Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.     

A dual role of SNAP‐25 as carrier and guardian of synaptic transmission

In this issue, Matteoli and colleagues show that SNAP-25 levels regulate the efficacy of presynaptic glutamate release and thereby alter short-term plasticity, with potential relevance for psychiatric diseases.EMBO reports(2013) 14 7, 645–651 doi:10.1038/embor.2013.75Control of exocytotic neurotransmitter release is essential for communication in the nervous system and for preventing synaptic abnormalities. The function of synaptosomal-associated protein of 25 kDa (SNAP-25) as a crucial component of the core machinery required for synaptic vesicle fusion is well established, but evidence is growing to suggest an additional modulatory role in neurotransmission. In this issue of EMBO reports, Antonucci et al show that the efficacy of evoked glutamate release is modulated by the expression levels of SNAP-25—a function that might relate to the ability of SNAP-25 to modulate voltage-gated calcium channels and presynaptic calcium ion concentration [1]. Altered synaptic transmission and short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disorders.Communication between neurons is essential for brain function and occurs through chemical neurotransmission at specialized cell–cell contacts termed ‘synapses''. Within the nerve terminal of the presynaptic neuron electrical stimuli cause the opening of voltage-gated calcium channels (VGCCs), which results in the influx of calcium ions. This triggers the exocytic release of neurotransmitter by fusion of synaptic vesicles with the presynaptic membrane. Released neurotransmitter molecules are detected by specific receptors expressed by the postsynaptic neuron.Calcium-induced synaptic vesicle fusion requires complex assembly between the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) synaptobrevin 2, located on the synaptic vesicle, and the abundant plasma membrane SNAREs SNAP-25 and syntaxin 1, on the opposing presynaptic plasma membrane. SNARE complex assembly is tightly regulated by Sec1/Munc18-like proteins [2]. Further regulatory factors such as the synaptic vesicle calcium-sensing protein synaptotagmin 1 couple the SNARE machinery to presynaptic calcium influx. SNARE-mediated neurotransmitter release occurs preferentially at the active zone—a presynaptic membrane domain specialized for exocytosis within which VGCCs are positioned close to docked synaptic vesicles through a proteinaceous cytomatrix and associated cell adhesion molecules [3,4].Altered short-term plasticity due to changes in SNAP-25 expression might have direct consequences for brain function and for the development of neuropsychiatric disordersAn unresolved conundrum in synaptic transmission remains—the observation that SNARE proteins, such as SNAP-25, are among the most highly expressed, in copy number, presynaptic proteins, whilst only a handful of SNARE complexes are needed to drive the fusion of a single synaptic vesicle [5]. Why, then, are SNAREs such as SNAP-25 so abundant? One possible explanation might be that SNARE proteins, in addition to forming trans-SNARE complexes, assemble with other proteins, and such partitioning might regulate neurotransmission. For example, SNAP-25 has been shown to negatively regulate VGCCs in glutamatergic but not in GABAergic neurons [6]. A secondary regulatory function of SNAP-25 is also supported by its genetic association with synaptic abnormalities such as schizophrenia and attention deficit hyperactivity disorder (ADHD) in humans [7]. SNAP-25 expression is reduced twofold in the hippocampus and frontal lobe from schizophrenic patients [8] and in animal models for ADHD [9]. Thus, SNAP-25 expression levels might crucially regulate normal synaptic function.A new study in this issue of EMBO reports by Antonucci and colleagues investigates the consequences of reduced SNAP-25 expression on synaptic function in SNAP-25^+/− heterozygous (Het) mutant mice. By using patch clamp electrophysiology, Antonucci et al revealed a selective enhancement of glutamatergic but not GABAergic neurotransmission as a result of reduced SNAP-25 expression. Several other parameters including the amplitude and frequency of miniature excitatory and inhibitory currents were unaffected. These data indicate that reduced levels of SNAP-25, an essential component of the fusion machinery, selectively enhance evoked release of glutamate whilst synaptic connectivity and postsynaptic glutamate receptor sensitivity remain unaltered. Further electrophysiological experiments in hippocampal neurons in culture