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Kimio Akagawa - One of the best experts on this subject based on the ideXlab platform.
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HPC-1/Syntaxin 1A and Syntaxin 1B play distinct roles in neuronal survival.
Journal of neurochemistry, 2014Co-Authors: Takefumi Kofuji, Tatsuya Mishima, Tomonori Fujiwara, Masumi Sanada, Kimio AkagawaAbstract:Two types of Syntaxin 1 isoforms, HPC-1/Syntaxin 1A (STX1A) and Syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A(-/-) mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B(-/-) mice using targeted gene disruption and investigated their phenotypes. STX1B(-/-) mice were born alive, but died before postnatal day 14, unlike STX1A(-/-) mice. Morphologically, brain development in STX1B(-/-) mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B(-/-) neurons was lower than that of WT or STX1A(-/-) neurons after 9 days. Interestingly, STX1B(-/-) neurons survived on WT or STX1A(-/-) glial feeder layers as well as WT neurons. However, STX1B(-/-) glial feeder layers were less effective at promoting survival of STX1B(-/-) neurons. Conditioned medium from WT or STX1A(-/-) glial cells had a similar effect on survival, but that from STX1B(-/-) did not promote survival. Furthermore, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 supported survival of STX1B(-/-) neurons. BDNF localization in STX1B(-/-) glial cells was disrupted, and BDNF secretion from STX1B(-/-) glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. Syntaxin 1A (STX1A) and Syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A(-/-) mice and STX1B(-/-) mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.
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Impairment of Catecholamine Systems during Induction of Long-Term Potentiation at Hippocampal CA1 Synapses in HPC-1/Syntaxin 1A Knock-out Mice
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2012Co-Authors: Tatsuya Mishima, Tomonori Fujiwara, Takefumi Kofuji, Kimio AkagawaAbstract:The membrane protein HPC-1/Syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/Syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/Syntaxin 1A knock-out (STX1A(-/-)) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A(-/-) mice. Because HPC-1/Syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A(-/-) mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/Syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/Syntaxin 1A causes impairment of LTP induction.
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loss of granuphilin and loss of Syntaxin 1A cause differential effects on insulin granule docking and fusion
Journal of Biological Chemistry, 2011Co-Authors: Hao Wang, Kimio Akagawa, Tomonori Fujiwara, Ray Ishizaki, Eri Kobayashi, Tetsuro IzumiAbstract:The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with Syntaxin-1A on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both Syntaxin-1A- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and Syntaxin-1A null pancreatic β cells derived from mice having the same genetic background. We found that loss of Syntaxin-1A does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/Syntaxin-1A double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of Syntaxin-1A null mice, including their granule-docking behavior. Because granuphilin binds to Syntaxin-2 and Syntaxin-3 as well as Syntaxin-1A, it likely mediates granule docking through interactions with those multiple Syntaxins on the plasma membrane.
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analysis of knock out mice to determine the role of hpc 1 Syntaxin 1A in expressing synaptic plasticity
The Journal of Neuroscience, 2006Co-Authors: Tomonori Fujiwara, Tatsuya Mishima, Takefumi Kofuji, Tomoki Chiba, Keiji Tanaka, Akitsugu Yamamoto, Kimio AkagawaAbstract:The protein HPC-1/Syntaxin 1A is abundantly expressed in neurons and localized in the neuronal plasma membrane. It forms a complex with SNAP-25 (25 kDa synaptosomal-associated protein) and VAMP-2 (vesicle-associated membrane protein)/synaptobrevin called SNARE (a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex, which is considered essential for synaptic vesicle exocytosis; thus, HPC-1/Syntaxin 1A is considered crucial for synaptic transmission. To examine the physiological function of HPC-1/Syntaxin 1A in vivo, we produced knock-out (KO) mice by targeted gene disruption. Although HPC-1/Syntaxin 1A expression was completely depleted without any effect on the expression of other SNARE proteins, the KO mice were viable. They grew normally, were fertile, and displayed no difference in appearance compared with control littermate. In cultured hippocampal neurons derived from the KO mice, the basic synaptic transmission in vitro was normal. However, the mutant mice had impaired long-term potentiation in the hippocampal slice. Also, although KO mice exhibited normal spatial memory in the hidden platform test, consolidation of conditioned fear memory was impaired. Interestingly, the KO mice had impaired conditioned fear memory extinction. These observations suggest that HPC-1/Syntaxin 1A may be closely related to synaptic plasticity.
