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Ethanol-sensitive Sites on the Human Dopamine Transporter

2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês

10.1074/jbc.m204914200

1083-351X

Rajani Maiya, Kari J. Buck, R. Adron Harris, R. Dayne Mayfield,

Receptor Mechanisms and Signaling

Previous studies have shown that ethanol enhanced [3H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity. Previous studies have shown that ethanol enhanced [3H]dopamine uptake in Xenopus oocytes expressing the dopamine transporter (DAT). This increase in DAT activity was mirrored by an increase in the number of transporters expressed at the cell surface. In the present study, ethanol potentiated the function of DAT expressed in HeLa cells but inhibited the function of the related norepinephrine transporter (NET). Chimeras generated between DAT and NET were examined for ethanol sensitivity and demonstrated that a 76-amino acid region spanning transmembrane domains (TMD) 2 and 3 was essential for ethanol potentiation of DAT function. The second intracellular loop between TMD 2 and 3 of DAT, which differs from that of NET by four amino acids, was explored for possible sites of ethanol action. Site-directed mutagenesis was used to replace each of these residues in DAT with the corresponding residue in NET, and the resulting cRNA were expressed in Xenopus oocytes. We found that mutations G130T or I137F abolished ethanol potentiation of DAT function, whereas the mutations F123Y and L138F had no significant effect. These results identify novel sites in the second intracellular loop that are important for ethanol modulation of DAT activity. dopamine DA transporter norepinephrine NE transporter transmembrane domain(s) 428, 2β-carbomethoxy-3β-(4-fluorophenyl)[3H] tropane protein kinase C The family of Na+ and Cl−-dependent transporters, which includes the dopamine (DA)1 and norepinephrine (NE) transporters (DAT and NET, respectively), functions to clear released neurotransmitters from the synaptic cleft (1Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (325) Google Scholar). DAT regulates the spatial and temporal aspects of dopaminergic synaptic transmission and is an integral part of the mesostriatal DA system. This system plays a central role in mediating the rewarding and reinforcing effects of various drugs of abuse, including ethanol (2Brodie M.S. Appel S.B. Alcohol. Clin. Exp. Res. 1998; 22: 236-244Crossref PubMed Scopus (120) Google Scholar, 3Brodie M.S. Shefner S.A. Dunwiddie T.V. Brain. Res. 1990; 508: 65-69Crossref PubMed Scopus (436) Google Scholar, 4Weiss F. Lorang M.T. Bloom F.E. Koob G.F. J. Pharmacol. Exp. Ther. 1993; 267: 250-258PubMed Google Scholar). DAT is also the site of action for various psychostimulants such as cocaine and amphetamine (1Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (325) Google Scholar). The function of monoamine transporters at the cell membrane is regulated by multiple second messenger systems; this regulation involves redistribution of the transporters at the cell surface rather than changes in rate of flux of substrate. For example, activation of protein kinase C (PKC) and drugs of abuse such as amphetamine inhibit DAT function by causing internalization of cell surface transporters in a dynamin- and clathrin-dependent manner (5Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-357801Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 6Melikian H.E. Bucley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar, 7Saunders C. Ferrer J.V. Shi L. Chen J. Merrill G. Lamb M.E. Leeb-Lundberg L.M. Cravelli L. Javitch J.A. Galli A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6850-6855Crossref PubMed Scopus (334) Google Scholar). Experiments carried out in human embryonic kidney (HEK-293) cells demonstrated that acute exposure to cocaine enhances DAT activity in a time-dependent manner by increasing the number of functional transporters at the cell surface (8Daws L.C. Callaghan P.D. Moron J.A. Kahlig K.M. Shippenberg T.S. Javitch J.A. Galli A. Biochem. Biophys. Res. Comm. 2002; 290: 1545-1550Crossref PubMed Scopus (148) Google Scholar). Cocaine also increases the number of DAT binding sites in neuro2A (N2A, derived from mouse neuroblastoma) cells by altering the intracellular trafficking of DAT (9Little K.Y. Elmer L.W. Zhong H. Scheys J.O. Zhang L. Mol. Pharmacol. 