N-Terminal Phosphorylation of the Dopamine Transporter Is Required for Amphetamine-Induced Efflux

Abstract

Amphetamine (AMPH) elicits its behavioral effects by acting on the dopamine (DA) transporter (DAT) to induce DA efflux into the synaptic cleft. We previously demonstrated that a human DAT construct in which the first 22 amino acids were truncated was not phosphorylated by activation of protein kinase C, in contrast to wild-type (WT) DAT, which was phosphorylated. Nonetheless, in all functions tested to date, which include uptake, inhibitor binding, oligomerization, and redistribution away from the cell surface in response to protein kinase C activation, the truncated DAT was indistinguishable from the full-length WT DAT. Here, however, we show that in HEK-293 cells stably expressing an N-terminal-truncated DAT (del-22 DAT), AMPH-induced DA efflux is reduced by approximately 80%, whether measured by superfusion of a population of cells or by amperometry combined with the patch-clamp technique in the whole cell configuration. We further demonstrate in a full-length DAT construct that simultaneous mutation of the five N-terminal serine residues to alanine (S/A) produces the same phenotype as del-22—normal uptake but dramatically impaired efflux. In contrast, simultaneous mutation of these same five serines to aspartate (S/D) to simulate phosphorylation results in normal AMPH-induced DA efflux and uptake. In the S/A background, the single mutation to Asp of residue 7 or residue 12 restored a significant fraction of WT efflux, whereas mutation to Asp of residues 2, 4, or 13 was without significant effect on efflux. We propose that phosphorylation of one or more serines in the N-terminus of human DAT, most likely Ser7 or Ser12, is essential for AMPH-induced DAT-mediated DA efflux. Quite surprisingly, N-terminal phosphorylation shifts DAT from a “reluctant” state to a “willing” state for AMPH-induced DA efflux, without affecting inward transport. These data raise the therapeutic possibility of interfering selectively with AMPH-induced DA efflux without altering physiological DA uptake.

MDMA (Ecstasy) and human dopamine, norepinephrine, and serotonin transporters: implications for MDMA-induced neurotoxicity and treatment

“…There are several consequences of enhanced 5-HT release that can potentially underlie the selective neurotoxic effects of MDMA. For example, N-terminal phosphorylation of the DAT is required for release of amphetamine but not for amphetamine transport (Khoshbouei et al 2004). If this process generalizes across all three transporters, the robust 5-HT releasing capacity of MDMA at the SERT may reflect a unique capacity of MDMA to promote phosphorylation at the SERT compared with the DAT or NET, enhancing carrier-mediated release.…”

C57BL/6J mice show greater amphetamine-induced locomotor activation and dopamine efflux in the striatum than 129S2/SvHsd mice

“…Phosphorylation of DAT is a prerequisite for AMPH-induced dopamine efflux (Khoshbouei et al, 2004). Protein kinase C (PKC) has been shown to contribute to AMPH-stimulated dopamine efflux (Cowell et al, 2000;Johnson et al, 2005;Kantor and Gnegy, 1998) and AMPH-stimulated locomotor activity (Browman et al, 1998).…”

Amphetamine induces dopamine efflux through a dopamine transporter channel

“…Therefore, we included 30 mM Na in the pipette, a concentration similar to that produced by AMPH in unclamped cells expressing DAT (22), thus making this the most physiological condition that we could choose for these experiments. The K m for intracellular DA for efflux has been determined to be Ϸ1.5 mM (12). In addition, in chromaffin cells, basal concentrations of intracellular catecholamine have been found to be as high as 50 M, and AMPH increases these basal levels 6-fold.…”