Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.1.107 (DAT)
 1,471 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have constructed stable cell lines expressing transporters for dopamine (DA), norepinephrine (NE), and serotonin (5-HT) by transfection with cloned cDNAs. The parental LLC-PK1 cell does not express any of these neurotransmitter transporters. Therefore, monoamine transport activities in each of these cell lines are due to the transfected DNA only, allowing comparison in the same background. Drug inhibition profiles for each cell line are distinct and as expected for each transporter. LLC-NET and LLC-DAT cells transported both NE and DA and both cell types exhibited a lower KM for DA transport than for NE transport. Analysis of Vmax data for LLC-NET cells suggests that substrate is bound to the NE transporter during the rate-limiting step(s) in transport. The cocaine analog 2-beta-carbomethoxy-3 beta-(4-[125I]iodophenyl)tropane binds to each cell type, and is displaced by transport substrate in each case. Binding and transport measurements on parallel cell cultures allowed estimation of turnover numbers for norepinephrine, dopamine, and serotonin transporters. All three transporters require external Na+ and Cl-. The Na+ concentration dependence suggests that a single Na+ ion is involved in transport catalyzed by norepinephrine and serotonin transporters while more than one Na+ ion participate in transport mediated by the dopamine transporter.
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PMID:Stable expression of biogenic amine transporters reveals differences in inhibitor sensitivity, kinetics, and ion dependence. 812 21

The successful generation and functional expression of a series of recombinant chimeric transporters, in which distinct functional properties of NET and DAT are exchanged, have allowed the assignment of a number of important functional properties of MPP+ and antidepressant-sensitive catecholamine transporters to specific domains within their primary structure. These studies are the first comprehensive structure-function analysis of members of the rapidly growing superfamily of Na+/Cl- carriers using chimeric transporters. This represents the first step in identifying the specific structural or regulatory determinants that differentiate NET and DAT. An appreciation of the potentially distinct sites for substrate recognition, translocation, and transport inhibition of NET and DAT may facilitate the development of more selective drugs for the treatment of stimulant addiction, human depression, and other affective disorders.
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PMID:Discrete structural domains and cell-specific expression determine functional selectivity of the dopamine and norepinephrine transporters. 878 50

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressing Xenopus laevis oocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate "transport-associated" current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na+ and 1 Cl- per DA+ molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na+ or Cl-, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like "DA-releasing" drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmission in vivo.
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PMID:Multiple ionic conductances of the human dopamine transporter: the actions of dopamine and psychostimulants. 899 51

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.
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PMID:Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters. 915 29

The monoamines, serotonin, dopamine, norepinephrine, epinephrine and histamine, play a critical role in the function of the hypothalamic-pituitary-adrenal axis and in the integration of information in sensory, limbic, and motor systems. The primary mechanism for termination of monoaminergic neurotransmission is through reuptake of released neurotransmitter by Na+, CI-dependent plasma membrane transporters. A second family of transporters packages monoamines into synaptic and secretory vesicles by exchange of protons. Identification of those cells which express these two families of neurotransmitter transporters is an initial step in understanding what adaptive strategies cells expressing monoamine transporters use to establish the appropriate level of transport activity and thus attain the appropriate efficiency of monoamine storage and clearance. The most recent advances in this field have yielded several surprises about their function, cellular and subcellular localization, and regulation, suggesting that these molecules are not static and most likely are the most important determinants of extracellular levels of monoamines. Here, information on the localization of mRNAs for these transporters in rodent and human brain is summarized along with immunohistochemical information at the light and electron microscopic levels. Regulation of transporters at the mRNA level by manipulation in rodents and differences in transporter site densities by tomographic techniques as an index of regulation in human disease and addictive states are also reviewed. These studies have highlighted the presence of monoamine neurotransmitter transporters in neurons but not in glia in situ. The norepinephrine transporter is present in all cells which are both tyrosine hydroxylase (TH)- and dopamine beta-hydroxylase-positive but not in those cells which are TH- and phenyl-N-methyltransferase-positive, suggesting that epinephrine cells may have their own, unique transporter. In most dopaminergic cells, dopamine transporter mRNA completely overlaps with TH mRNA-positive neurons. However, there are areas in which there is a lack of one to one correspondence. The serotonin transporter (5-HTT) mRNA is found in all raphe nuclei and in the hypothalamic dorsomedial nucleus where the 5-HTT mRNA is dramatically reduced following immobilization stress. The vesicular monoamine transporter 2 (VMAT2) is present in all monoaminergic neurons including epinephrine- and histamine-synthesizing cells. Immunohistochemistry demonstrates that the plasma membrane transporters are present along axons, soma, and dendrites. Subcellular localization of DAT by electron microscopy suggests that these transporters are not at the synaptic density but are confined to perisynaptic areas, implying that dopamine diffuses away from the synapse and that contribution of diffusion to dopamine signalling may vary between brain regions. Interestingly, the presence of VMAT2 in vesicles underlying dendrites, axons, and soma suggests that monoamines may be released at these cellular domains. An understanding of the regulation of transporter function may have important therapeutic consequences for neuroendocrine function in stress and psychiatric disorders.
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PMID:Localization and dynamic regulation of biogenic amine transporters in the mammalian central nervous system. 966 36

