Psychoactive substrates stimulate dopamine transporter phosphorylation and down-regulation by cocaine-sensitive and protein kinase C-dependent mechanisms

Dopamine transporters (DATs) undergo intracellular sequestration and functional down-regulation upon exposure to psychostimulant substrates. To investigate the potential mechanism underlying these responses, we examined the acute in vitro and in vivo effects of amphetamine and methamphetamine ( METH) on phosphorylation and down-regulation of rat DAT using wild type and N-terminal truncation mutants. Phosphorylation of DAT assessed by (PO4)-P-32 metabolic labeling was increased up to 2-fold by in vitro treatment of rDAT LLC-PK1 cells with amphetamine or METH and was similarly increased in rat striatal tissue by in vitro application or in vivo injection of METH. The dopamine transport blocker (-)-cocaine did not affect DAT phosphorylation but prevented the phosphorylation increase induced by METH. Phosphorylation of DAT induced by METH was also prevented by the protein kinase C blocker bisindoylmaleimide I and was absent in an N-terminally truncated protein that lacks the first 21 residues including 6 serines that also represent the site of phorbol ester induced phosphorylation. Down-regulation of transport induced by METH was also cocaine- and protein kinase C-dependent but was retained in the N-terminal truncation mutant. These results demonstrate that transport or binding of METH stimulates DAT phosphorylation and down-regulation by a mechanism that requires protein kinase C but that METH-induced down-regulation can occur independently of direct transporter phosphorylation. The finding that DAT phosphorylation is stimulated by amphetamines reveals a previously unknown effect of these drugs that is not produced by cocaine and may be related to reinforcement.