2002; 61: 436-445Crossref PubMed Scopus (129) Google Scholar). Ethanol has been shown to affect the function of several members of the Na+ and Cl−-dependent family of transporters. Experiments in HEK-293 cells stably transfected with glycine transporters (GLYT1 and GLYT2) have shown that relatively high concentrations of ethanol (100–200 mm) inhibit uptake of [3H]glycine by GLYT2 and potentiate [3H]glycine uptake by GLYT1 (10Nunez E. Lopez-Corcurea B. Martinez-Maza R. Aragon C. Br. J. Pharmacol. 2000; 129: 802-810Crossref PubMed Scopus (23) Google Scholar). Also, acute exposure to ethanol has been shown to enhance serotonin transporter activity in rat cortical, hippocampal, and brainstem synaptosomes (11Alexi T. Azmitia E.C. Brain Res. 1991; 544: 243-247Crossref PubMed Scopus (19) Google Scholar). Acute ethanol (10–100 mm) enhances DAT-mediated [3H]DA uptake and transporter-associated currents in a time- and concentration-dependent manner (12Mayfield R.D. Maiya R. Keller D. Zahnizer N.R. J. Neurochem. 2001; 79: 1070-1079Crossref PubMed Scopus (38) Google Scholar). This potentiation of transporter function was accompanied by an increase in the number of functional cell surface transporters, suggesting that ethanol affects transporter function by altering the steady state trafficking of DAT to the cell surface. In contrast, electrochemical experiments suggest that NET function is inhibited by ethanol (13Lin A.M. Bickford P.C. Palmer M.R. Gerhardt G.A. Neurosci. Lett. 1993; 164: 71-75Crossref PubMed Scopus (27) Google Scholar). DAT shares a high degree of sequence homology with NET (14Buck K.J. Lorang D.L. Amara S.G. Lee T.H.H. Molecular Approaches to Drug Abuse Research. National Institute on Drug Abuse research monograph, United States Department of Health and Human Services, Rockville, MD1996Google Scholar), but results outlined above suggest that ethanol may have different effects on DAT and NET function. In the present study, we have used the contrasting effects of ethanol on DAT and NET function to identify critical amino acids in DAT that are important for ethanol action. DAT/NET chimeras were expressed in HeLa cells to identify discrete structural domains on DAT and NET that are important for ethanol regulation of transporter function. Site-directed mutagenesis experiments were then carried out, and mutant transporters were functionally analyzed to pinpoint individual amino acid residues that may be crucial for ethanol enhancement of DAT function. Chimeras between the human NET (15Pacholczyk T. Blakely R.D. Amara S.G. Nature. 1991; 350: 350-354Crossref PubMed Scopus (783) Google Scholar) and rat DAT (1Nelson N. J. Neurochem. 1998; 71: 1785-1803Crossref PubMed Scopus (325) Google Scholar) were constructed using a restriction site-independent method as previously described (16Buck K.J. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12584-12588Crossref PubMed Scopus (194) Google Scholar, 17Buck K.J. Amara S.G. Mol. Pharmacol. 1995; 48: 1030-1037PubMed Google Scholar). Sequence analysis (partial) identified the precise location of each chimera junction and confirmed that the junction was in-frame. Previous data indicate that most junctions within conserved regions of DAT and NET are not disruptive of transporter function (18Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (363) Google Scholar, 19Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (158) Google Scholar). A graphic representation of each chimera is shown in Fig. 1. Wild type and chimeric transporter cDNA were expressed in HeLa cells using the vaccinia/T7 transient expression system as previously described (16Buck K.J. