Functional properties of dopamine transporter oligomers after copper linking

Although it is universally accepted that dopamine transporters ( DAT s) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine ( DA ), or whether each DAT protomer, the subunit of quaternary structure, functions independently in terms of DA translocation. In this study, copper phenanthroline (CuP) was used to selectively target surface DAT : increasing concentrations of CuP gradually cross‐linked natural DAT dimers in LLC ‐ PK 1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DAT s were inactivated. DAT s that were not cross‐linked by CuP showed normal DA uptake with DA K m at ~ 0.5 μM and DA efflux with basal and amphetamine‐induced DA efflux as much as control values. The cocaine analog 2β‐carbomethoxy‐3β‐[4‐fluorophenyl]‐tropane ( CFT ) was capable to bind to copper‐cross‐linked DAT s, albeit with an affinity more than fivefold decreased ( K d of CFT  = 109 nM after cross‐linking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for serotonin transporter and NET that only one protomer of an oligomer is active at the time, the present data indicated a functional activity of the DAT dimer of 0.74 relative to a monomer.