Dopamine transporter oligomerization: impact of combining protomers with differential cocaine analog binding affinities

Abstract

Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious “mixing” experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer.

Transporter oligomerization: form and function

“…Dominant negative mutants of the human dopamine transporter (DAT) have been shown to reduce the function of co-expressed WT transporters, without any effect on trafficking to the plasma membrane [22]. Further functional studies have supported co-operativity, or cross-talk, between individual protomers in DAT oligomers [23]. Additional supportive evidence for this cross-talk has come from studies on fusion proteins of the serotonin transporter (SERT) with GAT-1 [24].…”

Functional properties of dopamine transporter oligomers after copper linking

“…Our previous study demonstrated an influence of DAT protomer composition on the properties of a DAT inhibitor (Zhen et al . ). When DAT oligomers contained combinations of certain protomers with differential affinity for the cocaine phenyltropane analog CFT ((‐)‐2‐β‐carbomethoxy‐3‐β‐(4‐fluorophenyl) tropane = WIN 35 428), the resulting binding affinity differed from that found under conditions where heterooligomers were not present.…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…Whether oligomeric complexes are the predominant state of DAT orientation in vivo is unknown, but some evidence supports the existence of DAT tetramers within striatal membranes (Milner et al, 1994). However, the potential significance of oligomers has been demonstrated by recent work showing that, under certain conditions, DAT protomers within an oligomeric complex affect the conformational state of one another, altering DA transport activity and sensitivity to stimulants historically classified as DAT reuptake inhibitors such as cocaine (Zhen et al, 2015). The size, composition, and distribution of DAT oligomeric complexes may therefore influence the sensitivity of particular dopaminergic neurons to stimulants and any subsequent toxicity.…”