸⁵Asp or Pro¹⁸²Arg mutations of human SNAP-23 induced susceptibility toward BoNT/E or toward both BoNT/A and BoNT/E, respectively.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+

BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.     

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t- SNAREs participate in SV recycling in what may be functionally distinct forms.     

A Coiled Coil Trigger Site Is Essential for Rapid Binding of Synaptobrevin to the SNARE Acceptor Complex

Exocytosis from synaptic vesicles is driven by stepwise formation of a tight α-helical complex between the fusing membranes. The complex is composed of the three SNAREs: synaptobrevin 2, SNAP-25, and syntaxin 1a. An important step in complex formation is fast binding of vesicular synaptobrevin to the preformed syntaxin 1·SNAP-25 dimer. Exactly how this step relates to neurotransmitter release is not well understood. Here, we combined different approaches to gain insights into this reaction. Using computational methods, we identified a stretch in synaptobrevin 2 that may function as a coiled coil “trigger site.” This site is also present in many synaptobrevin homologs functioning in other trafficking steps. Point mutations in this stretch inhibited binding to the syntaxin 1·SNAP-25 dimer and slowed fusion of liposomes. Moreover, the point mutations severely inhibited secretion from chromaffin cells. Altogether, this demonstrates that the trigger site in synaptobrevin is crucial for productive SNARE zippering.     

Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling

SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. We found that the soluble domains of synaptobrevin, SNAP-25, and syntaxin 1 are unstructured. At higher concentrations, the SNARE motif of syntaxin 1 forms homooligomeric helical bundles with at least some of the alpha-helices aligned in parallel. In the assembled SNARE complex, mapping of thirty side chain positions yielded spectra which are in good agreement with the recently published crystal structure. The loop region of SNAP-25 that connects the two SNARE motifs is largely unstructured. C-terminal truncation of synaptobrevin resulted in complexes that are completely folded N-terminal of the truncation but become unstructured at the C-terminal end. The binary complex of syntaxin and SNAP-25 consists of a parallel four helix-bundle with properties resembling that of the ternary complex.     

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.     

Clostridial neurotoxins: new tools for dissecting exocytosis

Tetanus toxin and botulinal toxins are potent inhibitors of neuronal exocytosis. Within the past five years the protein sequences of all eight neurotoxins have been determined, their mode of action as metalloproteases has been established, and their intraneuronal targets have been identified. The toxins act by selectively proteolysing the synaptic vesicle protein synaptobrevin (VAMP) or the presynaptic membrane proteins syntaxin (HPC-1) and SNAP-25. These three proteins form the core of a complex that mediates fusion of carrier vesicles to target membranes. Tetanus and botulinal neurotoxins could serve in the future as tools to study membrane trafficking events, or even higher brain functions such as behaviour and learning.     

Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions

Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and thus may regulate SNARE function. Using purified proteins, we have performed a detailed analysis of the structure of complexin and of its interaction with SNARE proteins. NMR spectroscopy revealed that isolated complexins have no tertiary structure but contain an unusual alpha-helical middle domain of approximately 58 amino acids that overlaps with the most highly conserved region of the molecules. Complexins form a stable stoichiometric complex with the central domain of the ternary SNARE complex, whereas no binding was observed to monomeric SNAREs. Using a combination of limited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substitution of SNAP-25. In addition, a stretch of 12 amino acids in the middle of the SNARE motif of syntaxin 1A was able to confer binding activity to the non-binding relative syntaxin 4. Furthermore, disassembly of ternary complexes is not affected by complexins. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central alpha-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. Complexins may regulate the function of ternary complexes and control membrane fusion through this interaction.     

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.     

A molecular basis underlying differences in the toxicity of botulinum serotypes A and E

Botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of the proteins responsible for vesicle exocytosis. Paradoxically, two serotypes of BoNTs, A and E, cleave the same molecule, synaptosome-associated protein with relative molecular mass 25K (SNAP-25), and yet they cause synaptic blockade with very different properties. Here we compared the action of BoNTs A and E on the plasma membrane fusion machinery composed of syntaxin and SNAP-25. We now show that the BoNT/A-cleaved SNAP-25 maintains its association with two syntaxin isoforms in vitro, which is mirrored by retention of SNAP-25 on the plasma membrane in vivo. In contrast, BoNT/E severely compromises the ability of SNAP-25 to bind the plasma membrane syntaxin isoforms, leading to dissociation of SNAP-25. The distinct properties of botulinum intoxication, therefore, can result from the ability of shortened SNAP-25 to maintain its association with syntaxins-in the case of BoNT/A poisoning resulting in unproductive syntaxin/SNAP-25 complexes that impede vesicle exocytosis.     

SNAP-25/syntaxin 1A complex functionally modulates neurotransmitter gamma-aminobutyric acid reuptake

Neurotransmitter gamma-aminobutyric acid (GABA) release to the synaptic clefts is mediated by the formation of a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which includes two target SNAREs syntaxin 1A and SNAP-25 and one vesicle SNARE VAMP-2. The target SNAREs syntaxin 1A and SNAP-25 form a heterodimer, the putative intermediate of the SNARE complex. Neurotransmitter GABA clearance from synaptic clefts is carried out by the reuptake function of its transporters to terminate the postsynaptic signaling. Syntaxin 1A directly binds to the neuronal GABA transporter GAT-1 and inhibits its reuptake function. However, whether other SNARE proteins or SNARE complex regulates GABA reuptake remains unknown. Here we demonstrate that SNAP-25 efficiently inhibits GAT-1 reuptake function in the presence of syntaxin 1A. This inhibition depends on SNAP-25/syntaxin 1A complex formation. The H3 domain of syntaxin 1A is identified as the binding sites for both SNAP-25 and GAT-1. SNAP-25 binding to syntaxin 1A greatly potentiates the physical interaction of syntaxin 1A with GAT-1 and significantly enhances the syntaxin 1A-mediated inhibition of GAT-1 reuptake function. Furthermore, nitric oxide, which promotes SNAP-25 binding to syntaxin 1A to form the SNARE complex, also potentiates the interaction of syntaxin 1A with GAT-1 and suppresses GABA reuptake by GAT-1. Thus our findings delineate a further molecular mechanism for the regulation of GABA reuptake by a target SNARE complex and suggest a direct coordination between GABA release and reuptake.     

Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.