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identification of the carboxyl terminal membrane anchoring region of hpc 1 Syntaxin 1A with the substituted cysteine accessibility method and monoclonal antibodies
Journal of Biochemistry, 2003Co-Authors: Kei Suga, Tetsuo Yamamori, Kimio AkagawaAbstract:HPC-1/Syntaxin 1A is a member of the Syntaxin family, and functions at the plasma membrane during membrane fusion as the target-soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (t-SNARE). We identified the membrane-anchoring region of HPC-1/Syntaxin 1A, and examined its role in anchoring of a protein to the plasma membrane. A series of mutants was created from a cysteine-less mutant of HPC-1/Syntaxin 1A by substitution of each residue at the C-terminus with cysteine. The accessibility of the thiol-groups in each mutant was analyzed in vivo. The cysteine (C145) within the N-terminal cytosolic segment was labeled, but not that at C271 or C272, or any of those introduced at the C-terminus. The addition of additional residues to the C-terminal tail of HPC-1/Syntaxin 1A allowed labeling by thiol-specific reagents. A monoclonal antibody directed against the C-terminal tail peptide did not react with the protein located at the plasma membrane. In addition, subcellular fractionation and immunocytochemical analyses with various transmembrane mutants showed that the C-terminal tail comprising eight amino acids is essential for anchoring of HPC-1/Syntaxin 1A to the plasma membrane. These results indicate that the C-terminal membrane-anchoring region, which comprises 23 amino acids, does not traverse the lipid-bilayer and that the C-terminal tail is essential for anchoring of HPC-1/Syntaxin 1A to the plasma membrane.
Herbert Y Gaisano - One of the best experts on this subject based on the ideXlab platform.
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new roles of Syntaxin 1A in insulin granule exocytosis and replenishment
Journal of Biological Chemistry, 2017Co-Authors: Tao Liang, Youhou Kang, Michael B Wheeler, Lucy R Osborne, Tairan Qin, Li Xie, Subhankar Dolai, Dan Zhu, Kacey J Prentice, Herbert Y GaisanoAbstract:In type-2 diabetes (T2D), severely reduced islet Syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated β-cell-specific Syn-1A-KO (Syn-1A-βKO) mice to mimic β-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-βKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-βKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the β-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO β-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.
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Syntaxin 1A regulates surface expression of β cell atp sensitive potassium channels
American Journal of Physiology-cell Physiology, 2011Co-Authors: Pei Chun Chen, Herbert Y Gaisano, Cathrin E Bruederle, Show Ling ShyngAbstract:The pancreatic ATP-sensitive potassium (KATP) channel consisting of four inwardly rectifying potassium channel 6.2 (Kir6.2) and four sulfonylurea receptor SUR1 subunits plays a key role in insulin secretion by linking glucose metabolism to membrane excitability. Syntaxin 1A (Syn-1A) is a plasma membrane protein important for membrane fusion during exocytosis of insulin granules. Here, we show that Syn-1A and KATP channels endogenously expressed in the insulin-secreting cell INS-1 interact. Upregulation of Syn-1A by overexpression in INS-1 leads to a decrease, whereas downregulation of Syn-1A by small interfering RNA (siRNA) leads to an increase, in surface expression of KATP channels. Using COSm6 cells as a heterologous expression system for mechanistic investigation, we found that Syn-1A interacts with SUR1 but not Kir6.2. Furthermore, Syn-1A decreases surface expression of KATP channels via two mechanisms. One mechanism involves accelerated endocytosis of surface channels. The other involves decreased biogenesis and processing of channels in the early secretory pathway. This regulation is KATP channel specific as Syn-1A has no effect on another inward rectifier potassium channel Kir3.1/3.4. Our results demonstrate that in addition to a previously documented role in modulating KATP channel gating, Syn-1A also regulates KATP channel expression in β-cells. We propose that physiological or pathological changes in Syn-1A expression may modulate insulin secretion by altering glucose-secretion coupling via changes in KATP channel expression.
