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PubMed2004

Syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.

The secretion of lung surfactant in alveolar type II cells is a complex process involving the fusion of lamellar bodies with the plasma membrane. This process is somewhat different from the exocytosis of hormones and neurotransmitters. For example, it is a relatively slower process, and lamellar bodies are very large vesicles with a diameter of approximately 1 microm. SNARE proteins are the conserved molecular machinery of exocytosis in the majority of secretory cells. However, their involvement in surfactant secretion has not been reported. Here, we showed that syntaxin 2 and SNAP-23 are expressed in alveolar type II cells. Both proteins are associated with the plasma membrane, and to some degree with lamellar bodies. An antisense oligonucleotide complementary to syntaxin 2 decreased its mRNA and protein levels. The same oligonucleotide also inhibited surfactant secretion, independent of secretagogues. A peptide derived from the N-terminus of syntaxin 2 or the C-terminus of SNAP-23 significantly inhibited Ca(2+)- and GTPgammaS-stimulated surfactant secretion from permeabilized type II cells in a dose-dependent manner. Furthermore, introduction of anti-syntaxin 2 or anti-SNAP-23 antibodies into permeabilized type II cells also inhibited surfactant release. Our results suggest that syntaxin 2 and SNAP-23 are required for regulated surfactant secretion.

Amino Acid Sequence↗

PubMed2004

Activation of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, by phorbol 12-myristate 13-acetate (PMA) suppresses glucose transport into astroglioma cells via the glucose transporter-1 (GLUT-1).

Syntaxin 1C is an alternative splice variant lacking the transmembrane domain of HPC-1/syntaxin 1A. We found previously that syntaxin 1C is expressed as a soluble protein in human astroglioma (T98G) cells, and syntaxin 1C expression is enhanced by stimulation with phorbol 12-myristate 13-acetate (PMA). However, the physiological function of syntaxin 1C is not known. In this study, we examined the relationship between syntaxin 1C and glucose transport. First, we discovered that glucose transporter-1 (GLUT-1) was the primary isoform in T98G cells. Second, we demonstrated that glucose uptake in T98G cells was suppressed following an increase in endogenous syntaxin 1C after stimulation with PMA, which did not alter the expression levels of other plasma membrane syntaxins. We further examined glucose uptake and intracellular localization of GLUT-1 in cells that overexpressed exogenous syntaxin 1C; glucose uptake via GLUT-1 was inhibited without affecting sodium-dependent glucose transport. The value of Vmax for the dose-dependent uptake of glucose was reduced in syntaxin 1C-expressing cells, whereas there was no change in Km. Immunofluorescence studies revealed a reduction in the amount of GLUT-1 in the plasma membrane in cells that expressed syntaxin 1C. Based on these results, we postulate that syntaxin 1C regulates glucose transport in astroglioma cells by changing the intracellular trafficking of GLUT-1. This is the first report to indicate that a syntaxin isoform that lacks a transmembrane domain can regulate the intracellular transport of a plasma membrane protein.

Alternative Splicing↗

PubMed2004

One-trial aversive learning induces late changes in hippocampal CaMKIIalpha, Homer 1a, Syntaxin 1a and ERK2 protein levels.

Most studies regarding altered gene expression after learning are performed using multi-trial tasks, which do not allow a clear discrimination of memory acquisition, consolidation and retrieval. We screened for candidate memory-modulated genes in the hippocampus at 3 and 24 h after one-trial inhibitory avoidance (IA) training, using a cDNA array containing 1176 genes. While 33 genes were modulated by training (respect to shocked-only animals), most of them were upregulated (27 genes) and only 6 were downregulated. To confirm and extend these findings, we performed RT-PCRs and analyzed differences in protein levels in rat hippocampus using immunoblot assays. We found several proteins upregulated 24 h after training: extracellular signal-regulated kinase ERK2, Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha), Syntaxin 1a, c-fos and Homer 1a. The total level of none of these proteins were found to be altered when measured 3-h post-training. Several of the mRNAs corresponding to the upregulated proteins were changed at 3 h but not 24 h. Additionally, a number of other candidates were identified for the first time as modulated by learning. The results presented here suggest that single-trial tasks can expose previously unseen differences in dynamic regulation of gene expression after behavioral manipulations, both at the transcriptional and translational levels, and reveal a diversity of gene products modulated by this task, allowing deeper understanding of the molecular basis of memory formation.

