Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acid transporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product in Xenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na(+),Cl(-)-coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as l-proline, l-threonine, and l-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl(-). Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K(+) but preferring proline in the presence of Na(+).
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PMID:A novel electrogenic amino acid transporter is activated by K+ or Na+, is alkaline pH-dependent, and is Cl--independent. 1082 35

1. Carrier mediated uptake (uptake-1) transport of norepinephrine (NE) plays a key role in the regulation of sympathetic neurotransmission. Recent investigations indicate that nitric oxide (NO) may modulate uptake-1 activity, possibly in a cyclic GMP independent manner. 2. Carrier mediated transport of [(3)H-NE] and [(3)H-dopamine, DA] was examined in CHO cells transfected with cDNA for the NE and DA transporters (NET, DAT) respectively. 3. While exposure to the NO donor S-nitroso-N-acetylpenicillamine (100 microM, SNAP) significantly reduced [(3)H-NE] uptake (P<0.001), no effect on [(3)H-DA] transport was apparent. 4. Comparison of the amino acid sequences for NET and DAT identified cysteine residue 351 in NET, which was not present in DAT. Site-directed mutagenesis of Cys 351 to Ser produced a functional NET that was resistant to the inhibitory effects of SNAP. 5. The presence of SNAP mediated nitrosylation of the cysteine residue in an 8-mer model peptide based around Cys 351 in NET was confirmed by both biochemical and mass spectroscopic means. 6. These data indicate the potential regulatory role for NO in modulating sympathetic neurotransmission, and further confirm the importance of non-cyclic GMP dependent mechanisms in mediating the actions of NO.
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PMID:Nitric oxide mediated modulation of norepinephrine transport: identification of a potential target for S-nitrosylation. 1088 90

Presynaptic transporter proteins regulate the clearance of extracellular biogenic amines after release and are important targets for multiple psychoactive agents, including amphetamines, cocaine, and antidepressant drugs. Recent studies reveal that dopamine (DA), norepinephrine (NE), and serotonin (5-HT) transporters (DAT, NET, and SERT, respectively) are rapidly regulated by direct or receptor-mediated activation of cellular kinases, particularly protein kinase C (PKC). With SERTs, PKC activation results in activity-dependent transporter phosphorylation and sequestration. Protein phosphatase 1/2A (PP1/PP2A) inhibitors, such as okadaic acid (OA) and calyculin A, also promote SERT phosphorylation and functional downregulation. How kinase, phosphatase, and transporter activities are linked mechanistically is unclear. In the present study, we found that okadaic acid-sensitive phosphatase activity is enriched in SERT immunoprecipitates from human SERT stably transfected cells. Moreover, blots of these immunoprecipitates reveal the presence of PP2A catalytic subunit (PP2Ac), findings replicated using brain preparations. Whole-cell treatments with okadaic acid or calyculin A diminished SERT/PP2Ac associations. Phorbol esters, which trigger SERT phosphorylation, also diminish SERT/PP2Ac associations, effects that can be blocked by PKC antagonists as well as the SERT substrate 5-HT. Similar transporter/PP2Ac complexes were also observed in coimmunoprecipitation studies with NETs and DATs. Our findings provide evidence for the existence of regulated heteromeric assemblies involving biogenic amine transporters and PP2A and suggest that the dynamic stability of these complexes may govern transporter phosphorylation and sequestration.
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PMID:Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A. 1102 16

