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Robert H Edwards - One of the best experts on this subject based on the ideXlab platform.
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Is Aspartate an Excitatory Neurotransmitter
The Journal of Neuroscience, 2015Co-Authors: Bruce E. Herring, Katlin Silm, Robert H Edwards, Roger A. NicollAbstract:Recent evidence has resurrected the idea that the amino acid aspartate, a selective NMDA receptor agonist, is a neurotransmitter. Using a mouse that lacks the glutamate-selective Vesicular Transporter VGLUT1, we find that glutamate alone fully accounts for the activation of NMDA receptors at excitatory synapses in the hippocampus. This excludes a role for aspartate and, by extension, a recently proposed role for the sialic acid Transporter sialin in excitatory transmission. SIGNIFICANCE STATEMENT It has been proposed that the amino acid aspartate serves as a neurotransmitter. Although aspartate is a selective agonist for NMDA receptors, we find that glutamate alone fully accounts for neurotransmission at excitatory synapses in the hippocampus, excluding a role for aspartate.
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Evidence that certain retinal bipolar cells use both glutamate and GABA.
The Journal of comparative neurology, 2004Co-Authors: Yen-hong Kao, Robert H Edwards, Luisa Lassová, Tehilla Bar-yehuda, Peter Sterling, Noga VardiAbstract:Retinal bipolar neurons release the excitatory transmitter, glutamate. However, certain bipolar cells contain GABA, raising the question whether a neuron might release both transmitters and, if so, what function might the inhibitory transmitter play in a particular circuit? Here we identify a subset of cone bipolar cells in cat retina that contain glutamate, plus its Vesicular Transporter (VGLUT1), and GABA, plus its synthetic enzyme (GAD65) and its Vesicular Transporter (VGAT). These cells are negative for a marker of ON bipolar cells and restrict their axons to the OFF strata of the inner synaptic layer. They do not colocalize with the neurokinin 3 receptor that stains a type (or two) of OFF bipolar cells. By “targeted injection,” we identified two types of OFF bipolar cell with the machinery to make and package both transmitters. One of these types costratifies with a dopamine plexus. J. Comp. Neurol. 478:207–218, 2004. © 2004 Wiley-Liss, Inc. Indexing terms: VGLUT; VGAT; GAD; dopamine; dye injection
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Postnatal development of the glutamate Vesicular Transporter VGLUT1 in rat cerebral cortex.
Brain research. Developmental brain research, 2003Co-Authors: Andrea Minelli, Robert H Edwards, Tullio Manzoni, Fiorenzo ContiAbstract:Abstract The expression of the Vesicular glutamate Transporter VGLUT1 in the rat neocortex was studied during postnatal development using immunocytochemistry and Western blotting. At all ages, VGLUT immunoreactivity is localized to puncta that coexpress the presynaptic marker synaptophysin. VGLUT1 immunoreactivity is faint at birth, increases in the subplate during the first postnatal week, invades the supragranular layers in the second week and reaches the adult pattern at P20–P30. Its spatial and temporal maturation patterns suggest that VGLUT1 may be the Vesicular Transporter in developing corticocortical connections.
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Vesicular neurotransmitter transport and the presynaptic regulation of quantal size
Current opinion in neurobiology, 1998Co-Authors: Richard J. Reimer, Edward A. Fon, Robert H EdwardsAbstract:Specific transport activities package classical neurotransmitters into secretory vesicles for release by regulated exocytosis, but the proteins responsible for the Vesicular transport of neurotransmitters are still being identified. One family of proteins includes Vesicular Transporters for monoamines and acetylcholine. Genetic manipulation in cells and in mice now shows that changes in the expression of these proteins can alter the amount of neurotransmitter stored per synaptic vesicle, the amount released and behavior. Although the mechanisms responsible for regulating these Transporters in vivo remains unknown, recent work has demonstrated the potential for regulation by changes in intrinsic activity and in location. In addition, a recently identified Vesicular Transporter for GABA defines a novel family of proteins that mediates the packaging of amino acid neurotransmitters.
