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Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.
Zurawski Z, Page B, Chicka MC, Brindley RL, Wells CA, Preininger AM, Hyde K, Gilbert JA, Cruz-Rodriguez O, Currie KPM, Chapman ER, Alford S, Hamm HE
(2017) J Biol Chem 292: 12165-12177
MeSH Terms: Animals, Binding, Competitive, Calcium Signaling, Cattle, Cell Line, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Humans, Lipid Bilayers, Liposomes, Membrane Fusion, Models, Molecular, Mutation, Nerve Tissue Proteins, Peptide Fragments, Protein Conformation, Protein Interaction Domains and Motifs, Protein Multimerization, Rats, Recombinant Fusion Proteins, Recombinant Proteins, Synaptosomal-Associated Protein 25, Synaptotagmin I, Syntaxin 1
Show Abstract · Added July 12, 2017
G-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gβγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gβγ subunits with the soluble -ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gβγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gβγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gβγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gβγ for SNARE-binding sites in lipid environments. Mutant Gβγ subunits that were previously shown to be more efficacious at inhibiting Ca-triggered exocytotic release than wild-type Gβγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gβγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gβ and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gβγ inhibited Ca/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gβγ-SNARE interaction and show that the target of Gβγ, downstream of VGCC, is the membrane-embedded SNARE complex.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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24 MeSH Terms
TSH elevations as the first laboratory evidence for pseudohypoparathyroidism type Ib (PHP-Ib).
Molinaro A, Tiosano D, Takatani R, Chrysis D, Russell W, Koscielniak N, Kottler ML, Agretti P, De Marco G, Ahtiainen P, Christov M, Mäkitie O, Tonacchera M, Jüppner H
(2015) J Bone Miner Res 30: 906-12
MeSH Terms: Adult, Child, Preschool, Chromosomes, Human, Pair 20, Epigenesis, Genetic, Exons, Female, Humans, Infant, Infant, Newborn, Male, Pseudohypoparathyroidism, Syntaxin 16, Thyrotropin, Young Adult
Show Abstract · Added May 5, 2016
Hypocalcemia and hyperphosphatemia because of resistance toward parathyroid hormone (PTH) in the proximal renal tubules are the most prominent abnormalities in patients affected by pseudohypoparathyroidism type Ib (PHP-Ib). In this rare disorder, which is caused by GNAS methylation changes, resistance can occur toward other hormones, such as thyroid-stimulating hormone (TSH), that mediate their actions through G protein-coupled receptors. However, these additional laboratory abnormalities are usually not recognized until PTH-resistant hypocalcemia becomes clinically apparent. We now describe four pediatric patients, first diagnosed with subclinical or overt hypothyroidism between the ages of 0.2 and 15 years, who developed overt PTH-resistance 3 to 20 years later. Although anti-thyroperoxidase (anti-TPO) antibodies provided a plausible explanation for hypothyroidism in one of these patients, this and two other patients revealed broad epigenetic GNAS abnormalities, which included loss of methylation (LOM) at exons AS, XL, and A/B, and gain of methylation at exon NESP55; ie, findings consistent with PHP-Ib. LOM at GNAS exon A/B alone led in the fourth patient to the identification of a maternally inherited 3-kb STX16 deletion, a well-established cause of autosomal dominant PHP-Ib. Although GNAS methylation changes were not detected in additional pediatric and adult patients with subclinical hypothyroidism (23 pediatric and 39 adult cases), hypothyroidism can obviously be the initial finding in PHP-Ib patients. One should therefore consider measuring PTH, along with calcium and phosphate, in patients with unexplained hypothyroidism for extended periods of time to avoid hypocalcemia and associated clinical complications.
© 2014 American Society for Bone and Mineral Research.
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14 MeSH Terms
Gβγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.
Wells CA, Zurawski Z, Betke KM, Yim YY, Hyde K, Rodriguez S, Alford S, Hamm HE
(2012) Mol Pharmacol 82: 1136-49
MeSH Terms: Alanine, Animals, Binding Sites, Botulinum Toxins, Calcium, Cell Line, Exocytosis, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Lampreys, Mutation, Neurons, Peptides, Protein Binding, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins, SNARE Proteins, Sf9 Cells, Spodoptera, Synaptosomal-Associated Protein 25, Synaptotagmin I, Syntaxin 1
Show Abstract · Added December 10, 2013
Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein βγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gβ₁γ₁. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca²⁺ -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.
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22 MeSH Terms
Dopamine transporter/syntaxin 1A interactions regulate transporter channel activity and dopaminergic synaptic transmission.