Animals↗

PubMed2000

Definition of a minimal munc18c domain that interacts with syntaxin 4.

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein-protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225), but not munc18c(226-592), munc18c(1-100), munc18c(43-139) or munc18c(66-139), interacted with the cytoplasmic portion of syntaxin 4, Stx4(2-273), as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by beta-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx4(29-157), failed to interact with full-length munc18c(1-592), indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein-protein interaction studies between Stx4(2-273) and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c(1-592), munc18c(1-139) and munc18c(1-225) interacted with Stx4(2-273) whereas munc18c(1-100) did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

Animals↗

PubMed1997

Phosphorylation of the synaptic protein interaction site on N-type calcium channels inhibits interactions with SNARE proteins.

The synaptic protein interaction (synprint) site on the N-type calcium channel alpha1B subunit binds to the soluble N-ethylmaleimide-sensitive attachment factor receptor (SNARE) proteins syntaxin and synaptosomal protein of 25 kDa (SNAP-25), and this association may be required for efficient fast synaptic transmission. Protein kinase C (PKC) and calcium and calmodulin-dependent protein kinase type II (CaM KII) phosphorylated a recombinant his-tagged synprint site polypeptide rapidly to a stoichiometry of 3-4 mol of phosphate/mol, whereas cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) phosphorylated the synprint peptide more slowly to a stoichiometry of <1 mol/mol. Two-dimensional phosphopeptide mapping revealed similar patterns of phosphorylation of synprint polypeptides and native rat brain N-type calcium channel alpha1B subunits by PKC and Cam KII. Phosphorylation of the synprint peptide with PKC or CaM KII, but not PKA or PKG, strongly inhibited binding of recombinant syntaxin or SNAP-25, even at a level of free calcium (15 microM) that stimulates maximal binding. In contrast, phosphorylation of syntaxin and SNAP-25 with PKC and CaM KII did not affect interactions with the synprint site. Binding assays with polypeptides representing the N- and C-terminal halves of the synprint site indicate that the PKC- and CaM KII-mediated inhibition of binding involves multiple, disperse phosphorylation sites. PKC or CaM KII phosphorylation of the synprint peptide also inhibited its interactions with native rat brain SNARE complexes containing syntaxin and SNAP-25. These results suggest that phosphorylation of the synprint site by PKC or CaM KII may serve as a biochemical switch for interactions between N-type calcium channels and SNARE protein complexes.

Animals↗

PubMed2002

Mechanism of calcium-independent synaptotagmin binding to target SNAREs.

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, we purified all four proteins from brain membranes and analyzed their interactions. Our study reveals that, in the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. We now show that both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding.

Animals↗

PubMed2001

Mastoparan stimulates GABA release from MIN6 cells: relationship between SNARE proteins and mastoparan action.

We examined the action of mastoparan on beta cell exocytosis. Mastoparan stimulated GABA and insulin release from MIN6 beta cells. On the other hand, mastopraran-induced GABA release was decreased by expressing the tetanus toxin C1 light chain in MIN6 cells. We have then investigated the relationship between SNARE proteins and mastoparan action using adenovirus-mediated gene transfer system. Overexpression of t-SNAREs, syntaxin 1A, and SNAP-25 inhibited the mastoparan-induced insulin release by approximately half-fold of control levels, however, the mastoparan-induced GABA release was not affected by these t-SNAREs overexpression. The overexpression of mutant alpha-SNAP (1-285), which inhibits the wild-type alpha-SNAP function in a dominant negative manner, did not influence either mastoparan-induced GABA or insulin release in spite of its marked inhibition of glucose-stimulated insulin release. Our data indicate that mastoparan stimulates GABA exocytosis via vesicular transport; however, SNARE proteins are differently involved in the exocytosis of insulin and GABA.

