Abstract
The dopamine transporter (DAT) reversibly transports dopamine (DA) through a series of conformational transitions. Alanine (T62A) or aspartate (T62D) mutagenesis of Thr 62 revealed T62D-hDAT partitions in a predominately efflux-preferring conformation. Compared to wild type (WT), T62D-hDAT exhibits reduced [3H]DA uptake and enhanced baseline DA efflux, while 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 bidirectional, equal ion flow through T62D-hDAT. Utilization of the fluorescent DAT substrate, ASP+, revealed that T62D-hDAT accumulates substrate in 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 AMPH. We conclude that T62D-hDAT represents an efflux-willing, Na+-primed orientation-possibly representing an experimental model of the conformational impact of AMPH exposure to hDAT.
- Dopamine
- Biogenic Amine
- Molecular dynamics
- Func. analysis receptor/ion channel mutants
- Mutagenesis/Chimeric approaches
- Optical spectroscopy (fluorescence, DC, etc.)
- Receptor binding studies
- Patch clamp methods
- Amphetamines
- The American Society for Pharmacology and Experimental Therapeutics