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PubMed1997

Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c.

SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc 18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.

Adipocytes↗
PubMed2003

Glucose-stimulated insulin secretion is coupled to the interaction of actin with the t-SNARE (target membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein) complex.

The actin monomer sequestering agent latrunculin B depolymerized beta-cell cortical actin, which resulted in increased glucose-stimulated insulin secretion in both cultured MIN6 beta-cells and isolated rat islet cells. In perifused islets, latrunculin B treatment increased both first- and second-phase glucose-stimulated insulin secretion without any significant effect on total insulin content. This increase in secretion was independent of calcium regulation because latrunculin B also potentiated calcium-stimulated insulin secretion in permeabilized MIN6 cells. Confocal immunofluorescent microscopy revealed a redistribution of insulin granules to the cell periphery in response to glucose or latrunculin B, which correlated with a reduction in phalloidin staining of cortical actin. Moreover, the t-SNARE [target membrane soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins Syntaxin 1 and SNAP-25 coimmunoprecipitated polymerized actin from unstimulated MIN6 cells. Glucose stimulation transiently decreased the amount of actin coimmunoprecipitated with Syntaxin 1 and SNAP-25, and latrunculin B treatment fully ablated the coimmunoprecipitation. In contrast, the actin stabilizing agent jasplakinolide increased the amount of actin coimmunoprecipitated with the t-SNARE complex and prevented its dissociation upon glucose stimulation. These data suggest a mechanism whereby glucose modulates beta-cell cortical actin organization and disrupts the interaction of polymerized actin with the plasma membrane t-SNARE complex at a distal regulatory step in the exocytosis of insulin granules.

Actins↗
PubMed1996

A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina.

In contrast to conventional synapses, which release neurotransmitter transiently, ribbon synapses formed by photoreceptors and bipolar cells of the retina release neurotransmitter continuously and modulate the rate in response to light. Both modes of release are mediated by synaptic vesicles but probably differ in the regulation of docking and fusion of synaptic vesicles with the plasma membrane. We have found that syntaxin 1, an essential component of the core fusion complex in conventional synapses, is absent from ribbon synapses of the retina, raising the possibility that these synapses contain a different type of syntaxin or syntaxin-like protein. By immunoprecipitating syntaxin 1-depleted retina and brain extracts with a SNAP-25 antibody and microsequencing the precipitated proteins, syntaxin 3 was detected in retina complexed with SNAP-25, synaptobrevin, and complexin. Using an anti-syntaxin 3 antiserum, syntaxin 3 was demonstrated to be present at high levels in retina compared to brain. Immunofluorescent staining of rat retina sections confirmed that syntaxin 3 is expressed by photoreceptor and bipolar cells in the retina. Thus, in the retina, expression of syntaxin 3 is correlated with ribbon synapses and may play a role in the tonic release of neurotransmitter.

Adaptor Proteins, Vesicular Transport↗
PubMed2003

Syntaxin 4 and Synip (syntaxin 4 interacting protein) regulate insulin secretion in the pancreatic beta HC-9 cell.

Although syntaxin 1 is generally thought to function as the primary target-N-ethylmaleimide-sensitive factor attachment protein receptor required for pancreatic beta cell insulin secretion, we have observed that overexpression of a dominant-interfering syntaxin 4 mutant (syntaxin 4/DeltaTM) attenuated glucose-stimulated insulin secretion in betaHC-9 cells. Furthermore, these cells express the selective syntaxin 4-binding protein Synip (syntaxin 4 interacting protein), and Synip was specifically co-immunoprecipitated with syntaxin 4 but not syntaxin 1. Overexpression of the full-length Synip protein (Synip/wild type) inhibited VAMP2 association with syntaxin 4 and decreased glucose-stimulated insulin secretion. This did not occur with a Synip mutant (Synip/ DeltaEF) that was incapable of binding syntaxin 4. Consistent with a functional role of syntaxin 4 in this process, expression of syntaxin 4/DeltaTM also inhibited glucose-stimulated insulin secretion. Furthermore, analysis of first and second phase insulin secretion demonstrated that syntaxin 4/DeltaTM mainly suppressed the second phase of insulin secretion. In contrast, overexpression of Synip resulted in an inhibition of both the first and second phase of glucose-stimulated insulin secretion. These data demonstrate that syntaxin 4 plays a functional role on insulin release and granule fusion in beta cells and that this process is regulated by the syntaxin 4-specific binding protein Synip.

Antigens, Surface↗
PubMed2000

Munc-18 associates with syntaxin and serves as a negative regulator of exocytosis in the pancreatic beta -cell.

