The formation of oligomeric structures has been proposed for a large number of membrane proteins, including G-protein-coupled receptors and ion channels. Biochemical studies employing gel filtration, cross-linking or co-immunoprecipitation techniques showed that the serotonin [5-hydroxytryptamine (5-HT)] transporter is also capable of forming oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. To test this working hypothesis, we generated fusion proteins of hSERT and spectral variants of green fluorescent protein [cyan and yellow fluorescent proteins (CFP and YFP, respectively)]. When expressed in HeLa or HEK-293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were inserted into the plasma membrane and were indistinguishable from wild-type hSERT on functional testing (5-HT uptake assays, inhibition of 5-HT uptake by blockers such as imipramine). Oligomers were visualized by fluorescence resonance energy transfer (FRET) microscopy in living cells using complementary methods. Interestingly, oligomerization was not confined to hSERT; FRET was also observed between CFP-and YFP-labelled rat γ-aminobutyric acid transporter. Gel filtration experiments showed that most of the protein was recovered as higher molecular weight complexes; almost no monomeric form was detected. This indicates that the homo-oligomeric form is the favoured state of hSERT in living cells. The formation of oligomers was not significantly affected by co-incubation with transporter substrates or blockers. Based on our observations, oligomer formation might not be essential for the physiological function of the transporter protein, the re-uptake of substrates. Furthermore, we conclude that constitutive oligomer formation might be a general property of Na+/Cl−-dependent neurotransmitter transporters.
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Conference Article|
November 01 2001
Impact of oligomerization on the function of the human serotonin transporter
J. A. Schmid;
J. A. Schmid
*Department of Vascular Biology and Thrombosis Research, Vienna International Research Co-operation Center, Brunnerstrasse 59, A-1234 Vienna, Austria
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H. Just;
H. Just
†University of Vienna, Austria Institute of Pharmacology, Währingerstrasse 13a, A-1090 Vienna, Austria
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H. H. Sitte
H. H. Sitte
1
†University of Vienna, Austria Institute of Pharmacology, Währingerstrasse 13a, A-1090 Vienna, Austria
1To whom correspondence should be addressed (e-mail harald.sitte@univie.ac.at).
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Publisher: Portland Press Ltd
Received:
June 14 2001
Online ISSN: 1470-8752
Print ISSN: 0300-5127
© 2001 Biochemical Society
2001
Biochem Soc Trans (2001) 29 (6): 732–736.
Article history
Received:
June 14 2001
Citation
J. A. Schmid, H. Just, H. H. Sitte; Impact of oligomerization on the function of the human serotonin transporter. Biochem Soc Trans 1 November 2001; 29 (6): 732–736. doi: https://doi.org/10.1042/bst0290732
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