Amphetamine regulation of dopamine transport. Combined measurements of transporter currents and transporter imaging support the endocytosis of an active carrier.
Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na(+)/Cl(-)-dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPH-induced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane.
Kahlig
Kristopher M
KM
Department of Molecular Physiology and Biophysics, Center for Molecular Neuroscience, Vanderbilt University, Nashville, Tennessee 37232-8548, USA.
Javitch
Jonathan A
JA
Galli
Aurelio
A
D000661:Amphetamine / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*
D050483:Dopamine Plasma Membrane Transport Proteins; D018765:Dopamine Uptake Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; C493214:SLC6A3 protein, human; D000661:Amphetamine; D004298:Dopamine