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Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

ABSTRACT: 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 (PIP₂) stabilized the oligomers, the stability of mGFP-hDAT dimers was PIP₂ independent.

SUBMITTER: Das AK

PROVIDER: S-EPMC6462504 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

Das Anand Kant AK Kudlacek Oliver O Baumgart Florian F Jaentsch Kathrin K Stockner Thomas T Sitte Harald H HH Schütz Gerhard J GJ

The Journal of biological chemistry 20190131 14

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 exac ...[more]

PMID: 30705091

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Project description:BackgroundPhosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a critically important regulatory phospholipid found in the plasma membrane of all eukaryotic cells. In addition to being a precursor of important second messengers, PtdIns(4,5)P2 also regulates ion channels and transporters and serves the endocytic machinery by recruiting clathrin adaptor proteins. Visualization of the localization and dynamic changes in PtdIns(4,5)P2 levels in living cells is critical to understanding the biology of PtdIns(4,5)P2. This has been mostly achieved with the use of the pleckstrin homology (PH) domain of PLCdelta1 fused to GFP. Here we report on a comparative analysis of several recently-described yeast PH domains as well as the mammalian Tubby domain to evaluate their usefulness as PtdIns(4,5)P2 imaging tools.ResultsAll of the yeast PH domains that have been previously shown to bind PtdIns(4,5)P2 showed plasma membrane localization but only a subset responded to manipulations of plasma membrane PtdIns(4,5)P2. None of these domains showed any advantage over the PLCdelta1PH-GFP reporter and were compromised either in their expression levels, nuclear localization or by causing peculiar membrane structures. In contrast, the Tubby domain showed high membrane localization consistent with PtdIns(4,5)P2 binding and displayed no affinity for the soluble headgroup, Ins(1,4,5)P3. Detailed comparison of the Tubby and PLCdelta1PH domains showed that the Tubby domain has a higher affinity for membrane PtdIns(4,5)P2 and therefore displays a lower sensitivity to report on changes of this lipid during phospholipase C activation.ConclusionThese results showed that both the PLCdelta1PH-GFP and the GFP-Tubby domain are useful reporters of PtdIns(4,5)P2 changes in the plasma membrane, with distinct advantages and disadvantages. While the PLCdelta1PH-GFP is a more sensitive reporter, its Ins(1,4,5)P3 binding may compromise its accuracy to measure PtdIns(4,5)P2 changes. The Tubby domain is more accurate to report on PtdIns(4,5)P2 but its higher affinity and lower sensitivity may limit its utility when phospholipase C activation is only moderate. These studies also demonstrated that similar changes in PtdIns(4,5)P2 levels in the plasma membrane can differentially regulate multiple effectors if they display different affinities to PtdIns(4,5)P2.

Dataset Information

Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

Publications

Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate-independent manner.

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