Oligomerization of the human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells.
Recent biochemical studies indicate that the serotonin transporter can form oligomers. We investigated whether the human serotonin transporter (hSERT) can be visualized as an oligomer in the plasma membrane of intact cells. For this purpose, we generated fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP, respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were efficiently inserted into the plasma membrane and were functionally indistinguishable from wild-type hSERT. Oligomers were visualized by fluorescence resonance energy transfer microscopy in living cells using two complementary methods, i.e. ratio imaging and donor photobleaching. Interestingly, oligomerization was not confined to hSERT; fluorescence resonance energy transfer was also observed between CFP- and YFP-labeled rat gamma-aminobutyric acid transporter. The bulk of serotonin transporters was recovered as high molecular weight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we conclude that constitutive oligomer formation might be a general property of Na(+)/Cl(-)-dependent neurotransmitter transporters.
Schmid
J A
JA
Institute of Pharmacology, University of Vienna Medical School, Währingerstrasse 13a, A-1090 Vienna, Austria.
Scholze
P
P
Kudlacek
O
O
Freissmuth
M
M
Singer
E A
EA
Sitte
H H
HH
D000818:Animals; D001704:Biopolymers; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D004735:Energy Transfer; D050485:GABA Plasma Membrane Transport Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; D051381:Rats; D050486:Serotonin Plasma Membrane Transport Proteins; D013050:Spectrometry, Fluorescence
D001704:Biopolymers; D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D027361:Organic Anion Transporters; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C493232:Slc6a4 protein, rat