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ABSTRACT: 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.
Molecular Brain Research 03/2001; 87(1):124-43. · 2.00 Impact Factor
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ABSTRACT: The human dopamine transporter (DAT) gene contains a variable number tandem repeat (VNTR; 40 bases/3 to >11 repeats) in the 3'-untranslated region (3'-UTR), resulting in multiple alleles categorized by length. The 10-copy allele has been associated with attention deficit hyperactivity disorder (ADHD), yet it accounts for only a small proportion of symptom variance. We investigated whether the rhesus monkey DAT gene contains a repeat sequence similar to the human and whether this region differs in the five most hyperactive and the five most sedate animals selected from a behaviorally characterized cohort (n = 22). A fixed number tandem repeat (FNTR; 39 bases/12 repeats) was observed in all animals. Accordingly, this FNTR is unbefitting an association of DAT transcript length with hyperactivity. However, sequence analysis revealed potential single nucleotide polymorphisms (SNPs), one of which affects a Bst1107I restriction site. We screened the entire cohort, confirmed that all the rhesus monkeys had repeat regions of the same length, and demonstrated that digestion with Bst1107I was sufficient to distinguish two distinct FNTR alleles. Bst1107I genotype was suggestive but not predictive of hyperactive behavior. Based on these data, we speculated that SNPs may exist in human DAT VNTR alleles. To support this hypothesis, we cloned a portion of a novel 10-repeat allele from the human gene containing an SNP that abolishes a DraI restriction site. We conclude that SNPs create a diversity of DAT alleles between individuals that may be greater than previously identified based solely on the length of the VNTR region, and that alleles of specific sequence may contribute to dopamine-related disorders.
Molecular Psychiatry 02/2001; 6(1):50-8. · 13.67 Impact Factor
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ABSTRACT: The D(4) dopamine receptor has been investigated for its potential role in neuropsychiatric disorders, "novelty-seeking" behaviors, and effects produced by some psychostimulants. An accurate map of D(4) distribution and density in brain is essential to clarify the role of this receptor subtype in normal brain function and in neuropsychiatric disorders. We investigated the autoradiographic distribution of D(4) receptors in non-human primate (Macaca mulatta) brain (N = 3) with the novel D(4) receptor probe [(3)H]PNU-101958. Quantification of [(3)H]PNU-101958 binding sites in 77 brain regions revealed dense levels of D(4) receptors in several cortical areas, especially in prefrontal cortex, uncus, hypothalamic median eminence, hippocampal formation, and distinct thalamic nuclei, but were significantly lower in striatum. The results correspond well with previous reports of brain distribution of D(4) receptors using other radiolabeled probes, and of D(4) mRNA localization (with some exceptions). Overall, this study reveals that [(3)H]PNU-101958 binding sites in non-human primate brain appear to reflect D(4) dopamine receptor distribution. The significance of a dense localization of D(4) receptors in prefrontal cortex and hippocampus, and broad distribution in other brain areas, allows for investigation of the relationship of these receptors to specific neuropsychiatric disorders and effects produced by psychostimulants.
Synapse 10/2000; 37(3):232-44. · 2.94 Impact Factor
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ABSTRACT: Drug development in psychopharmacology has adhered to the unwritten precept that compounds targeting monoamine transporters must contain an amine nitrogen in the molecular structure. A series of non-amine-bearing aryloxatropanes that are potent inhibitors of the dopamine transporter (DAT) challenged this precept. In the present study, we investigated the brain distribution of a selective, high-affinity DAT non-amine, [(3)H]tropoxene (2-carbomethoxy-3, 4dichloro-3-aryl-8-oxabicyclo[3.2.1] octene), which binds to the DAT in monkey striatum. The autoradiographic distribution of [(3)H]tropoxene was conducted in tissue sections of rhesus (Macaca mulatta) monkey brain. Highest accumulation of the radioligand was detected in the putamen and caudate nucleus, with significant levels also observed in the nucleus accumbens and substantia nigra. Moderate to low levels of [(3)H]tropoxene binding were noted in the hypothalamus, amygdala, ventral tegmental area, and thalamus. The distribution of [(3)H]tropoxene was restricted to brain regions previously identified as expressing DAT, and the relative densities of [(3)H]tropoxene binding sites in various brain regions corresponded to those observed with other selective monoamine radioligands for the DAT. This is the first report to demonstrate that transporter-selective compounds that bear no amine nitrogen in their structure bind selectively to brain regions rich in the transporter. The results support our conclusion that an amine nitrogen is not necessary for compounds to bind to monoamine transporters and distribute in brain according to the known distribution of transporters. The findings provide further incentives to investigate the pharmacological potential of transport inhibitors lacking an amine nitrogen in the molecular structure.
Synapse 11/1999; 34(1):20-7. · 2.94 Impact Factor
