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At least 469 records · Page 26Linked to original sources
PubMed2001

G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry.

The nervous system can modulate neurotransmitter release by neurotransmitter activation of heterotrimeric GTP-binding protein (G protein)-coupled receptors. We found that microinjection of G protein betagamma subunits (Gbetagamma) mimics serotonin's inhibitory effect on neurotransmission. Release of free Gbetagamma was critical for this effect because a Gbetagamma scavenger blocked serotonin's effect. Gbetagamma had no effect on fast, action potential-evoked intracellular Ca2+ release that triggered neurotransmission. Inhibition of neurotransmitter release by serotonin was still seen after blockade of all classical Gbetagamma effector pathways. Thus, Gbetagamma blocked neurotransmitter release downstream of Ca2+ entry and may directly target the exocytotic fusion machinery at the presynaptic terminal.

Action Potentials↗
PubMed2003

Fusion of cells by flipped SNAREs.

The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis suggests that pairs of proteins known as vesicle (v-) SNAREs and target membrane (t-) SNAREs interact specifically to control and mediate intracellular membrane fusion events. Here, cells expressing the interacting domains of v- and t-SNAREs on the cell surface were found to fuse spontaneously, demonstrating that SNAREs are sufficient to fuse biological membranes.

Animals↗
PubMed1992

Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.

Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35 proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.

Amino Acid Sequence↗
PubMed2001

Control of fusion pore dynamics during exocytosis by Munc18.

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. All vesicle transport steps also have an essential requirement for a member of the Sec1 protein family, including the neuronal Munc18-1 (also known as nSec1) in regulated exocytosis. Here, in adrenal chromaffin cells, we expressed a Munc18 mutant with reduced affinity for syntaxin, which specifically modified the kinetics of single-granule exocytotic release events, consistent with an acceleration of fusion pore expansion. Thus, Munc18 functions in a late stage in the fusion process, where its dissociation from syntaxin determines the kinetics of postfusion events.

Action Potentials↗
PubMed1993

Characterization of a presynaptic glutamate receptor.

Glutamate receptors mediate excitatory neurotransmission in the brain and are important in the formation of memory and in some neurodegenerative disorders. A complementary DNA clone that encoded a 33-kilodalton protein (GR33) was obtained by screening a library with an antibody generated against glutamate binding proteins. The sequence of GR33 is identical to that of the recently reported presynaptic protein syntaxin. When GR33 was expressed in Xenopus oocytes, it formed glutamate-activated ion channels that are pharmacologically similar to those of N-methyl-D-aspartate receptors but with different electrophysiological properties. Mutation of the leucine 278 residue in the single putative transmembrane segment of GR33 affects the properties of the channel. Thus, in vivo GR33 may be a presynaptic glutamate receptor.

Animals↗
PubMed2002

Granuphilin modulates the exocytosis of secretory granules through interaction with syntaxin 1a.

The molecular mechanism for the regulated exocytosis of dense-core granules in endocrine cells remains relatively uncharacterized compared to that of synaptic vesicles in neurons. A novel set of Rab and its effector, Rab27a/granuphilin, which is localized on insulin granules in pancreatic beta cells, was recently identified. Here we demonstrate that granuphilin directly binds to syntaxin 1a on the plasma membrane, and this interaction is regulated by Rab27a. Granuphilin shows affinity to syntaxin 1a with a closed conformation but not to mutant syntaxin 1a, which adopts an open conformation constitutively. Overexpression of granuphilin significantly enhances basal insulin secretion but profoundly inhibits high K(+)-induced insulin secretion. The effect of granuphilin on insulin secretion was impaired by its mutation that disrupts the binding to either Rab27a or syntaxin 1a. Thus, granuphilin is the first regulator in the exocytotic pathway that functions by directly connecting two critical vesicle transport proteins, Rab and SNARE.

Animals↗
PubMed2004

Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells.

Exocytic insertion of H(+)-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H(+)-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H(+)-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 +/- 7% and increased the interaction between GFP-syntaxin 1A and H(+)-ATPase by 170 +/- 23%. Apical membrane Munc-18-2 decreased by 27.5 +/- 4.6% and H(+)-ATPase increased by 246 +/- 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H(+)-ATPase. In a pull-down assay of H(+)-ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H(+)-ATPase pulled down by 64 +/- 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H(+)-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H(+)-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H(+)-ATPase vesicles.

