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Functional properties of dopamine transporter oligomers after copper linking
Author(s) -
Zhen Juan,
Reith Maarten E. A.
Publication year - 2018
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.14259
Subject(s) - dopamine transporter , chemistry , dimer , dopamine , dopamine plasma membrane transport proteins , tropane , monomer , transporter , oligomer , efflux , biophysics , biochemistry , stereochemistry , biology , neuroscience , organic chemistry , gene , polymer
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.