Membrane Cholesterol Modulates the Outward Facing Conformation of the Dopamine Transporter and Alters Cocaine Binding
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-β-cyclodextrin), we observed an increase in DAT binding Bmax values for cocaine analogs [3H]WIN35428 and [125I]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. 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-β-cyclodextrin), we observed an increase in DAT binding Bmax values for cocaine analogs [3H]WIN35428 and [125I]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. IntroductionThe dopamine transporter (DAT) 2The abbreviations used are: DATdopamine transporterDAdopaminewsCholwater-soluble cholesterolMβCDmethyl-β-cyclodextrinEAATexcitatory amino acid transporterTMtransmembrane domainPEO2polyethylene oxideWIN35428(−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropaneRTI-55(−)-2β-carbomethoxy-3-β-(4-iodophenyl)tropaneNHSN-hydroxysuccinimide. 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 (1Ritz M.C. Lamb R.J. Goldberg S.R. Kuhar M.J. Science. 1987; 237: 1219-1223Crossref PubMed Scopus (2018) Google Scholar, 2Torres G.E. Gainetdinov R.R. Caron M.G. Nat. Rev. Neurosci. 2003; 4: 13-25Crossref PubMed Scopus (720) Google Scholar, 3Mortensen O.V. Amara S.G. Eur. J. Pharmacol. 2003; 479: 159-170Crossref PubMed Scopus (132) Google Scholar).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 (4Brown D.A. London E. Annu. Rev. Cell. Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Early reports have shown that the sodium-dependent uptake of γ-aminobutyric acid (GABA) by brain synaptosomes or reconstituted proteoliposomes requires membrane cholesterol (6North P. Fleischer S. J. Biol. Chem. 1983; 258: 1242-1253Abstract Full Text PDF PubMed Google Scholar, 7Shouffani A. Kanner B.I. J. Biol. Chem. 1990; 265: 6002-6008Abstract Full Text PDF PubMed Google Scholar). Recent studies on the serotonin transporter, norepinephrine transporter, glycine transporter, and excitatory amino acid transporters (EAATs) have demonstrated that they can be associated with cholesterol-rich membrane domains in brain tissues or transfected cell lines (8Jayanthi L.D. Samuvel D.J. Ramamoorthy S. J. Biol. Chem. 2004; 279: 19315-19326Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 9Magnani F. Tate C.G. Wynne S. Williams C. Haase J. J. Biol. Chem. 2004; 279: 38770-38778Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Butchbach M.E. Tian G. Guo H. Lin C.L. J. Biol. Chem. 2004; 279: 34388-34396Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 11Núñez E. Alonso-Torres P. Fornés A. Aragón C. López-Corcuera B. J. Neurochem. 2008; 105: 2080-2090Crossref PubMed Scopus (30) Google Scholar). These reports have focused on the regulation of transporters under conditions in which membrane cholesterol is decreased by cholesterol-extracting agents such as methyl-β-cyclodextrin (MβCD). The substrate transport activity of these carriers is generally inhibited by cholesterol depletion. Association of DAT with cholesterol-rich membrane domains has been proposed to regulate the trafficking and lateral mobility of DAT in the membrane (12Adkins E.M. Samuvel D.J. Fog J.U. Eriksen J. Jayanthi L.D. Vaegter C.B. Ramamoorthy S. Gether U. Biochemistry. 2007; 46: 10484-10497Crossref PubMed Scopus (110) Google Scholar, 13Foster J.D. Adkins S.D. Lever J.R. Vaughan R.A. J. Neurochem. 2008; 105: 1683-1699Crossref PubMed Scopus (102) Google Scholar).Our laboratory has been interested in the modulation of neurotransmitter transporters by the surrounding lipid milieu and has shown previously that the electrophysiological properties of EAAT4 and DAT are modulated by arachidonate and polyunsaturated fatty acids (14Fairman W.A. Sonders M.S. Murdoch G.H. Amara S.G. Nat. Neurosci. 1998; 1: 105-113Crossref PubMed Scopus (58) Google Scholar, 15Ingram S.L. Amara S.G. J. Neurosci. 