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snareing voltage gated k and atp sensitive k channels tuning β cell excitability with Syntaxin 1A and other exocytotic proteins
Endocrine Reviews, 2007Co-Authors: Yuk Man Leung, Youhou Kang, Edwin P Kwan, Herbert Y GaisanoAbstract:The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, Syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca(2+) channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet beta-cells, ATP-sensitive K(+) (K(ATP)) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca(2+) channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K(+) (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that Syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in beta-cells) and the sulfonylurea receptor subunit of beta-cell K(ATP) channel to modify their gating behaviors. A model has proposed that the conformational changes of Syntaxin-1A during exocytosis induce distinct functional modulations of K(ATP) and Kv2.1 channels in a manner that optimally regulates cell excitability and insulin secretion. Other proteins involved in exocytosis, such as Munc-13, tomosyn, rab3a-interacting molecule, and guanyl nucleotide exchange factor II, have also been implicated in direct or indirect regulation of beta-cell ion channel activities and excitability. This review discusses this interesting aspect that exocytotic proteins not only promote secretion per se, but also fine-tune beta-cell excitability via modulation of ion channel gating.
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snareing voltage gated k and atp sensitive k channels tuning β cell excitability with Syntaxin 1A and other exocytotic proteins
Endocrine Reviews, 2007Co-Authors: Yuk Man Leung, Youhou Kang, Edwin P Kwan, Herbert Y GaisanoAbstract:The three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, Syntaxin, SNAP25 (synaptosome-associated protein of 25 kDa), and synaptobrevin, constitute the minimal machinery for exocytosis in secretory cells such as neurons and neuroendocrine cells by forming a series of complexes prior to and during vesicle fusion. It was subsequently found that these SNARE proteins not only participate in vesicle fusion, but also tether with voltage-dependent Ca2+ channels to form an excitosome that precisely regulates calcium entry at the site of exocytosis. In pancreatic islet β-cells, ATP-sensitive K+ (KATP) channel closure by high ATP concentration leads to membrane depolarization, voltage-dependent Ca2+ channel opening, and insulin secretion, whereas subsequent opening of voltage-gated K+ (Kv) channels repolarizes the cell to terminate exocytosis. We have obtained evidence that Syntaxin-1A physically interacts with Kv2.1 (the predominant Kv in β-cells) and the sulfonylurea recep...
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the actions of a novel potent islet β cell specific atp sensitive k channel opener can be modulated by Syntaxin 1A acting on sulfonylurea receptor 1
Diabetes, 2007Co-Authors: Youhou Kang, Chadwick L Elias, Huanli Xie, John Bondo Hansen, Philip Wahl, Herbert Y GaisanoAbstract:Islet β-cell–specific ATP-sensitive K + (K ATP ) channel openers thiadiazine dioxides induce islet rest to improve insulin secretion, but their molecular basis of action remains unclear. We reported that Syntaxin-1A binds nucleotide binding folds of sulfonylurea receptor 1 (SUR1) in β-cells to inhibit K ATP channels. As a strategy to elucidate the molecular mechanism of action of these K ATP channel openers, we explored the possibility that 6-chloro-3-(1-methylcyclobutyl)amino-4 H -thieno[3,2- e ]-1,2,4-thiadiazine 1,1-dioxide (NNC55-0462) might influence Syntaxin-1A–SUR1 interactions or vice versa. Whole-cell and inside-out patch-clamp electrophysiology was used to examine the effects of glutathione S-transferase (GST)-Syntaxin-1A dialysis or green fluorescence protein/Syntaxin-1A cotransfection on NNC55-0462 actions. In vitro pull-down binding studies were used to examine NNC55-0462 influence on Syntaxin-1A–SUR1 interactions. Dialysis of GST–Syntaxin-1A into the cell cytoplasm reduced both potency and efficacy of extracellularly perfused NNC55-0462 in a HEK cell line stably expressing Kir6.2/SUR1 (BA8 cells) and in rat islet β-cells. Moreover, inside-out membrane patches excised from BA8 cells showed that both GST–Syntaxin-1A and its H3 domain inhibited K ATP channels previously activated by NNC55-0462. This action on K ATP channels is isoform-specific to Syntaxin-1A because Syntaxin-2 was without effect. Furthermore, the parent compound diazoxide showed similar sensitivity to GST–Syntaxin-1A inhibition. NNC55-0462, however, did not influence Syntaxin-1A–SUR1 binding interaction. Our results demonstrated that Syntaxin-1A interactions with SUR1 at its cytoplasmic domains can modulate the actions of the K ATP channel openers NNC55-0462 and diazoxide on K ATP channels. The reduced levels of islet Syntaxin-1A in diabetes would thus be expected to exert a positive influence on the therapeutic effects of this class of K ATP channel openers.
