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. F or this purpose, we generated fusion proteins of hSERT and spectral variant s of the green fluorescent protein (cyan and yellow fluorescent proteins, C FP and YFP, respectively). When expressed in human embryonic kidney 293 cel ls, the resulting fusion proteins (CFP-hSERT and PFP-hSERT) were efficientl y 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, oligomerizatio n was not confined to hSERT; fluorescence resonance energy transfer was als o observed between CFP- and YFP-labeled rat gamma -aminobutyric acid transp orter, The bulk of serotonin transporters was recovered as high molecular w eight complexes upon gel filtration in detergent solution. In contrast, the monomers of CFP-hSERT and YFP-hSERT were essentially undetectable, This in dicates that the homo-oligomeric form is the favored state of hSERT in livi ng cells, which is not significantly affected by coincubation with transpor ter substrates or blockers. Based on our observations, we conclude that con stitutive oligomer formation might be a general property of Na+/Cl--depende nt neurotransmitter transporters.