Adenosine Triphosphatases↗
PubMed1999

Syntaxin 1A inhibits regulated CFTR trafficking in xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302-305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopus oocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin's effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

Animals↗
PubMed2001

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells.

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.

Animals↗
PubMed1998

Characterization of rat epimorphin/syntaxin 2 expression suggests a role in crypt-villus morphogenesis.

The rodent intestinal mucosa undergoes a remarkable morphogenesis as the crypt-villus axis is formed. Endoderm-mesenchymal interactions play a critical role in this process. Epimorphin is a mesenchymal protein postulated to play a role in lung and skin morphogenesis. The rat homologue, syntaxin 2, belongs to a family of integral membrane proteins that function in vesicle docking and fusion. To clarify its role in fetal gut morphogenesis, epimorphin expression was examined during ontogeny, in an isograft model of ischemic injury and mucosal repair, and during intestinal adaptation after small bowel resection. Epimorphin/syntaxin 2 mRNA levels were increased in fetal gut during lumen formation and villus morphogenesis. mRNA levels remained elevated in the first 2 wk after birth and then declined at weaning. In situ hybridization showed epimorphin/syntaxin 2 mRNA in gestational day 14 (G14) and G15 intestinal mesenchymal cells and in the mucosal lamina propria during villus formation. Epimorphin/syntaxin 2 mRNA expression increased during villus repair in the isograft. In contrast, in the early stages of intestinal adaptation after small bowel resection, epimorphin/syntaxin 2 mRNA expression was suppressed in the adapting gut. We conclude the cell-specific and temporal patterns of epimorphin expression in the models used in this study suggest a role in the morphogenesis of the crypt-villus axis.

Aging↗
PubMed1999

Syntaxin 1A is transiently expressed in fetal lung mesenchymal cells: potential developmental roles.

Lung development is a complex process in which epithelial-mesenchymal interactions play a key role. A conserved secretory apparatus, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, is essential for exocytosis in many cell types. Syntaxins, located on the terminal plasma membrane (T-SNAREs), are a critical component of the secretosomal complex involved in vesicular docking, fusion, and exocytosis. We analyzed syntaxin 1A mRNA and protein in fetal rat lung ontogeny, demonstrating peak expression on about day 19 of embryonic development, immediately preceding type II pneumocyte differentiation. Syntaxin 1A is predominantly expressed by lipofibroblasts, which are required for bombesin-like peptide-induced surfactant phospholipid synthesis (choline uptake) by isolated type II cells. In organ cultures, anti-syntaxin 1A antibody HPC-1 blocks choline uptake both at baseline and when induced by bombesin-like peptide or dexamethasone. HPC-1 also promotes thymidine uptake in parallel in a dose-dependent fashion. These observations indicate a potential role for syntaxin 1A during fetal lung development, possibly through involvement in secretion of mesenchymal cell-derived factors that induce terminal type II cell differentiation.

Animals↗
PubMed2002

SNARE expression and localization in renal epithelial cells suggest mechanism for variability of trafficking phenotypes.

The apical- and basolateral-specific distribution of target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (t-SNAREs) of the syntaxin family appear to be critical for polarity in epithelial cells. To test whether differential SNARE expression and/or subcellular localization may contribute to the known diversity of trafficking phenotypes of epithelial cell types in vivo, we have investigated the distribution of syntaxins 2, 3, and 4 in epithelial cells along the renal tubule. Syntaxins 3 and 4 are restricted to the apical and basolateral domains, respectively, in all cell types, indicating that their mutually exclusive localizations are important for cell polarity. The expression level of syntaxin 3 is highly variable, depending on the cell type, suggesting that it is regulated in concert with the cellular requirement for apical exocytic pathways. While syntaxin 4 localizes all along the basal and lateral plasma membrane domains in vivo, it is restricted to the lateral membrane in Madin-Darby canine kidney (MDCK) cells in two-dimensional monolayer culture. When cultured as cysts in collagen, however, MDCK cells target syntaxin 4 correctly to the basal and lateral membranes. Unexpectedly, the polarity of syntaxin 2 is inverted between different tubule cell types, suggesting a role in establishing plasticity of targeting. The vesicle-associated (v)-SNARE endobrevin is highly expressed in intercalated cells and colocalizes with the H(+)-ATPase in alpha- but not beta-intercalated cells, suggesting its involvement in H(+)-ATPase trafficking in the former cell type. These results suggest that epithelial membrane trafficking phenotypes in vivo are highly variable and that different cell types express or localize SNARE proteins differentially as a mechanism to achieve this variability.

