Dopamine transporter forms stable dimers in the live cell plasma membrane in a phosphatidylinositol 4,5-bisphosphate–independent manner

Abstract

The human dopamine transporter (hDAT) regulates the level of the neurotransmitter dopamine (DA) in the synaptic cleft and recycles DA for storage in the presynaptic vesicular pool. Many neurotransmitter transporters exist as oligomers, but the physiological role of oligomerization remains unclear; for example, it has been speculated to be a prerequisite for amphetamine-induced release and protein trafficking. Previous studies point to an oligomeric quaternary structure of hDAT; however, the exact stoichiometry and the fraction of co-existing oligomeric states are not known. Here, we used single-molecule brightness analysis to quantify the degree of oligomerization of heterologously expressed hDAT fused to monomeric GFP (mGFP–hDAT) in Chinese hamster ovary (CHO) cells. We observed that monomers and dimers of mGFP–hDAT co-exist and that higher-order molecular complexes of mGFP–hDAT are absent at the plasma membrane. The mGFP–hDAT dimers were stable over several minutes, and the fraction of dimers was independent of the mGFP–hDAT surface density. Furthermore, neither oxidation nor depletion of cholesterol had any effect on the fraction of dimers. Unlike for the human serotonin transporter (hSERT), in which direct binding of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) stabilized the oligomers, the stability of mGFP–hDAT dimers was PIP 2 independent.

Direct coupling of oligomerization and oligomerization-driven endocytosis of the dopamine transporter to its conformational mechanics and activity

“…These trimers apparently represent only a small fraction of the plasma membrane DAT in untreated cells based on western blotting detection and cross-linking with BS 3 , and are highly specific to DAT. By contrast, single-molecule microscopy analysis showed that about 35% of the plasma membrane DAT is constitutively dimeric (24). We did not observe SDS-resistant or BS 3 -linked dimer species of wt DAT detectable by western blotting, and our modeling predicts AIM-100/AL stabilization of DAT trimers by strong hydrophobic interactions, which may explain SDS-resistance of trimers ( Figure 7).…”

“…We did not observe SDS-resistant or BS 3 -linked dimer species of wt DAT detectable by western blotting, and our modeling predicts AIM-100/AL stabilization of DAT trimers by strong hydrophobic interactions, which may explain SDS-resistance of trimers ( Figure 7). Furthermore, DAT dimers were detected by single-molecule imaging due to their lateral mobility in the plasma membrane (24). By contrast, AIM-100 oligomers were mostly immobile (25), and therefore would not be detected by the method used in (24).…”

“…For example while Drosophila DAT was crystallized as a monomer (12), biochemical and microscopy studies provide evidence for dimerization, high order oligomerization and cluster assembly of mammalian DAT expressed in non-neuronal cells and endogenous rodent DAT in dopaminergic neurons (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Furthermore, the molecular mechanisms mediating and regulating DAT oligomerization, and their relation to substrate transport properties and intracellular trafficking remain largely unknown.…”

Monoamine transporters: structure, intrinsic dynamics and allosteric regulation

“…Published work has shown that trimerization of hDAT is promoted by the binding of a heterocyclic drug (furopyrimidine AIM-100) 25 . DAT forms stable dimers in a PIP 2 - and CHOL-independent manner 22 , whereas for SERT, the formation of oligomers is promoted by the binding of PIP 2 (ref. 24 ).…”

Structure and Gating Dynamics of Na+/Cl– Coupled Neurotransmitter Transporters

“…Computational analyses identified that this interaction can stabilize additional intermediates in the ion-release pathway thereby facilitating altered Na + release pathways leading to dopamine transport (Razavi et al, 2018). Also, PIP2 interactions are suggested to mediate stable oligomer formation with hSERT (Anderluh et al, 2017) although hDAT oligomerization is observed to be PIP2 independent in single-molecule studies (Das et al, 2019). Most of the interactions with PIP2 were suggested to happen at the N-termini of the transporters, which are not part of the experimental structures as they have been removed due to their disordered characteristics.…”