2000; 20: 550-557Crossref PubMed Google Scholar). In the current study, we examined whether altering the membrane cholesterol content can affect the function of DAT. We showed that moderate increases in the membrane cholesterol content in rat striatal synaptosomes and in cells transfected with DAT result in an increase in the number of binding sites for radiolabeled cocaine analogs and enhanced sulfhydryl accessibility of cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT. Using the substituted cysteine scanning method, we further demonstrated that the accessibility of cysteine residues introduced at multiple domains of DAT is influenced by changes in the membrane cholesterol content in a physiologically relevant range. Thus, we propose that in cholesterol-rich membrane microdomains DAT adopts an outward facing conformation.DISCUSSIONMany integral membrane proteins and glycosylphosphatidylinositol-anchored proteins have been shown to be present in specialized membrane domains rich in cholesterol and sphingolipids. As a cell biological criterion, “lipid rafts” are resistant to extraction by non-ionic detergents such as Triton X-100 at a low temperature. However, the more general category of lipid microdomains defined as detergent-resistant membranes have been shown to exhibit heterogeneous sensitivities to different detergent extractions (24Pike L.J. Biochem. J. 2004; 378: 281-292Crossref PubMed Scopus (612) Google Scholar, 25Schuck S. Honsho M. Ekroos K. Shevchenko A. Simons K. Proc. Nat. Acad. Sci. U.S.A. 2003; 100: 5795-5800Crossref PubMed Scopus (551) Google Scholar). Our observations indicate that DAT is partially distributed within cholesterol-rich membrane domains resistant to extraction by a mild detergent, Brij-58. However, the association of DAT with detergent-resistant membranes does not appear to fulfill the strict criterion for lipid rafts. When treated with cold Triton X-100, DAT was almost completely solubilized (supplemental Fig. 1). In early reports, lower concentrations of Triton X-100 (0.1 or 0.5%) were used to show the association of DAT with detergent-resistant membranes (12Adkins E.M. Samuvel D.J. Fog J.U. Eriksen J. Jayanthi L.D. Vaegter C.B. Ramamoorthy S. Gether U. Biochemistry. 2007; 46: 10484-10497Crossref PubMed Scopus (110) Google Scholar, 13Foster J.D. Adkins S.D. Lever J.R. Vaughan R.A. J. Neurochem. 2008; 105: 1683-1699Crossref PubMed Scopus (102) Google Scholar). We speculate that the association of DAT with cholesterol-rich membranes may be similar to that of SNARE proteins, which are known to be concentrated in cholesterol-dependent clusters in PC12 cells but are largely susceptible to cold Triton X-100 extraction (26Lang T. Bruns D. Wenzel D. Riedel D. Holroyd P. Thiele C. Jahn R. EMBO J. 2001; 20: 2202-2213Crossref PubMed Scopus (531) Google Scholar).In this study, we used moderate doses of wsChol, cholesterol-rich lipids, and MβCD, which increased or decreased the cholesterol content by up to 50% in cells and synaptosomes. Such changes in the cholesterol content potentially fall in the physiologically relevant range, considering the wide variations of plasma cholesterol levels detected among the human population (27Brown M.S. Kovanen P.T. Goldstein J.L. Science. 1981; 212: 628-635Crossref PubMed Scopus (600) Google Scholar). In cases where cholesterol homeostasis in the body is severely impaired such as familial hypercholesterolemia and Niemann-Pick diseases the patients' plasma cholesterol levels are severalfold higher than normal.Using the substituted cysteine accessibility method and methanethiosulfonate reagents, it was previously demonstrated that Cys-90 and Cys-306 of DAT were accessible from the extracellular side and that cocaine binding affected the reactivity of Cys-90 (20Ferrer J.V. Javitch J.A. Proc. Nat. Acad. Sci. U.S.A. 1998; 95: 9238-9243Crossref PubMed Scopus (110) Google Scholar). Others have shown that benztropine and cocaine differentially protected cysteine mutants from modification by methanethiosulfonate reagents, suggesting that the two drugs induce different conformational changes in the DAT (28Reith M.E. Berfield