Dopamine transporter oligomerization involves the scaffold domain, but spares the bundle domain

Abstract

The human dopamine transporter (hDAT) is located on presynaptic neurons, where it plays an essential role in limiting dopaminergic signaling by temporarily curtailing high neurotransmitter concentration through rapid re-uptake. Transport by hDAT is energized by transmembrane ionic gradients. Dysfunction of this transporter leads to disease states, such as Parkinson’s disease, bipolar disorder or depression. It has been shown that hDAT and other members of the monoamine transporter family exist in oligomeric forms at the plasma membrane. Several residues are known to be involved in oligomerization, but interaction interfaces, oligomer orientation and the quarternary arrangement in the plasma membrane remain poorly understood. Here we examine oligomeric forms of hDAT using a direct approach, by following dimerization of two randomly-oriented hDAT transporters in 512 independent simulations, each being 2 μs in length. We employed the DAFT (docking assay for transmembrane components) approach, which is an unbiased molecular dynamics simulation method to identify oligomers, their conformations and populations. The overall ensemble of a total of >1 ms simulation time revealed a limited number of symmetric and asymmetric dimers. The identified dimer interfaces include all residues known to be involved in dimerization. Importantly, we find that the surface of the bundle domain is largely excluded from engaging in dimeric interfaces. Such an interaction would typically lead to inhibition by stabilization of one conformation, while substrate transport relies on a large scale rotation between the inward-facing and the outward-facing state.

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

“…Oligomers could also have deleterious effects on cells as observed in the case of neurodegenerative disorders (12–14). For a large number of proteins, there are specific binding domains that facilitate or modulate oligomerization (15, 16). Lipids have also been found to be important in membrane protein folding and oligomerization (17–19).…”

“…In addition, cross-linking experiments suggest the involvement of TMHs 4 and 6 in the interfaces of symmetrical hDAT dimers (31, 35, 36). More recently, unbiased molecular dynamics simulations followed the dimerization of two randomly oriented hDAT monomers and reported the existence of a limited number of symmetric and asymmetric dimers (16). Additionally, the bundle domain comprising of TMHs 1, 2, 6, and 7 was largely excluded from the dimer interface, whereas the scaffold domain formed a significant part of the dimer interface (16).…”

Transporter oligomerisation: roles in structure and function

“…This is a little surprising given that TMH6, together with 1, 2 and 7, forms part of the so-called bundle domain responsible for transport. A more recent analysis suggested that DAT forms a range of different dimer interfaces involving virtually all the protein surface, except the vast majority of TMHs 1, 2, 6 and 7 (Figure 2A,B) [29]. The authors of this more recent study do not rule out a dimer interface involving the intermolecular disulfide bridge mentioned above; however, if a covalent interaction was involved in mediating the interactions between individual protomers, this should be captured in the crystal structure.…”

“…( B ) DAT monomer coloured and labelled as in ( A ) and shown from the extracellular side of the membrane. The black curve indicates all the regions predicted to be able form DAT:DAT interactions by MD simulations [29]. ( C ) LeuT dimer (PDB: 3TT1 [31]) looking through the membrane.…”

“…The different predicted dimer interfaces would allow the formation of higher oligomeric states [29]. The fact that there are a range of possible dimer interfaces, rather than a single defined interaction surface, means that the multiple weak interactions between individual protomers could be equally as likely to occur.…”