Tomonori Fujiwara - One of the best experts on this subject based on the ideXlab platform.
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HPC-1/Syntaxin 1A and Syntaxin 1B play distinct roles in neuronal survival.
Journal of neurochemistry, 2014Co-Authors: Takefumi Kofuji, Tatsuya Mishima, Tomonori Fujiwara, Masumi Sanada, Kimio AkagawaAbstract:Two types of Syntaxin 1 isoforms, HPC-1/Syntaxin 1A (STX1A) and Syntaxin 1B (STX1B), are thought to have similar functions in exocytosis of synaptic vesicles. STX1A(-/-) mice which we generated previously develop normally, possibly because of compensation by STX1B. We produced STX1B(-/-) mice using targeted gene disruption and investigated their phenotypes. STX1B(-/-) mice were born alive, but died before postnatal day 14, unlike STX1A(-/-) mice. Morphologically, brain development in STX1B(-/-) mice was impaired. In hippocampal neuronal culture, the cell viability of STX1B(-/-) neurons was lower than that of WT or STX1A(-/-) neurons after 9 days. Interestingly, STX1B(-/-) neurons survived on WT or STX1A(-/-) glial feeder layers as well as WT neurons. However, STX1B(-/-) glial feeder layers were less effective at promoting survival of STX1B(-/-) neurons. Conditioned medium from WT or STX1A(-/-) glial cells had a similar effect on survival, but that from STX1B(-/-) did not promote survival. Furthermore, brain-derived neurotrophic factor (BDNF) or neurotrophin-3 supported survival of STX1B(-/-) neurons. BDNF localization in STX1B(-/-) glial cells was disrupted, and BDNF secretion from STX1B(-/-) glial cells was impaired. These results suggest that STX1A and STX1B may play distinct roles in supporting neuronal survival by glia. Syntaxin 1A (STX1A) and Syntaxin 1B (STX1B) are thought to have similar functions as SNARE proteins. However, we found that STX1A and STX1B play distinct roles in neuronal survival using STX1A(-/-) mice and STX1B(-/-) mice. STX1B was important for neuronal survival, possibly by regulating the secretion of neurotrophic factors, such as BDNF, from glial cells.
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Impairment of Catecholamine Systems during Induction of Long-Term Potentiation at Hippocampal CA1 Synapses in HPC-1/Syntaxin 1A Knock-out Mice
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2012Co-Authors: Tatsuya Mishima, Tomonori Fujiwara, Takefumi Kofuji, Kimio AkagawaAbstract:The membrane protein HPC-1/Syntaxin 1A is believed to play a key role in synaptic vesicle exocytosis, and it was recently suggested to be required for synaptic plasticity. Despite evidence for the function of HPC-1/Syntaxin 1A in synaptic plasticity, the underlying cellular mechanism is unclear. We found that although fast synaptic transmission and long-term depression were unaffected, HPC-1/Syntaxin 1A knock-out (STX1A(-/-)) mice showed impaired long-term potentiation (LTP) in response to theta-burst stimulation in CA1 hippocampal slices. The impairment in LTP was rescued by the application of forskolin, an adenylyl cyclase activator, or more robust stimulation, suggesting that cAMP/protein kinase A signaling was suppressed in these mice. In addition, catecholamine release from the hippocampus was significantly reduced in STX1A(-/-) mice. Because HPC-1/Syntaxin 1A regulates exocytosis of dense-core synaptic vesicles, which contain neuromodulatory transmitters such as noradrenaline, dopamine and 5-HT, we examined the effect of neuromodulatory transmitters on LTP induction. Noradrenaline and dopamine enhanced LTP induction in STX1A(-/-) mice, whereas catecholamine depletion reduced LTP induction in wild-type mice. Theses results suggest that HPC-1/Syntaxin 1A regulates catecholaminergic systems via exocytosis of dense-core synaptic vesicles, and that deletion of HPC-1/Syntaxin 1A causes impairment of LTP induction.
