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...
In this study, we analysed the agonist-promoted trafficking of human B2 (B2R) and B1 (B1R) bradykinin (BK) receptors using wild-type and green fluorescent protein (GFP)-tagged receptors in HEK293 cells. B2R was sequestered to a major extent upon exposure to BK, as determined by the loss of cell-surface B2R using radioligand binding and by imaging of B2R–GFP using laser-scanning confocal fluorescence microscopy. Concurrent BK sequestration was revealed by the appearance of acid-resistant specific BK receptor binding. The same techniques showed that B1R was sequestered to a considerably lesser extent upon binding of des-Arg10-kallidin. B2R sequestration was rapid (half-life ∼ 5min) and reached a steady-state level that was significantly lower than that of BK sequestration. B2R sequestration was minimally inhibited by K44A dynamin (22.4±3.7%), and was insensitive to arrestin-(319–418), which are dominant-negative mutants of dynamin I and β-arrestin respectively. Furthermore, the B2R-mediated sequestration of BK was completely insensitive to both mutants, as was the association of BK with a caveolae-enriched fraction of the cells. On the other hand, agonist-promoted sequestration of the β2-adrenergic receptor was dramatically inhibited by K44A dynamin (81.2±16.3%) and by arrestin-(319–418) (36.9±4.4%). Our results show that B2R is sequestered to a significantly greater extent than is B1R upon agonist treatment in HEK293 cells. Furthermore, B2R appears to be recycled in the process of sequestering BK, and this process occurs in a dynamin- and β-arrestin-independent manner and, at least in part, involves caveolae.
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