Presenting Author University Hospital Basel Basel, Basel-Stadt, Switzerland
Background: Monoamine transporters are the principal regulators of extracellular monoamine levels in the brain. At the plasma membrane, monoamine transporters form oligomers, which may play an important role in transporter trafficking, drug-induced monoamine efflux, and drug tolerance development. Using single molecule microscopy, we previously reported that the serotonin transporter (SERT) co-exists as monomers, dimers, and higher-order oligomers, while the dopamine transporter (DAT) forms monomers and dimers only. Furthermore, whereas SERT oligomers are kinetically trapped at the plasma membrane by the phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), DAT dimerization is PIP2 independent. The subunit stoichiometry of the norepinephrine transporter (NET) is currently unknown. Based on a high degree of sequence homology between DAT and NET, we aimed to test the hypothesis that NET oligomerization resembles the one of DAT.
Methods: We fused monomeric green fluorescent protein (mGFP) to the N-terminus of human NET and expressed the construct in Chinese hamster ovary cells. We assessed NET oligomerization using single molecule total internal reflection fluorescence (TIRF) microscopy in live cells. In order to determine the role of cell membrane lipids in NET oligomerization, we pharmacologically depleted the cell membrane of cholesterol and PIP2 before imaging.
Results: Single molecule brightness analysis revealed that NET co-exists as monomers (~60%) and dimers (~40%) at the plasma membrane. NET oligomerization displayed signs of transporter density dependence and subunit exchange. Cholesterol depletion did not affect NET dimerization, whereas PIP2 depletion resulted in a small but significant decrease in the average oligomeric state (Students t-test, Plt;0.05).
Conclusion: NET forms monomers and dimers but no higher-order oligomers, thereby resembling the oligomerization of DAT. However, unlike DAT, NET dimers exchange subunits over time and dimerization is affected by the transporter density and PIP2 concentrations. This study therefore demonstrates that the oligomerization behavior of each monoamine transporters has unique features.
This work was supported by the Swiss National Science Foundation (grant No. P400PM_191032 to D.L.).