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loss of granuphilin and loss of Syntaxin 1A cause differential effects on insulin granule docking and fusion
Journal of Biological Chemistry, 2011Co-Authors: Hao Wang, Kimio Akagawa, Tomonori Fujiwara, Ray Ishizaki, Eri Kobayashi, Tetsuro IzumiAbstract:The Rab27 effector granuphilin/Slp4 is essential for the stable attachment (docking) of secretory granules to the plasma membrane, and it also inhibits subsequent fusion. Granuphilin is thought to mediate these processes through interactions with Rab27 on the granule membrane and with Syntaxin-1A on the plasma membrane and its binding partner Munc18-1. Consistent with this hypothesis, both Syntaxin-1A- and Munc18-1-deficient secretory cells, as well as granuphilin null cells, have been observed to have a deficit of docked granules. However, to date there has been no direct comparative analysis of the docking defects in those mutant cells. In this study, we morphometrically compared granule-docking states between granuphilin null and Syntaxin-1A null pancreatic β cells derived from mice having the same genetic background. We found that loss of Syntaxin-1A does not cause a significant granule-docking defect, in contrast to granuphilin deficiency. Furthermore, we newly generated granuphilin/Syntaxin-1A double knock-out mice, characterized their phenotypes, and found that the double mutant mice represent a phenocopy of granuphilin null mice and do not represent phenotypes of Syntaxin-1A null mice, including their granule-docking behavior. Because granuphilin binds to Syntaxin-2 and Syntaxin-3 as well as Syntaxin-1A, it likely mediates granule docking through interactions with those multiple Syntaxins on the plasma membrane.
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analysis of knock out mice to determine the role of hpc 1 Syntaxin 1A in expressing synaptic plasticity
The Journal of Neuroscience, 2006Co-Authors: Tomonori Fujiwara, Tatsuya Mishima, Takefumi Kofuji, Tomoki Chiba, Keiji Tanaka, Akitsugu Yamamoto, Kimio AkagawaAbstract:The protein HPC-1/Syntaxin 1A is abundantly expressed in neurons and localized in the neuronal plasma membrane. It forms a complex with SNAP-25 (25 kDa synaptosomal-associated protein) and VAMP-2 (vesicle-associated membrane protein)/synaptobrevin called SNARE (a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex, which is considered essential for synaptic vesicle exocytosis; thus, HPC-1/Syntaxin 1A is considered crucial for synaptic transmission. To examine the physiological function of HPC-1/Syntaxin 1A in vivo, we produced knock-out (KO) mice by targeted gene disruption. Although HPC-1/Syntaxin 1A expression was completely depleted without any effect on the expression of other SNARE proteins, the KO mice were viable. They grew normally, were fertile, and displayed no difference in appearance compared with control littermate. In cultured hippocampal neurons derived from the KO mice, the basic synaptic transmission in vitro was normal. However, the mutant mice had impaired long-term potentiation in the hippocampal slice. Also, although KO mice exhibited normal spatial memory in the hidden platform test, consolidation of conditioned fear memory was impaired. Interestingly, the KO mice had impaired conditioned fear memory extinction. These observations suggest that HPC-1/Syntaxin 1A may be closely related to synaptic plasticity.
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Syntaxin 1A is expressed in airway epithelial cells where it modulates cftr cl currents
Journal of Clinical Investigation, 2000Co-Authors: Anjaparavanda P Naren, Kimio Akagawa, Tomonori Fujiwara, Estelle Cormetboyaka, Prosper N Boyaka, Jerry R Mcghee, Weihong Zhou, Ulrich Thome, John F Engelhardt, Deborah J. NelsonAbstract:The CFTR Cl(-) channel controls salt and water transport across epithelial tissues. Previously, we showed that CFTR-mediated Cl(-) currents in the Xenopus oocyte expression system are inhibited by Syntaxin 1A, a component of the membrane trafficking machinery. This negative modulation of CFTR function can be reversed by soluble Syntaxin 1A peptides and by the Syntaxin 1A binding protein, Munc-18. In the present study, we determined whether Syntaxin 1A is expressed in native epithelial tissues that normally express CFTR and whether it modulates CFTR currents in these tissues. Using immunoblotting and immunofluorescence, we observed Syntaxin 1A in native gut and airway epithelial tissues and showed that epithelial cells from these tissues express Syntaxin 1A at >10-fold molar excess over CFTR. Syntaxin 1A is seen near the apical cell surfaces of human bronchial airway epithelium. Reagents that disrupt the CFTR-Syntaxin 1A interaction, including soluble Syntaxin 1A cytosolic domain and recombinant Munc-18, augmented cAMP-dependent CFTR Cl(-) currents by more than 2- to 4-fold in mouse tracheal epithelial cells and cells derived from human nasal polyps, but these reagents did not affect CaMK II-activated Cl(-) currents in these cells.