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12584-12588Crossref PubMed Scopus (194) Google Scholar, 17Buck K.J. Amara S.G. Mol. Pharmacol. 1995; 48: 1030-1037PubMed Google Scholar). This method employed a recombinant vaccinia virus strain that encodes a bacteriophage T7 RNA polymerase and allows rapid high level expression of proteins encoded by plasmids bearing T7 promoters (18Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (363) Google Scholar,19Blakely R.D. Clark J.A. Rudnick G. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref PubMed Scopus (158) Google Scholar). Briefly, HeLa cells were plated (2 × 105cells/well) into 24-well tissue culture plates and infected the following day. The recombinant vaccinia virus strain VTF-7 was used to infect cells at 10 plaque-forming units/cell in 100 μl of growth medium. T7 promoter-driven plasmids with cDNA inserts encoding wild type NET, DAT, or chimeric transporters were added 30 min later as liposome suspensions (1 μg of DNA and 3 μg of Lipofectin; Invitrogen) in a total volume of 350 μl/well. Sixteen hours after transfection, the virus/liposome suspension was removed by aspiration, and the cells were washed once with 37 °C KRTH medium containing (in mm): 120 NaCl, 4.7 KCl, 2.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 5 Tris, 10 HEPES, pH 7.4. Cells were preincubated for 1 min at 37 °C in 500 μl of KRTH in the absence or presence of ethanol (20, 40, 60, or 100 mm). Uptake was initiated by the addition of [3H]DA or [3H]NE (10, 100, or 1000 nm) in KRTH containing l-ascorbate (100 μm final). Uptake was terminated after 20 min at 37 °C by washing twice with 1 ml of ice-cold KRTH medium. Cells were solubilized with 0.5 n NaOH, and the accumulated radioactivity was determined by scintillation spectrometry. Nonspecific transport was determined by assays of cells transfected with the plasmid vector (pBluescript SKII) on the same plate and subtracted from total uptake. cDNA encoding the human DAT was provided by S. G. Amara and M. S. Sonders (Vollum Institute, Oregon Health & Science University, Portland, OR). Capped cRNA were transcribed from linearized plasmids using standard in vitro transcription reactions (Stratagene). After manual isolation, Xenopus laevis oocytes were injected with water-diluted cRNA (∼10 ng/oocyte) and maintained in Frog Ringers Buffer (FRB) containing (in mm): 96 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.5, supplemented with 2.5 mm Na pyruvate, 0.5 mmtheophylline, 100 units/ml penicillin, 100 μg/ml streptomycin, and 50 μg/ml gentamycin. Uninjected or water-injected oocytes were used to define nonspecific uptake and binding in all experiments. Site-directed mutagenesis of DAT was performed on cDNA subcloned in pBK-CMV vector (Stratagene) using the QuikChange site-directed mutagenesis kit (Stratagene). Mutagenesis was verified by sequence analysis. For [3H]DA uptake assays, DAT-expressing oocytes were incubated in 0.5 ml of FRB containing 100 nm[3H]DA for 10 min at 21 °C. Oocytes were exposed to 100 mm ethanol (12Mayfield R.D. Maiya R. Keller D. Zahnizer N.R. J. Neurochem. 