Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain "SNARE motifs" which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.     

Cholecystokinin-regulated exocytosis in rat pancreatic acinar cells is inhibited by a C-terminus truncated mutant of SNAP-23

INTRODUCTION: Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined. AIMS: To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted. METHODOLOGY AND RESULTS: High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin-phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE-stimulated amylase release from adenovirus-cytomegalovirus (AdCMV)-SNAP-23 or AdCMV-SNAP-25-infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE-evoked enzyme secretion from AdCMV-SNAP-23deltaC8- and AdCMV-SNAP-25(1-197)-infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV-SNAP-25AC8- or AdCMV-SNAP-25(1-197)-infected acini were co-infected with wild-type SNAP-23 or SNAP-25. CONCLUSION: From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.     

Replacing SNAP-25b with SNAP-25a expression results in metabolic disease

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a key molecule in the soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE) complex mediating fast Ca²⁺-triggered release of hormones and neurotransmitters, and both splice variants, SNAP-25a and SNAP-25b, can participate in this process. Here we explore the hypothesis that minor alterations in the machinery mediating regulated membrane fusion can increase the susceptibility for metabolic disease and precede obesity and type 2 diabetes. Thus, we used a mouse mutant engineered to express normal levels of SNAP-25 but only SNAP-25a. These SNAP-25b–deficient mice were exposed to either a control or a high-fat/high-sucrose diet. Monitoring of food intake, body weight, hypothalamic function, and lipid and glucose homeostases showed that SNAP-25b–deficient mice fed with control diet developed hyperglycemia, liver steatosis, and adipocyte hypertrophy, conditions dramatically exacerbated when combined with the high-fat/high-sucrose diet. Thus, modified SNARE function regulating stimulus-dependent exocytosis can increase the vulnerability to and even provoke metabolic disease. When combined with a high-fat/high-sucrose diet, this vulnerability resulted in diabesity. Our SNAP-25b–deficient mouse may represent a diabesity model.On-going lifestyle changes, including oversized meals with excessive amounts of sugar and fat, have led to a worldwide pandemic of obesity and type 2 diabetes (T2D) (1). These diseases and their comorbidities cause individual suffering and represent a heavy financial burden on society (2, 3). The term “diabesity” is used to define the coincidence of obesity with T2D under conditions of exaggerated intake of energy-dense diets (4–6). Moreover, genomewide association studies (GWAS) have identified polymorphisms associated with obesity and T2D, indicating that genetic factors predispose certain individuals to diabesity (7–11).Signs of metabolic diseases include impaired regulated release of hormones, particularly insulin as in T2D (4, 12). Likewise, the secretion of inflammatory markers and other peripheral bioactive peptides is either increased (e.g., leptin, resistin, and adipsin) or decreased (e.g., ghrelin and adiponectin) (13–16). Synaptic and nonsynaptic transmission involving the release of neuropeptides and neurotransmitters, especially in hypothalamic areas controlling eating behavior and energy balance, are involved too (17–19).In excitable cells, the release of messenger molecules is a consequence of regulated membrane fusion and involves soluble N-ethylmaleimide–sensitive factor attachment proteins (SNAREs) (20, 21), including synaptosomal-associated protein of 25 kDa (SNAP-25). SNAP-25 is expressed as two developmentally regulated and alternatively spliced isoforms, SNAP-25a and SNAP-25b, which differ in only 9 of 206 amino acids (22, 23). In the mouse brain, SNAP-25a precedes SNAP-25b expression during development, but by the second postnatal week SNAP-25b becomes the major splice variant, concomitantly with a dramatic increase in total SNAP-25 expression (23). In endocrine and neuroendocrine cells SNAP-25a appears to be the dominant isoform throughout life, although the expression patterns of the two splice variants are not dedicated exclusively to certain cell types (24, 25). Either SNAP-25a or SNAP-25b can participate in the core SNARE complex, which in addition comprises the two proteins syntaxin and vesicle-associated membrane protein (VAMP) (21, 26). Normal expression levels of total SNAP-25 appear to be critical for precise synaptic function (27, 28), and a reduction affects SNARE protein assembly and synaptic plasticity and even can cause neurodegeneration (28, 29). Even if both SNAP-25 splice variants mediate fast Ca²⁺-triggered release, SNAP-25b–containing SNARE complexes are believed to have a higher degree of stability and therefore increase the pool of primed vesicles, resulting in release dynamics different from those involving SNAP-25a (26, 30).Polymorphisms in the human Snap25 gene have been associated with weight gain after antipsychotic treatment, with altered levels of serum triglycerides (31, 32), and also with the severity of the metabolic syndrome in T2D (33). Furthermore, polymorphisms in genes expressing proteins directly interacting with SNAP-25 have been implicated in childhood obesity, impaired glucose metabolism and obesity, age of onset of T2D, and insulin requirement in T2D (34–36). Moreover, polymorphisms in the human L-type channel (37) and the K_ATP channel (38) have been directly associated with T2D. These channels are indispensable for glucose-stimulated insulin secretion from pancreatic β cells and thus directly influence SNARE protein function and efficiency. Thus, emerging evidence suggests that direct or indirect alterations of SNARE protein function can increase the susceptibility and predisposition to serious metabolic illness.To test this hypothesis experimentally, we used a genetically engineered mutant mouse expressing normal levels of SNAP-25 but with the nine amino acids specific for the SNAP-25b splice variant converted, by knockout/knockin replacement, to the SNAP-25a sequence (30). Using these mice, we studied the interplay between modified SNARE function and metabolic parameters during a 7-wk diet intervention involving a control standard food diet (CoD) and a high-fat/high-sucrose diet (Western diet, WeD). Our results demonstrate that SNAP-25b deficiency alone predisposes to metabolic disease and that the progressing pathology is accelerated dramatically by a high-fat/high-sucrose diet, leading to diabesity.     

SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts ( approximately 25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.     

Involvement of SNAP-23 and syntaxin 6 in human neutrophil exocytosis

To understand the molecular basis of exocytosis in human neutrophils, the role of syntaxin 6 and SNAP-23 in neutrophil degranulation was examined. Human syntaxin 6 was cloned and identified as a 255-amino acid protein with a carboxy-terminal transmembrane region and two coiled-coil domains. Syntaxin 6 was localized mainly in the plasma membrane of human resting neutrophils, whereas SNAP-23 was located primarily in the mobilizable tertiary and specific granules. SNAP-23 was translocated to the cell surface, colocalizing with syntaxin 6, on neutrophil activation. In vitro binding studies established that SNAP-23 binds to syntaxin 6. Coimmunoprecipitation assays indicated that SNAP-23 interacts with syntaxin 6 in vivo, and this interaction was dramatically increased on neutrophil activation. Antibodies against SNAP-23 inhibited Ca(++) and GTP-gamma-S-induced exocytosis of CD67-enriched specific granules, but they hardly affected exocytosis of the CD63-enriched azurophilic granules, when introduced into electropermeabilized neutrophils. Anti-syntaxin 6 antibodies prevented exocytosis of both CD67- and CD63-enriched granules in electropermeabilized neutrophils. These data show that syntaxin 6 and SNAP-23 are involved in human neutrophil exocytosis, demonstrating that vesicle SNAP receptor-target SNAP receptor (v-SNARE- t-SNARE) interactions modulate neutrophil secretion. Syntaxin 6 acts as a target for secretion of specific and azurophilic granules, whereas SNAP-23 mediates specific granule secretion.     

The SNARE protein SNAP-25 is linked to fast calcium triggering of exocytosis

Synchronous neurotransmission depends on the tight coupling between Ca(2+) influx and fusion of neurotransmitter-filled vesicles with the plasma membrane. The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein synaptobrevin 2 and the plasma membrane SNAREs syntaxin 1 and synaptosomal protein of 25 kDa (SNAP-25) are essential for calcium-triggered exocytosis. However, the link between calcium triggering and SNARE function remains elusive. Here we describe mutations in two sites on the surface of the SNARE complex formed by acidic and hydrophilic residues of SNAP-25 and synaptobrevin 2, which were found to coordinate divalent cations in the neuronal SNARE complex crystal structure. By reducing the net charge of the site in SNAP-25 we identify a mutation that interferes with calcium triggering of exocytosis when overexpressed in chromaffin cells. Exocytosis was elicited by photorelease of calcium from a calcium cage and evaluated by using patch-clamp capacitance measurements at millisecond time resolution. We present a method for monitoring the dependence of exocytotic rate upon calcium concentration at the release site and demonstrate that the mutation decreased the steepness of this relationship, indicating that the number of sequential calcium-binding steps preceding exocytosis is reduced by one. We conclude that the SNARE complex is linked directly to calcium triggering of exocytosis, most likely in a complex with auxiliary proteins.     

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin,SNAP-23, and syntaxin 2

Morphologic studies have demonstrated a process by which alpha-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for alpha-granule secretion. These observations raise the possibility that morphologic features of alpha-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins-human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca(++)-induced alpha-granule secretion by approximately 50%. Inhibition of alpha-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of alpha-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of alpha-granule secretion that include homotypic alpha-granule fusion and the fusion of alpha-granules with the open canalicular system and plasma membrane.     

Determinants of liposome fusion mediated by synaptic SNARE proteins

Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT/E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin/VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step.     