J.L. Wang L.C. Ferrer J.V. Javitch J.A. J. Biol. Chem. 2001; 276: 29012-29018Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Here we used maleimide-PEO2-biotin, which selectively and irreversibly reacts with sulfhydryl groups, and because its 29-Å spacer contains hydrophilic polyethylene oxide moieties, it is membrane-impermeant. Our results indicate that it specifically reacts with Cys-306, but not Cys-90, in human DAT. Although Cys-90 has been shown to react with smaller methanethiosulfonate reagents, the orientation of its thiol group in the DAT tertiary structure model (based on its prokaryotic ortholog LeuTAa) may be difficult for maleimide-PEO2-biotin to access. In contrast, Cys-306 is located in a more open area near the extracellular side of TM6a with its sulfhydryl more exposed.Because the C306A mutant of DAT does not react with externally applied maleimide-PEO2-biotin, we engineered a series of substituted cysteine mutants within the C306A background. The sulfhydryl accessibility of these mutants suggests that in cholesterol-rich membranes the conformational rearrangements of DAT involve multiple domains. Specifically, it appears that the tilting of TM6a is influenced by membrane cholesterol because Cys-306 on the top of TM6a exhibited enhanced reactivity, whereas Thr-316 in the middle of TM6a showed reduced sulfhydryl reactivity. The mutant K92C, located at the extracellular end of TM1b, displayed increased sulfhydryl accessibility by either cholesterol loading or cocaine binding. Previous work on serotonin transporter and the GABA transporter probed the accessibility of substituted cysteine residues and suggested that TM1 and TM6 of SLC6 transporters may form part of the substrate permeation pathway (29Zhou Y. Bennett E.R. Kanner B.I. J. Biol. Chem. 2004; 279: 13800-13808Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 30Henry L.K. Adkins E.M. Han Q. Blakely R.D. J. Biol. Chem. 2003; 278: 37052-37063Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 31Rosenberg A. Kanner B.I. J. Biol. Chem. 2008; 283: 14376-14383Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). The altered accessibility of residues in TM6a and TM1b in our assays also hints that these domains could undergo structural rearrangements in response not only to inhibitor binding but also to surrounding lipid composition. Indeed, a recent study has resolved the structure of LeuTAa with a bound competitive inhibitor and postulated a model in which TM1b, TM2a, and TM6a of the prokaryotic transporter undergo substantial conformational changes (32Singh S.K. Piscitelli C.L. Yamashita A. Gouaux E. Science. 2008; 322: 1655-1661Crossref PubMed Scopus (345) Google Scholar).We explored the possibility that membrane cholesterol regulates the dynamic equilibrium between “outward facing” and “inward facing” conformations of DAT in the membrane using radioligand binding assays. Following membrane cholesterol loading at 37 °C, the binding assays were performed on ice to limit the conformational transitions of the DAT in cholesterol-rich membranes. Our results show that binding of cocaine analogs [125I]RTI-55 and [3H]WIN35428 in striatal synaptosomes and HEK-DAT cells is significantly increased when the membrane cholesterol content is augmented by treatment with wsChol or cholesterol-rich lipids. Competitive binding assays using DAT inhibitors and substrates indicated that the effect was specifically attributable to DAT, and it results from a change in Bmax, not Kd, values for DAT radioligands. The increase in Bmax by wsChol pretreatment was also observed when the binding assays was performed at RT. However, Bmax values for either vehicle- or wsChol-treated samples were reduced to approximately half of those from assays done on ice, a condition at which protein degradation was minimized. As the recent LeuTAa structure predicts that competitive inhibitors bind and trap the transporter in the “open-to-out” conformation (32Singh S.K. Piscitelli C.L. Yamashita A. Gouaux E. Science. 