2001; 79: 1070-1079Crossref PubMed Scopus (38) Google Scholar) for different time periods in tightly sealed 12-well plates. Oocytes were washed three times in FRB, and [3H]DA uptake into individual oocytes was quantitated by liquid scintillation spectroscopy. Whole cell radioligand binding was performed in 0.5 ml of FRB containing 4 nm [3H]WIN 35,428 for 15 min at 4 °C. Radioactivity was quantitated using liquid scintillation spectroscopy. Uninjected oocytes were used to specify nonspecific binding. Oocyte homogenates were prepared by sonicating six DAT (wild type or mutant)-expressing oocytes in 0.5 ml of ice-cold FRB. Binding to oocyte homogenates was performed in 0.5 ml of FRB containing 4 nm [3H]WIN 35,428. Nonspecific binding was determined using 3-PPP (R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride). Binding was terminated by rapid filtration and washing using a vacuum manifold. Wild type DAT and NET were expressed in HeLa cells, and the effect of ethanol on [3H]DA and [3H]NE accumulation into these cells was measured. In DAT-expressing cells, ethanol (20–100 mm) significantly increased [3H]DA uptake in a concentration-dependent manner, with 60 mm ethanol producing an ∼50% increase in DA uptake (Fig. 2A). In contrast, in NET-expressing cells, ethanol (40–100 mm) inhibited [3H]NE uptake by as much as 22% (Fig.2B) but had no effect on NET-mediated [3H]DA uptake (Table I).Table IEffect of ethanol on [3H]DA uptake in HeLa cells expressing wild type DAT, NET, and chimeric transportersEnhancement of DA uptake by ethanolKmDAKm NE% of controlμmμmWT DAT178 ± 191-ap < .05 as compared to respective controls.3.05.0WT NET103 ± 50.20.4NET/DAT1147 ± 151-ap < .05 as compared to respective controls.3.03.0DAT/NET199 ± 50.20.4NET/DAT3101 ± 70.81.7DAT/NET3143 ± 161-ap < .05 as compared to respective controls.1.02.0NET/DAT10112 ± 200.60.9DAT/NET9172 ± 251-ap < .05 as compared to respective controls.2.02.0NET/DAT11116 ± 210.30.4DAT/NET10158 ± 181-ap < .05 as compared to respective controls.2.02.01-a p < .05 as compared to respective controls. Open table in a new tab DAT and NET chimeras were generated and eight chimeras (i.e. four sets of approximately reciprocal chimeras) with junctions in or near TMD 1, 3, 9, and 10 were used in this study (Fig.1). The chimeras were referred to as NET/DAT or DAT/NET according to their relative orientations and were numbered to indicate a TMD near their junction. Maximally effective concentrations of ethanol (40–100 mm) were chosen for each chimera. DA uptake was similar to that observed for wild type transporters (Vmax> 90% of wild type DAT) in most of the chimeras used in this study. However, chimeras NET/DAT3, DAT/NET1, and DAT/NET3 showed DA uptake that was 60–80% of that observed for wild type DAT (16Buck K.J. Amara S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12584-12588Crossref PubMed Scopus (194) Google Scholar, 17Buck K.J. Amara S.G. Mol. Pharmacol. 1995; 48: 1030-1037PubMed Google Scholar). Functional analyses of the chimeric transporters indicate that chimeras that possess DAT sequence elements within the region including TMD 1–3 all demonstrate enhancement of [3H]DA uptake by ethanol similar to DAT (Table I). In contrast, in chimeras with sequence elements within TMD 1–3 from NET, [3H]DA uptake was not enhanced by ethanol and thus resembled NET in this regard. These results indicate that ethanol enhancement of DAT function requires the TMD 1–3 region of DAT. Chimeras NET/DAT1 and DAT/NET1 with junctions in TMD1 (F78 and W63, respectively) were also tested for ethanol effects. The ethanol responsiveness of these two chimeras was similar to DAT and NET, respectively (Table I). Furthermore, we also tested chimeras DAT/NET9, DAT/NET10, NET/DAT10, and NET/DAT11 with junctions downstream of TMD3 (TMD9, TMD10, and TMD11, respectively) for ethanol effects. These chimeras demonstrated ethanol sensitivities comparable with that of NET/DAT3 and DAT/NET3 and further delineated the TMD 1–3 region to 76 amino acid residues spanning positions 78–154. Mutagenesis studies were carried out in human DAT to pinpoint sites of ethanol action within the TMD 1–3 region. Because intracellular loops are more accessible for proteins that modulate trafficking (20Quick M.W. Corey J.L. Davidson N. Lester H.A. J. Neurosci. 