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distribution of the putative Vesicular Transporter for acetylcholine in the rat central nervous system
Neuroscience, 1997Co-Authors: Arshia Shirzadi, Ali Roghani, Larry L Butcher, Robert H EdwardsAbstract:Abstract In order to develop another selective marker for cholinergic cell bodies and fibres, we have raised a highly specific polyclonal antibody against a peptide derived from the C-terminus of a recently cloned putative Vesicular acetylcholine Transporter. This antibody recognizes the Vesicular acetylcholine Transporter protein on Western blots of membranes from transfected monkey fibroblast COS cells as well as from various rat brain regions but not from untransfected COS cells or rat liver. In separate mapping studies, the antibody was found to stain cell bodies and fibres in all of the regions of the nervous system known to be cholinergic, including (i) the various nuclei of the basal nuclear complex and their projections to the hippocampus, amygdala, and cerebral cortex, (ii) the caudate–putamen nucleus, accumbens nucleus, olfactory tubercle, and islands of calleja complex, (iii) the medial habenula, (iv) the mesopontine cholinergic complex and its projections to the thalamus, extrapyramidal motor nuclei, basal forebrain, cingulate cortex, raphe and reticular nuclei, and some cranial nerve nuclei, and (v) the somatic motor and autonomic nuclei of the cranial and spinal nerves. In many of these cholinergic neurons, it is possible to detect immunoreactivity for the Vesicular acetylcholine Transporter in proximal portions of processes and their branches, as well as in numerous puncta in close association with them. Some of these puncta are large and surround cell bodies and processes of neurons in several regions, including the somatic motor neurons of cranial nerve nuclei in the brainstem and in the ventral horn of the spinal cord. Double immunofluorescence studies indicated that neurons positive for the Vesicular acetylcholine Transporter also stained for the biosynthetic enzyme of acetylcholine, choline acetyltransferase. We conclude that antibody against the C-terminus of the putative Vesicular acetylcholine Transporter provides another marker for cholinergic neurons that, unlike in situ hybridization procedures, labels terminals as well as cell bodies. Therefore this antibody has the potential to reveal changes in number and morphology of cholinergic cell bodies and their terminal varicosities that occur in both physiologic and pathologic conditions.
Gonzalo E Torres - One of the best experts on this subject based on the ideXlab platform.
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the orphan Transporter rxt1 ntt4 slc6a17 functions as a synaptic vesicle amino acid Transporter selective for proline glycine leucine and alanine
Molecular Pharmacology, 2008Co-Authors: Leonardo A Parra, Salah El Mestikawy, Tracy Baust, Marisol Quiroz, Beth Hoffman, Jack M Haflett, Gonzalo E TorresAbstract:Rxt1/NTT4 (SLC6A17) belongs to a gene family of “orphan Transporters” whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane Transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a Vesicular Transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H+-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a Vesicular Transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.
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The Orphan Transporter Rxt1/NTT4 (SLC6A17) Functions as a Synaptic Vesicle Amino Acid Transporter Selective for Proline, Glycine, Leucine, and Alanine
Molecular pharmacology, 2008Co-Authors: Leonardo A Parra, Salah El Mestikawy, Tracy Baust, Marisol Quiroz, Beth Hoffman, Jack M Haflett, Jeffrey K Yao, Gonzalo E TorresAbstract:Rxt1/NTT4 (SLC6A17) belongs to a gene family of “orphan Transporters” whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane Transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a Vesicular Transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H+-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a Vesicular Transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.
Erik M. Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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UNC-46 is required for trafficking of the Vesicular GABA Transporter.
Nature neuroscience, 2007Co-Authors: Kim Schuske, Mark T. Palfreyman, Shigeki Watanabe, Erik M. JorgensenAbstract:Mutations in unc-46 in Caenorhabditis elegans cause defects in all behaviors that are mediated by GABA. Here we show that UNC-46 is a sorting factor that localizes the Vesicular GABA Transporter to synaptic vesicles. The UNC-46 protein is related to the LAMP (lysosomal associated membrane protein) family of proteins and is localized at synapses. In unc-46 mutants, the Vesicular Transporter is not found specifically in synaptic vesicles but rather is diffusely spread along the axon. Mislocalization of the Transporter severely reduces the frequency of miniature currents, but the remaining currents are normal in amplitude. Because the number of synaptic vesicles is not depleted, it is likely that only a fraction of vesicles harbor the Transporter in unc-46 mutants. Our data indicate that the Transporter and UNC-46 have mutual roles in sorting. The Vesicular GABA Transporter recruits UNC-46 to synaptic vesicle precursors in the cell body, and UNC-46 sorts the Transporter at the cell body and during endocytosis at the synapse.