2008; 322: 1655-1661Crossref PubMed Scopus (345) Google Scholar), our results suggest that cholesterol-rich membranes provide an environment that favors an outward facing conformation of DAT, thus increasing the number of binding sites for radiolabeled inhibitors.This hypothesis is further strengthened when we compared the effects of wsChol with zinc, which has been reported to stabilize an outward facing conformation of DAT (21Liang Y.J. Zhen J. Chen N. Reith M.E. J. Neurochem. 2009; 109: 981-994Crossref PubMed Scopus (23) Google Scholar, 22Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Crossref PubMed Scopus (134) Google Scholar). There was a striking similarity between the effects of zinc and wsChol in our assays. First, both zinc and wsChol increased Bmax values but did not change Kd values in radioligand binding assays done in HEK-DAT cells. Second, both treatments enhanced the cysteine sulfhydryl reactivity of DAT when probed by maleimide-PEO2-biotin.We also observed a reduced [3H]DA uptake rate in wsChol-treated HEK-DAT cells when the assay was done in 1.5-min duration at RT. It is plausible that following wsChol incubation the membrane microenvironment promotes the outward facing conformation of DAT and/or creates an energy barrier that limits the conversion of DAT to its inward facing conformation, resulting in an apparent decrease of substrate translocation rates. Surprisingly, no significant reduction of Vmax values was seen when [3H]DA uptake assays were done with longer durations such as 5 min at RT. It can be speculated that at RT the membrane dynamics of the cell may quickly alter the cholesterol-rich microenvironment surrounding DAT within minutes, thus diluting the initial effects imposed by wsChol. Such rapid effects by membrane cholesterol may be examined by electrophysiological or that can on of [3H]WIN35428 binding in HEK-DAT cells is We observed values of the of cocaine for [3H]WIN35428 in a similar to those in reports that [3H]WIN35428 exhibited high and low affinity sites in and rat R.D. J.L. R.A. Pharmacol. Google Scholar, J. Pharmacol. Google and in cells transfected with rat DAT L. A. G. Kuhar M.J. PubMed Scopus Google Scholar). Our binding in striatal synaptosomes using [125I]RTI-55, were by a model of cholesterol The between DAT binding of these two could be to the different membrane lipid environment between native tissues and transfected cells, which further the that membrane lipid milieu can modulate the conformational of DAT and affect the binding of DAT inhibitors such as the reports have suggested that cholesterol is for the function of membrane The from was in and exhibited a of membrane cholesterol for M.G. PubMed Scopus Google Scholar, J. Neurochem. 2007; PubMed Scopus Google Scholar). dynamics predicts that cholesterol molecules are embedded in multiple sites of and are to stabilize the native structure of G. J. R. R. Proc. Nat. Acad. Sci. U.S.A. 2008; 105: PubMed Scopus Google Scholar). In the of the cholesterol molecules were present in the S.G. P. Science. 2007; PubMed Scopus Google or in a by transmembrane and to stabilize the conformation of the protein C.B. J. P. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). of reconstituted GABA transport in by of cholesterol suggested a structural of GABA transporter for cholesterol A. Kanner B.I. J. Biol. Chem. 1990; 265: 6002-6008Abstract Full Text PDF PubMed Google Scholar). In this study, we demonstrate that membrane cholesterol the protein conformation of DAT and significantly cocaine binding to DAT. cholesterol binding a role in the native conformation of The possibility of such between cholesterol and DAT may be resolved in assays and when the of neurotransmitter transporters and associated IntroductionThe dopamine transporter (DAT) 2The abbreviations used are: DATdopamine transporterDAdopaminewsCholwater-soluble cholesterolMβCDmethyl-β-cyclodextrinEAATexcitatory amino acid transporterTMtransmembrane domainPEO2polyethylene oxideWIN35428(−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropaneRTI-55(−)-2β-carbomethoxy-3-β-(4-iodophenyl)tropaneNHSN-hydroxysuccinimide. 