1997; 17: 2967-2979Crossref PubMed Google Scholar), we explored the intracellular loop between TMD 2 and TMD 3 for possible sites of ethanol action. The second intracellular loop differs between DAT and NET by only four amino acids, at positions 123, 130, 137, and 138. Each of these amino acids in DAT was replaced with the corresponding amino acid from NET. The resulting cDNA were transcribed in vitro and expressed in Xenopus oocytes. Oocytes expressing wild type and G130T mutant DAT were exposed to ethanol for 1 or 4 h, and [3H]DA uptake was measured. The time periods were chosen to examine the effects of ethanol on [3H]DA uptake as a function of ethanol exposure time. Basal [3H]DA uptake in this mutant was 1.2 fmol/sec/oocyte, which was comparable with that of wild type DA uptake (1.0 fmol/sec/oocyte). Wild type DAT-expressing oocytes showed a 50% potentiation of [3H]DA uptake after 4 h of ethanol exposure. In contrast, the potentiating effects of ethanol were abolished in the G130T DAT mutant after preincubation with ethanol for 4 h (Fig. 3). Amino acids phenylalanine at position 123, isoleucine at position 137, and leucine at position 138 were also mutated to the corresponding amino acids in NET (F123Y, I137F, and L138F, respectively). [3H]DA uptake in these mutants was comparable with that of wild type DAT (data not shown). Maximum potentiation of transporter function was observed after 1 h of ethanol exposure in oocytes expressing the wild type transporter (Fig. 4). After exposure to 100 mm ethanol for 1 h, potentiation of [3H]DA uptake was not observed in the mutant I137F (Fig.4). The mutant F123Y showed ethanol sensitivity comparable with that of wild type DAT (Fig. 4). Ethanol sensitivity of the mutant L138F was reduced but not significantly, compared with that of wild type DAT (Fig. 4).FIG. 4Effects of ethanol on F123Y, I137F, and L138F mutants. [3H]DA uptake was measured in oocytes expressing wild type versus mutant DAT. Oocytes expressing WT and mutant transporters were exposed to 100 mm ethanol for 1 h, and [3H]DA (100 nm) uptake was measured. Enhancement of [3H]DA uptake was observed in wild type (white bar) and F123Y (black bar)-expressing oocytes but not in the mutants I137F and L138F. Mean values ± S.E. are shown for n = 8–10 oocytes per condition from 4–5 batches of oocytes. *, p < 0.05 as compared with wild type potentiation, Student's t test.View Large Image Figure ViewerDownload (PPT) Previously, we showed that ethanol potentiation of DAT activity was mirrored by an increase in the density of cell surface transporters (12Mayfield R.D. Maiya R. Keller D. Zahnizer N.R. J. Neurochem. 2001; 79: 1070-1079Crossref PubMed Scopus (38) Google Scholar). Upon exposure to ethanol for 1 and 4 h, [3H]WIN 35,428 binding was significantly increased by 40 and 53%, respectively (12Mayfield R.D. Maiya R. Keller D. Zahnizer N.R. J. Neurochem. 2001; 79: 1070-1079Crossref PubMed Scopus (38) Google Scholar). We investigated cell surface transporter numbers in the mutant G130T DAT in the presence and absence of ethanol by measuring [3H]WIN 35,428 binding. G130T DAT cell surface density was ∼20 fmol/oocyte. Ethanol had no significant effect on the number of cell surface transporters in the mutant G130T (Fig. 5). To test whether ethanol inhibits DAT function when the second intracellular loop is replaced with that of NET, we substituted the second intracellular loop of DAT with that of NET by sequentially mutating positions Phe-123, Gly-130, Ile-137, and Leu-138 to the corresponding amino acids in NET. This loop replacement mutant is termed IGLF. [3H]DA uptake and [3H] WIN 35,428 binding were measured in this mutant and compared with wild type. [3H]DA uptake was not observed in the mutant IGLF (Fig. 6A). [3H]WIN 35,428 binding in the mutant IGLF was significantly reduced as compared with that of wild type DAT (Fig.6B). We next carried out [3H]WIN 35,428 binding studies on oocyte homogenates (see “Experimental Procedures”). We found that the mutant IGLF is synthesized but not inserted to the cell surface (Fig. 6C). Previous studies carried out in the Xenopus oocyte expression system have shown that acute ethanol (10–100 mm) enhances [3H]DA uptake and transporter-associated currents in a time- and concentration-dependent manner. Ethanol-induced increases in cell surface DAT were not associated with increased protein synthesis (12Mayfield R.D. Maiya R. Keller D. Zahnizer N.R. J. Neurochem. 