Amphetamine-induced loss of human dopamine transporter activity: An internalization-dependent and cocaine-sensitive mechanism

Abstract

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of Dopamine (DA) signaling in the central nervous system mediates a wide variety of physiologic functions such as movement, motivational control of voluntary behavior, and lactation (1, 2). The magnitude and duration of DA signaling is defined by the amount of vesicular release, the sensitivity of the DA receptors, and the efficiency of DA clearance. The DA transporter (DAT) is largely responsible for regulating DA clearance (3).Psychostimulants, such as cocaine and amphetamine (AMPH), induce DA overflow into the synaptic cleft by acting on the DAT, thereby enhancing dopaminergic transmission (4). Cocaine acts by inhibiting the re-uptake of released DA (5, 6). AMPH-like drugs, however, are thought to promote the release of the transmitter (carrier-mediated efflux) as well as to inhibit its uptake (7,8). Repeated administration of AMPH has been shown to sensitize monoaminergic synapses to subsequent psychostimulant challenge (9). Furthermore, administration of a single, high dose of AMPH acutely (1 h) decreased DAT function in vivo as assessed in striatal synaptosomes prepared from drug-treated rats (10). In contrast, administration of a high dose of cocaine had no effect on subsequent transporter activity (10).To explore the mechanism for the differential effects of AMPH and cocaine on the homeostatic uptake capacity of the human DAT (hDAT), we stably expressed a FLAG-tagged hDAT in EM4 cells (see Materials and Methods). The use of the FLAG fusion protein has provided the opportunity for confocal microscopy analysis of trafficking of the transporter in cells. Here, we report that AMPH caused the hDAT to redistribute intracellularly in a dynamindependent manner, consequently reducing subsequent DA transport capacity. These results provide a mechanism for the AMPHinduced elevation of synaptic DA mediated through a reduction of the number of transporters on the cell surface. Materials and MethodsCell Culture. We created a synthetic hDAT gene, which was tagged at the amino terminus with a FLAG epitope. The gene encodes a protein with an amino acid sequence identical to that of wild-type hDAT with the Met at position 1 replaced by MDYKDDDDKA, but the nucleotide sequence was altered to increase the number of unique restriction sites and to optimize codon utilization. The nucleotide sequence of this construct and its creation will be described elsewhere. The FLAG-tagged syntheticDAT was subcloned into a bicistronic expression vector that expresses the syntheticDAT from a cytomegalovirus promoter and the hygromycin resista...

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

“…Thus, the magnified MDMA-induced release of 5-HT can lock SERT on the surface, prevent SERT internalization, and enhance intracellular accumulation of MDMA, neurotransmitters, and toxic metabolites, leading to neuron terminal adaptation or toxicity. In contrast to the SERT, DAT internalization is promoted by amphetamine and DA, thereby reducing transport capacity and diminishing DAT cell-surface availability (Saunders et al 2000;Sandoval et al 2001;Hansen et al 2002). NET is also susceptible to internalization and downregulation, but the effects of NE or MDMA on NET trafficking are not known (Jayanthi et al 2004).…”

Individual Differences in Cocaine- and Amphetamine-Induced Activation of Male Sprague–Dawley Rats: Contribution of the Dopamine Transporter

“…Likewise, regardless of AB/AA classification, AMPH-induced locomotor activity and [ 3 H]DA uptake were not correlated. The lack of AMPH-induced regulation of DAT activity was surprising since AMPH exposure has been shown to downregulate DAT surface expression in cells (Saunders et al, 2000). However, only a dose of 10 mg/kg has been reported to produce downregulation measured ex vivo (Fleckenstein et al, 1997(Fleckenstein et al, , 1999.…”

“…It is well documented that psychomotor stimulants that are DAT substratesFsuch as AMPH, methamphetamine, and methylenedioxymethamphetamineFinduce rapid downregulation of DAT cell surface expression in vitro or ex vivo (Kokoshka et al, 1998;Fleckenstein et al, 2000;Saunders et al, 2000;Hansen et al, 2001;Gulley et al, 2002;. Typically, COC has been shown to have the opposite effect on DAT, increasing its cell surface expression (Daws et al, 2002;Little et al, 2002; but see Chi and Reith, 2003).…”

Anticipatory postural adjustments stabilise the whole upper-limb prior to a gentle index finger tap

“…dopaminergic drugs will eventually down-regulate, rather than up-regulate, the surface expression of DAT and NET through internalization of the transporters [27,28]. Accordingly, the average FP-CIT binding values in the LC remained enhanced when data were L-Dopa weighted for equivalent daily dose and L-Dopa daily dose.…”