Carvelli L, Blakely RD, DeFelice LJ
(2008) Proc Natl Acad Sci U S A 105: 14192-7
MeSH Terms: Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Dopamine, Dopamine Plasma Membrane Transport Proteins, Electrophysiology, Genes, Reporter, Patch-Clamp Techniques, Protein Binding, Recombinant Fusion Proteins, Swimming, Synaptic Transmission, Syntaxin 1
Show Abstract · Added July 10, 2013
The Caenorhabditis elegans (C. elegans) dopamine (DA) transporter (DAT-1) regulates DA signaling through efficient DA reuptake following synaptic release. In addition to its DA transport function, DAT-1 generates detectable DA-gated currents that may influence neuronal excitability. Previously, we provided evidence that single Cl-channel events underlie DAT-1 currents. In these studies, we identified a distinct population of altered DAT-1 currents arising from DAT-1 transgenic constructs bearing an N-terminal GFP fusion. The presence of these channels suggested disruption of an endogenous regulatory mechanism that modulates occupancy of DAT-1 channel states. A leading candidate for such a regulator is the SNARE protein syntaxin 1A (Syn1A), previously found to interact with homologous transporters through N-terminal interactions. Here we establish that UNC-64 (C. elegans Syn1A homologue) associates with DAT-1 and suppresses transporter channel properties. In contrast, GFP::DAT-1 is unable to form stable transporter/UNC-64 complexes that limit channel states. Although DAT-1 and GFP::DAT-1 expressing DA neurons exhibit comparable DA uptake, GFP::DAT-1 animals exhibit swimming-induced paralysis (SWIP), a phenotype associated with excess synaptic DA release and spillover. We propose that loss of UNC-64/DAT-1 interactions leads to enhanced synaptic DA release, providing a novel mechanism for DA neuron sensitization that may be relevant to mechanisms of DA-associated disorders.
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13 MeSH Terms
Syntaxin 1A interaction with the dopamine transporter promotes amphetamine-induced dopamine efflux.
Binda F, Dipace C, Bowton E, Robertson SD, Lute BJ, Fog JU, Zhang M, Sen N, Colbran RJ, Gnegy ME, Gether U, Javitch JA, Erreger K, Galli A
(2008) Mol Pharmacol 74: 1101-8
MeSH Terms: Amino Acid Sequence, Amphetamine, Animals, Cell Line, Cell Membrane, Cells, Cultured, Corpus Striatum, Dopamine, Dopamine Plasma Membrane Transport Proteins, Glutathione Transferase, Humans, Kidney, Mesencephalon, Mice, Mice, Transgenic, Molecular Sequence Data, Neurons, Recombinant Fusion Proteins, Synaptosomes, Syntaxin 1, Transfection
Show Abstract · Added December 10, 2013
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein syntaxin 1A (SYN1A) interacts with and regulates the function of transmembrane proteins, including ion channels and neurotransmitter transporters. Here, we define the first 33 amino acids of the N terminus of the dopamine (DA) transporter (DAT) as the site of direct interaction with SYN1A. Amphetamine (AMPH) increases the association of SYN1A with human DAT (hDAT) in a heterologous expression system (hDAT cells) and with native DAT in murine striatal synaptosomes. Immunoprecipitation of DAT from the biotinylated fraction shows that the AMPH-induced increase in DAT/SYN1A association occurs at the plasma membrane. In a superfusion assay of DA efflux, cells overexpressing SYN1A exhibited significantly greater AMPH-induced DA release with respect to control cells. By combining the patch-clamp technique with amperometry, we measured DA release under voltage clamp. At -60 mV, a physiological resting potential, AMPH did not induce DA efflux in hDAT cells and DA neurons. In contrast, perfusion of exogenous SYN1A (3 microM) into the cell with the whole-cell pipette enabled AMPH-induced DA efflux at -60 mV in both hDAT cells and DA neurons. It has been shown recently that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by AMPH and regulates AMPH-induced DA efflux. Here, we show that AMPH-induced association between DAT and SYN1A requires CaMKII activity and that inhibition of CaMKII blocks the ability of exogenous SYN1A to promote DA efflux. These data suggest that AMPH activation of CaMKII supports DAT/SYN1A association, resulting in a mode of DAT capable of DA efflux.