Catecholaminergic neurotransmission is normally terminated by rapid re-uptake of the neurotransmitter by a high-affinity Na+/Cl--dependent plasma membrane transporter. Specific transporters have been cloned for both dopamine (DAT) and noradrenaline (NAT) in the rat. While DAT has been studied extensively, NAT expression has received less attention, particularly at the protein level. We used an antibody generated against a 49 residue segment of an extracellular loop region of NAT to study expression of the transporter protein throughout the rat pons and medulla oblongata. NAT was expressed in over 95% of noradrenergic neurones in the A1, A2/area postrema, A5, A6/locus subcoeruleus, and A7 noradrenergic groups. Approximately 10% of C1 adrenergic neurones located in the rostral ventrolateral medulla (RVL) also expressed NAT. Expression of NAT mRNA in bulbospinal C1 cells was confirmed using single-cell reverse transcription polymerase chain reaction (RT-PCR) of acutely isolated RVL neurones. Spinally projecting neurones were identified by retrograde labelling with rhodamine beads, and C1 neurones were identified by RT-PCR using primers specific for tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT) mRNAs. Thirteen percent of adrenergic bulbospinal neurones tested expressed NAT mRNA. C1 neurones are potentially important in cardiovascular control and blood pressure regulation, and the identification of NAT expression in a sub-population of these neurones provides further evidence for the heterogeneity of this neuronal population.
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PMID:Noradrenaline transporter expression in the pons and medulla oblongata of the rat: localisation to noradrenergic and some C1 adrenergic neurones. 979 40

The mechanism of release mediated by the human dopamine and norepinephrine transporter (DAT and NET, respectively) was studied by a superfusion technique in human embryonic kidney 293 cells stably transfected with the respective transporter cDNA and loaded with the metabolically inert substrate [(3)H]1-methyl-4-phenylpyridinium. Release was induced by amphetamine, dopamine, and norepinephrine or by lowering the sodium or chloride concentration in the superfusion buffer (iso-osmotic replacement by lithium and isethionate, respectively). Efflux of [(3)H]1-methyl-4-phenylpyridinium was analyzed at 30-s time resolution. In both transporters, release induced by the substrates amphetamine, dopamine, and norepinephrine followed the same time course as release induced by the removal of chloride and was faster than that caused by the removal of sodium. In the presence of low sodium (DAT: 10 mM; NET: 5 mM) none of the substrates was able to induce release from either type of cell, but adding back sodium to control conditions promptly restored the releasing action. In the presence of low chloride (DAT: 3 mM; NET: 2 mM), however, amphetamine as well as the catecholamines stimulated release from both types of cell. In contrast with the ion dependence of release observed in superfusion experiments, uptake initial rates of substrates at concentrations used in release experiments were the same or even higher at low sodium than at low chloride. The results indicate a decisive role of extracellular sodium for carrier-mediated release unrelated to the sodium-dependent uptake of the releasing substrate, and suggest a release mechanism different from simple exchange diffusion considering only the amines as substrates.
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PMID:Ion dependence of carrier-mediated release in dopamine or norepinephrine transporter-transfected cells questions the hypothesis of facilitated exchange diffusion. 1053 12