showed that elevated glutamatergic transmission was probably due to increased release probability rather than changes in the number of fusion-prone, so-called ‘readily releasable synaptic vesicles''. This effect was occluded by pharmacologically induced calcium entry bypassing VGCCs, suggesting that altered calcium influx might underlie the differences in evoked glutamate release between wild-type and SNAP-25 Het neurons. As schizophrenia and ADHD are associated with changes in short-term plasticity, a paradigm reflecting presynaptic function, Antonucci et al analysed neurotransmission by paired-pulse stimulation—a protocol whereby two closely paired stimuli are applied within a 50 ms time interval. Wild-type neurons showed significant short-term facilitation, that is, a stronger response to the second stimulus as a result of increased calcium levels in the presynaptic compartment. By contrast, Het neurons had a reduced response to the second stimulus. Such paired-pulse depression is commonly viewed as a sign of increased release probability, which occurs when the first stimulus induces a partial depletion of release-ready synaptic vesicles during paired stimulation. As a consequence, the second stimulus evokes a comparably reduced response [3]. The switch from paired-pulse facilitation to depression was not fully reproduced in hippocampal slices from wild-type and Het mice, although facilitation seemed to be attenuated in SNAP-25 Het slices. One possible explanation for the apparent discrepancy between cultured neurons taken from newborn animals and acute slices from adult mice is the constant postnatal increase in SNAP-25 expression in SNAP-25 Het mice [10], which might partly counteract the defects caused by heterozygosity. Consistent with this explanation are data from rescue experiments by Antonucci et al, which showed that altered neurotransmission and defects in short-term plasticity in Het neurons can be gradually recovered in parallel with increased SNAP-25 expression. Moreover, cultured neurons show substantially higher levels of endogenous activity compared with acute slice preparations, leading to possible changes in the partitioning of SNAP-25 between SNARE complexes and association with VGCCs. Further experiments are clearly required to resolve these issues. Irrespective of these potential caveats, the combined data support the hypothesis that alterations in SNAP-25 expression underlie regulatory changes in neurotransmission, resulting in altered short-term plasticity and possibly disease.Many open questions remain. In particular, the precise mechanisms underlying elevated glutamatergic transmission and presynaptic plasticity under conditions of reduced SNAP-25 expression remain elusive. It has been shown before that free SNAP-25 inhibits Cav2.1-type VGCCs [6], an effect reversed by overexpression of synaptotagmin 1, which might associate with SNAP-25. Conversely, SNAP-25 occludes negative regulation of Cav2.2 VGCCs by free syntaxin 1 [3]. Hence, it is tempting to speculate that differential partitioning of SNAP-25 between free, SNARE-, synaptotagmin 1- and VGCC-complexed forms could regulate evoked neurotransmission (Fig 1). In this scenario, reduced SNAP-25 expression in Het animals and in schizophrenic and ADHD patients would be sufficient to sustain SNARE-mediated synaptic vesicle fusion but partially releases VGCCS from SNAP-25-mediated inhibition. This would result in elevated calcium influx and facilitated neurotransmission. Additional levels of regulation could be imposed by developmental switching between alternatively spliced ‘a'' and ‘b'' isoforms of SNAP-25 [11], age-dependent alterations in presynaptic protein turnover and post-translational modifications.Open in a separate windowFigure 1Effect of presynaptic SNAP-25 levels on calcium-induced glutamate release. Top: in wild-type (WT) neurons, SNARE-mediated calcium-triggered synaptic vesicle fusion is negatively regulated by complex formation between SNAP-25 and VGCCs. Bottom: reduced SNAP-25 expression in heterozygotes (Het;^+/−) partly releases VGCCs from SNAP-25-mediated clamping, resulting in elevated calcium influx through VGCCs and increased glutamate release through SNARE-mediated calcium-triggered synaptic vesicle fusion. Note that many key exocytotic proteins have been omitted for clarity. SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, soluble NSF attachment protein receptor; VGCC. voltage-gated calcium channel.Future studies need to address these possibilities, and their relationship to cognitive impairments and synaptic diseases, such as schizophrenia and ADHD.     