Animals↗

PubMed2004

Association between schizophrenia and the syntaxin 1A gene.

BACKGROUND: Both microarray and candidate molecule studies have demonstrated that protein and mRNA expression of syntaxin and other genes involved in synaptic function are altered in the cerebral cortex of patients with schizophrenia. METHODS: Genetic association between polymorphic markers in the syntaxin 1A gene and schizophrenia was assessed in a matched case-control sample of 192 pairs, and in an independent sample of 238 nuclear families. RESULTS: In the family-based sample, a significant genetic association was found between schizophrenia and one of the four single nucleotide polymorphisms (SNPs) tested: an intron 7 SNP (transmission disequilibrium test [TDT] chi(2) = 5.898; df = 1; p =.015, family-based association test [FBAT] z = 2.280, p =.023). When the results for the TDT and case-control analyses were combined, the association was stronger (n = 430; z(c) = 2.859; p =.004). Haplotype analysis supported the association with several significant values that appear to be driven by the intron 7 SNP. CONCLUSIONS: The results should be treated with caution until replicated, but this is the first report of a genetic association between syntaxin 1A and schizophrenia.

Adult↗

PubMed2003

Structure of the GAT domain of human GGA1: a syntaxin amino-terminal domain fold in an endosomal trafficking adaptor.

The Golgi-associated, gamma-adaptin homologous, ADP-ribosylation factor (ARF)-interacting proteins (GGAs) are adaptors that sort receptors from the trans-Golgi network into the endosomallysosomal pathway. The GGAs and TOM1 (GAT) domains of the GGAs are responsible for their ARF-dependent localization. The 2.4-A crystal structure of the GAT domain of human GGA1 reveals a three-helix bundle, with a long N-terminal helical extension that is not conserved in GAT domains that do not bind ARF. The ARF binding site is located in the N-terminal extension and is separate from the core three-helix bundle. An unanticipated structural similarity to the N-terminal domain of syntaxin 1a was discovered, comprising the entire three-helix bundle. A conserved binding site on helices 2 and 3 of the GAT domain three-helix bundle is predicted to interact with coiled-coil-containing proteins. We propose that the GAT domain is descended from the same ancestor as the syntaxin 1a N-terminal domain, and that both protein families share a common function in binding coiled-coil domain proteins.

ADP-Ribosylation Factor 1↗

PubMed2000

Expression of synaptic vesicle trafficking proteins in the developing rat pineal gland.

Adult mammalian pinealocytes contain several synaptic membrane proteins that are probably involved in the regulation of targeting and exocytosis of synaptic-like microvesicles (SLMVs). Immunohistochemical techniques have now demonstrated the spatiotemporal expression pattern of some of these proteins during rat pineal ontogenesis. Various synaptic vesicle trafficking proteins are detectable in proliferating epithelial cells of the pineal anlage even at embryonic day 17.5 (E 17.5), with the exception of syntaxin I (weakly expressed from E 19.5) and dynamin I (whose levels increase markedly during the first postnatal week). Numerous cells exhibiting strong immunoreactivity for synaptobrevin II, SNAP-25, synaptophysin, and munc-18-1 are distributed throughout the increasingly compact gland at E 19.5 and E 20.5; however, their number declines toward the proximal deep part of the organ. Groups of postmitotic cells situated at the surface of the developing gland exhibit marked immunoreactivity for the aforementioned proteins and lie close to the laminin-immunoreactive outer limiting basement membrane or to its remnants in regions of basement membrane dissolution. We also show that synthesis of vimentin and S-antigen seems to begin earlier during pineal development than previously recognized. Thus, synaptic vesicle trafficking proteins are the earliest molecular markers of pinealocyte differentiation known to date, being expressed well before the onset of rhythmic hormone secretion in the pineal gland, where they may play a role in morphogenetic events. Components of the extracellular matrix such as laminin may be critically involved in the upregulation of synaptic membrane protein expression. The dynamin immunostaining pattern indicates that SLMVs of pinealocytes begin to undergo regulated cycles of exo/endocytosis during postnatal week 1.

