Lipid Mediated Oligomeric Assembly of the Serotonin Transporter at the Plasma Membrane

Abstract

The human serotonin transporter (hSERT) is responsible for the termination of synaptic serotonergic signaling. Using single molecule microscopy, we have shown previously that SERT forms higher oligomers at the plasma membrane of living cells; we found a variety of aggregation 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, indicating a stable interaction of the subunits. We developed a special bleaching protocol to decipher the interaction kinetics of SERT oligomers; the complexes remained stable over several minutes in the live cell plasma membrane. Together, the results indicate kinetic trapping of SERT oligomers at the plasma membrane, subsequent to an equilibration which occurs at an unknown subcellular organelle.By evaluating the subunit stoichiometry of SERT in the endoplasmic reticulum (ER) we show here that the oligomerization is indeed chemically equilibrated at ER membranes; after trafficking to the plasma membrane, the SERT stoichiometry remains fixed. Furthermore, we show that the stabilization of the transporter complexes can be ascribed to the differences in the lipid composition of the plasma membrane and the ER. Upon alteration of the lipid composition the SERT complexes are liberated from kinetic trapping at the plasma membrane and their oligomeric size is significantly reduced. In conjunction with the use of SERT mutants exhibiting altered lipid binding capability, we could thus show that stable oligomerization of SERT at the plasma membrane is lipid-mediated.