2001; 79: 1070-1079Crossref PubMed Scopus (38) Google Scholar) but were associated with increased cell surface binding. These results suggest that ethanol enhancement of transporter function may involve redistribution of DAT at the cell surface. The goal of this study was to determine regions on DAT that are critical for ethanol action. NET, which also belongs to the Na+ and Cl−-dependent family of neurotransmitter transporters, shares a high degree of homology (64%) with DAT (14Buck K.J. Lorang D.L. Amara S.G. Lee T.H.H. Molecular Approaches to Drug Abuse Research. National Institute on Drug Abuse research monograph, United States Department of Health and Human Services, Rockville, MD1996Google Scholar), but in vivo electrochemical studies have shown that ethanol inhibits rather than enhances NET function (13Lin A.M. Bickford P.C. Palmer M.R. Gerhardt G.A. Neurosci. Lett. 1993; 164: 71-75Crossref PubMed Scopus (27) Google Scholar). We used chimeras between DAT and NET and site-directed mutagenesis studies to define critical ethanol-sensitive sites in the second intracellular loop of the transporter. The kinetic parameters of DA uptake (Km and Vmax) in most of these chimeras were nearly identical to wild type DAT. These chimeras showed that ethanol enhancement of DAT-mediated [3H]DA uptake required the presence of TMD 1–3. Replacing this region of DAT with the corresponding region from NET resulted in abolition of ethanol effects. Chimeras NET/DAT1 and DAT/NET1 with junctions in TMD1 had ethanol sensitivities comparable with DAT and NET, respectively. In contrast, chimeras NET/DAT3 and DAT/NET3 with junctions at TMD3 have ethanol sensitivities comparable with NET and DAT. Also, chimeras with junctions downstream of TMD3 (NET/DAT10 and -11, DAT/NET9 and -10) resemble NET/DAT3 and DAT/NET3 in their ethanol sensitivities. Taken together, these results indicate that a 76-amino acid region spanning TMD 1–3, including the second intracellular loop, is critical for mediating ethanol enhancement of DAT function. Because intracellular loops are accessible at all times to modifying enzymes and accessory proteins that modulate trafficking (20Quick M.W. Corey J.L. Davidson N. Lester H.A. J. Neurosci. 1997; 17: 2967-2979Crossref PubMed Google Scholar), we explored the second intracellular loop between TMD2 and TMD3 for possible sites of ethanol action. This loop in DAT differs from that of NET by four amino acids. Site-directed mutagenesis was carried out to substitute glycine at position 130 for threonine, the corresponding amino acid in NET. [3H]DA uptake in this mutant was comparable with that of the wild type DAT. However, ethanol potentiation of [3H]DA uptake was abolished completely in this mutant. Using [3H]WIN 35,428 binding, we examined cell surface expression of this mutant before and after ethanol exposure. [3H]WIN 35,428 binding to the wild type DAT is Na+-dependent, and low intracellular concentrations of sodium ions in the oocyte prevent [3H]WIN 35,428 from binding to intracellular DAT (21Barish M.E. J. Physiol. (Lond). 1983; 342: 309-325Crossref Scopus (504) Google Scholar,22Reith M.E. Coffey L.L. J. Neurochem. 