CSPα promotes SNARE-complex assembly by chaperoning SNAP-25 during synaptic activity

A neuron forms thousands of presynaptic nerve terminals on its axons, far removed from the cell body. The protein CSPα resides in presynaptic terminals, where it forms a chaperone complex with Hsc70 and SGT. Deletion of CSPα results in massive neurodegeneration that impairs survival in mice and flies. In CSPα-knockout mice, levels of presynaptic SNARE complexes and the SNARE protein SNAP-25 are reduced, suggesting that CSPα may chaperone SNARE proteins, which catalyse synaptic vesicle fusion. Here, we show that the CSPα-Hsc70-SGT complex binds directly to monomeric SNAP-25 to prevent its aggregation, enabling SNARE-complex formation. Deletion of CSPα produces an abnormal SNAP-25 conformer that inhibits SNARE-complex formation, and is subject to ubiquitylation and proteasomal degradation. Even in wild-type mouse terminals, SNAP-25 degradation is regulated by synaptic activity; this degradation is decreased by CSPα overexpression, and enhanced by CSPα deletion. Thus, SNAP-25 function is maintained during rapid SNARE cycles by equilibrium between CSPα-dependent chaperoning and ubiquitin-dependent degradation, revealing unique protein quality-control machinery within the presynaptic compartment.     

Differential Roles for Snapin and Synaptotagmin in the Synaptic Vesicle Cycle

Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin null mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.     

Endogenous SNAP-25 Regulates Native Voltage-gated Calcium Channels in Glutamatergic Neurons

In addition to its primary role as a fundamental component of the SNARE complex, SNAP-25 also modulates voltage-gated calcium channels (VGCCs) in various overexpression systems. Although these studies suggest a potential negative regulatory role of SNAP-25 on VGCC activity, the effects of endogenous SNAP-25 on native VGCC function in neurons are unclear. In the present study, we investigated the VGCC properties of cultured glutamatergic and GABAergic rat hippocampal neurons. Glutamatergic currents were dominated by P/Q-type channels, whereas GABAergic cells had a dominant L-type component. Also, glutamatergic VGCC current densities were significantly lower with enhanced inactivation rates and shifts in the voltage dependence of activation and inactivation curves compared with GABAergic cells. Silencing endogenous SNAP-25 in glutamatergic neurons did not alter P/Q-type channel expression or localization but led to increased VGCC current density without changes in the VGCC subtype proportions. Isolation of the P/Q-type component indicated that increased current in the absence of SNAP-25 was correlated with a large depolarizing shift in the voltage dependence of inactivation. Overexpressing SNAP-25 in GABAergic neurons reduced current density without affecting the VGCC subtype proportion. Accordingly, VGCC current densities in glutamatergic neurons from Snap-25^+/− mice were significantly elevated compared with wild type glutamatergic neurons. Overall, this study demonstrates that endogenous SNAP-25 negatively regulates native VGCCs in glutamatergic neurons which could have important implications for neurological diseases associated with altered SNAP-25 expression.     

Are neuronal SNARE proteins Ca2+ sensors?

The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.     

Targeting Synaptic Pathology with a Novel Affinity Mass Spectrometry Approach

We report a novel strategy for studying synaptic pathology by concurrently measuring levels of four SNARE complex proteins from individual brain tissue samples. This method combines affinity purification and mass spectrometry and can be applied directly for studies of SNARE complex proteins in multiple species or modified to target other key elements in neuronal function. We use the technique to demonstrate altered levels of presynaptic proteins in Alzheimer disease patients and prion-infected mice.One prominent pathological feature of neuropsychiatric disorders such as Alzheimer disease (AD)¹ is severe synaptic loss (1–3). Previous reports of AD patients have shown that presynaptic dysfunction might occur early in the disease process (1, 4). Cortical synapse pathology has also been shown to correlate to the severity of dementia more closely than other pathological hallmarks of AD such as plaques and neurofibrillary tangles (5, 6). The SNARE proteins are essential components for the regulation of neurotransmitter exocytosis at the presynaptic site (7). Animal models suggest that changed expression or modification of SNARE complex proteins (synaptosomal-associated protein 25 (SNAP-25), syntaxin-1, and vesicle-associated membrane protein (VAMP)) alters synaptic function and is an interesting target for the development of therapeutics for neuropsychiatric illness (8, 9). The constituents of the SNARE complex are either localized in synaptic vesicles (VAMPs) or anchored at the presynaptic plasma membrane (SNAP-25 and syntaxin). The SNARE proteins are tightly assembled, and subsequent neurotransmitter release of the complex is quickly dissociated by N-ethylmaleimide-sensitive factor (7, 10–12). Because they are both strongly associated into complexes and membrane associated, the SNARE proteins are difficult to analyze via mass spectrometry, which is incompatible with most detergents necessary for the solubilization of proteins. Each SNARE complex protein exists in several isoforms that are differently distributed within the central nervous system (13–18). Post-translational modifications and truncated variants of the SNARE proteins make investigation of the protein expression even more complicated.In this study we developed an approach for the characterization and concurrent quantification of SNARE complex proteins that combines affinity purification by immunoprecipitation and mass spectrometry (IP-MS). We used precipitation with monoclonal antibodies against SNAP-25 to target the SNARE complex proteins and nanoflow LC–tandem mass spectrometry (LC-MS/MS) to characterize the co-immunoprecipitated interaction partners. Selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer coupled to a microflow LC system was used for quantification of the SNARE proteins. To demonstrate the usability of the IP-MS method, we performed a comparison of SNARE complex protein levels in brain tissue from AD patients and age-matched controls, as well as a study of SNARE complex protein levels in brain tissue from prion-infected mice.     

