Syntaxin 3 is required for cAMP-induced acid secretion: streptolysin O-permeabilized gastric gland model

2002 ◽  
Vol 282 (1) ◽  
pp. G23-G33 ◽  
Author(s):  
David A. Ammar ◽  
Rihong Zhou ◽  
John G. Forte ◽  
Xuebiao Yao
Keyword(s):  
Plasma Membrane ◽  
Acid Secretion ◽  
Gastric Gland ◽  
Apical Plasma Membrane ◽  
Streptolysin O ◽  
Acid Accumulation ◽  
Hcl Secretion ◽  
Inhibited Acid ◽  
Syntaxin 3 ◽  
Gland System

Gastric gland stimulation triggers H+,K+-ATPase translocation from cytoplasmic tubulovesicles to apical plasma membrane in parietal cells, resulting in HCl secretion. We studied the mechanisms involved in tubulovesicle translocation with a permeabilized gland system. Streptolysin O (SLO)-treated glands were permeabilized such that exogenous fluorescently labeled actin incorporated into cytoskeleton in a pattern mimicking endogenous F-actin. As shown by accumulation of the weak base aminopyrine (AP), SLO-permeabilized glands are stimulated to secrete acid by addition of cAMP and ATP and inhibited by proton pump inhibitors. Direct visualization with the fluorescent pH probe Lysosensor showed acid accumulation in glandular lumen and parietal cell canaliculi. ME-3407, an antiulcer drug with inhibitory action implicated to involve ezrin, inhibited AP uptake in and effectively released ezrin from intact and SLO-permeabilized glands. In contrast, wortmannin, an effective secretion inhibitor in intact glands, had minimal effects on ezrin or AP accumulation in SLO-permeabilized glands. The finding that SNARE protein syntaxin 3 is associated with H+,K+-ATPase-containing tubulovesicles suggested that it is involved in membrane fusion. Addition of recombinant syntaxin 3, but not syntaxin 5 or heat-denatured syntaxin 3, dose-dependently inhibited acid secretion. Our studies are consistent with a membrane recycling hypothesis that activation of protein kinase cascades leads to SNARE-mediated fusion of H+,K+-ATPase-containing tubulovesicles to apical plasma membrane.

Stimulation of gastric acid secretion by cAMP in a novel alpha-toxin-permeabilized gland model

1996 ◽  
Vol 271 (1) ◽  
pp. C61-C73 ◽  
Author(s):  
X. Yao ◽  
S. M. Karam ◽  
M. Ramilo ◽  
Q. Rong ◽  
A. Thibodeau ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Cell Activation ◽  
Gastric Gland ◽  
Secretory Activity ◽  
Apical Plasma Membrane ◽  
Alpha Toxin ◽  
Maximal Stimulation

It is generally believed that histamine-stimulated gastric acid secretion involves a transient elevation of intracellular Ca2+ and the adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) cascade through phosphorylation, whose actions ultimately effect the fusion of H(+)-K(+)-adenosinetriphosphatase (ATPase)-containing vesicles to the apical plasma membrane of parietal cells. To dissect the signaling events underlying gastric acid secretion, we have developed a permeabilized gastric gland model using Staphylococcus alpha-toxin. The advantage of this model is its ability to retain cytosolic components that are required for the secretory machinery. Here we show that acid secretion in alpha-toxin-permeabilized glands is a cAMP-dependent process, reaching a maximal stimulation at 100 microM cAMP. The cAMP-elicited acid secretion, as monitored by the accumulation of the weak base aminopyrine (AP), required functional mitochondria or exogenously supplied ATP. Maximal stimulation elicited by cAMP for AP uptake by permeabilized glands was 51-85% of intact glands. Moreover, secretory activity was potentiated by 0.1 mM ATP. The recruitment of H(+)-K(+)-ATPase-rich tubulovesicles into the apical plasma membrane was measured using biochemical and morphological assays, thus validating the cell activation processes in response to cAMP. From this permeabilized model, [gamma-32P]ATP was used to directly phosphorylate target proteins. A number of proteins whose phosphorylation-dephosphorylation is specifically modulated by cAMP were found. These studies establish the first permeabilized gland model in which the resting-to-secreting transition can be triggered and show that cAMP-mediated phosphorylation is correlated with secretory activity.

scholarly journals A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

1998 ◽  
Vol 9 (6) ◽  
pp. 1437-1448 ◽  
Author(s):  
Thierry Galli ◽  
Ahmed Zahraoui ◽  
Vadakkanchery V. Vaidyanathan ◽  
Graça Raposo ◽  
Jian Min Tian ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Apical Plasma Membrane ◽  
Domain Specific ◽  
Tetanus Neurotoxin ◽  
Synaptic Vesicle Exocytosis ◽  
Clostridial Neurotoxins ◽  
Vesicle Exocytosis ◽  
Syntaxin 3

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

scholarly journals Syntaxin specificity of aquaporins in the inner medullary collecting duct