Kevin L. Kirk - One of the best experts on this subject based on the ideXlab platform.
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The interaction between Syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl- channels is mechanistically distinct from Syntaxin 1A-SNARE interactions.
The Journal of biological chemistry, 2002Co-Authors: Radhika Ganeshan, Michael W. Quick, Deborah J. Nelson, Kevin L. KirkAbstract:Abstract Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels and synaptic Ca2+ channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between Syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of Syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the Syntaxin regulatory protein Munc-18a. None of these mutations affected Syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the Syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca2+ channel regulation. Thus, CFTR channel regulation by Syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca2+ channel regulation by this t-SNARE.
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cftr chloride channels are regulated by a snap 23 Syntaxin 1A complex
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Estelle Cormetboyaka, Deborah J. Nelson, Anjaparavanda P Naren, Steven Y Chang, Albert Tousson, Kevin L. KirkAbstract:Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., Syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and Syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on Syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.
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mechanisms of cftr regulation by Syntaxin 1A and pka
Journal of Cell Science, 2002Co-Authors: Steven Y Chang, Anjaparavanda P Naren, Kevin L. Kirk, Clive H Palfrey, Deborah J. NelsonAbstract:Activation of the chloride selective anion channel CFTR is stimulated by cAMP-dependent phosphorylation and is regulated by the target membrane t-SNARE Syntaxin 1A. The mechanism by which SNARE proteins modulate CFTR in secretory epithelia is controversial. In addition, controversy exists as to whether PKA activates CFTR-mediated Cl- currents (ICFTR) by increasing the number of channels in the plasma membrane and/or by stimulating membrane-resident channels. SNARE proteins play a well known role in exocytosis and have recently been implicated in the regulation of ion channels; therefore this investigation sought to resolve two related issues: (a) is PKA activation or SNARE protein modulation of CFTR linked to changes in membrane turnover and (b) does Syntaxin 1A modulate CFTR via direct effects on the gating of channels residing in the plasma membrane versus alterations in membrane traffic. Our data demonstrate that Syntaxin 1A inhibits CFTR as a result of direct protein-protein interactions that decrease channel open probability (Po) and serves as a model for other SNARE protein-ion channel interactions. We also show that PKA activation can enhance membrane trafficking in some epithelial cell types, and this is independent from CFTR activation or Syntaxin 1A association.
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Syntaxin 1A inhibits cftr chloride channels by means of domain specific protein protein interactions
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Anjaparavanda P Naren, Michael W. Quick, Deborah J. Nelson, James F Collawn, Kevin L. KirkAbstract:Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, Syntaxin 1A and Munc-18. Here we report that Syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein–protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by Syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on Syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl− currents are effectively inhibited by a minimal Syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type Syntaxin 1A in membrane fusion reactions. We also show that Syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant ΔF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with Syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and Syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.
Shinya Nagamatsu - One of the best experts on this subject based on the ideXlab platform.