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Vesicular glutamate Transporter - Shooting blanks
Science (New York N.Y.), 2004Co-Authors: Kim Schuske, Erik M. JorgensenAbstract:Brain activity depends on the release of the neurotransmitter glutamate by exocytosis of synaptic vesicles. In their Perspective, Schuske and Jorgensen comment on two studies ( Fremeau et al ., Wocjik et al .) that find that mice with the main Vesicular glutamate Transporter knocked out survive for several months. However, the two studies present different models for subcellular localization of the Vesicular Transporter and for vesicle loading.
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Identification and characterization of the Vesicular GABA Transporter
Nature, 1997Co-Authors: Steven L. Mcintire, Robert H Edwards, Kim Schuske, Richard J. Reimer, Erik M. JorgensenAbstract:Synaptic transmission involves the regulated exocytosis of vesicles filled with neurotransmitter. Classical transmitters are synthesized in the cytoplasm, and so must be transported into synaptic vesicles. Although the Vesicular Transporters for monoamines and acetylcholine have been identified, the proteins responsible for packaging the primary inhibitory and excitatory transmitters, γ-aminobutyric acid (GABA) and glutamate remain unknown^ 1 ,^ 2 . Studies in the nematode Caenorhabditis elegans have implicated the gene unc-47 in the release of GABA^ 3 . Here we show that the sequence of unc-47 predicts a protein with ten transmembrane domains, that the gene is expressed by GABA neurons, and that the protein colocalizes with synaptic vesicles. Further, a rat homologue of unc-47 is expressed by central GABA neurons and confers Vesicular GABA transport in transfected cells with kinetics and substrate specificity similar to those previously reported for synaptic vesicles from the brain. Comparison of this Vesicular GABA Transporter (VGAT) with a Vesicular Transporter for monoamines shows that there are differences in the bioenergetic dependence of transport, and these presumably account for the differences in structure. Thus VGAT is the first of a new family of neurotransmitter Transporters.
Jeffrey D. Erickson - One of the best experts on this subject based on the ideXlab platform.
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presynaptic regulation of quantal size by the Vesicular glutamate Transporter vglut1
The Journal of Neuroscience, 2005Co-Authors: Nathan R Wilson, Hélène Varoqui, Jeffrey D. Erickson, Jiansheng Kang, Emily V Hueske, Tony Leung, Jonathan G Murnick, Guosong LiuAbstract:A fundamental question in synaptic physiology is whether the unitary strength of a synapse can be regulated by presynaptic characteristics and, if so, what those characteristics might be. Here, we characterize a newly proposed mechanism for altering the strength of glutamatergic synapses based on the recently identified Vesicular glutamate Transporter VGLUT1. We provide direct evidence that filling in isolated synaptic vesicles is subject to a dynamic equilibrium that is determined by both the concentration of available glutamate and the number of Vesicular Transporters participating in loading. We observe that changing the number of Vesicular Transporters expressed at hippocampal excitatory synapses results in enhanced evoked and miniature responses and verify biophysically that these changes correspond to an increase in the amount of glutamate released per vesicle into the synaptic cleft. In addition, we find that this modulation of synaptic strength by Vesicular Transporter expression is endogenously regulated, both across development to coincide with a maturational increase in vesicle cycling and quantal amplitude and by excitatory and inhibitory receptor activation in mature neurons to provide an activity-dependent scaling of quantal size via a presynaptic mechanism. Together, these findings underscore that Vesicular Transporter expression is used endogenously to directly regulate the extent of glutamate release, providing a concise presynaptic mechanism for controlling the quantal efficacy of excitatory transmission during synaptic refinement and plasticity.
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Functional identification of Vesicular monoamine and acetylcholine Transporters.