Animals↗
PubMed2004

Inhibition of ROMK potassium channel by syntaxin 1A.

ROMK potassium channels are present in the cortical collecting duct (CCD) of the kidney and serve as apical exit pathways for K+ secretion in this nephron segment. K+ secretion in the CCD is regulated by multiple factors. In this study, we show that syntaxin 1A, but not syntaxin 3 or 4, inhibited whole cell ROMK currents in Xenopus laevis oocytes. Syntaxin 1A, but not syntaxin 3 or 4, interacted with the COOH-terminal cytoplasmic domain of ROMK in intro. Coexpression with synaptobrevin 2 reversed inhibition of whole cell ROMK currents by syntaxin 1A. In excised inside-out membranes of oocytes, application of fusion proteins containing the cytoplasmic region of syntaxin 1A to the cytoplasmic face caused a dose-dependent inhibition of ROMK. We further examined regulation of the K+ channels in the CCD by syntaxin 1A. Application of botulinum toxin C1 to the excised inside-out membranes of the CCD caused an increase in the activity of K+ channels. In contrast, application of toxin B had no effects. These results suggest that syntaxin 1A causes a tonic inhibition of the K+ channels in the apical membrane of the CCD. Binding of synaptobrevin 2 to syntaxin 1A during docking and fusion of transport vesicles to the plasma membranes of CCD may lead to activation of these channels.

Animals↗
PubMed2004

ENaC subunit-subunit interactions and inhibition by syntaxin 1A.

Amiloride-sensitive epithelial Na(+) channels (ENaCs) are subject to modulation by many factors. Recent data have also linked the N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery to this regulation of ENaC, but the molecular mechanisms that underlie this modulation are poorly understood. In this study, we demonstrate that syntaxin 1A physically interacts with ENaC and functionally regulates ENaC activity. Syntaxin 1A was able to coimmunoprecipitate in vitro-translated gamma-ENaC, but not alpha- or beta-ENaC. Also, using antibodies raised against alpha-, beta-, or gamma-ENaC, we detected syntaxin 1A in immunoprecipitates from Madin-Darby canine kidney cells stably transfected with alphabetagamma-ENaC. In bilayers, syntaxin 1A inhibited ENaC, and this syntaxin 1A modulation of ENaC activity was eliminated by truncations of cytoplasmic domains of the ENaC subunits. Our findings provide evidence for a direct physical interaction between ENaC and syntaxin 1A and suggest involvement of ENaC's cytoplasmic domains in functional modulation of ENaC activity by syntaxin 1A.

Animals↗
PubMed1997

Expression of syntaxins in rat kidney.

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.

Amino Acid Sequence↗
PubMed2005

Epimorphin overexpression in the mouse mammary gland promotes alveolar hyperplasia and mammary adenocarcinoma.

Epimorphin/syntaxin-2 (EPM) is a plasma membrane-anchored protein that has at least two distinct functions depending on its membrane topology: vesicle fusion when localized to the cytoplasmic surface and morphogenic signaling when localized to the extracellular surface. Transgenic mice that express full-length extracellular EPM fused to the NH2-terminal signal sequence of interleukin-2, under the control of the whey acidic protein (WAP) gene promoter, exhibit aberrant mammary gland morphogenesis associated with increased expression of CCAAT enhancer binding protein beta (C/EBPbeta). Here we report that aged nulliparous and uniparous female WAP-EPM transgenic mice develop alveolar hyperplasias and well-differentiated adenocarcinomas that express high levels of C/EBPbeta, keratin-14, matrix metalloproteinase-3, and beta-catenin. This study reveals another pathway in which overexpression and alteration of a normal morphogenic process promote the development of cancer in the mammary gland.

Adenocarcinoma↗
PubMed1998

Hemizygous deletion of the HPC-1/syntaxin 1A gene (STX1A) in patients with Williams syndrome.

HPC-1/syntaxin 1A is a membrane protein that plays an important role in exocytosis of neurotransmitters from neuronal cells. We previously mapped the human HPC-1/syntaxin 1A gene (STX1A) to chromosome 7q11.2, which is within the Williams syndrome (WS) region. Here, we performed FISH analysis on 46 patients with WS to examine the relationship between STX1A and WS. Our results showed a hemizygous deletion of the HPC-1/syntaxin 1A gene in each patient, suggesting that the neurological symptoms of WS may be related to the hemizygous deletion of STX1A.

Adolescent↗