FIGURE 1.

DAT is associated with cholesterol-rich membrane domains. A, rat striatal synaptosomes (1.8 mg of total protein) were treated with 1% Brij-58 at 4 °C for 1 h and subjected to discontinuous sucrose gradient flotation centrifugation. Twenty-four fractions were collected. Every other fraction was analyzed for protein (▴) and cholesterol (○) concentrations and immunoblotted for DAT, flotillin-1, and EAAT3. B, manipulation of the membrane cholesterol content in HEK-DAT cells shifts the distribution of DAT between detergent-solubilized and detergent-resistant cholesterol-rich fractions. Confluent cells in 10-cm dishes were treated with 5 mm MβCD or 0.25 mg/ml wsChol in culturing medium at 37 °C for 0.5 h and solubilized with 1% Brij-58 at 4 °C for 1 h. Band densities of DAT were quantified and converted to percentage of total signals within each treatment. C and D, measurement of cholesterol concentrations in HEK-DAT cell and striatal synaptosomes after a brief incubation with MβCD or wsChol. A dose of 0.1 mg/ml wsChol contains ∼2 mm MβCD carrier. Cholesterol concentrations in lysates were normalized to percentage of vehicle control. Shown are averages ± S.D. (error bars) of triplicate samples from a representative assay.