Animals↗

PubMed1999

Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation.

Various proteins regulating neurotransmission release and synaptic vesicle exocytosis have been implicated in axonal elongation and synaptic maturation. In the present study, immunohistochemistry to the presynaptic membrane proteins syntaxin-I and synaptosomal-associated protein of 25 kDa (SNAP-25) synaptic vesicle-associated proteins synaptophysin and synapsin-I and the neuronal maturation and axonal growth-related protein GAP-43, has been carried out in the normal developing cerebellum and following a single dose of ionizing radiation (2 Gy gamma-rays) at postnatal day 1. Our aim has been to learn about the morphological and possible functional modalities that occur during the progression of neuronal connectivity in normal and abnormal development. Expression of all these proteins is associated with the arrival of afferents in the subcortical white matter and with the maturation of the internal granule cell layer and molecular layer during normal development. In addition, SNAP-25 and GAP-43 are strongly expressed in granule cells of the external granule cell layer, thus suggesting that these proteins are involved in cell elongation of granule cells. Apoptosis appears at 3 h and peaks at 6 h following ionizing radiation. Radiation-induced apoptosis in the external granule cell layer produces a transient decrease in the expression of SNAP-25 and GAP-43 in the external granule cell layer. The external granule cell layer recovers at 48 h and external granule cells of proliferating cells also express SNAP-25 and GAP-43, thus indicating that proliferating cells in this layer are equipped with proteins involved in cell elongation. Furthermore, expression of synaptophysin, synapsin-I, syntaxin-I and SNAP-25 is the same in the cerebellum of irradiated and normal rats from this time to adulthood (3 months). These results point to the likelihood that recovery of the cerebellar cortex occurs following a single exposure of ionizing radiation during postnatal development.

Aging↗

PubMed2004

Altered interaction and expression of proteins involved in neurosecretion in scrapie-infected GT1-1 cells.

Prions cause transmissible and fatal diseases that are associated with spongiform degeneration, astrogliosis, and loss of axon terminals in the brains. To determine the expression of proteins involved in neurosecretion and synaptic functions after prion infection, gonadotropin-releasing hormone neuronal cell line subclone (GT1-1) was infected with the RML scrapie strain and analyzed by Western blotting, real time PCR, and immunohistochemistry. As revealed by Western blotting of lysates exposed to different temperatures, the levels of complexed SNAP-25, syntaxin 1A, and synaptophysin were decreased in scrapie-infected GT1-1 cells (ScGT1-1), whereas the level of monomeric forms of these proteins was increased and correlated to the level of scrapie prion protein (PrPSc). However, when complex formation was prevented by prolonged heating of samples in SDS, the levels of monomeric SNAP-25, syntaxin 1A and synaptophysin in ScGT1-1 cells were decreased in comparison to GT1-1 cells. The reduced level of SNAP-25 was observed as early as 32 days postinfection. Increased mRNA levels of both splice variants SNAP-25a and -b in ScGT1-1 cells were seen. No difference in the morphology, neuritic outgrowth or distribution of SNAP-25, syntaxin 1A, or synaptophysin could be observed in ScGT1-1 cells. Treatment with quinacrine or pentosan polysulfate cleared the PrPSc from the ScGT1-1 cell cultures, and the increase in levels of monomeric SNAP-25 and synaptophysin was reversible. These results indicate that a scrapie infection can cause changes in the expression of proteins involved in neuronal secretion, which may be of pathogenetic relevance for the axon terminal changes seen in prion-infected brains.