2009 ◽  
Vol 297 (2) ◽  
pp. F292-F300 ◽  
Author(s):  
Abinash C. Mistry ◽  
Rickta Mallick ◽  
Janet D. Klein ◽  
Thomas Weimbs ◽  
Jeff M. Sands ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Snare Complex ◽  
Apical Plasma Membrane ◽  
Transport Activity ◽  
Inner Medullary Collecting Duct ◽  
Syntaxin 4 ◽  
Medullary Collecting Duct ◽  
Syntaxin 3

Proper targeting of the aquaporin-2 (AQP2) water channel to the collecting duct apical plasma membrane is critical for the urine concentrating mechanism and body water homeostasis. However, the trafficking mechanisms that recruit AQP2 to the plasma membrane are still unclear. Snapin is emerging as an important mediator in the initial interaction of trafficked proteins with target soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (t-SNARE) proteins, and this interaction is functionally important for AQP2 regulation. We show that in AQP2-Madin-Darby canine kidney cells subjected to adenoviral-mediated expression of both snapin and syntaxins, the association of AQP2 with both syntaxin-3 and syntaxin-4 is highly enhanced by the presence of snapin. In pull-down studies, snapin detected AQP2, syntaxin-3, syntaxin-4, and SNAP23 from the inner medullary collecting duct. AQP2 transport activity, as probed by AQP2's urea permeability, was greatly enhanced in oocytes that were coinjected with cRNAs of SNARE components (snapin+syntaxin-3+SNAP23) over those injected with AQP2 cRNA alone. It was not enhanced when syntaxin-3 was replaced by syntaxin-4 (snapin+syntaxin-4+SNAP23). On the other hand, the latter combination significantly enhanced the transport activity of the related AQP3 water channel while the presence of syntaxin-3 did not. This AQP-syntaxin interaction agrees with the polarity of these proteins' expression in the inner medullary collecting duct epithelium. Thus our findings suggest a selectivity of interactions between different aquaporin and syntaxin isoforms, and thus in the regulation of AQP2 and AQP3 activities in the plasma membrane. Snapin plays an important role as a linker between the water channel and the t-SNARE complex, leading to the fusion event, and the pairing with specific t-SNAREs is essential for the specificity of membrane recognition and fusion.

scholarly journals Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

2017 ◽  
Vol 28 (21) ◽  
pp. 2843-2853 ◽  
Author(s):  
Adrian J. Giovannone ◽  
Elena Reales ◽  
Pallavi Bhattaram ◽  
Alberto Fraile-Ramos ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Apical Plasma Membrane ◽  
Late Endosomes ◽  
Cargo Protein ◽  
Dominant Negative Inhibitor ◽  
Specific Proteins ◽  
Basolateral Plasma Membrane ◽  
Syntaxin 3

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion.

scholarly journals Apical targeting of syntaxin 3 is essential for epithelial cell polarity

2006 ◽  
Vol 173 (6) ◽  
pp. 937-948 ◽  
Author(s):  
Nikunj Sharma ◽  
Seng Hui Low ◽  
Saurav Misra ◽  
Bhattaram Pallavi ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cell ◽  
Membrane Trafficking ◽  
Cell Polarization ◽  
Apical Plasma Membrane ◽  
Membrane Expression ◽  
Tight Junction Formation ◽  
Necessary And Sufficient ◽  
Syntaxin 3

In polarized epithelial cells, syntaxin 3 localizes to the apical plasma membrane and is involved in membrane fusion of apical trafficking pathways. We show that syntaxin 3 contains a necessary and sufficient apical targeting signal centered around a conserved FMDE motif. Mutation of any of three critical residues within this motif leads to loss of specific apical targeting. Modeling based on the known structure of syntaxin 1 revealed that these residues are exposed on the surface of a three-helix bundle. Syntaxin 3 targeting does not require binding to Munc18b. Instead, syntaxin 3 recruits Munc18b to the plasma membrane. Expression of mislocalized mutant syntaxin 3 in Madin-Darby canine kidney cells leads to basolateral mistargeting of apical membrane proteins, disturbance of tight junction formation, and loss of ability to form an organized polarized epithelium. These results indicate that SNARE proteins contribute to the overall specificity of membrane trafficking in vivo, and that the polarity of syntaxin 3 is essential for epithelial cell polarization.

scholarly journals The SNARE Machinery Is Involved in Apical Plasma Membrane Trafficking in MDCK Cells

1998 ◽  
Vol 141 (7) ◽  
pp. 1503-1513 ◽  
Author(s):  
Seng Hui Low ◽  
Steven J. Chapin ◽  
Christian Wimmer ◽  
Sidney W. Whiteheart ◽  
László G. Kömüves ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Membrane Trafficking ◽  
Mdck Cells ◽  
Membrane Traffic ◽  
Cellular Membrane ◽  
Apical Plasma Membrane ◽  
Sensitive Factor ◽  
Syntaxin 3

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide–sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against α-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide–sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and α-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.

