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At least 289 records · Page 16Linked to original sources
PubMed2004

R-type voltage-gated Ca(2+) channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of Xenopus oocytes.

It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.

Animals↗
PubMed2006

C-type natriuretic peptide modulates glutamate receptors on cultured rat retinal amacrine cells.

C-type natriuretic peptide, widely distributed in the CNS, may work as a neuromodulator. In this work, we investigated modulation by C-type natriuretic peptide of functional properties of glutamate receptors in rat retinal GABAergic amacrine cells in culture. Immunocytochemical data revealed that natriuretic peptide receptor-B was strongly expressed on the membrane of cultured GABAergic amacrine cells. By whole cell recording techniques we further identified the glutamate receptor expressed on the GABAergic amacrine cells as an AMPA-preferring subtype. Incubation with C-type natriuretic peptide suppressed the AMPA receptor-mediated current of these cells in a dose-dependent manner by decreasing the efficacy and apparent affinity for glutamate. The effect of C-type natriuretic peptide was reversed by HS-142-1, a guanylyl cyclase-coupled natriuretic peptide receptor-A/B antagonist. Meanwhile, the selective natriuretic peptide receptor-C agonist cANF did not change the glutamate current. In conjunction with the immunocytochemical data, these results suggest that the C-type natriuretic peptide effect may be mediated by natriuretic peptide receptor-B. Furthermore, incubation of retinal cultures in the C-type natriuretic peptide-containing medium elevated cGMP immunoreactivity in the GABAergic amacrine cells, and the C-type natriuretic peptide effect on the glutamate current was mimicked by application of 8-Br-cGMP. It is therefore concluded that C-type natriuretic peptide may modulate the glutamate current by increasing the intracellular concentration of cGMP in these cells via activation of natriuretic peptide receptor-B.

Amacrine Cells↗
PubMed2006

Cholesterol is critical to the integrity of neuronal porosome/fusion pore.

Secretion is one of the most fundamental cellular processes. Porosomes have been demonstrated as the universal secretory machinery in cells. Earlier studies determine the presence of a number of proteins in porosomes, among them the N- and P/Q-type calcium channels, actin, syntaxin-1, synaptotagmin-1, vimentin, the N-ethylmaleimide-sensitive factor (NSF), the chloride channel CLC-3, and the alpha subunit of the heterotrimeric GTP-binding protein G(o). Studies demonstrate that t-SNAREs localize at the base of porosomes, and directly interact with calcium channels. In the present study, we demonstrate that Syntaxin-1 co-localizes with cholesterol in solubilized synaptosomal membrane preparations. Depletion of cholesterol, results in the dissociation of both Syntaxin-1 and N-type calcium channel from neuronal porosomes. Thus, cholesterol participates as an integral component of the neuronal porosome complex, and is required for its stability.

Animals↗
PubMed2000

Interaction of SNARE proteins in rat parotid acinar cells.

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.

Adenosine Triphosphatases↗
PubMed2003

Protein disregulation in red blood cell membranes of type 2 diabetic patients.

The proteomics analysis was used to search for the membrane proteins related to the type 2 diabetes in human red blood cell (RBC). To improve the solubilization and separation for membrane proteins during two-dimensional electrophoresis (2-DE), several types of chaotropes and surfactants were tested. The optimized condition was then screened. About 1000 protein spots from RBC membranes can be resolved on the 2-D gel. To compare the 2-DE patterns between RBC membranes of type 2 diabetic patients and healthy controls, a total of 42 proteins that were differentially expressed were found. The analysis shows that flotillin-1, a recently discovered membrane protein of RBC lipid rafts, appears to be affected in the disease. The result would be quite interesting because flotillin-1 in adipocytes functions is related to stimulate activation of glucose transporter 4 in response to insulin. Additionally, syntaxin 1C and arginase were also disregulated in patient RBC membranes.

Aged↗
PubMed2001

Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.

According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.

Anions↗
PubMed2003

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins.

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.

Antigens, Surface↗
PubMed2003

Mechanical coupling via the membrane fusion SNARE protein syntaxin 1A: a molecular dynamics study.