1-Methyl-3-isobutylxanthine↗

PubMed1998

Pth1/Vam3p is the syntaxin homolog at the vacuolar membrane of Saccharomyces cerevisiae required for the delivery of vacuolar hydrolases.

The PEP12 homolog Pth1p (Pep twelve homolog 1) is predicted to be similar in size to Pep12p, the endosomal syntaxin homolog that mediates docking of Golgi-derived transport vesicles and, like other members of the syntaxin family, is predicted to be a cytoplasmically oriented, integral membrane protein with a C-terminal transmembrane domain. Kinetic analyses indicate that deltapth1/vam3 mutants fail to process the soluble vacuolar hydrolase precursors and that PrA, PrB and most of CpY accumulate within the cell in their Golgi-modified P2 precursor forms. This is in contrast to a pep12 mutant in which P2CpY is secreted from the cell. Furthermore, pep12 is epistatic to pth1/vam3 with respect to the CpY secretion phenotype. Alkaline phosphatase, a vacuolar membrane hydrolase, accumulates in its precursor form in the deltapth1/vam3 mutant. Maturation of pro-aminopeptidase I, a hydrolase precursor delivered directly to the vacuole from the cytoplasm, is also blocked in the deltapth1/vam3 mutant. Subcellular fractionation localizes Pth1/Vam3p to vacuolar membranes. Based on these data, we propose that Pth1/Vam3p is the vacuolar syntaxin/t-SNARE homolog that participates in docking of transport vesicles at the vacuolar membrane and that the function of Pth1/Vam3p impinges on at least three routes of protein delivery to the yeast vacuole.

Amino Acid Sequence↗

PubMed2002

Spatio-temporal heterogeneity and cell-specificity of long-term potentiation-induced mRNA expression in the dentate gyrus in vivo.

Gene expression in neurones can vary in response to neuronal activation. In this study, to analyse the spatio-temporal dynamics of the transcriptional response of three genes following the induction of long-term potentiation within the entire dentate gyrus in vivo, two new complementary approaches based on in situ hybridisation were developed: three-dimensional reconstruction of the pattern of mRNA expression within the entire dentate gyrus; and radioactive co-detection of two mRNA species allowing quantification of two different mRNAs in the same brain section. Zif268, Homer and syntaxin 1B genes were studied, and their regulated expression was examined three times after the induction of long-term potentiation. Constitutive expression of each gene under control conditions was homogeneous, but the spatial distribution of mRNA was heterogeneous along the rostro-caudal axis of the dentate gyrus following the induction of long-term potentiation, and different for each gene. In addition, the intensity of each gene-specific pattern of expression varied over time following the induction of long-term potentiation. Our results reveal that long-term potentiation differentially modulates the expression of mRNA species in cells of the dentate gyrus depending on their position along the rostro-caudal axis, on the gene and on time. We suggest that there are several molecular mechanisms of long-term potentiation, differing from one cluster of cells of the dentate gyrus to another, or that the different signaling pathways involved in long-term potentiation are used with varying efficiencies by different cells.

Animals↗

PubMed2003

Syntaxin 1A binds to the cytoplasmic C terminus of Kv2.1 to regulate channel gating and trafficking.