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 (1Ritz M.C. Lamb R.J. Goldberg S.R. Kuhar M.J. Science. 1987; 237: 1219-1223Crossref PubMed Scopus (2018) Google Scholar, 2Torres G.E. Gainetdinov R.R. Caron M.G. Nat. Rev. Neurosci. 2003; 4: 13-25Crossref PubMed Scopus (720) Google Scholar, 3Mortensen O.V. Amara S.G. Eur. J. Pharmacol. 2003; 479: 159-170Crossref PubMed Scopus (132) Google Scholar).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 (4Brown D.A. London E. Annu. Rev. Cell. Dev. Biol. 1998; 14: 111-136Crossref PubMed Scopus (2542) Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8019) Google Scholar). Early reports have shown that the sodium-dependent uptake of γ-aminobutyric acid (GABA) by brain synaptosomes or reconstituted proteoliposomes requires membrane cholesterol (6North P. Fleischer S. J. Biol. Chem. 1983; 258: 1242-1253Abstract Full Text PDF PubMed Google Scholar, 7Shouffani A. Kanner B.I. J. Biol. Chem. 1990; 265: 6002-6008Abstract Full Text PDF PubMed Google Scholar). Recent studies on the serotonin transporter, norepinephrine transporter, glycine transporter, and excitatory amino acid transporters (EAATs) have demonstrated that they can be associated with cholesterol-rich membrane domains in brain tissues or transfected cell lines (8Jayanthi L.D. Samuvel D.J. Ramamoorthy S. J. Biol. Chem. 2004; 279: 19315-19326Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 9Magnani F. Tate C.G. Wynne S. Williams C. Haase J. J. Biol. Chem. 2004; 279: 38770-38778Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 10Butchbach M.E. Tian G. Guo H. Lin C.L. J. Biol. Chem. 2004; 279: 34388-34396Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 11Núñez E. Alonso-Torres P. Fornés A. Aragón C. López-Corcuera B. J. Neurochem. 2008; 105: 2080-2090Crossref PubMed Scopus (30) Google Scholar). These reports have focused on the regulation of transporters under conditions in which membrane cholesterol is decreased by cholesterol-extracting agents such as methyl-β-cyclodextrin (MβCD). The substrate transport activity of these carriers is generally inhibited by cholesterol depletion. Association of DAT with cholesterol-rich membrane domains has been proposed to regulate the trafficking and lateral mobility of DAT in the membrane (12Adkins E.M. Samuvel D.J. Fog J.U. Eriksen J. Jayanthi L.D. Vaegter C.B. Ramamoorthy S. Gether U. Biochemistry. 2007; 46: 10484-10497Crossref PubMed Scopus (110) Google Scholar, 13Foster J.D. Adkins S.D. Lever J.R. Vaughan R.A. J. Neurochem. 2008; 105: 1683-1699Crossref PubMed Scopus (102) Google Scholar).Our laboratory has been interested in the modulation of neurotransmitter transporters by the surrounding lipid milieu and has shown previously that the electrophysiological properties of EAAT4 and DAT are modulated by arachidonate and polyunsaturated fatty acids (14Fairman W.A. Sonders M.S. Murdoch G.H. Amara S.G. Nat. Neurosci. 1998; 1: 105-113Crossref PubMed Scopus (58) Google Scholar, 15Ingram S.L. Amara S.G. J. Neurosci. 2000; 20: 550-557Crossref PubMed Google Scholar). In the current study, we examined whether altering the membrane cholesterol content can affect the function of DAT. We showed that moderate increases in the membrane cholesterol content in rat striatal synaptosomes and in cells transfected with DAT result in an increase in the number of binding sites for radiolabeled cocaine analogs and enhanced sulfhydryl accessibility of cysteine 306, a juxtamembrane residue on transmembrane domain 6 (TM6) of DAT. Using the substituted cysteine scanning method, we further demonstrated that the accessibility of cysteine residues introduced at multiple domains of DAT is influenced by changes in the membrane cholesterol content in a physiologically relevant range. Thus, we propose that in cholesterol-rich membrane microdomains DAT adopts an outward facing