Single Molecule Analysis Reveals Coexistence of Stable Serotonin Transporter Monomers and Oligomers in the Live Cell Plasma Membrane

Abstract

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. The formation of SERT oligomers in the plasma membrane has already been indicated by various approaches, including optical methods such as Förster resonance energy transfer (FRET) and classical biochemical ensemble analysis. However, neither application is suitable to yield quantitative interpretation and decipher the exact configuration of the oligomerization states; additionally, biochemical methods do not take the influence of the membrane environment into account. Here we used single molecule fluorescence microscopy to obtain the oligomerization state of SERT via brightness analysis of single diffraction limited fluorescent spots. The techniques applied in this study allow for identification and quantitative evaluation of subpopulations of SERT complexes exhibiting different degrees of oligomerization in a living cell. We found a variety of oligomerization states of membrane-associated transporters, revealing molecular associations at least up to pentamers and demonstrating the coexistence of different degrees of oligomerization in a single cell. The oligomerization was found to be independent of SERT surface density, and the interactions were stable over several minutes. Together, these results indicate kinetic trapping of preformed SERT oligomers at the plasma membrane. Next, we developed a strategy for single molecule analysis at the membrane of the endoplasmic reticulum. By evaluating the oligomerization of SERT in the ER we found that the oligomerization process is chemically equilibrated at ER membranes; after trafficking to the plasma membrane, the SERT stoichiometry remains fixed. This work was supported by the Austrian Science Fund FWF, the NIDA-IRP, and the NIH.