Expression and regulation of SNAP-25 and synaptotagmin VII in developing mouse ovarian follicles via the FSH receptor

Soluble-NSF attachment protein receptor (SNARE) proteins play a role in vesicle fusion, exocytosis, and intracellular trafficking in neuronal cells as well as in fertilization and embryogenesis. We investigated the expression patterns of two SNARE proteins, SNAP-25 and synaptotagmin VII (SytVII), and their regulation by pregnant mare serum gonadotropin (PMSG) during mouse ovarian follicular development. Ovaries were obtained at 0, 12, 24, 36, and 48 h post-PMSG injection of immature mice. SNAP-25 and SytVII mRNA expression levels increased gradually in a time-dependant manner. However, protein levels revealed different patterns of expression, suggesting different translational regulation following PMSG stimulation. SNAP-25 and SytVII expression was closely associated with thickening of the granulosa cell (GC) layer and follicle morphological changes from a flattened to a cuboidal shape. To explore follicle stimulating hormone receptor (FSHR)-mediated regulation of their expression, GCs from preantral follicles were cultured to examine the effects of FSHR siRNA knockdown. FSHR siRNA abolished upregulation of the SNAREs in both PMSG and FSH-stimulated GCs. This abolished gene expression was rescued by adding dibutyryl cyclic AMP to the cultures. These results suggest that SNAP-25 and SytVII expression is regulated via the FSHR-cAMP pathway during follicular development.     

Differential control of the releasable vesicle pools by SNAP-25 splice variants and SNAP-23

The SNARE complex, consisting of synaptobrevin, syntaxin, and SNAP-25, is essential for calcium-triggered exocytosis in neurosecretory cells. Little is known, however, about how developmentally regulated isoforms and other cognate SNARE components regulate vesicular fusion. To address this question, we examined neuroexocytosis from chromaffin cells of Snap25 null mice rescued by the two splice variants SNAP-25a and SNAP-25b and the ubiquitously expressed homolog SNAP-23. In the absence of SNAP-25, vesicle docking persisted, but primed vesicle pools were empty and fast calcium-triggered release abolished. Single vesicular fusion events showed normal characteristics, except for a shorter duration of the fusion pore. Overexpression of SNAP-25a, SNAP-25b, and SNAP-23 resulted in three distinct phenotypes; SNAP-25b induced larger primed vesicle pools than SNAP-25a, whereas SNAP-23 did not support a standing pool of primed vesicles. We conclude that three alternative SNARE components support exocytosis, but they differ in their ability to stabilize vesicles in the primed state.     

Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal–truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.     

