Faculty Bibliography

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

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine

The present study addresses the effect of intracellular Na(+) and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K(i)) of DA in inhibiting the binding of cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na(+) to reduce DA K(i). No substantial effect on DA K(i) was observed upon gramicidin treatment in Na(+)-free buffer, membrane depolarization with high [K(+)](o), or elevation of [Na(+)](i) with monensin under non-depolarizing conditions. Elevation of DA K(i) was greater at more positive potentials when [Na(+)](i) was raised to a similar level, or at higher [Na(+)](i) when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K(i) was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA K(i) in cell-free membranes was insensitive to Na(+), gramicidin, and D313N mutation. The data suggest that (i) intracellular Na(+) plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na(+) and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA

The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity

The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins

Information is available on the structure-activity relationships for dopamine as a substrate for uptake by the dopamine transporter. However, dopamine transport is a complex process involving substrate binding, translocation, release as well as transporter reorientation. The present study examines only the substrate recognition step by assessment of the potency of various dopamine-related compounds in inhibiting the binding of the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([3H]WIN 35,428) to human dopamine transporters expressed in HEK-293 cells. alpha-Methylation of the side chain, the presence of the amine, and the 2-carbon-length of the side chain were found to be important for binding affinity, whereas beta-hydroxylation of the side chain and methoxylation at the phenyl ring generated weaker compounds. In addition, the presence of both m- and p-OH at the phenyl ring bestowed an increase in potency but the presence of p-OH alone a decrease. N-alkylation (propylation or methylation) had little or an even slightly beneficial effect on affinity, whereas alpha-carbonylation and alpha-methanoylation reduced affinity. Amino naphthalene compounds with a fused benzenoid ring system retained some potency consonant with the extended (i.e. beta-rotameric) trans (=anti) form of the side chain in dopamine when interacting with the transporter. In a second series of experiments, the interaction between dopamine and structural variants was assessed by monitoring the capability of a compound to shift the dopamine inhibition curve to the right as expected for a competitive inhibitor acting at the same site. Appreciable deviation from competitive interaction was observed by removal of the amine from the side chain, by alpha-carbonylation, and by alpha-methanoylation. Two blocker-type compounds, semi-rigid variants of cocaine, also displayed significant deviation. A substrate-based compound, inhibiting cocaine analog binding without interfering with dopamine recognition, could be a cocaine antagonist allowing conformational changes to occur during dopamine uptake

Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 degrees C. When there is sufficient Na+ in the medium (> or =25 mM), [3H]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 degrees C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, D-amphetamine, p-tyramine, and DL-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 degrees C

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 microM) followed by washout caused reductions in subsequent dopamine uptake (reflected in Vmax) with effective dopamine concentrations in the 10 to 100 microM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 microM 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 microM) also caused reductions in DAT uptake activity (Vmax), 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

Advances have been made in characterizing the relationship between Na+ and the substrate permeation pathway in the dopamine transporter. This review covers the role of Na+ in co-transport with dopamine as well as in the recognition of dopamine. Apparent recognition depends on the preparation studied: it differs between intact cells heterologously expressing the dopamine transporter and membranes prepared from these cells. In our search for amino acid residues in the transporter involved in Na+ action, W84 and D313 were found to play a special role in cation interaction, with evidence for regulation of both Na+ and H+ sensitivity. Mutation of D313 to N appeared to decrease the affinity for the dopamine transporter in intact cells, not by altering recognition per se. A model is proposed in which access of dopamine, not recognition itself, is regulated by D313 and Na+. Thus, the role of external Na+ in intact cell preparations is to turn dopamine transporters to the externally facing form, allowing access of dopamine to its binding site

Because much evidence implicates the dopamine transporter in the reinforcing effects of cocaine, development of potential medications for cocaine dependence has included the dopamine transporter as a target. The present overview covers progress in the drug development area regarding several classes of dopamine uptake inhibitors, with an emphasis on structure-activity relationships that enhance potency and selectivity at transporters for dopamine compared with those for serotonin or norepinephrine. The following categories of compounds are covered: tropane, benztropine, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR), methylphenidate, mazindol, and phencyclidine analogs. Activity at transporters as well as on behavior is highlighted