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Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner
The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; f...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462504/ https://www.ncbi.nlm.nih.gov/pubmed/30705091 http://dx.doi.org/10.1074/jbc.RA118.006178 |
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author | Das, Anand Kant Kudlacek, Oliver Baumgart, Florian Jaentsch, Kathrin Stockner, Thomas Sitte, Harald H. Schütz, Gerhard J. |
author_facet | Das, Anand Kant Kudlacek, Oliver Baumgart, Florian Jaentsch, Kathrin Stockner, Thomas Sitte, Harald H. Schütz, Gerhard J. |
author_sort | Das, Anand Kant |
collection | PubMed |
description | The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP–hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP–hDAT co-exist and that higher-order molecular complexes of mGFP–hDAT are absent at the plasma membrane. The mGFP–hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP–hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilized the oligomers, the stability of mGFP–hDAT dimers was PIP(2) independent. |
format | Online Article Text |
id | pubmed-6462504 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-64625042019-04-15 Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner Das, Anand Kant Kudlacek, Oliver Baumgart, Florian Jaentsch, Kathrin Stockner, Thomas Sitte, Harald H. Schütz, Gerhard J. J Biol Chem Membrane Biology The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP–hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP–hDAT co-exist and that higher-order molecular complexes of mGFP–hDAT are absent at the plasma membrane. The mGFP–hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP–hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) stabilized the oligomers, the stability of mGFP–hDAT dimers was PIP(2) independent. American Society for Biochemistry and Molecular Biology 2019-04-05 2019-01-31 /pmc/articles/PMC6462504/ /pubmed/30705091 http://dx.doi.org/10.1074/jbc.RA118.006178 Text en © 2019 Das et al. Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license (http://creativecommons.org/licenses/by/4.0) . |
spellingShingle | Membrane Biology Das, Anand Kant Kudlacek, Oliver Baumgart, Florian Jaentsch, Kathrin Stockner, Thomas Sitte, Harald H. Schütz, Gerhard J. Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title | Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title_full | Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title_fullStr | Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title_full_unstemmed | Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title_short | Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
title_sort | dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner |
topic | Membrane Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462504/ https://www.ncbi.nlm.nih.gov/pubmed/30705091 http://dx.doi.org/10.1074/jbc.RA118.006178 |
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