The Munc-18 protein (mammalian homologue of the unc-18 gene; also called nSec1 or rbSec1) has been identified as an essential component of the synaptic vesicle fusion protein complex. The cellular and subcellular localization and functional role of Munc-18 protein in pancreatic beta-cells was investigated. Subcellular fractionation of insulin-secreting HIT-T15 cells revealed a 67-kDa protein in both cytosol and membrane fractions. Immunohistochemistry showed punctate Munc-18 immunoreactivity in the cytoplasm of rat pancreatic islet cells. Direct double-labeling immunofluorescence histochemistry combined with confocal laser microscopy revealed the presence of Munc-18 immunoreactivity in insulin-, glucagon-, pancreatic polypeptide-, and somatostatin-containing cells. Syntaxin 1 immunoreactivity was detected in extracts of HIT-T15 cells, which were immunoprecipitated using Munc-18 antiserum, suggesting an intimate association of Munc-18 with syntaxin 1. Administration of Munc-18 peptide or Munc-18 antiserum to streptolysin O-permeabilized HIT-T15 cells resulted in significantly increased insulin release, but did not have any significant effect on voltage-gated Ca(2+) channel activity. The findings taken together show that the Munc-18 protein is present in insulin-secreting beta-cells and implicate Munc-18 as a negative regulator of the insulin secretory machinery via a mechanism that does not involve syntaxin-associated Ca(2+) channels.

Animals↗
PubMed1999

Cultured glial cells express the SNAP-25 analogue SNAP-23.

Astrocytes release glutamate and aspartate in response to elevated intracellular calcium levels, and it has been proposed that this occurs by a vesicular release mechanism, in which SNARE proteins are implicated. Although syntaxin, synaptobrevin, and cellubrevin have been shown to be expressed by cultured astrocytes, SNAP-25 has not been detected. By using immunocytochemical, immunoblotting, and polymerase chain reaction techniques, the present study demonstrates that SNAP-23, an analogue of SNAP-25, is expressed by astrocytes both in culture and in rat cerebellum. These findings provide additional evidence that astrocytes release excitatory amino acids by a vesicular mechanism involving SNARE proteins. SNAP-23 and also syntaxin 1 and cellubrevin were found to be expressed in glial precursor cells, oligodendrocytes, and microglia. These data suggest that the t-SNAREs SNAP-23 and syntaxin 1 and the v-SNARE cellubrevin participate in general membrane insertion mechanisms involved in diverse glial cell functions such as secretion, phagocytosis, and myelinogenesis.

Animals↗
PubMed2004

Genetic segregation of brain gene expression identifies retinaldehyde binding protein 1 and syntaxin 12 as potential contributors to ethanol preference in mice.

Genetic strains of mice represent an important resource for research on the biological determinants of complex diseases and behavioral phenotypes. To date, the approaches used have had little success in identifying causal genes. We have evaluated brain gene expression in C57BL/6J (B6) and DBA/2J (D2) inbred mouse strains using differential display to identify a number of sequences showing significant expression differences between the two strains. These differences were confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Knowing that B6 and D2 mouse strains differ for a number of behavioral phenotypes, we asked whether this difference in brain gene expression could explain any of these traits. Here, we show that the expression of two of these genes, retinaldehyde binding protein 1 (Rlbp1) and syntaxin 12 (Stx12), co-segregate with the ethanol preference phenotype in a B6D2 F2 population. Our results suggest a potential role for Rlbp1 and Stx12 in ethanol preference in mice, a conclusion supported by the location of these genes in quantitative trait loci (QTL) regions for this phenotype. This experimental approach has the potential for a broad application in the assessment of the roles of differentially expressed genes in a variety of complex phenotypes, with the advantage of identifying novel and potentially causal candidate genes directly.

Animals↗
PubMed1997

Association of syntaxin 3 and vesicle-associated membrane protein (VAMP) with H+/K(+)-ATPase-containing tubulovesicles in gastric parietal cells.

H+/K(+)-ATPase is the proton pump in the gastric parietal cell that is responsible for gastric acid secretion. Stimulation of acid secretion is associated with a reorganization of the parietal cells resulting in the incorporation of H+/K(+)-ATPase from a cytoplasmic membrane pool, the tubulovesicle compartment, into the apical canalicular membrane. To better characterize the role of membrane trafficking events in the morphological and physiological changes associated with acid secretion from parietal cells, we have characterized the expression and localization of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in these cells. Each of the six different SNARE proteins examined [syntaxins 1 through 4 of 25-kDa synaptosome-associated protein, and vesicle-associated membrane protein] were found to be expressed in parietal cells. Furthermore, two of these SNAREs, vesicle-associated membrane protein and syntaxin 3, were associated with H+/K(+)-ATPase-containing tubulovesicles while the remainder were excluded from this compartment. The expression of syntaxin 1 and synaptosome-associated protein of 25 kDa in parietal cells, two SNAREs previously thought to be restricted to neuroendocrine tissues, suggests that parietal cells may utilize membrane trafficking machinery that is similar to that utilized for regulated exocytosis in neurons. Furthermore, the localization of syntaxin 3, a putative target membrane SNARE, to the tubulovesicle compartment indicates that syntaxin 3 may have an alternative function. These observations support a role for intracellular membrane trafficking events in the regulated recruitment of H+/K(+)-ATPase to the plasma membrane after parietal cell stimulation.