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21 MeSH Terms
Gbetagamma interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
Yoon EJ, Gerachshenko T, Spiegelberg BD, Alford S, Hamm HE
(2007) Mol Pharmacol 72: 1210-9
MeSH Terms: Animals, Calcium, Cattle, Exocytosis, GTP-Binding Protein beta Subunits, GTP-Binding Protein gamma Subunits, Lampreys, SNARE Proteins, Serotonin, Spinal Cord, Synapses, Synaptosomal-Associated Protein 25, Synaptotagmin I, Syntaxin 1, Vesicle-Associated Membrane Protein 2
Show Abstract · Added December 10, 2013
Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of Gbetagamma with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of Gbetagamma requires the carboxyl terminus of the 25-kDa synaptosome-associated protein (SNAP25) and is downstream of the well known inhibition of Ca2+ entry through voltage-gated calcium channels. We propose a mechanism in which Gbetagamma and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the Gbetagamma interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the Gbetagamma- and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-sensitive manner with binding of Gbetagamma to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca2+ concentrations, Ca2+-synaptotagmin I can displace Gbetagamma binding and the Gbetagamma-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via Gbetagamma to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and Gbetagamma-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between Gbetagamma and synaptotagmin I for SNARE binding. This type of dynamic regulation may represent a novel mechanism for modifying transmitter release in a graded manner based on the history of action potentials that increase intracellular Ca2+ concentrations and of inhibitory signals through G(i)-coupled GPCRs.
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15 MeSH Terms
Calcium-dependent interactions of the human norepinephrine transporter with syntaxin 1A.
Sung U, Blakely RD
(2007) Mol Cell Neurosci 34: 251-60
MeSH Terms: Animals, Calcium, Cells, Cultured, Cerebral Cortex, Cricetinae, Cricetulus, Drug Interactions, Enzyme Activation, Humans, Models, Biological, Neurons, Norepinephrine Plasma Membrane Transport Proteins, Phorbol Esters, Protein Kinase C, Protein Transport, Synaptosomes, Syntaxin 1, Time Factors, Transfection
Show Abstract · Added July 10, 2013
The norepinephrine (NE) transporter (NET) terminates noradrenergic signaling by clearing released NE at synapses. The activity of NET can be rapidly regulated by depolarization and receptor activation via Ca2+ and kinase/phosphatase-linked pathways. The SNARE protein syntaxin 1A (SYN1A) interacts with NET and influences transporter surface trafficking and catalytic activity. In this study, we establish a link between changes in intracellular Ca2+ and SYN1A/NET interactions. SYN1A influenced NE transport only in the presence of Ca2+ in brain cortical synaptosomes. Although NET/SYN1A associations were sensitive to manipulations of Ca2+ in CHO cells, in vitro binding experiments using purified NET and SYN1A fusion proteins demonstrated a lack of direct Ca2+ sensitivity. Disruption of NET/SYN1A interaction abolished inhibition of NE transport by phorbol ester (PMA) to activate protein kinase C (PKC), but had no effect on transport inhibition by the Ca2+ calmodulin kinase (CaMK) inhibitor KN93. Furthermore, PMA enhanced Ca2+-dependent modulation of NE transport in synaptosomes. Our data reveal roles for SYN1A in the Ca2+-dependent regulation of NET, likely reliant on regulation by PKC signaling, but independent of CaMK.
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19 MeSH Terms
Amphetamine induces a calcium/calmodulin-dependent protein kinase II-dependent reduction in norepinephrine transporter surface expression linked to changes in syntaxin 1A/transporter complexes.
Dipace C, Sung U, Binda F, Blakely RD, Galli A
(2007) Mol Pharmacol 71: 230-9
MeSH Terms: Amphetamine, Biotinylation, Calcium, Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium-Calmodulin-Dependent Protein Kinases, Cell Line, Cell Membrane, Enzyme Activation, Gene Expression Regulation, Humans, Kinetics, Norepinephrine Plasma Membrane Transport Proteins, Syntaxin 1
Show Abstract · Added December 10, 2013
Norepinephrine (NE) transporters (NETs) are high-affinity transport proteins that mediate the synaptic clearance of NE after vesicular release. NETs represent a major therapeutic target for antidepressants and are targets of multiple psychostimulants including amphetamine (AMPH) and cocaine. Recently, we demonstrated that syntaxin 1A (SYN1A) regulates NET surface expression and, through binding to the transporter's NH(2) terminus, regulates transporter catalytic function. AMPH induces NE efflux and may also regulate transporter trafficking. We monitored NET distribution and function in catecholaminergic cell lines (CAD) stably transfected with either full-length human NET (CAD-hNET) or with an hNET N-terminal deletion (CAD-hNETDelta(28-47) cells). In hNET-CAD cells, AMPH causes a slow and small reduction of surface hNET with a modest increase in hNET/SYN1A associations at the plasma membrane. In contrast, in CAD-hNETDelta(28-47) cells, AMPH induces a rapid and substantial reduction in surface hNETDelta(28-47) accompanied by a large increase in plasma membrane hNETDelta(28-47)/SYN1A complexes. We also found that AMPH in CAD-hNETDelta(28-47) cells induces a robust increase in cytosolic Ca2+ and concomitant activation of calcium/calmodulin-dependent protein kinase II (CaMKII). Inhibition of either the increase in intracellular Ca2+ or CaMKII activity blocks AMPH-stimulated hNETDelta(28-47) trafficking and the formation of hNETDelta(28-47)/SYN1A complexes. Here, we demonstrate that AMPH stimulation of CAMKII stabilizes an hNET/SYN1A complex. This hNET/SYN1A complex rapidly redistributes, upon AMPH treatment, when mechanisms supported by the transporter's NH2 terminus are eliminated.
