Abstract
The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62D-human (h)DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [3H]DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influx-preferring conformation. We now interrogate the basis of the mutants’ altered function with respect to membrane conductance and Na+ sensitivity. The hDAT constructs were expressed in Xenopus oocytes to investigate if heightened membrane potential would explain the efflux characteristics of T62D-hDAT. In the absence of substrate, all constructs displayed identical resting membrane potentials. Substrate-induced inward currents were present in oocytes expressing WT- and T62A-hDAT but not T62D-hDAT, suggesting equal bidirectional ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate ASP+ [4-(4-(dimethylamino)styryl)-N-methylpyridinium] revealed that T62D-hDAT accumulates substrate in human embryonic kidney (HEK)-293 cells when the substrate is not subject to efflux. Extracellular sodium (Na+e) replacement was used to evaluate sodium gradient requirements for DAT transport functions. The EC50 for Na+e stimulation of [3H]DA uptake was identical in all constructs expressed in HEK-293 cells. As expected, decreasing [Na+]e stimulated [3H]DA efflux in WT- and T62A-hDAT cells. Conversely, the elevated [3H]DA efflux in T62D-hDAT cells was independent of Na+e and commensurate with [3H]DA efflux attained in WT-hDAT cells, either by removal of Na+e or by application of amphetamine. We conclude that T62D-hDAT represents an efflux-willing, Na+-primed orientation—possibly representing an experimental model of the conformational impact of amphetamine exposure to hDAT.
Footnotes
- Received February 3, 2014.
- Accepted April 21, 2014.
R.F. and Y.C. contributed equally to this work.
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants 5R01-DA011697 (to M.E.G.), 5RC1-DA028112 and 5R01-DA026947 (to L.J.D.), and 5T32-DA007281 (to R.F.)].
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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