Abstract
The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Although there is solid evidence that SERT forms oligomeric complexes, the exact stoichiometry of the complexes and the fractions of different coexisting oligomeric states still remain enigmatic. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of the SERT via brightness analysis of single diffraction-limited fluorescent spots. Heterologously expressed SERT was labeled either with the fluorescent inhibitor JHC 1-64 or via fusion to monomeric GFP. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. Furthermore, oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane.
Highlights
The serotonin transporter (SERT) terminates synaptic signaling by reuptake of the neurotransmitter serotonin
Oligomerization was found to be independent of SERT surface density, and oligomers remained stable over several minutes in the live cell plasma membrane
We first evaluated the subunit stoichiometry of an mGFPSERT fusion construct heterologously expressed in HEK 293 cells
Summary
The serotonin transporter (SERT) terminates synaptic signaling by reuptake of the neurotransmitter serotonin. Significance: The results reveal the first evidence for kinetic trapping of preformed neurotransmitter transporter oligomers. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations larger than dimers and demonstrating the coexistence of different degrees of oligomerization in a single cell; the data are in agreement with a linear aggregation model. The mentioned studies have provided an important contribution to obtain a deeper understanding of the oligomerization of NSSs, these methods cannot determine the exact stoichiometry of the complexes, the distribution of different oligomeric states, and its interaction kinetics. We used a single molecule approach based on brightness analysis of diffraction-limited spots (19 –22) to unravel the subunit stoichiometry of fluorescently labeled SERT in the native plasma membrane of living HEK 293 cells. Using a novel photobleaching protocol, we found that the formed complexes are stable on the minute time scale, indicating kinetic trapping of preformed oligomers
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