RT Journal Article
SR Electronic
T1 An N-Terminal Threonine Mutation Produces an Efflux-Favorable, Sodium-Primed Conformation of the Human Dopamine Transporter
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 76
OP 85
DO 10.1124/mol.114.091926
VO 86
IS 1
A1 Rheaclare Fraser
A1 Yongyue Chen
A1 Bipasha Guptaroy
A1 Kathryn D. Luderman
A1 Stephanie L. Stokes
A1 Asim Beg
A1 Louis J. DeFelice
A1 Margaret E. Gnegy
YR 2014
UL http://molpharm.aspetjournals.org/content/86/1/76.abstract
AB 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.