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13 MeSH Terms
The N-terminus of the norepinephrine transporter regulates the magnitude and selectivity of the transporter-associated leak current.
Binda F, Lute BJ, Dipace C, Blakely RD, Galli A
(2006) Neuropharmacology 50: 354-61
MeSH Terms: Amino Acid Sequence, Analysis of Variance, Animals, Biological Transport, Active, Cell Line, Chlorides, Dose-Response Relationship, Drug, Dose-Response Relationship, Radiation, Electric Stimulation, Humans, Membrane Potentials, Mice, Mutagenesis, Site-Directed, Norepinephrine, Norepinephrine Plasma Membrane Transport Proteins, Patch-Clamp Techniques, Protein Structure, Tertiary, Sodium, Syntaxin 1, Transfection
Show Abstract · Added December 10, 2013
The norepinephrine (NE) transporter (NET) mediates the removal of NE from synaptic spaces and is a major target for antidepressants, amphetamine and cocaine. Previously, we have shown that syntaxin 1A (SYN 1A) supports human NET (hNET) cell surface expression, that hNET/SYN 1A interactions are direct and mediated by the hNET N-terminus, and that the hNET/SYN 1A association limits substrate-induced hNET-associated currents [Sung, U., Apparsundaram, S., Galli, A., Kahlig, K.M., Savchenko, V., Schroeter, S., Quick, M.W., Blakely, R.D., 2003. A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J. Neurosci. 23, 1697-1709]. These data raise the possibility that the hNET N-terminus, and potentially its interaction with SYN 1A, might regulate other hNET conductance states, including the hNET-mediated leak current. Importantly for monoamine transporters, the leak conductance has been shown to play a critical role in regulating cell membrane potential and possibly neuronal excitability [Quick, M.W., 2003. Regulating the conducting states of a mammalian serotonin transporter. Neuron 40, 537-549]. Here we demonstrate that deletion of the binding domain for SYN 1A in the NET N-terminus robustly enhances the NET-mediated leak current as well as its selectivity for Cl- permeation under particular intracellular ionic compositions. In addition, we show that the NET N-terminus coordinates the ability of intracellular Na+ and Cl- to regulate the leak conductance. These data suggest that the NET N-terminus regulates and defines the ionic specificity of the NET-mediated leak current.
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20 MeSH Terms
A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity.
Sung U, Apparsundaram S, Galli A, Kahlig KM, Savchenko V, Schroeter S, Quick MW, Blakely RD
(2003) J Neurosci 23: 1697-709
MeSH Terms: Animals, Antidepressive Agents, Antigens, Surface, Botulinum Toxins, Brain Chemistry, Catecholamines, Cells, Cultured, Enzyme Activation, Enzyme Activators, Enzyme Inhibitors, Humans, Male, Membrane Proteins, Mice, Mice, Inbred C57BL, Nerve Tissue Proteins, Neurons, Norepinephrine, Norepinephrine Plasma Membrane Transport Proteins, Oligonucleotides, Antisense, Patch-Clamp Techniques, Protein Binding, Protein Structure, Tertiary, Protein Transport, Rats, Recombinant Fusion Proteins, SNARE Proteins, Sequence Deletion, Symporters, Synaptosomes, Syntaxin 1, Vas Deferens, Vesicular Transport Proteins
Show Abstract · Added December 10, 2013
Norepinephrine (NE) transporters (NETs) terminate noradrenergic synaptic transmission and represent a major therapeutic target for antidepressant medications. NETs and related transporters are under intrinsic regulation by receptor and kinase-linked pathways, and clarification of these pathways may suggest candidates for the development of novel therapeutic approaches. Syntaxin 1A, a presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein, interacts with NET and modulates NET intrinsic activity. NETs colocalize with and bind to syntaxin 1A in both native preparations and heterologous systems. Protein kinase C activation disrupts surface NET/syntaxin 1A interactions and downregulates NET activity in a syntaxin-dependent manner. Syntaxin 1A binds the NH(2) terminal domain of NET, and a deletion of this domain both eliminates NET/syntaxin 1A associations and prevents phorbol ester-triggered NET downregulation. Whereas syntaxin 1A supports the surface trafficking of NET proteins, its direct interaction with NET limits transporter catalytic function. These two contradictory roles of syntaxin 1A on NET appear to be linked and reveal a dynamic cycle of interactions that allow for the coordinated control between NE release and reuptake.
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33 MeSH Terms