Voltage-gated K(+) (Kv) 2.1 is the dominant Kv channel that controls membrane repolarization in rat islet beta-cells and downstream insulin exocytosis. We recently showed that exocytotic SNARE protein SNAP-25 directly binds and modulates rat islet beta-cell Kv 2.1 channel protein at the cytoplasmic N terminus. We now show that SNARE protein syntaxin 1A (Syn-1A) binds and modulates rat islet beta-cell Kv2.1 at its cytoplasmic C terminus (Kv2.1C). In HEK293 cells overexpressing Kv2.1, we observed identical effects of channel inhibition by dialyzed GST-Syn-1A, which could be blocked by Kv2.1C domain proteins (C1: amino acids 412-633, C2: amino acids 634-853), but not the Kv2.1 cytoplasmic N terminus (amino acids 1-182). This was confirmed by direct binding of GST-Syn-1A to the Kv2.1C1 and C2 domains proteins. These findings are in contrast to our recent report showing that Syn-1A binds and modulates the cytoplasmic N terminus of neuronal Kv1.1 and not by its C terminus. Co-expression of Syn-1A in Kv2.1-expressing HEK293 cells inhibited Kv2.1 surfacing, which caused a reduction of Kv2.1 current density. In addition, Syn-1A caused a slowing of Kv2.1 current activation and reduction in the slope factor of steady-state inactivation, but had no affect on inactivation kinetics or voltage dependence of activation. Taken together, SNAP-25 and Syn-1A mediate secretion not only through its participation in the exocytotic SNARE complex, but also by regulating membrane potential and calcium entry through their interaction with Kv and Ca(2+) channels. In contrast to Ca(2+) channels, where these SNARE proteins act on a common synprint site, the SNARE proteins act not only on distinct sites within a Kv channel, but also on distinct sites between different Kv channel families.

Animals↗

PubMed1997

Botulinum neurotoxin B inhibits insulin-stimulated glucose uptake into 3T3-L1 adipocytes and cleaves cellubrevin unlike type A toxin which failed to proteolyze the SNAP-23 present.

Types A, B, and C1 botulinum neurotoxin (BoNT), a group of selective Zn2+-dependent endoproteases, have been instrumental in demonstrating that their respective substrates [synaptosomal-associated protein with Mr = 25 kDa (SNAP-25), synaptobrevin (Sbr), and syntaxin] are essential for regulated exocytosis from nerve terminals and neuroendocrine cells. The colocalization of Sbr, or its homologue cellubrevin (Cbr), in the majority of the glucose transporter-isotype 4 (GLUT4)-containing vesicles from adipocytes implicates their involvement in insulin-stimulated glucose uptake, which results in part from enhanced fusion of these vesicles with the plasmalemma. In this study, exposure of cultured 3T3-L1 adipocytes to BoNT/B in a low-ionic strength medium was found to block insulin-evoked glucose uptake by up to 64%. BoNT/B was shown by immunoblotting to cause extensive proteolysis of Cbr and Sbr resulting in a significant blockade of the insulin-stimulated translocation of GLUT4 to the plasmalemma. This establishes that these two toxin substrates contribute to the insulin-regulated fusion of GLUT4-containing vesicles with the plasmalemma, at least in this differentiated 3T3-L1 clone. Although SNAP-25 was not detectable in the differentiated adipocytes, its functional homologue SNAP-23 is abundant and largely confined to the plasmalemma. SNAP-23 proved to be resistant to cleavage by BoNT/A. Consistent with these results, type A did not block insulin-induced glucose uptake, precluding a demonstration of its likely importance in this process.

3T3 Cells↗

PubMed1999

Vesicle-associated proteins and P2X receptor clusters at single sympathetic varicosities in mouse vas deferens.

