Membrane Cholesterol Modulates the Outward Facing Conformation of the Dopamine Transporter and Alters Cocaine Binding

Abstract

Clearance of synaptically released dopamine is regulated by the plasmalemmal dopamine transporter (DAT), an integral membrane protein that resides within a complex lipid milieu. Here we demonstrate that cholesterol, a major component of the lipid bilayer, can modulate the conformation of DAT and alter cocaine binding to DAT. In striatal synaptosomes and transfected cells, DAT was in cholesterol-rich membrane fractions after mild detergent extraction. After increasing the membrane cholesterol content by treatment of water-soluble cholesterol (cholesterol mixed with methyl-␤-cyclodextrin), we observed an increase in DAT binding B max values for cocaine analogs [3 H]WIN35428 and [ 125 I]RTI-55, but similar levels of DAT proteins on the cell surface were shown by surface biotinylation assays. Membrane cholesterol addition also markedly enhanced the accessibility of cysteine sulfhydryl moieties in DAT as probed by a membrane-impermeable maleimide-biotin conjugate. We identified cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT, as the intrinsic residue exhibiting enhanced reactivity. Similar effects on DAT cysteine accessibility and radioligand binding were observed with addition of zinc, a reagent known to promote the outward facing conformation of DAT. Using substituted cysteine mutants on various positions likely to be extracellular, we identified additional residues located on TM1, TM6, TM7, and TM12 of DAT that are sensitive to alterations in the membrane cholesterol content. Our findings in transfected cells and native tissues support the hypothesis that DAT adopts an outward facing conformation in a cholesterol-rich membrane environment, suggesting a novel modulatory role of the surrounding membrane lipid milieu on DAT function. The dopamine transporter (DAT)2 has a primary function to reaccumulate dopamine (DA) molecules into the presynaptic nerve terminal, thus limiting the extent and duration of DA signaling. Cocaine, a natural alkaloid from the leaves of the coca plant, is among the most widely abused drugs in modern society. Cocaine binds to DAT with high affinity and inhibits the transport of DA, resulting in prolonged DA neurotransmission in the brain. The enhanced DA signaling in the mesolimbic system is believed to be the main mechanism of action for cocaine addiction (1-3).The DAT is a multispan integral membrane protein embedded in the plasmalemmal lipid bilayer, which comprises three main components: phospholipids, sphingolipids, and cholesterol. Cholesterol-rich domains within the membrane have been proposed to serve as a dynamic platform for membrane protein compartmentalization and organization (4, 5). Early reports have shown that the sodium-dependent uptake of ␥-aminobutyric acid (GABA) by brain synaptosomes or reconstituted proteoliposomes requires membrane cholesterol (6, 7). Recent studies on the serotonin transporter, norepinephrine transporter, glycine transporter, and excitatory amino acid transporters (EAATs) have demonstrated that they can be associated with ...

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2-Substituted 3 -Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations

“…The lysates were incubated with 60 ml of Pierce NeutrAvidin Agarose beads (ThermoFisher Scientific, Grand Island, NY) overnight at 4°C. After beads were washed three times with TNE buffer, biotinylated proteins were eluted with sodium dodecyl sulfate sample buffer, separated by polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with rabbit antisera for DAT (Hong and Amara, 2010). Mean densities of chemiluminescent DAT bands were quantified using the NIH ImageJ software and normalized to percent of vehicle.…”