Role of vesicle-associated membrane protein 2 in exocytosis of glucagon-like peptide-1 from the murine intestinal L cell

Aims/hypothesis

Glucagon-like peptide-1 (GLP-1), secreted by the enteroendocrine L cell, is an incretin hormone that potently stimulates insulin secretion. Although signalling pathways promoting GLP-1 release are well characterised, the mechanisms by which GLP-1-containing granules fuse to the L cell membrane are unknown. As soluble NSF attachment proteins (SNAREs) are known to mediate granule–membrane fusion, the role of vesicle-associated membrane proteins (VAMPs) in GLP-1 exocytosis was examined.

Methods

SNARE expression was determined in murine GLUTag L cells by RT-PCR and immunoblot and in primary murine L cells by immunofluorescence. Co-immunoprecipitation was used to examine SNARE interactions, while tetanus toxin (TetX)-mediated cleavage of VAMP was used with a GLP-1 secretion assay and total internal reflection fluorescence microscopy to determine the role of VAMP2 in exocytosis.

Results

VAMP2 was expressed in murine L cells and localised to secretory granules in GLUTag cells. VAMP1/3 and the core membrane proteins syntaxin1a and synaptosomal-associated protein 25 kDa (SNAP25) were also detected. TetX cleaved VAMPs in GLUTag cells. However, only VAMP2 interacted with syntaxin1a, as did SNAP25 and Munc18-1. TetX treatment of GLUTag cells prevented glucose-dependent insulinotrophic peptide- and oleic-acid-stimulated GLP-1 secretion (p?<?0.05–0.01), as well as K⁺-stimulated single-cell exocytosis (p?<?0.05–0.001), while TetX-resistant VAMP2 expression rescued GLP-1 secretion (p?<?0.01–0.001).

Conclusions/interpretation

Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag L cell in response to a variety of established secretagogues. An improved understanding of the mechanisms governing the release of GLP-1 may lead to new therapeutic approaches to enhance the levels of this incretin hormone in patients with type 2 diabetes.     

A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.     

Rat and gerbil pinealocytes contain the synaptosomal-associated protein 25 (SNAP-25)

Abstract: It has recently been established that the neuroendocrine pinealocytes of mammals contain several synaptic membrane proteins that are involved in the regulation of vesicle trafficking in the nerve terminal. In the present study, we have conducted immunoblot and immunocytochemical analyses to demonstrate that another key component of the presynaptic plasmalemma, i.e., protein SNAP-25 (synaptosomal-associated protein 25 kDa), can be detected in pinealocytes. Immunostaining of serial semi-thin sections of plastic-embedded rat and gerbil pineals with monoclonal SNAP-25 antibodies showed that SNAP-25 was present in pinealocytes of both species. We proved its coexpression with other synaptic membrane proteins (synaptophysin, synaptotagmin I, synaptobrevin II, and syntaxin I) at the single cell level. Thus, pinealocytes obviously are endowed with the major proteins that are thought to regulate the targeting and exocytosis of secretory vesicles, in particular of synaptic -like microvesicles.     

Vesicle-associated membrane protein 3 (VAMP-3) and VAMP-8 are present in human platelets and are required for granule secretion

Secretion of platelet granules is necessary for normal hemostasis. Platelet secretion requires soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) complex formation between different members of the syntaxin, SNAP-25, and vesicle-associated membrane protein (VAMP) gene families. Using microcapillary reverse-phase high-performance liquid chromatography-nano-electrospray tandem mass spectrometry, we identified VAMP-3 and VAMP-8 as VAMP isoforms coimmunoprecipitated from platelets with syntaxin 4. Immunoblotting experiments confirmed the presence of VAMP-3 and VAMP-8 but not VAMP-1 or VAMP-2 in platelets. To examine the effect of VAMP proteins on platelet secretion, soluble recombinant (r) VAMP-2, rVAMP-3, and rVAMP-8 were incubated with streptolysin O-permeabilized platelets. Secretion of alpha granules (monitored by flow cytometric measurement of P-selectin) was blocked, and dense-granule secretion (assessed by release of carbon 14-serotonin) was almost completely inhibited by rVAMP-3, whereas rVAMP-8 inhibited secretion of dense granules but not alpha granules. In contrast, rVAMP-2, which formed SNARE complexes in vitro, had no effect on platelet exocytosis. We conclude that VAMP-3 and VAMP-8 form SNARE complexes with platelet syntaxin 4 and are required for platelet granule secretion.     

Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that regulates neurotransmission by the formation of a complex with syntaxin 1 and synaptobrevin/VAMP2. SNAP-25 also reduces neuronal calcium responses to stimuli, but neither the functional relevance nor the molecular mechanisms of this modulation have been clarified. In this study, we demonstrate that hippocampal slices from Snap25^+/− mice display a significantly larger facilitation and that higher calcium peaks are reached after depolarization by Snap25^−/− and Snap25^+/− cultured neurons compared with wild type. We also show that SNAP-25b modulates calcium dynamics by inhibiting voltage-gated calcium channels (VGCCs) and that PKC phosphorylation of SNAP-25 at ser187 is essential for this process, as indicated by the use of phosphomimetic (S187E) or nonphosphorylated (S187A) mutants. Neuronal activity is the trigger that induces the transient phosphorylation of SNAP-25 at ser187. Indeed, enhancement of network activity increases the levels of phosphorylated SNAP-25, whereas network inhibition reduces the extent of protein phosphorylation. A transient peak of SNAP-25 phosphorylation also is detectable in rat hippocampus in vivo after i.p. injection with kainate to induce seizures. These findings demonstrate that differences in the expression levels of SNAP-25 impact on calcium dynamics and neuronal plasticity, and that SNAP-25 phosphorylation, by promoting inhibition of VGCCs, may mediate a negative feedback modulation of neuronal activity during intense activation.     

Three SNARE complexes cooperate to mediate membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.     

Munc18/SNARE proteins’ regulation of exocytosis in guinea pig duodenal Brunner’s gland acini

AIM： To examine the molecular mechanism of exocytosis in the Brunner’s gland acinar cell. METHODS： We used a submucosal preparation of guinea pig duodenal Brunner’s gland acini to visualize the dilation of the ductal lumen in response to cholinergic stimulus. We correlated this to electron microscopy to determine the extent of exocytosis of the mucin-filled vesicles. We then examined the behavior of SNARE and interacting Munc18 proteins by confocal microscopy. RESULTS： One and 6 μmol/L carbachol evoked a dose-dependent dilation of Brunner’s gland acini lumen, which correlated to the massive exocytosis of mucin. Munc18c and its cognate SNARE proteins Syntaxin-4 and SNAP-23 were localized to the apical plasma membrane, and upon cholinergic stimulation, Munc18c was displaced into the cytosol leaving Syntaxin-4 and SNAP-23 intact. CONCLUSION： Physiologic cholinergic stimulation induces Munc18c displacement from the Brunner’s gland acinar apical plasma membrane, which enables apical membrane Syntaxin-4 and SNAP-23 to form a SNARE complex with mucin-filled vesicle SNARE proteins to affect exocytosis.     

CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.     

Identification of exocytotic membrane proteins, syntaxin-1 and SNAP-25, in Entamoeba histolytica from hamster liver

Entamoeba histolytica is a protozoan parasite causing dysentery and in some cases liver abscesses. These effects have been attributed to cytolytic substances released by exocytosis. In this study, the presence of the proteins syntaxin-1 and SNAP-25, which are assumed to be involved in exocytosis, were examined by immunohistochemistry, immunoelectron microscopy and western blot analysis. Syntaxin-1 and SNAP-25 were expressed in the vesicular, vacuolar and plasma membranes of E. histolytica trophozoites. It can be concluded that these proteins might be involved in exocytosis processes.     

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions

Ca²⁺-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca²⁺-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P₂ in acceptor liposomes and was independent of Ca²⁺, but Ca²⁺ dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.     

Molecular mechanisms of platelet exocytosis: role of SNAP-23 and syntaxin 2 in dense core granule release

To characterize the molecular mechanisms of platelet secretion, we focused on the calcium-induced exocytosis of dense core granules. Platelets contain several known t-SNAREs (soluble N-ethylmaleimide sensitive factor [NSF] attachment protein receptors) such as syntaxins 2, 4, and 7 and SNAP-23 (synaptosomal associated protein 23). By using an in vitro exocytosis assay, we have been able to assign roles for some of these t-SNAREs in dense core granule release. This calcium-induced secretion relies on the SNARE proteins because it is stimulated by the addition of recombinant alpha-SNAP and inhibited by a dominant negative alpha-SNAP-L294A mutant or by anti-alpha-SNAP and anti-NSF antibodies. SNAP-23 antibodies and an inhibitory C-terminal SNAP-23 peptide both blocked dense core granule release, demonstrating a role for SNAP-23. Unlike other cell types, platelets contain a significant pool of soluble SNAP-23, which does not partition into Triton X-114. Of the anti-syntaxin antibodies tested, only anti-syntaxin 2 antibody inhibited dense core granule release. Immunoprecipitation studies showed that the 2 t-SNAREs syntaxin 2 and SNAP-23 do form a complex in vivo. These data clearly show that SNAPs, NSF, and specific t-SNAREs are used for dense core granule release; these data provide a greater understanding of regulated exocytosis in platelets.