1. Antibodies against vesicle-associated proteins of the SNARE complex (syntaxin (AbS), SNAP 25 (AbS25), synaptobrevin (VAMP; AbV) and the alpha1B subunit of calcium channels (Abalpha(1B)) were located with respect to sympathetic varicosities (labelled with the ubiquitous vesicle proteoglycan antibody AbSV2) and to clusters of P2X receptor subunits (labelled with antibodies AbP2X(1) to AbP2X(6)). In addition, these receptor clusters were located with respect to Schwann cells labelled with antibodies to S100 (AbS100).2. The spatial relation between proteins of the SNARE complex and calcium channels was determined. AbS25 patches ranged from 250-500 nm in size and were often colocalised with smaller AbS patches (250-350 nm). Abalpha(1B) patches (300-700 nm diameter) were always coincidental with AbS patches. AbV patches (400-1000 nm in diameter) also coincided with AbS patches.3. The spatial relation between different P2X subunit clusters and varicosities labelled with AbSV2 was ascertained. Large (500-700 nm diameter) AbP2X(1) receptor clusters were found colocalised with many (91%) AbSV2 labelled varicosities, although small diameter (250-350 nm) AbP2X(1) clusters occurred at random over the muscle. Small AbP2X(2) clusters were found uniquely in the vicinity of AbSV2 labelled varicosities, but were not entirely coincidental with these. Small AbP2X(3) receptor clusters were found in close association with AbSV2 labelled nerves. Small diameter AbP2X(4) clusters (250-350 nm) were found throughout the muscle with some of these coincidental with AbSV2 labelling. Small diameter AbP2X(5) (250-350 nm) cluster labelling was found in juxtaposition to strings of AbSV2 labelled varicosities but were not coincidental with these. Small (250-350 nm) diameter AbP2X(6) clusters were also found in close juxtaposition to AbSV2 labelled nerves.4. The spatial relation between different P2X subtype clusters and Schwann cells labelled with AbS100 was examined. Both AbP2X(1) and AbP2X(3) receptor clusters were found in close apposition with AbS100, with clusters of the former sometimes coincidental with patches of the latter. On the other hand AbP2X(2) was found in association with AbS100 at low levels while AbP2X(4) labelling was generally not coincidental with AbS100. AbP2X(5) and AbP2X(6) labelling was often colocalised with AbS100 labelling.5. The spatial relation between proteins of the SNARE complex and P2X(1) receptors was determined. Large AbP2X(1) clusters were often found apposed by AbS, AbV and Abalpha(1B) labelled patches.6. Destruction of the sympathetic varicosities with 6-hydroxydopamine led to the virtual disappearance of AbP2X(2) labelling, but to a large increase in the number of small AbP2X(1) receptor clusters and a reduction in the number of large AbP2X(1) clusters. AbS100 Schwann cell labelling was largely unaffected.7. These observations are interpreted as showing that most terminal sympathetic varicosities possess active zones about 250-700 nm diameter, delineated by syntaxin, SNAP 25 and N-type calcium channels and that synaptic vesicles are concentrated at these sites as indicated by the localisation of VAMP. Most of these terminal varicosities possess active zones that are precisely apposed to large clusters of P2X(1) receptors. However small clusters of P2X(2) to P2X(6) receptors can be found that are near the strings of varicosities but not usually coincidental with them except P2X(3). The functional significance of this arrangement of vesicle-associated proteins and P2X receptors for the generation of synaptic potentials at the autonomic neuromuscular junction is discussed.

Animals↗

PubMed2004

[Methodological consideration in studying the exocytosis mechanisms using microdialysis].

Microdialysis has become an effective and frequently used technique to study the extracellular levels of monoamine, i.e. dopamine, serotonin and norepinephrine in the central nervous system. However, the detailed exocytosis mechanisms of monoamine using microdialysis has remained to be clarified. The present report introduces methods for administration of voltage-sensitive calcium channel (VSCC) inhibitors and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) inhibitors to clarify the mechanisms of monoamine exocytosis using in vivo microdialysis. The N-type and P-type VSCCs are inhibited by perfusion with omega-conotoxin GVIA and omega-agatoxin IVA, respectively; however, their diffusion rate from internal to external spaces of the microdialysis probe is lower than 1%. Unlike VSCC inhibitors, SNAP-25, synaptobrevin and syntaxin can be cleavaed with botulinum toxin type A, B and C, respectively. These toxins (with molecular weights over 500,000) bind specifically to the presynaptic membrane via the heavy chain, while the light chain enters the cytosol, where it displays zinc-endopeptidase activity directed to proteins of the neuroexocytosis apparatus. Therefore, to prevent SNARE activity, botulinum toxins are microinjected. These two methods, perfusion with VSCC inhibitors and microinjection with botulinum toxins, can contribute to the clarification of the mechanisms of monoaminergic exocytosis.

Animals↗