title: Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Dental Pharmacology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Sogawa
authors.forename: Chieko
authors.initials: C
authors.lastname: Mitsuhata
authors.forename: Kei
authors.initials: K
authors.lastname: Kumagai-Morioka
authors.forename: Norio
authors.initials: N
authors.lastname: Sogawa
authors.forename: Kazumi
authors.initials: K
authors.lastname: Ohyama
authors.forename: Katsuya
authors.initials: K
authors.lastname: Morita
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Kozai
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Dohi
authors.forename: Shigeo
authors.initials: S
authors.lastname: Kitayama
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002522:Chlorocebus aethiops; D050483:Dopamine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D020033:Protein Isoforms