Animals↗
PubMed2005

Chronic antidepressants reduce depolarization-evoked glutamate release and protein interactions favoring formation of SNARE complex in hippocampus.

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mm KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr286-phosphorylated alphaCaM kinase II (alpha-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals.

Animals↗
PubMed1995

A novel ubiquitous form of Munc-18 interacts with multiple syntaxins. Use of the yeast two-hybrid system to study interactions between proteins involved in membrane traffic.

Munc-18-1 is a 67-kDa neuronal protein that binds tightly to syntaxin 1 and functions in synaptic vesicle exocytosis (Hata, Y., Slaughter, C.A., and Südhof, T.C. (1993a) Nature 366, 347-351). We have now characterized a new Munc-18 isoform, Munc-18-2, that exhibits 63% amino acid sequence identity with Munc-18-1. Munc-18-2 is expressed in most tissues, whereas Munc-18-1 is primarily expressed in brain. Using recombinant Munc-18-1 and Munc-18-2 produced in COS cells, we show that both forms of Munc-18 bind tightly to syntaxins 1A, 2, and 3 but not to syntaxin 4. In an independent approach to study the binding specificities of Munc-18-1 and Munc-18-2, we used the yeast two-hybrid system. This assay system depends on protein-protein interactions in the cell nucleus. We validated its utility for studying membrane trafficking proteins by testing well characterized interactions between cytosolic proteins that are known to be physiologically important in exocytosis. Strong interactions, such as the binding of syntaxins 1-4 with SNAP-25, were effectively detected by the yeast two-hybrid assay, but weak binding, such as the binding of syntaxins to synaptotagmin or of synaptotagmin to neurexins, was not. Studies on full-length and truncated forms of Munc-18s by the yeast two-hybrid system confirmed their interactions with syntaxins. Both the N and the C terminus of Munc-18 were essential for binding. Munc-18-1 and Munc-18-2 bind only to syntaxins 1A, 2, and 3 but not 4 and 5 by yeast-two hybrid system assays. Our studies demonstrate that neural and non-neural tissues have distinct forms of Munc-18, which may function in different types of exocytosis. The lack of specificity of the interactions between syntaxins and Munc-18s indicates that specificity of membrane trafficking reactions is not dependent on this interaction.

Amino Acid Sequence↗
PubMed1998

Interactions between presynaptic calcium channels and proteins implicated in synaptic vesicle trafficking and exocytosis.

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized omega conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.

Animals↗
PubMed2000

Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin.

Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca(2+) could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca(2+)-dependent calmodulin binding (K(d) = 500 nM) to glutathione S-transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP(77-94), immediately C-terminal to the tetanus toxin cleavage site (Q(76)-F(77)). In isolated synaptic vesicles, Ca(2+)/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a (35)S-SNAP-25, syntaxin 1 GST-VAMP(1-96) complex was inhibited by Ca(2+)/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca(2+) may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.

Amino Acid Sequence↗
PubMed2001

Spring, a novel RING finger protein that regulates synaptic vesicle exocytosis.

The synaptosome-associated protein of 25 kDa (SNAP-25) interacts with syntaxin 1 and vesicle-associated membrane protein 2 (VAMP2) to form a ternary soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex that is essential for synaptic vesicle exocytosis. We report a novel RING finger protein, Spring, that specifically interacts with SNAP-25. Spring is exclusively expressed in brain and is concentrated at synapses. The association of Spring with SNAP-25 abolishes the ability of SNAP-25 to interact with syntaxin 1 and VAMP2 and prevents the assembly of the SNARE complex. Overexpression of Spring or its SNAP-25-interacting domain reduces Ca(2+)-dependent exocytosis from PC12 cells. These results indicate that Spring may act as a regulator of synaptic vesicle exocytosis by controlling the availability of SNAP-25 for the SNARE complex formation.

Amino Acid Sequence↗
PubMed1997

Mints, Munc18-interacting proteins in synaptic vesicle exocytosis.

