Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6.
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...
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doaj-670a8e5b939e4fe5ac3434a7b83d12df2020-11-25T02:21:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-01-0158e1194510.1371/journal.pone.0011945Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6.Chiharu SogawaChieko MitsuhataKei Kumagai-MoriokaNorio SogawaKazumi OhyamaKatsuya MoritaKatsuyuki KozaiToshihiro DohiShigeo KitayamaBACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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.http://europepmc.org/articles/PMC2916826?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Chiharu Sogawa Chieko Mitsuhata Kei Kumagai-Morioka Norio Sogawa Kazumi Ohyama Katsuya Morita Katsuyuki Kozai Toshihiro Dohi Shigeo Kitayama |
spellingShingle |
Chiharu Sogawa Chieko Mitsuhata Kei Kumagai-Morioka Norio Sogawa Kazumi Ohyama Katsuya Morita Katsuyuki Kozai Toshihiro Dohi Shigeo Kitayama Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. PLoS ONE |
author_facet |
Chiharu Sogawa Chieko Mitsuhata Kei Kumagai-Morioka Norio Sogawa Kazumi Ohyama Katsuya Morita Katsuyuki Kozai Toshihiro Dohi Shigeo Kitayama |
author_sort |
Chiharu Sogawa |
title |
Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
title_short |
Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
title_full |
Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
title_fullStr |
Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
title_full_unstemmed |
Expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
title_sort |
expression and function of variants of human catecholamine transporters lacking the fifth transmembrane region encoded by exon 6. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2010-01-01 |
description |
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. METHODOLOGY/PRINCIPAL FINDINGS: 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/SIGNIFICANCE: 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. |
url |
http://europepmc.org/articles/PMC2916826?pdf=render |
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