The hypophysis controls expression of SNAP-25 and other SNAREs in the adrenal gland

SNAP-25 (Synaptosomal Associated Protein of 25 kDa), in association with two other SNARE (soluble NSF attachment protein receptor) proteins, syntaxin and Vesicle Associated Membrane Protein, VAMP, is implicated in regulated and constitutive exocytosis in neurones and neuroendocrine cells. Our previous studies have shown that it is expressed more by noradrenergic than adrenergic chromaffin cells in the rat adrenal gland. Since certain hormones under hypophyseal control play an essential role in determining chromaffin cell phenotype, the present study examined the effect of hypophysectomy on SNAP-25 expression. Hypophysectomy was found by immunoblotting and RT-PCR analysis to increase adrenal gland SNAP-25, syntaxin-1 and VAMP-2 levels, without modifying the relative expression of SNAP-25 isoforms: immunocytochemistry showed a dramatic increase in SNAP-25 expression in former adrenergic chromaffin cells. Since adrenal glucocorticoids are considerably reduced by hypophysectomy, the effect of corticosterone replacement therapy was investigated. This did not change levels of SNAP-25, syntaxin-1 or VAMP-2. SNARE expression was also unmodified in pheochromocytoma cells treated with a synthetic glucocorticoid. In contrast, subcutaneous injection of hypophysectomized rats with thyroid hormone decreased adrenal SNAP-25, demonstrating the potential importance of the pituitary-thyroid axis. The current data thus demonstrate that the hypophysis exerts an inhibitory control on adrenal gland SNARE proteins. They suggest that glucocorticoids are unlikely to be directly responsible for this but provide evidence that thyroid hormones are implicated in this phenomenon. The putative role of hormonal regulation on SNARE function is discussed.     

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.     

Plasma membrane targeting of exocytic SNARE proteins

SNARE proteins play a central role in the process of intracellular membrane fusion. Indeed, the interaction of SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Eukaryotic cells express a large number of SNARE isoforms, and the function of individual SNAREs is required for specific intracellular fusion events. Exocytosis, the fusion of secretory vesicles with the plasma membrane, employs the proteins syntaxin and SNAP-25 as plasma membrane SNAREs. As a result, exocytosis is dependent upon the targeting of these proteins to the plasma membrane; however, the mechanisms that underlie trafficking of exocytic syntaxin and SNAP-25 proteins to the cell surface are poorly understood. The intracellular trafficking itinerary of these proteins is particularly intriguing as syntaxins are tail-anchored (or Type IV) membrane proteins, whereas SNAP-25 is anchored to membranes via a central palmitoylated domain-there is no common consensus for the trafficking of such proteins within the cell. In this review, we discuss the plasma membrane targeting of these essential exocytic SNARE proteins.     

Docking of liposomes to planar surfaces mediated by trans-SNARE complexes

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.     

Deletion of SNAP-23 results in pre-implantation embryonic lethality in mice

SNARE-mediated membrane fusion is a pivotal event for a wide-variety of biological processes. SNAP-25, a neuron-specific SNARE protein, has been well-characterized and mouse embryos lacking Snap25 are viable. However, the phenotype of mice lacking SNAP-23, the ubiquitously expressed SNAP-25 homolog, remains unknown. To reveal the importance of SNAP-23 function in mouse development, we generated Snap23-null mice by homologous recombination. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23(Δ/wt) matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. Thus these data reveal a critical role for SNAP-23 during embryogenesis.     

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~ 10 k_BT, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).     

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.     

SNAP-25 modulation of calcium dynamics underlies differences in GABAergic and glutamatergic responsiveness to depolarization

SNAP-25 is a component of the SNARE complex implicated in synaptic vesicle exocytosis. In this study, we demonstrate that hippocampal GABAergic synapses, both in culture and in brain, lack SNAP-25 and are resistant to the action of botulinum toxins type A and E, which cleave this SNARE protein. Relative to glutamatergic neurons, which express SNAP-25, GABAergic cells were characterized by a higher calcium responsiveness to depolarization. Exogenous expression of SNAP-25 in GABAergic interneurons lowered calcium responsiveness, and SNAP-25 silencing in glutamatergic neurons increased calcium elevations evoked by depolarization. Expression of SNAP-25(1-197) but not of SNAP-25(1-180) inhibited calcium responsiveness, pointing to the involvement of the 180-197 residues in the observed function. These data indicate that SNAP-25 is crucial for the regulation of intracellular calcium dynamics and, possibly, of network excitability. SNAP-25 is therefore a multifunctional protein that participates in exocytotic function both at the mechanistic and at the regulatory level.