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rab27 effector granuphilin promotes the plasma membrane targeting of insulin granules via interaction with Syntaxin 1A
Journal of Biological Chemistry, 2004Co-Authors: Seiji Torii, Shinya Nagamatsu, Toshiyuki Takeuchi, Tetsuro IzumiAbstract:Secretory vesicle exocytosis is a highly regulated process involving vesicle targeting, priming, and membrane fusion. Rabs and SNAREs play a central role in executing these processes. We have shown recently that Rab27a and its effector, granuphilin, are involved in the exocytosis of insulin-containing secretory granules through a direct interaction with the plasma membrane Syntaxin 1A in pancreatic beta cells. Here, we demonstrate that fluorescence-labeled insulin granules are peripherally accumulated in cells overexpressing granuphilin. The peripheral location of granules is well overlapped with both localizations of granuphilin and Syntaxin 1A. The plasma membrane targeting of secretory granules is promoted by wild-type granuphilin but not by granuphilin mutants that are defective in binding to either Rab27a or Syntaxin 1A. Granuphilin directly binds to the H3 domain of Syntaxin 1A containing its SNARE motif. Moreover, introduction of the H3 domain into beta cells induces a dissociation of the native granuphilin-Syntaxin complex and a marked reduction of newly docked granules. These results indicate that granuphilin plays a role in tethering insulin granules to the plasma membrane by an interaction with both Rab27a and Syntaxin 1A. The complex formation of these three proteins may contribute to the specificity of the targeting process during the exocytosis of insulin granules.
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overexpressed Syntaxin 1A hpc 1 inhibits insulin secretion via a regulated pathway but does not influence glucose metabolism and intracellular ca2 in insulinoma cell line βtc3 cells
Biochemical and Biophysical Research Communications, 1997Co-Authors: Shinya Nagamatsu, Yoko Nakamichi, Hiroki Sawa, Kazuhiko Yamaguchi, Kimio AkagawaAbstract:We have previously established a stable βTC3 cell line that overexpresses Syntaxin 1A, designated βTC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using βTC-hpc1 cells, we aimed to determine whether Syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. βTC3 and βTC-hpc1 cells were simultaneously pulse-labeled with3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from βTC-hpc1 cells was lower than that from βTC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in βTC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that Syntaxin 1A preferentially functions in the regulated insulin release pathway in βTC3 cells. In order to clarify the effect of overexpressed Syntaxin 1A on glucose metabolism and intracellular Ca2+we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of βTC-hpc1 cells were not different from those of βTC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between βTC3 and βTC-hpc1 cells. [Ca2+]imeasured by using fura 2 demonstrated that there was no difference in [Ca2+]ibetween βTC3 and βTC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that Syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]iin βTC3 cells.
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overexpressed Syntaxin 1A hpc 1 inhibits insulin secretion via a regulated pathway but does not influence glucose metabolism and intracellular ca2 in insulinoma cell line βtc3 cells
Biochemical and Biophysical Research Communications, 1997Co-Authors: Shinya Nagamatsu, Yoko Nakamichi, Hiroki Sawa, Kazuhiko Yamaguchi, Kimio AkagawaAbstract:We have previously established a stable beta TC3 cell line that overexpresses Syntaxin 1A, designated beta TC-hpc1 cells, in which glucose-stimulated insulin release was decreased. Using beta TC-hpc1 cells, we aimed to determine whether Syntaxin 1A functions in the regulatory or constitutive pathway of insulin release. We therefore examined the secretion of phorbol-12-myristate-13-acetate (TPA)-stimulated newly synthesized proinsulin/insulin and total immunoreactive insulin. beta TC3 and beta TC-hpc1 cells were simultaneously pulse-labeled with 3H-leucine for 30 min in 11 mM glucose and chased for 1 h in one of a number of different concentrations of TPA in 11 mM glucose. Total immunoreactive insulin release (IRI) by both cell types during the chase period was markedly increased by the addition of TPA in a dose-dependent manner; however, the IRI from beta TC-hpc1 cells was lower than that from beta TC3 cells. The secretion of newly synthesized proinsulin/insulin from both cell types, which in beta TC3 cells is thought to occur via a constitutive pathway, was in the same range under any condition. Thus, the evidence indicates that Syntaxin 1A preferentially functions in the regulated insulin release pathway in beta TC3 cells. In order to clarify the effect of overexpressed Syntaxin 1A on glucose metabolism and intracellular Ca2+ we analyzed the glucose transport system, glucose phosphorylation activity, and cytosolic concentration of free Ca2+ ([Ca2+]i). 2-Deoxy-glucose uptake and the content of GLUT1 protein in the plasma membrane fractions of beta TC-hpc1 cells were not different from those of beta TC3 cells. Radiometric assays of glucose phosphorylation activity showed that there were no differences in hexokinase activity and glucokinase activity between beta TC3 and beta TC-hpc1 cells. [Ca2+]i measured by using fura 2 demonstrated that there was no difference in [Ca2+]i between beta TC3 and beta TC-hpc 1 cells under glucose-stimulated conditions. The present experiments indicate that Syntaxin 1A plays a central role in a late step of the regulatory insulin release pathway without a change in glucose metabolism and [Ca2+]i in beta TC3 cells.