The norepinephrine (NET) and dopamine (DAT) transporters are highly homologous proteins, displaying many pharmacological similarities. Both transport dopamine with higher affinity than norepinephrine and are targets for the psychostimulants cocaine and amphetamine. However, they strikingly contrast in their affinities for tricyclic antidepressants (TCA). Previous studies, based on chimeric proteins between DAT and NET suggest that domains ranging from putative transmembrane domain (TMD) 5 to 8 are involved in the high affinity binding of TCA to NET. We substituted 24 amino acids within this region in the human NET with their counterparts in the human DAT, resulting in 22 different mutants. Mutations of residues located in extra- or intracytoplasmic loops have no effect on binding affinity of neither TCA nor cocaine. Three point mutations in TMD6 (F316C), -7 (V356S), and -8 (G400L) induced a loss of TCA binding affinity of 8-, 5-, and 4-fold, respectively, without affecting the affinity of cocaine. The triple mutation F316C/V356S/G400L produced a 40-fold shift in desipramine affinity. These three residues are strongly conserved in all TCA-sensitive transporters cloned in mammalian and nonmammalian species. A strong shift in TCA affinity (IC(50)) was also observed for double mutants F316C/D336T (35-fold) and S399P/G400L (80-fold for nortriptyline and 1000-fold for desipramine). Reverse mutations P401S/L402G in hDAT did not elicit any gain in TCA affinities, whereas C318F and S358V resulted in a 3- and 10-fold increase in affinity, respectively. Our results clearly indicate that two residues located in TMD6 and -7 of hNET may play an important role in TCA interaction and that a critical region in TMD8 is likely to be involved in the tertiary structure allowing the high affinity binding of TCA.
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PMID:Determination of residues in the norepinephrine transporter that are critical for tricyclic antidepressant affinity. 1109 98

We used RT-PCR to clone monoamine transporters from Macaca mulatta, Macaca fasicularis and Saimiri sciureus (dopamine transporter; DAT) and Macaca mulatta (norepinephrine transporter; NET and serotonin transporter; SERT). Monkey DAT, NET and SERT proteins were >98% homologous to human and, when expressed in HEK-293 cells, displayed drug affinities and uptake kinetics that were highly correlated with monkey brain or human monoamine transporters. In contrast to reports of other species, we discovered double (leucine for phenylalanine 143 and arginine for glutamine 509; Variant I) and single (proline for leucine 355; Variant II) amino acid variants of DAT. Variant I displayed dopamine transport kinetics and binding affinities for various DAT blockers (including cocaine) versus [3H] CFT (WIN 35, 428) that were identical to wild-type DAT (n=7 drugs; r(2)=0.991). However, we detected a six-fold difference in the affinity of cocaine versus [3H] cocaine between Variant I (IC(50): 488+/-102 nM, SEM, n=3) and wild-type DAT (IC(50): 79+/-8.2 nM, n=3, P<0.05). Variant II was localized intracellularly in HEK-293 cells, as detected by confocal microscopy, and had very low levels of binding and dopamine transport. Also discovered was a novel exon 5 splice variant of NET that displayed very low levels of transport and did not bind cocaine. With NetPhos analysis, we detected a number of highly conserved putative phosphorylation sites on extracellular as well as intracellular loops of the DAT, NET, and SERT, which may be functional for internalized transporters. The homology and functional similarity of human and monkey monoamine transporters further support the value of primates in investigating the role of monoamine transporters in substance abuse mechanisms, neuropsychiatric disorders and development of diagnostic and therapeutic agents.
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PMID:Cloning of dopamine, norepinephrine and serotonin transporters from monkey brain: relevance to cocaine sensitivity. 1122 67

Biogenic amine transporters, namely the dopamine (DA), norepinephrine (NE) and 5-hydroxytryptamine (serotonin, 5-HT) transporters (DAT, NET and 5-HTT, respectively) appear to be the key elements in regulating biogenic amine neurotransmission. These proteins therefore represent a primary target for therapeutic intervention in the treatment of numerous psychiatric disorders, such as depression, anxiety and perhaps even schizophrenia as well as drug abuse. The cloning of DAT, NET and 5-HTT and development of selective radioligands for them over the last decade has dramatically increased our understanding of their location, structure and function. These breakthroughs have also enabled remarkable progress in determining how biogenic amine transporters are regulated under not only normal conditions but also when confronted with acute or chronic exposure to a variety of stimuli including psychotherapeutic drugs. Because of the important therapeutic consequences of a better understanding of these transporters, the present review discusses recent advances in defining their mechanism of action, location and regulation and the implications of the newer data for neuropsychopharmacology.
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PMID:New views of biogenic amine transporter function: implications for neuropsychopharmacology. 1128 47