Methods in enzymology, 1998Co-Authors: Hélène Varoqui, Jeffrey D. EricksonAbstract:Publisher Summary Active transport of monoamines by Vesicular monoamine Transporter (VMAT1) and VMAT2 is performed in permeabilized fibroblasts transiently expressing these proteins by the recombinant T7 vaccinia virus system. Active transport of acetylcholine (ACh) by human Vesicular ACh Transporter (VAChT) is performed in postnuclear homogenates containing secretory vesicles from stably transfected neuroendocrine PC-12 cells. Neurotransmission depends on the regulated release of transmitter molecules, such as the biogenic amines and ACh. This requires the packaging of these molecules into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific Vesicular Transporters. Active transport of neurotransmitters into secretory organelles requires the presence of a transmembrane H + electrochemical gradient, established and maintained by a vacuolar-type H + -ATPase and a Vesicular Transporter molecule, which catalyzes the exchange of H + ions for neurotransmitter. A characteristic feature of the VMATs is their broad range of substrate specificity. The chapter describes the development of active transport and binding assay in permeabilized cells.
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Vesicular neurotransmitter Transporters
Molecular Neurobiology, 1997Co-Authors: Hélène Varoqui, Jeffrey D. EricksonAbstract:Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific Vesicular Transporters. The family of genes encoding the Vesicular Transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about Vesicular transport bioenergetics, substrate feature recognition by each Transporter, and the role of Vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of Vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined Vesicular transport function is critical for survival and have enabled further evaluation of the role of Vesicular neurotransmitter Transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in Vesicular Transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these Transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine Vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.
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Vesicular neurotransmitter Transporters potential sites for the regulation of synaptic function
Molecular Neurobiology, 1997Co-Authors: Hélène Varoqui, Jeffrey D. EricksonAbstract:Neurotransmission depends on the regulated release of chemical transmitter molecules. This requires the packaging of these substances into the specialized secretory vesicles of neurons and neuroendocrine cells, a process mediated by specific Vesicular Transporters. The family of genes encoding the Vesicular Transporters for biogenic amines and acetylcholine have recently been cloned. Direct comparison of their transport characteristics and pharmacology provides information about Vesicular transport bioenergetics, substrate feature recognition by each Transporter, and the role of Vesicular amine storage in the mechanism of action of psychopharmacologic and neurotoxic agents. Regulation of Vesicular transport activity may affect levels of neurotransmitter available for neurosecretion and be an important site for the regulation of synaptic function. Gene knockout studies have determined Vesicular transport function is critical for survival and have enabled further evaluation of the role of Vesicular neurotransmitter Transporters in behavior and neurotoxicity. Molecular analysis is beginning to reveal the sites involved in Vesicular Transporter function and the sites that determine substrate specificity. In addition, the molecular basis for the selective targeting of these Transporters to specific vesicle populations and the biogenesis of monoaminergic and cholinergic synaptic vesicles are areas of research that are currently being explored. This information provides new insights into the pharmacology and physiology of biogenic amine and acetylcholine Vesicular storage in cardiovascular, endocrine, and central nervous system function and has important implications for neurodegenerative disease.
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Molecular biology of the Vesicular ACh Transporter
Trends in neurosciences, 1995Co-Authors: Ted B. Usdin, Lee E. Eiden, Tom I. Bonner, Jeffrey D. EricksonAbstract:Abstract The cholinergic synapse has long been a model for biochemical studies of neurotransmission. The molecules that are responsible for synaptic transmission are being identified rapidly. The Vesicular Transporter for ACh, which is responsible for the concentration of ACh within synaptic vesicles, has been characterized recently, both at the molecular and functional level. Definitive identification of the cloned gene involved genetics of Caenohabditis elegans , the specialized Torpedo electromotor system, and expression in mammalian tissue culture. Comparison of the Vesicular Transporter for ACh with the Vesicular Transporters for monoamines demonstrates a new gene family. Gene mapping has demonstrated a unique relationship between the genes for the Vesicular ACh Transporter and for choline acetyltransferase.
David E. Krantz - One of the best experts on this subject based on the ideXlab platform.
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Vesicular neurotransmitter Transporter trafficking in vivo moving from cells to flies
Fly, 2010Co-Authors: Anna Grygoruk, Hao Fei, Richard W. Daniels, Bradley R. Miller, Audrey Chen, Aaron Diantonio, David E. KrantzAbstract:During exocytosis, classical and amino acid neurotransmitters are released from the lumen of synaptic vesicles to allow signaling at the synapse. The storage of neurotransmitters in synaptic vesicles and other types of secretory vesicles requires the activity of specific Vesicular Transporters. Glutamate and monoamines such as dopamine are packaged by VGLUTs and VMATs respectively. Changes in the localization of either protein have the potential to up- or down regulate neurotransmitter release, and some of the mechanisms for sorting these proteins to secretory vesicles have been investigated in cultured cells in vitro. We have used Drosophila molecular genetic techniques to study Vesicular Transporter trafficking in an intact organism and have identified a motif required for localizing Drosophila VMAT (DVMAT) to synaptic vesicles in vivo. In contrast to DVMAT, large deletions of Drosophila VGLUT (DVGLUT) show relatively modest deficits in localizing to synaptic vesicles, suggesting that DVMAT and DVGLUT m...
