The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V max , and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type.Biogenic amine carriers include the dopamine transporter (DAT), 1 the norepinephrine transporter, and the serotonin transporter, all part of the larger family of Na ϩ ,Cl Ϫ -dependent neurotransmitter transporters (1), which also includes the nonbiogenic amine subfamily of ␥-aminobutyric acid-related transporters (2). In the brain, the DAT is responsible for the clearance of extraneuronal dopamine thought to be the crucial process for terminating dopamine action (3). The DAT is a heavily glycosylated protein with sugars attached to asparagine (4 -6). In general, carbohydrate units of glycoproteins can play several roles, such as controlling protein folding, stabilizing protein conformation, protecting against proteolysis, and regulating intracellular and surface trafficking (7). Early on, for the DAT, glycosylation has been proposed to alter uptake function (6); thus, the generally higher K m values for dopamine uptake in cell systems (micromolar) as compared with striatal synaptosomes (submicromolar) is speculated to be due to environment-specific differences in glycosylation, both in extent and type of sugars.So far, only limited information is available to answer the question as to whether or how glycosylation affects DAT function. In a preliminary report, we describe the generation of three N-glycosylation mutants in which the three putative N-linked glycosylation sites in human DAT are removed: the single mutant N181Q, the double mutant N181Q,N188Q, and the tripl...

Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT (HEK-hDAT), pretreatment with dopamine (0.1-100 M) followed by washout caused reductions in subsequent dopamine uptake (reflected in V max ) with effective dopamine concentrations in the 10 to 100 M range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 M dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine (10 and 100 M) also caused reductions in DAT uptake activity (V max ), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue.In the brain, the dopamine transporter (DAT) clears extraneuronal dopamine, thereby terminating dopamine action (Iversen, 1971). It is generally thought that the local density of DAT is one factor that determines how much dopamine can be cleared, just as the density of dopamine receptors is crucial for the intensity of dopamine receptor-mediated signaling. In this context, many studies over the past decade have addressed long-term regulation of DAT density in chronic drug studies (for reviews, see Kuhar and Pilotte, 1996;Zahniser and Doolen, 2001). The idea that DAT is also susceptible to short-term regulation on a scale of minutes, through changes in its presence at the cell surface, is more recent and originates in studies searching for a link between transporter activity and phosphorylation states. Thus, depending on the mode of treatment, the protein kinase C (PKC) activator ␤-phorbol 12-myristate 13-acetate (PMA) rapidly increases (Quick et al., 1997) or decreases (Beckman et al., 1999) surface-resident GABA transporter 1 (GAT1) in oocytes or primary hippocampal cultures. Down-regulation of GAT1 is probably the physiologically relevant effect of PKC activation (Robinson, 2002). In addition, phorbol esters down-regulate, or reduce surface activity, of monoamine transporters for serotonin (Qian et al., 1997), norepinephrine (Apparsundaram et al

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