scholarly journals Mono-ubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

2017 ◽  
Author(s):  
Adrian J. Giovannone ◽  
Elena Reales ◽  
Pallavi Bhattaram ◽  
Alberto Fraile-Ramos ◽  
Thomas Weimbs
Keyword(s):  
Plasma Membrane ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
Apical Plasma Membrane ◽  
Late Endosomes ◽  
Cargo Protein ◽  
Dominant Negative Inhibitor ◽  
Specific Proteins ◽  
Basolateral Plasma Membrane ◽  
Syntaxin 3

AbstractSyntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes / lysosomes though how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes mono - ubiquitination in a conserved polybasic domain. Stx3 present at the basolateral – but not the apical - plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs) and excreted in exosomes. A non - ubiquitinatable mutant of Stx3 (Stx3 - 5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV / exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3 - 5R mutant acts as a dominant - negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3 - 5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploit this pathway for virion excretion.

scholarly journals Syntaxin 3 is necessary for cAMP- and cGMP-regulated exocytosis of CFTR: implications for enterotoxigenic diarrhea

2010 ◽  
Vol 299 (6) ◽  
pp. C1450-C1460 ◽  
Author(s):  
Anne Collaco ◽  
Jai Marathe ◽  
Hannes Kohnke ◽  
Dmitri Kravstov ◽  
Nadia Ameen
Keyword(s):  
Plasma Membrane ◽  
Cyclic Nucleotide ◽  
Chloride Channels ◽  
Fluid Secretion ◽  
Apical Plasma Membrane ◽  
Rat Jejunum ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Syntaxin 3

Enterotoxins elaborated by Vibrio cholerae and Escherichia coli cannot elicit fluid secretion in the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels. After enterotoxin exposure, CFTR channels are rapidly recruited from endosomes and undergo exocytic insertion into the apical plasma membrane of enterocytes to increase the number of channels on the cell surface by at least fourfold. However, the molecular machinery that orchestrates exocytic insertion of CFTR into the plasma membrane is largely unknown. The present study used immunofluorescence, immunoblotting, surface biotinylation, glutathione S-transferase (GST) pulldown assays, and immunoprecipitation to identify components of the exocytic soluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) vesicle fusion machinery in cyclic nucleotide-activated exocytosis of CFTR in rat jejunum and polarized intestinal Caco-2BBe cells. Syntaxin 3, an intestine-specific SNARE, colocalized with CFTR on the apical domain of enterocytes in rat jejunum and polarized Caco-2BBe cells. Coimmunoprecipitation and GST binding studies confirmed that syntaxin 3 interacts with CFTR in vivo. Moreover, heat-stable enterotoxin (STa) activated exocytosis of both CFTR and syntaxin 3 to the surface of rat jejunum. Silencing of syntaxin 3 by short hairpin RNA (shRNA) interference abrogated cyclic nucleotide-stimulated exocytosis of CFTR in cells. These observations reveal a new and important role for syntaxin 3 in the pathophysiology of enterotoxin-elicited diarrhea.

Expression of syntaxins in rat kidney

1997 ◽  
Vol 273 (5) ◽  
pp. F718-F730 ◽  
Author(s):  
Béatrice Mandon ◽  
Søren Nielsen ◽  
Bellamkonda K. Kishore ◽  
Mark A. Knepper
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Kidney Cortex ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Rt Pcr ◽  
Acid Base ◽  
Syntaxin 4 ◽  
Renal Collecting Duct ◽  
Syntaxin 3

Previously, we demonstrated that a putative vesicle-targeting protein, syntaxin-4, is expressed in renal collecting duct principal cells and is localized to the apical plasma membrane, suggesting a role in targeting aquaporin-2-containing vesicles to the apical plasma membrane. To investigate whether other syntaxin isoforms are present in the renal collecting duct, we determined the intrarenal localization of syntaxin-2 and -3. Reverse transcription-polymerase chain reaction (RT-PCR) experiments using total RNA extracted from kidney and various organs revealed that both syntaxin-2 and -3 are expressed in kidney cortex and medulla. RT-PCR experiments using microdissected tubules and vascular structures from the kidney revealed that syntaxin-3 mRNA, but not syntaxin-2, is expressed in collecting duct cells. Syntaxin-3 mRNA was also relatively abundant in the thick ascending limb of Henle’s loop and in vasa recta. Syntaxin-2 mRNA was found chiefly in glomeruli. To investigate the localization of syntaxin-3 protein, a peptide-derived polyclonal antibody was raised in rabbits. In immunoblotting experiments, this antibody labeled a 37-kDa protein in inner medulla that was most abundant in plasma membrane-enriched subcellular fractions. Immunoperoxidase labeling of thin cryosections combined with immunogold electron microscopy showed that, in contrast to the labeling seen for syntaxin-4, syntaxin-3 labeling in medullary collecting duct was predominantly in the basolateral plasma membrane of intercalated cells. These results suggest the possibility that syntaxin-3 may be involved in selective targeting of acid-base transporters and/or in basolateral membrane remodeling in response to systemic acid-base perturbations.

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