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A tyrosine-based motif localizes a Drosophila Vesicular Transporter to synaptic vesicles in vivo
The Journal of biological chemistry, 2010Co-Authors: Anna Grygoruk, Hao Fei, Richard W. Daniels, Bradley R. Miller, Aaron Diantonio, David E. KrantzAbstract:Vesicular neurotransmitter Transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize Vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the Vesicular monoamine and glutamate Transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.
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a splice variant of the drosophila Vesicular monoamine Transporter contains a conserved trafficking domain and functions in the storage of dopamine serotonin and octopamine
Journal of Neurobiology, 2005Co-Authors: Christina L Greer, Anna Grygoruk, Aaron Diantonio, Rafael Romerocalderon, David E Patton, Brett Ley, Huiyun Chang, Roozbeh Houshyar, Roland J Bainton, David E. KrantzAbstract:Vesicular monoamine Transporters (VMATs) mediate the transport of dopamine (DA), serotonin (5HT), and other monoamines into secretory vesicles. The regulation of mammalian VMAT and the related Vesicular acetylcholine Transporter (VAChT) has been proposed to involve membrane trafficking, but the mechanisms remain unclear. To facilitate a genetic analysis of Vesicular Transporter function and regulation, we have cloned the Drosophila homolog of the Vesicular monoamine Transporter (dVMAT). We identify two mRNA splice variants (DVMAT-A and B) that differ at their C-terminus, the domain responsible for endocytosis of mammalian VMAT and VAChT. DVMAT-A contains trafficking motifs conserved in mammals but not C. elegans, and internalization assays indicate that the DVMAT-A C-terminus is involved in endocytosis. DVMAT-B contains a divergent C-terminal domain and is less efficiently internalized from the cell surface. Using in vitro transport assays, we show that DVMAT-A recognizes DA, 5HT, octopamine, tyramine, and histamine as substrates, and similar to mammalian VMAT homologs, is inhibited by the drug reserpine and the environmental toxins 2,2,4,5,6-pentachlorobiphenyl and heptachlor. We have developed a specific antiserum to DVMAT-A, and find that it localizes to dopaminergic and serotonergic neurons as well as octopaminergic, type II terminals at the neuromuscular junction. Surprisingly, DVMAT-A is co-expressed at type II terminals with the Drosophila Vesicular glutamate Transporter. Our data suggest that DVMAT-A functions as a Vesicular Transporter for DA, 5HT, and octopamine in vivo, and will provide a powerful invertebrate model for the study of Transporter trafficking and regulation.
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A molecular analysis of Vesicular amine transport.
Behavioural Brain Research, 1995Co-Authors: Doris Peter, David E. Krantz, Andrew Merickel, J. Patrick Finn, Robert H EdwardsAbstract:Abstract To package classical neurotransmitters into vesicles so that their release can be regulated by activity, neuronal cells express a set of specific Vesicular transport proteins. We have used selection in MPP + to clone the cDNAs encoding two Vesicular monoamine Transporters, the first members of this novel gene family that now also includes the Vesicular Transporter for acetylcholine. The sequences show similarity to several bacterial antibiotic resistance proteins, further supporting a role in detoxification and possibly Parkinson's disease. The two Vesicular amine Transporters show differences in their affinity for substrates, their turnover number and their pharmacology. In particular, the proteins differ in their interactions with the potent inhibitor tetrabenazine and with amphetamines, accounting for several classic pharmacological observations. Since the subcellular localization of the transport proteins determines the site of monoamine storage and the site of monoamine storage appears to differ from other classical transmitters, we have also raised polyclonal antibodies to the Transporters and used these to demonstrate localization in dense core vesicles rather than synaptic vesicles. In addition to the implications for monoamine release, these observations also indicate a Vesicular amine Transporter as the first integral membrane protein restricted to the regulated secretory pathway.