1993; 61: 167-177Crossref PubMed Scopus (56) Google Scholar). Ethanol had no effect on the number of cell surface G130T mutant transporters, indicating that this amino acid is critical for ethanol-mediated increases in [3H]DA uptake. This suggests that ethanol affects DAT function by altering cell surface distribution of the transporters. We also mutated amino acids Phe-123, Ile-137, and Leu-138 to the corresponding amino acids in NET. We found that I137F abolished ethanol potentiation of [3H]DA uptake, whereas the other mutations demonstrated ethanol sensitivities comparable with the wild type DAT. Robust changes in ethanol sensitivities were observed in the G130T and I137F mutants, which are non-conservative amino acid changes. No change in ethanol sensitivity was observed in the F123Y mutant, which is a conservative amino acid change. However, the mutant L138F did not demonstrate significant attenuation of ethanol sensitivity despite being a non-conservative mutation. We generated the mutant transporter IGLF to investigate whether replacing the second intracellular loop of DAT with that of NET would result in ethanol inhibition of [3H]DA uptake. However, this mutant transporter does not demonstrate [3H]DA uptake and expresses a significantly lower number of functional transporters at the cell surface. Oocyte fractionation studies indicate that the transporters are synthesized but not trafficked to the cell surface. This result, though unexpected, supports the hypothesis that the second intracellular loop is important for steady state insertion of DAT to the cell surface. There is now an emerging literature on mutations that affect ethanol actions on ion channels, and it is of interest to compare our analysis of catecholamine transporters with studies of other proteins. Discrete amino acid residues located either in TMD or in the cytoplasmic regions are required for ethanol action on ion channels (23Harris R.A. Alcohol. Clin. Exp. Res. 1999; 23: 1563-1570PubMed Google Scholar). For example, mutation of Ser-267 at TMD2 of the glycine receptor α1-subunit to isoleucine results in an alcohol-insensitive receptor (24Mihic S.J., Ye, Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1108) Google Scholar). A transmembrane phenylalanine residue is important for ethanol inhibition of N-methyl-d-aspartate receptors (25Ronald K.M. Mirshahi T. Woodward J.J. J. Biol. Chem. 2001; 276: 44729-44735Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Alcohol modulation of the G-protein-coupled inwardly rectifying the potassium channel (GIRK) function requires a 48-amino acid region in the intracellular C-terminal region of the protein (26Lewohl J.M. Wilson W.R. Mayfield R.D. Brozowski S.J. Morrisett R.A. Harris R.A. Nat. Neurosci. 1999; 2: 1084-1090Crossref PubMed Scopus (210) Google Scholar). Similarly, ethanol action on voltage-sensitive potassium channels (Kv1) requires the presence of a discrete amino acid residue in the putative cytoplasmic region of the protein (27Covarrubias M. Vyas T.B. Escobar L. Wei A. J. Biol. Chem. 1995; 270: 19408-19416Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Ethanol indirectly affects the function of metabotrophic glutamate receptors (mGluR1) by enhancing PKC-mediated receptor phosphorylation (28Minami K. Gereau R.W., IV Minami M. Heinemann S.F. Harris R.A. Mol. Pharmacol. 1998; 53: 148-156Crossref PubMed Scopus (110) Google Scholar). Mutation of a consensus PKC phosphorylation site, Ser-890, abolishes ethanol regulation of receptor function. None of these sites appears to be important for protein trafficking; in contrast, our results identify novel amino acid residues in the second intracellular loop of DAT that are necessary for ethanol modulation of DAT function by a mechanism that is consistent with altered trafficking of the protein to the cell surface. A common theme in the functional regulation of the Na+/Cl−-dependent family of transporters is the redistribution of cell surface transporters. Several agonists and antagonists of γ-aminobutyric acid and serotonin transporters have been shown to alter subcellular distribution of these transporters (29Whitworth T.L. Quick M.W. J. Biol. Chem. 2001; 276: 42932-42937Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 30Whitworth T.L. Herndon L.C. Quick M.W. J. Neurosci. 