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.
Highlights
The dopamine transporter (DAT) plays a critical role in the synaptic clearance of dopamine (DA) by mediating the reuptake of DA released into the presynaptic terminal (Amara and Kuhar 1993; Giros and Caron 1993)
AMPH-induced DA efflux is thought to be mediated by a facilitated exchange diffusion process, in which inward transport of substrates increases the availability of inwardfacing binding sites of the transporter (Fischer and Cho 1979), which leads thereby to increased efflux of cytosolic substrates
Indicates that inward and outward transport of monoamines may differ in more fundamental ways. It appears that AMPHinduced DA efflux does not rely exclusively on the ability of AMPH to increase the availability of inward-facing DATs (Chen and Justice 2000) and relates to the ability of AMPH to induce uncoupled currents (Sitte et al 1998) and to increase intracellular sodium (Khoshbouei et al 2003) and kinase activity (Kantor and Gnegy 1998)
Summary
Habibeh Khoshbouei1,[1, Namita Sen2,[1, Bipasha Guptaroy, L’Aurelle Johnson, David Lund, Margaret E. 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. 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. N-terminal phosphorylation shifts DAT from a ‘‘reluctant’’ state to a ‘‘willing’’ state for AMPHinduced 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
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