AB1766 Sigma-AldrichAnti-Dopamine Transporter Antibody, CT

The transporters for dopamine (DAT) and norepinephrine (NET) are members of the Na+- and Cl--dependent neurotransmitter transporter family SLC6. There is a line of evidence that alternative splicing results in several isoforms of neurotransmitter transporters including NET. However, its relevance to the physiology and pathology of the neurotransmitter reuptake system has not been fully elucidated.We found novel isoforms of human DAT and NET produced by alternative splicing in human blood cells (DAT) and placenta (NET), both of which lacked the region encoded by exon 6. RT-PCR analyses showed a difference in expression between the full length (FL) and truncated isoforms in the brain and peripheral tissues, suggesting tissue-specific alternative splicing. Heterologous expression of the FL but not truncated isoforms of DAT and NET in COS-7 cells revealed transport activity. However, immunocytochemistry with confocal microscopy and a cell surface biotinylation assay demonstrated that the truncated as well as FL isoform was expressed at least in part in the plasma membrane at the cell surface, although the truncated DAT was distributed to the cell surface slower than FL DAT. A specific antibody to the C-terminus of DAT labeled the variant but not FL DAT, when cells were not treated with Triton for permeabilization, suggesting the C-terminus of the variant to be located extracellulary. Co-expression of the FL isoform with the truncated isoform in COS-7 cells resulted in a reduced uptake of substrates, indicating a dominant negative effect of the variant. Furthermore, an immunoprecipitation assay revealed physical interaction between the FL and truncated isoforms.The unique expression and function and the proposed membrane topology of the variants suggest the importance of isoforms of catecholamine transporters in monoaminergic signaling in the brain and peripheral tissues.

The Coffin-Lowry syndrome, a rare syndromic form of X-linked mental retardation, is caused by loss-of-function mutations in the hRSK2 (RPS6KA3) gene. To further investigate RSK2 (90-kDa ribosomal S6 kinase) implication in cognitive processes, a mrsk2_KO mouse has previously been generated as an animal model of Coffin-Lowry syndrome. The aim of the present study was to identify possible neurochemical dysregulation associated with the behavioral and morphological abnormalities exhibited by mrsk2_KO mice. A cortical dopamine level increase was found in mrsk2_KO mice that was accompanied by an over-expression of dopamine receptor of type 2 and the dopamine transporter. We also detected an increase of total and phosphorylated extracellular regulated kinase that may be responsible for the increased level of tyrosine hydroxylase phosphorylation also observed. By taking into consideration previously reported data, our results strongly suggest that the dopaminergic dysregulation in mrsk2_KO mice may be caused, at least in part, by tyrosine hydroxylase hyperactivity. This cortical hyperdopaminergia may explain some non-cognitive but also cognitive alterations exhibited by mrsk2_KO mice.

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, P0.05). 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.