SNARE trans complexes between membranes likely promote membrane fusion. For the t-SNARE syntaxin 1A involved in synaptic transmission, the secondary structure and bending stiffness of the five-residue juxtamembrane linker is assumed to determine the required mechanical energy transfer from the cytosolic core complex to the membrane. These properties have here been studied by molecular dynamics and annealing simulations for the wild-type and a C-terminal-prolongated mutant within a neutral and an acidic bilayer, suggesting linker stiffnesses above 1.7 but below 50 x 10(-3) kcal mol(-1) deg(-2). The transmembrane helix was found to be tilted by 15 degrees and tightly anchored within the membrane with a stiffness of 4-5 kcal mol(-1) A(-2). The linker turned out to be marginally helical and strongly influenced by its lipid environment. Charged lipids increased the helicity and H3 helix tilt stiffness. For the wild type, the linker was seen embedded deeply within the polar region of the bilayer, whereas the prolongation shifted the linker outward. This reduced its helicity and increased its average tilt, thereby presumably reducing fusion efficiency. Our results suggest that partially unstructured linkers provide considerable mechanical coupling; the energy transduced cooperatively by the linkers in a native fusion event is thus estimated to be 3-8 kcal/mol, implying a two-to-five orders of magnitude fusion rate increase.

Antigens, Surface↗
PubMed1996

Enhancement of neurite-sprouting by suppression of HPC-1/syntaxin 1A activity in cultured vertebrate nerve cells.

HPC-1/syntaxin 1A is a C-terminal anchored neuronal membrane protein, of which all of the N-terminal regions are located on the intracellular side, and it interacts with presynaptic membrane proteins, synaptic vesicle proteins and soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs). HPC-1/syntaxin 1A has been proposed to act as a target SNAP receptor (t-SNARE) in the neuron and contributes to the vesicle docking/fusion process during the fast exocytosis at the presynaptic active zone. However, studies using an electron-microscope revealed that HPC-1/syntaxin 1A distributed not only at the presynaptic region but throughout the whole axonal membrane, and the functions of this axonal HPC-1/syntaxin 1A remain completely unknown. To investigate its physiological role, we attempted to inhibit the function of HPC-1/syntaxin 1A in cultured neural cells by following two methods. First, de novo synthesis of HPC-1/syntaxin 1A was inhibited by an application of antisense oligonucleotide in cultured adult rat dorsal root ganglion (DRG) neurons. Second, antibody against HPC-1/syntaxin 1A was applied intra-axonally in the cultured chick retinal ganglion neuron. Both treatments, which were expected to downregulate the function of HPC-1/syntaxin 1A, consistently elicited an enhancement of the axonal sprouting. These results suggest that the axonal HPC-1/syntaxin 1A would physiologically suppress the excess axon-collateral sprouting. Downregulation of HPC-1/syntaxin 1A expression may underlie the control of collateral sprouting and synapse formation during development and memory processes.

Animals↗
PubMed2000

Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A.

In this study we have examined the effect of the SNARE protein syntaxin 1A on the glycine transporters GLYT1 and GLYT2. Our results demonstrate a functional and physical interaction between both glycine transporters and syntaxin 1A. Co-transfection of syntaxin 1A with GLYT1 or GLYT2 in COS cells resulted in approximately 40% inhibition in glycine transport. This inhibition was reversed by the syntaxin 1A-binding protein, Munc18. Furthermore, immunoprecipitation studies showed a physical interaction between syntaxin 1A and both transporters in COS cells and in rat brain tissue. Finally, we conclude that this physical interaction resulted in a partial removal of the glycine transporters from the plasma membrane as demonstrated by biotinylation studies.

Amino Acid Transport Systems, Neutral↗
PubMed2002

Cellular munc18c levels can modulate glucose transport rate and GLUT4 translocation in 3T3L1 cells.

Munc18c has been shown to bind syntaxin 4 and to play a role in GLUT4 translocation and glucose transport, although this role is as yet poorly defined. In the present study, the effects of modulating the available level of munc18c on glucose transport and GLUT4 translocation were examined. Over-expression of munc18c in 3T3L1 adipocytes inhibited insulin-stimulated glucose transport by approximately 50%. Basal glucose transport rates were also decreased by approximately 25%. In contrast, microinjection of a munc18c polyclonal antibody stimulated GLUT4 translocation by approximately 60% over basal levels without affecting insulin-stimulated GLUT4 levels. Microinjection of a control antibody had no effect. These data are consistent with the likelihood that antibody microinjection sequesters munc18c enabling translocation/fusion of GLUT4 vesicles. Mutagenesis of a potential proline-directed kinase phosphorylation site in munc18c, T569, that in previous studies of its neuronal counterpart munc18a caused its dissociation from its complex with syntaxin 1a, had no effect on munc18c's association with syntaxin 4 or its inhibition of glucose transport, indicative that phosphorylation of this residue is not important for insulin regulation of glucose transport. The over-expression and microinjection sequestration data support an inhibitory role for munc18c on translocation/fusion of GLUT4 vesicles. They further show that altering the level of available munc18c in 3T3L1 cells can modulate glucose transport rates, indicating its potential as a target for therapeutics in diabetes.

