An N-Terminal Threonine Mutation Produces an Efflux-Favorable, Sodium-Primed Conformation of the Human Dopamine Transporter

作者：Fraser Rheaclare; Chen Yongyue; Guptaroy Bipasha; Luderman Kathryn D; Stokes Stephanie L; Beg Asim; DeFelice Louis J; Gnegy Margaret E^*

来源：Molecular Pharmacology, 2014, 86(1): 76-85.

DOI：10.1124/mol.114.091926

The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr62 revealed T62Dhuman (h) DAT partitions in a predominately efflux-preferring conformation. Compared with wild-type (WT), T62D-hDAT exhibits reduced [H-3] DA uptake and enhanced baseline DA efflux, whereas T62A-hDAT and WT-hDAT function in an influxpreferring conformation. We now interrogate the basis of the mutants%26apos; 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.