Munc18-1 is a neuronal protein that interacts with syntaxin 1 and is required for synaptic vesicle exocytosis. We have now identified two Munc18-1-interacting proteins called Mint1 and Mint2 that may mediate the function of Munc18-1. Mint proteins are detectable only in brain and are composed of an N-terminal sequence that binds Munc18-1, a middle phosphotyrosine-binding domain, and two C-terminal PDZ domains thought to attach proteins to the plasma membrane. In brain, Mint proteins are part of a multimeric complex containing Munc18-1 and syntaxin that likely functions as an intermediate in synaptic vesicle docking/fusion. The phosphotyrosine-binding domain specifically binds to phosphatidylinositol phosphates known to be produced during vesicle exocytosis (Hay, J. C., Fisette, P. L., Jenkins, G. H., Fukami, K., Takonawa, T., Anderson, R. A., and Martin, T. F. J. (1995) Nature 374, 173-177). Our data suggest a model whereby local production of phosphatidylinositol phosphates may trigger the binding of vesicles to the active zone via the Mint.Munc18-1 complex in conjunction with syntaxin 1.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Synaptobrevin isoforms in secretory granules and synaptic-like microvesicles in anterior pituitary cells.

A set of synaptic proteins have been shown to be essential for the life cycle and exocytosis of synaptic vesicles at the nerve terminal. Recently, these proteins have also been identified in certain endocrine cells. Here we analysed the presence and location of some of these synaptic proteins in anterior pituitary cells. Immunoblotting data demonstrated that Rab3a, synaptotagmin, cellubrevin, synaptobrevin 2, syntaxin 1, SNAP-25 and synaptophysin were well represented in anterior pituitary cells as well as in the corticotroph cell line AtT-20. Cellubrevin was the most abundant synaptobrevin isoform present in pituitary cells. Moreover, both cellubrevin and synaptobrevin 2 took part of a protein complex involved in the fusion process in adenohypophyseal cells. Immunocytochemical and subcellular fractionation showed that cellubrevin, synaptobrevin 2, Rab3a and synaptotagmin were located in both secretory granules and synaptic-like microvesicles fractions. In contrast, SNAP-25 and syntaxin 1 were mainly associated with plasma membrane fractions. Therefore, these results suggest similar secretory mechanisms for synaptic vesicles and secretory organelles in both neuronal and endocrine cells.

Animals↗
PubMed2002

Convergence and divergence in the mechanism of SNARE binding by Sec1/Munc18-like proteins.

Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs. We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 N-terminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.

Animals↗
PubMed1999

Redistribution of presynaptic proteins during alpha-latrotoxin-induced release of neurotransmitter and membrane retrieval at the frog neuromuscular junction.

Calcium-dependent exocytosis at the nerve terminal involves the synaptic core (SNARE) complex composed of the t-SNAREs syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), and the v-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), a stable heterotrimer which can associate with the putative calcium sensor protein, synaptotagmin. The distribution of these proteins at the frog neuromuscular junction was examined by immunofluorescent staining and confocal microscopy following exocytosis induced by alpha-latrotoxin. Experiments were performed under conditions in which synaptic vesicle recycling was either maintained in balance with exocytosis, or completely blocked, or during recovery from block of endocytosis. When endocytosis was maintained, protein distribution was essentially identical to that of unstimulated nerve terminals, in which syntaxin 1 and SNAP-25 are localized to the presynaptic active zones coincident with the postsynaptic folds that contain a high density of acetylcholine receptors (AChRs). Block of endocytosis led to complete incorporation of vesicle proteins into the plasmalemma, and t-SNARE distribution was no longer restricted to active zones. Five minutes after the onset of recovery, both synaptic vesicle proteins and t-SNARE proteins were concentrated into small spots, in a similar pattern to that obtained following endocytosis of the vital styryl dye FM1-43. These findings are consistent with a model in which following sustained exocytosis, t-SNARE trafficking involves internalization and transit via a vesicular compartment before recycling to the presynaptic plasma membrane.

Animals↗
PubMed2005

Cellular localization and stimulation-associated distribution dynamics of syntaxin-1 and syntaxin-3 in gastric parietal cells.

Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin-1 and syntaxin-3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin-1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin-3 was found predominantly in the H,K-ATPase-rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP-syntaxin-1 and CFP-syntaxin-3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin-1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin-1 in a subadjacent cytoplasmic compartment. In resting, non-secreting parietal cells, syntaxin-3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin-3 translocated to the apical membrane vacuoles, there co-localizing with H,K-ATPase, syntaxin-1 and F-actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.

1-Methyl-3-isobutylxanthine↗