Cloning and functional characterization of a cocaine‐sensitive dopamine transporter

Giros, Bruno; Mestikawy, Salah El; Bertrand, Lucie; Caron, Marc G.

doi:10.1016/0014-5793(91)81406-x

Cited by 326 publications

(144 citation statements)

References 21 publications

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“…DAT is selectively expressed in the dopaminergic ventral midbrain neurons of the ventral tegmental area and substantia nigra compacta (20). Mice received one of the following: a 2-week infusion of an in vitro validated DAT-targeting siRNA, a corresponding mmRNA, a selective DAT inhibitor (3 and 30 g͞day GBR-12909), or vehicle.…”

Section: Sirna-induced Knockdown Of An Endogenous Gene In the Brainmentioning

confidence: 99%

“…The specificity, temporality, and magnitude of EGFP knockdown were evaluated after infusion of an EGFP-targeting siRNA in the dorsal third ventricle. Further, we tested the validity of this RNAi approach by targeting an endogenous gene that codes for the dopamine transporter (DAT) within the ventral midbrain neurons, far distal from the siRNA infusion site (20). DAT controls the temporal and spatial activity of dopamine released into the synapse by facilitating a rapid uptake of the neurotransmitter into presynpatic terminals.…”

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confidence: 99%

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Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

Thakker

Natt²,

Hüsken³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

198

164

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Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.T he burgeoning use of microarray analyses to detect potential target genes relevant to neuropsychiatric disorders necessitates the validation of such targets in vivo (1-3). The approach of genetically modifying animals (knockouts or transgenics) for target validation often is limited by developmental adaptations and genetic compensation that may mask the establishment of a clear phenotype. Further, such methodology is laborious and timeconsuming and not applicable for high-throughput in vivo validation of the large number of hits generated from modern microarray and proteomic target-identification strategies. Additional approaches using ribozymes and antisense oligodeoxynucleotides for somatic gene manipulation also suffer from drawbacks of eliciting nonspecific actions. RNA interference (RNAi) recently has emerged as a potentially superior alternative to the traditional approaches for assessing gene function in adult animals (4).RNAi is a cellular surveillance mechanism that responds to double-stranded RNA (dsRNA) by destroying cytoplasmic mRNAs containing sequences homologous to the dsRNA trigger (5). dsRNAs longer than 30 nt introduced in mammalian cells can induce an undesirable IFN response to produce cell death (6, 7). Conversely, short interfer...

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Section: Sirna-induced Knockdown Of An Endogenous Gene In the Brainmentioning

confidence: 99%

mentioning

confidence: 99%

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

Thakker

Natt²,

Hüsken³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

198

164

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“…74,75 At present there is only one study 74 which examined all three stimulants (methylphenidate, dextroamphetamine and cocaine) on the cloned dopamine transporter, and a summary of these data is given in Table 3.…”

Section: The Occupancy Of the Dopamine Transporter By The Stimulant Dmentioning

confidence: 99%

Anti-hyperactivity medication: methylphenidate and amphetamine

Seeman¹,

1998

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How do 'stimulants' reduce hyperactivity in children and adults? How can drugs which raise extracellular dopamine result in psychomotor slowing of hyperactive children when dopamine is known to enhance motor activity, such as in Parkinson's disease? These apparent paradoxes are the focus of this brief review on the mechanism of action of stimulant medications used in the treatment of children, and of an increasing number of adults who meet diagnostic criteria for attention deficit hyperactivity disorder.

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“…T he dopamine transporter (DAT) is responsible for the reuptake of dopamine released into the synaptic cleft, and thus it represents the major mechanism for the termination of dopaminergic neurotransmission (1,2). DAT is a member of the family of Na ϩ -and Cl Ϫ -dependent neurotransmitter transporters that includes transporters for other neurotransmitters, norepinephrine, serotonin, ␥-aminobutyric acid (GABA), glycine, and a number of other small molecules (3,4).…”

mentioning

confidence: 99%

Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment

Hastrup

Karlin

Javitch

2001

Proc. Natl. Acad. Sci. U.S.A.

181

224

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There is evidence both for and against Na ؉ -and Cl ؊ -dependent neurotransmitter transporters forming oligomers. We found that cross-linking the human dopamine transporter (DAT), which is heterologously expressed in human embryonic kidney 293 cells, either with copper phenanthroline (CuP) or the bifunctional reagent bis-(2-methanethiosulfonatoethyl)amine hydrochloride (bis-EA) increased the apparent molecular mass determined with nonreducing SDS͞PAGE from Ϸ85 to Ϸ195 kDa. After cross-linking, but not before, coexpressed, differentially epitope-tagged DAT molecules, solubilized in Triton X-100, were coimmunoprecipitated. Thus, the 195-kDa complex was a homodimer. Cross-linking of DAT did not affect tyramine uptake. Replacement of Cys-306 with Ala prevented cross-linking. Replacement of all of the non-disulfidebonded cysteines in the extracellular and membrane domains, except for Cys-306, did not prevent cross-linking. We conclude that the cross-link is between Cys-306 at the extracellular end of TM6 in each of the two DATs. The motif GVXXGVXXA occurs at the intracellular end of TM6 in DAT and is found in a number of other neurotransmitter transporters. This sequence was originally found at the dimerization interface in glycophorin A, and it promotes dimerization in model systems. Mutation of either glycine disrupted DAT expression and function. The intracellular end of TM6, like the extracellular end, is likely to be part of the dimerization interface.T he dopamine transporter (DAT) is responsible for the reuptake of dopamine released into the synaptic cleft, and thus it represents the major mechanism for the termination of dopaminergic neurotransmission (1, 2). DAT is a member of the family of Na ϩ -and Cl Ϫ -dependent neurotransmitter transporters that includes transporters for other neurotransmitters, norepinephrine, serotonin, ␥-aminobutyric acid (GABA), glycine, and a number of other small molecules (3, 4). These transporters couple the movement of sodium down its concentration gradient to the transport of substrate, but the molecular mechanism of this process is understood only in general terms. The Na ϩ -and Cl Ϫ -dependent neurotransmitter transporters are thought to have 12 transmembrane segments (TMs) with intracellular N and C termini, and this general topology has been supported by the accessibility of putative extracellular and intracellular loop residues to chemical modification (5, 6). Although the packing of the 12 TMs is unknown, some distance constraints have been established in DAT through the identification of an endogenous zinc-binding site and subsequent use of engineered metalbinding sites (7,8). These constraints place the large extracellular loop between TM3 and TM4 in proximity to the extracellular ends of TM7 and TM8.The serotonin transporter (SERT) has been inferred to be a homo-oligomer, based in part on coimmunoprecipitation studies of differentially epitope-tagged SERT constructs (9). In addition, in cells in which two SERT constructs, only one of which was sensitive to chemical mo...

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Cloning and functional characterization of a cocaine‐sensitive dopamine transporter

Cited by 326 publications

References 21 publications

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference

Anti-hyperactivity medication: methylphenidate and amphetamine

Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment

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