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Expression and Functional Role of Syntaxin 1/HPC-1 in Pancreatic β Cells: Syntaxin 1A, BUT NOT 1B, PLAYS A NEGATIVE ROLE IN REGULATORY INSULIN RELEASE PATHWAY
The Journal of biological chemistry, 1996Co-Authors: Shinya Nagamatsu, Takashi Watanabe, Tomonori Fujiwara, Yoko Nakamichi, Hiroshi Katahira, Hiroki Sawa, Kimio AkagawaAbstract:Abstract Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated Syntaxin 1 expression in pancreatic β cells and the functional role of Syntaxin 1 in the insulin release mechanism. Expression of Syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat Syntaxin 1/HPC-1 antibody demonstrated Syntaxin 1A protein with an apparent molecular mass of ∼35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that Syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of Syntaxin 1 in pancreatic β-cells, rat Syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either Syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by Syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by Syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed Syntaxin 1A and used them to evaluate the effect of Syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in Syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by Syntaxin 1A overexpression, indicating a specific role of Syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that Syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of Syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that Syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.
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expression and functional role of Syntaxin 1 hpc 1 in pancreatic beta cells Syntaxin 1A but not 1b plays a negative role in regulatory insulin release pathway
Journal of Biological Chemistry, 1996Co-Authors: Shinya Nagamatsu, Takashi Watanabe, Tomonori Fujiwara, Yoko Nakamichi, Hiroshi Katahira, Hiroki Sawa, Kimio AkagawaAbstract:Syntaxin 1/HPC-1 is an integral membrane protein, which is thought to be implicated in the regulation of synaptic neurotransmitter release. We investigated Syntaxin 1 expression in pancreatic β cells and the functional role of Syntaxin 1 in the insulin release mechanism. Expression of Syntaxin 1A, but not 1B, was detected in mouse isolated islets by the reverse transcriptase-polymerase chain reaction procedure. An immunoprecipitation study of metabolically labeled islets with an anti-rat Syntaxin 1/HPC-1 antibody demonstrated Syntaxin 1A protein with an apparent molecular mass of ∼35 kDa. Immunohistochemistry of the mouse pancreas demonstrated that Syntaxin 1/HPC-1 was present in the plasma membranes of the islets of Langerhans. In order to determine the functional role of Syntaxin 1 in pancreatic β-cells, rat Syntaxin 1A or 1B was overexpressed in mouse βTC3 cells using the transient transfection procedure. Transfection of βTC3 cells with either Syntaxin 1 resulted in approximately 7-fold increases in their immunodetectable protein levels. Glucose-stimulated insulin release by Syntaxin 1A-overexpressing cells was suppressed to about 50% of the level in control cells, whereas insulin release by Syntaxin 1B-overexpressing and control cells did not differ. Next, we established stable βTC3 cell lines that overexpressed Syntaxin 1A and used them to evaluate the effect of Syntaxin 1A on the regulatory insulin release pathway. Two insulin secretogogues, 4-β-phorbol 12-myristate 13-acetate or forskolin, increased insulin release by untransfected βTC3 cells markedly, but their effects were diminished in Syntaxin 1A-overexpressing βTC3 cells. Glucose-unstimulated insulin release and the proinsulin biosynthetic rate were not affected by Syntaxin 1A overexpression, indicating a specific role of Syntaxin 1A in the regulatory insulin release pathway. Finally, in vitro binding assays showed that Syntaxin 1A binds to insulin secretory granules, indicating an inhibitory role of Syntaxin 1A in insulin exocytosis via its interaction with vesicular proteins. These results demonstrate that Syntaxin 1A is expressed in the islets of Langerhans and functions as a negative regulator in the regulatory insulin release pathway.