3T3 Cells↗
PubMed2003

Expression of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, is enhanced by phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway.

Syntaxin 1C is an alternative splice variant of HPC-1/syntaxin 1A; the latter participates in neurotransmitter release and is assigned to the gene domain responsible for Williams' syndrome (WS). It is expressed in the soluble fraction extracted from human astroglioma cell lines T98G and U87MG. Quantitative immunoblot and indirect immunofluorescence analyses revealed that the expression of syntaxin 1C was upregulated by phorbol 12-myristate 13-acetate (PMA), but not by forskolin. A protein kinase C (PKC) inhibitor suppressed this enhancement. These results suggest that syntaxin 1C expression is regulated via the PKC signal pathway. This is the first report of a signal transduction system that directly affects the expression of syntaxin protein.

Alternative Splicing↗
PubMed1998

Stimulation of NSF ATPase activity during t-SNARE priming.

N-Ethylmaleimide-sensitive factor (NSF) plays a key role in vesicular traffic by disassembling and priming SNARE proteins for their function in docking and fusion. We demonstrate that the ATPase activity of NSF is activated by alpha-soluble NSF attachment protein (alpha-SNAP) in a complex with syntaxin 1A. In addition, we show that a construct consisting of the H3 domain of syntaxin IA (GST-synt(195-263), which does not support NSF disassembly in the presence of MgATP gave a larger stimulation. NSF ATPase activation was specific and did not occur using mutant alpha-SNAPs unable to bind GST-synt or with mutated C-termini. We suggest that activation of NSF ATPase activity in the SNARE complex may be essential to allow SNARE priming.

Adenosine Triphosphate↗
PubMed2000

Mechanisms underlying regulated CFTR trafficking.

Stimulation of membrane capacitance and cell surface labeling of epitope-tagged CFTR provide evidence of cAMP-regulated CFTR trafficking. Co-expression of syntaxin 1A inhibits cAMP-stimulated current and capacitance changes in CFTR expressing cells and blocks cAMP-induced increases in cell surface CFTR. Inhibition of CFTR trafficking by syntaxin over-expression suggests a role for SNARE proteins in this process. CFTR phosphorylation may alter physical interactions with SNARE proteins to regulate plasma membrane CFTR density.

Cyclic AMP↗
PubMed2005

Functional analysis of Rab27a effector granuphilin in insulin exocytosis.

Granuphilin is specifically expressed on dense-core granules in a defined set of secretory cells such as insulin-producing pancreatic beta-cells. It preferentially binds the GTP-bound form of Rab27a and regulates the exocytosis of secretory granules. Furthermore, granuphilin directly interacts with syntaxin-la, the plasma-membrane-anchored SNARE protein, and with Munc18-1, a Sec1/Munc18 protein. We previously reported evidence that granuphilin mediates the docking of secretory granules onto the plasma membrane through these protein-protein interactions. This chapter details the methods and protocols we use to analyze the function of granuphilin with particular attention to the assays for detecting the expression, protein interactions, and effects on exocytosis of secretory granules in pancreatic beta-cells and their derivative cell lines.

Amino Acid Sequence↗
PubMed1999

Inhibition of SNARE complex assembly differentially affects kinetic components of exocytosis.

In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.

Adrenal Glands↗
PubMed1998

Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A.

Syntaxin 1A plays a central role in neurotransmitter release through multiple protein-protein interactions. We have used NMR spectroscopy to identify an autonomously folded N-terminal domain in syntaxin 1A and to elucidate its three-dimensional structure. This 120-residue N-terminal domain is conserved in plasma membrane syntaxins but not in other syntaxins, indicating a specific role in exocytosis. The domain contains three long alpha helices that form an up-and-down bundle with a left-handed twist. A striking residue conservation is observed throughout a long groove that is likely to provide a specific surface for protein-protein interactions. A highly acidic region binds to the C2A domain of synaptotagmin I in a Ca2+-dependent interaction that may serve as an electrostatic switch in neurotransmitter release.

Animals↗