The dopamine (DA) transporter (DAT) regulates DA neurotransmission by recycling DA back into neurons. Drugs that interfere with DAT function, e.g., cocaine and amphetamine, can have profound behavioral effects. The kinetics of DA transport by DAT in isolated synaptosomal or single cell preparations have been previously studied. To investigate how DA transport is regulated in intact tissue and to examine how amphetamine affects the DAT, the kinetics of DA uptake by the DAT were examined in tissue slices of the mouse caudate-putamen with fast-scan cyclic voltammetry. The data demonstrate that inward DA transport is saturable and sodium-dependent. Elevated levels of cytoplasmic DA resulting from disruption of vesicular storage by incubation with 10 microM Ro 4-1284 did not generate DA efflux or decrease its uptake rate. However, incubation with 10 microM amphetamine reduced the net DA uptake rate and increased extracellular DA levels due to DA efflux through the DAT. In addition, a new, elevated steady-state level of extracellular DA was established after electrically stimulated DA release in the presence of amphetamine, norepinephrine, and exogenous DA. These results from intact tissue are consistent with a kinetic model of the DAT established in more purified preparations in which amphetamine and other transported substances make the inwardly facing DAT available for outward transport of intracellular DA.
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PMID:Dopamine neuronal transport kinetics and effects of amphetamine. 1058

The serotonin transporters (SERT) are the primary binding sites for selective serotonin reuptake inhibitors, commonly used antidepressants such as fluoxetine, sertraline, and paroxetine. Imaging of SERT with positron emission tomography and single photon emission computed tomography in humans would provide a useful tool for understanding how alterations of this system are related to depressive illnesses and other psychiatric disorders. In this article the synthesis and characterization of [(125)I]ODAM [(5-iodo-2-(2-dimethylaminomethylphenoxy)-benzyl alcohol, 9)] as an imaging agent in the evaluation of central nervous system SERT are reported. A new reaction scheme was developed for the preparation of compound 9, ODAM, and the corresponding tri-n-butyltin derivative 10. Upon reacting 10 with hydrogen peroxide and sodium[(125)I]iodide, the radiolabeled [(125)95%). In an initial binding study using cortical membrane homogenates of rat brain, ODAM displayed a good binding affinity with a value of K(i) = 2.8 +/- 0.88 nM. Using LLC-PK(1) cells specifically expressing the individual transporter (i.e. dopamine [DAT], norepinephrine [NET], and SERT, respectively), ODAM showed a strong inhibition on SERT (K(i) = 0.12 +/- 0.02 nM). Inhibition constants for the other two transporters were lower (K(i) = 3.9 +/- 0.7 microM and 20.0 +/- 1.9 nM for DAT and NET, respectively). Initial biodistribution study in rats after an intravenous (IV) injection of [(125)I]ODAM showed a rapid brain uptake and washout (2.03, 1.49, 0.79, 0.27, and 0.07% dose/organ at 2, 30, 60, 120, and 240 min, respectively). The hypothalamus region where the serotonin neurons are located exhibited a high specific uptake. Ratios of hypothalamus-cerebellum/cerebellum based on percent dose per gram of these two regions showed values of 0.35, 0. 86, 0.86, 0.63, and 0.34 at 2, 30, 60, 120, and 240 min, post-IV injection, respectively. The specific uptake in hypothalamus can be effectively blocked by pretreatment of known SERT ligands. The results suggest that this novel ligand displays desirable in vitro and in vivo properties as a potential SERT imaging agent.
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PMID:A novel serotonin transporter ligand: (5-iodo-2-(2-dimethylaminomethylphenoxy)-benzyl alcohol. 1077 46

Following exocytotic release, the biogenic amine neurotransmitters, norepinephrine, dopamine, and serotonin are removed from the synaptic cleft by the respective transporter, NET, DAT, and SERT, located on the plasma membrane and then re-stored into synaptic vesicles by vesicular monoamine transporter, VMAT. The molecular cloning of these transporters revealed that NET, DAT, and SERT are members of a sodium-dependent neurotransmitter transporter gene family, while VMATs arise from proton-dependent transporter gene family. Structural features common to NET, DAT, and SERT reveal a putative 12 transmembrane-spanning domain structure with cytosolic N- and C-terminal regions. Recent evidence suggest the regulation of the functional expression of these transporters via phosphorylation, which include direct phosphorylation of transporter proteins and/or of associated proteins that may control transporter function/expression. In addition, the substrates and inhibitors for these transporters appear capable of regulating transporter cell surface expression, thereby suggesting both activity-dependent and pharmacological regulatory mechanisms for transporter expression. Analyses of the genes provide new insight into their relation to neuronal diseases since NET, DAT and SERT are the molecular targets for many antidepressants as well as drugs of abuse such as cocaine and amphetamine. The availability of cDNAs of these and vesicular transporters has permitted detailed pharmacological studies in heterologous expression systems, and thus would promise the development of novel drugs with diverse chemical structures.
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PMID:[Pharmacology of monoamine neurotransmitter transporters]. 1249 7


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