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Importance of cholesterol in dopamine transporter function.

ABSTRACT: The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared with non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl-?-cyclodextrin (m?CD), which effectively depletes total membrane cholesterol--uniformly affecting cholesterol-DAT interactions in both raft and non-raft membrane domains--reduced both DA uptake and efflux rate. In contrast, disruption of raft-localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft-localized DAT in substrate uptake or efflux. Supranormal repletion of cholesterol-depleted cells with the analog desmosterol, a non-raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn(2+) and the conformationally biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward-facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol-DAT interactions in regulating DAT function.

SUBMITTER: Jones KT

PROVIDER: S-EPMC3517300 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Importance of cholesterol in dopamine transporter function.

Jones Kymry T KT Zhen Juan J Reith Maarten E A ME

Journal of neurochemistry 20121011 5

The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared with non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode volt ...[more]

PMID: 22957537

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Project description:A rat model of diet-induced obesity (DIO) was used to determine dopamine transporter (DAT) function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity.To evaluate neurobehavioral alterations following the development of DIO induced by an 8-week high-fat diet (HF) exposure, striatal D2-receptor density, DAT function and expression, extracellular dopamine concentrations, impulsivity, and motivation for high- and low-fat reinforcers were determined. To determine predictors of DIO, neurobehavioral antecedents including impulsivity, motivation for high-fat reinforcers, DAT function and extracellular dopamine were evaluated before the 8-week HF exposure.Striatal D2-receptor density was determined by in vitro kinetic analysis of [(3)H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [(3)H]dopamine uptake, methamphetamine-evoked [(3)H]dopamine overflow and no-net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule, respectively.Relative to obesity-resistant (OR) rats, obesity-prone (OP) rats exhibited 18% greater body weight following an 8-week HF-diet exposure, 42% lower striatal D2-receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine and twofold greater methamphetamine-evoked [(3)H]dopamine overflow. OP rats exhibited higher motivation for food, and surprisingly, were less impulsive relative to OR rats. Impulsivity, in vivo DAT function and extracellular dopamine concentration did not predict DIO. Importantly, motivation for high-fat reinforcers predicted the development of DIO.Human studies are limited by their ability to determine if impulsivity, motivation and DAT function are causes or consequences of DIO. The current animal model shows that motivation for high-fat food, but not impulsive behavior, predicts the development of obesity, whereas decreases in striatal DAT function are exhibited only after the development of obesity.

Project description:BACKGROUND:The effects of estrogens on dopamine (DA) transport may have important implications for the increased incidence of neurological disorders in women during life stages when hormonal fluctuations are prevalent, e.g. during menarche, reproductive cycling, pregnancy, and peri-menopause. RESULTS:The activity of the DA transporter (DAT) was measured by the specific uptake of 3H-DA. We found that low concentrations (10(-14) to 10(-8) M) of 17beta-estradiol (E2) inhibit uptake via the DAT in PC12 cells over 30 minutes, with significant inhibition taking place due to E2 exposure during only the last five minutes of the uptake period. Such rapid action suggests a non-genomic, membrane-initiated estrogenic response mechanism. DAT and estrogen receptor-alpha (ERalpha) were elevated in cell extracts by a 20 ng/ml 2 day NGFbeta treatment, while ERbeta was not. DAT, ERalpha and ERbeta were also detectable on the plasma membrane of unpermeabilized cells by immunocytochemical staining and by a fixed cell, quantitative antibody (Ab)-based plate assay. In addition, PC12 cells contained RNA coding for the alternative membrane ER GPR30; therefore, all 3 ER subtypes are candidates for mediating the rapid nongenomic actions of E2. At cell densities above 15,000 cells per well, the E2-induced inhibition of transport was reversed. Uptake activity oscillated with time after a 10 nM E2 treatment; in a slower room temperature assay, inhibition peaked at 9 min, while uptake activity increased at 3 and 20-30 min. Using an Ab recognizing the second extracellular loop of DAT (accessible only on the outside of unpermeabilized cells), our immunoassay measured membrane vs. intracellular/nonvesicular DAT; both were found to decline over a 5-60 min E2 treatment, though immunoblot analyses demonstrated no total cellular loss of protein. CONCLUSION:Our results suggest that physiological levels of E2 may act to sequester DAT in intracellular compartments where the transporter's second extramembrane loop is inaccessible (inside vesicles) and that rapid estrogenic actions on this differentiated neuronal cell type may be regulated via membrane ERs of several types.

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Importance of cholesterol in dopamine transporter function.

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Importance of cholesterol in dopamine transporter function.

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