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine (DAT), serotonin (SERT), and norepinephrine (NET). Cocaine reward/reinforcement has been linked to actions at DAT or to blockade of SERT. However, knockouts of neither DAT, SERT, or NET reduce cocaine reward/reinforcement, leaving substantial uncertainty about cocaine's molecular mechanisms for reward. Conceivably, the molecular bases of cocaine reward might display sufficient redundancy that either DAT or SERT might be able to mediate cocaine reward in the other's absence. To test this hypothesis, we examined double knockout mice with deletions of one or both copies of both the DAT and SERT genes. These mice display viability, weight gain, histologic features, neurochemical parameters, and baseline behavioral features that allow tests of cocaine influences. Mice with even a single wild-type DAT gene copy and no SERT copies retain cocaine reward/reinforcement, as measured by conditioned place-preference testing. However, mice with no DAT and either no or one SERT gene copy display no preference for places where they have previously received cocaine. The serotonin dependence of cocaine reward in DAT knockout mice is thus confirmed by the elimination of cocaine place preference in DAT/SERT double knockout mice. These results provide insights into the brain molecular targets necessary for cocaine reward in knockout mice that develop in their absence and suggest novel strategies for anticocaine medication development.
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PMID:Molecular mechanisms of cocaine reward: combined dopamine and serotonin transporter knockouts eliminate cocaine place preference. 1132 Feb 58

There is evidence for an association between polymorphisms of monoamine transporter genes and temperamental personality traits. Recent findings have shown that interaction of allelic variants of the different genes may contribute to the personality factors. We studied the association between temperamental personality dimensions measured with the Temperament and Character Inventory (TCI) and polymorphisms of the dopamine (DAT), norepinephrine (NET) and serotonin (5-HTT) transporter genes in 127 healthy Polish volunteers. There were no significant differences between means of TCI temperamental dimensions (novelty seeking, reward dependence, persistence and harm avoidance) and the transporter genes compared by ANOVA. There were some significant associations between genotypes and TCI subdimensions. Individuals carrying the A9/A9 DAT genotype have lower RD4 scores (dependence vs. independence) than A10/A10 individuals (3.0 +/- 1.4 vs. 3.5 +/- 1.3); p = 0.01. Examining 5-HTT gene promoter polymorphism, heterozygous individuals (l/s) and individuals with 44-bp deletion (s/s) scored significantly lower in the HA1 subdimension (anticipatory worry and pessimism vs. uninhibited optimism; 4.3 +/- 2.3 vs. 5.5 +/- 2.6) in comparison with individuals without deletion (l/l); p = 0.021. The NET transporter gene polymorphism showed no significant association with any of the temperamental TCI subdimensions.
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PMID:Polymorphisms in the dopamine, serotonin, and norepinephrine transporter genes and their relationship to temperamental dimensions measured by the Temperament and Character Inventory in healthy volunteers. 1134 Mar 64

The synthesis of novel 4beta-aryl-1-methyl-3alpha-(3-substituted-1,2,4-oxadiazol-5-yl)piperidines, bioisosteres of ester (+)-1, is described. The synthesized oxadiazoles were evaluated for their affinity to the DAT and their ability to inhibit monoamine reuptake at the DAT, NET, and 5HTT. The results show that affinity to the DAT and ability to inhibit the reuptake at the DAT, NET, and 5HTT is a function of the size of the substituent in the oxadiazole ring. (+)-(3R,4S)-4beta-(4-Chlorophenyl)-1-methyl-3alpha-(3-methyl-1,2,4-oxadiazol-5-yl)piperidine [(+)-2a], which is structurally and pharmacologically most similar to the ester (+)-1 in this series, showed at least a 2-fold longer duration of action when compared to ester (+)-1.
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PMID:Sar studies of piperidine-based analogues of cocaine. Part 3: oxadiazoles. 1151 43


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