2001; 22: RC1921Google Scholar). PKC activators such as phorbol 12-myristate 13-acetate down-regulate DAT function by causing internalization of cell surface DAT (5Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-357801Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 6Melikian H.E. Bucley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar, 31Zhu S.J. Kavanaugh M.P. Sonders M.S. Amara S.G. Zahniser N.R. J. Neurosci. 1997; 282: 1358-1365Google Scholar). Protein tyrosine kinase A inhibitors have been shown to inhibit DAT function by internalization of cell surface DAT (32Doolen S. Zahniser N.R. J. Pharmacol. Exp. Ther. 2001; 296: 931-938PubMed Google Scholar). Drugs of abuse have also been shown to alter cell surface DAT densities in some but not all studies. For example, amphetamine decreases cell surface DAT levels by endocytosing the transporter in a dynamin- and clathrin-dependent manner in HEK-293 cells (7Saunders C. Ferrer J.V. Shi L. Chen J. Merrill G. Lamb M.E. Leeb-Lundberg L.M. Cravelli L. Javitch J.A. Galli A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6850-6855Crossref PubMed Scopus (334) Google Scholar). More recently two independent studies have shown that cocaine causes an increase in cell surface DAT levels (8Daws L.C. Callaghan P.D. Moron J.A. Kahlig K.M. Shippenberg T.S. Javitch J.A. Galli A. Biochem. Biophys. Res. Comm. 2002; 290: 1545-1550Crossref PubMed Scopus (148) Google Scholar, 9Little K.Y. Elmer L.W. Zhong H. Scheys J.O. Zhang L. Mol. Pharmacol. 2002; 61: 436-445Crossref PubMed Scopus (129) Google Scholar). These studies, carried out in N2A and HEK-293 cells, demonstrate that cocaine-induced enhancement in cell surface DAT levels is due to increased rates of insertion of DAT at the cell surface. Also, dopamine D2receptor activation affects DAT function by increasing the number of functional cell surface transporters (33Mayfield R.D. Zahniser N.R. Mol. Pharmacol. 2001; 59: 113-121Crossref PubMed Scopus (90) Google Scholar). Our results, which suggest that ethanol-mediated functional regulation of DAT involves redistribution of cell surface transporters and that this redistribution involves discrete regions on the transporter, fit very well with this common theme in transporter regulation. The absence of consensus sites for PKC or for protein tyrosine kinase A phosphorylation in the second intracellular loop suggests that ethanol regulation of DAT function may not be due to increased phosphorylation of the protein. Furthermore, it has been demonstrated that direct phosphorylation of the transporter is not required for functional regulation by PKC (34Chang M.Y. Lee S.H. Kim J.H. Lee K.H. Kim Y.S. Son H. Lee Y.S. J. Neurochem. 2001; 77: 754-761Crossref PubMed Scopus (57) Google Scholar). It is likely that accessory proteins aid in the functional regulation of DAT, but these remain largely unknown. Recent studies using yeast two-hybrid techniques have identified candidate proteins that interact with the C-terminal tail of DAT and regulate transporter function. For example, α-synuclein interacts with DAT in neurons, and the resulting DAT-α-synuclein complex has been shown to be essential for clustering of DAT at the cell surface and thereby accelerating cellular DA uptake and DA-induced apoptosis (35Lee F.J.S. Liu F. Pristupa Z.B. Niznik H.B. FASEB J. 2001; 15: 916-926Crossref PubMed Scopus (391) Google Scholar). The C-terminal tail of DAT also contains a conserved PDZ domain that interacts with the protein PICK1; this interaction is crucial for proper targeting and functioning of DAT (36Torres G.E. Yao W. Mohn A.R. Quan H. Kim K. Levey A.I. Staudinger J. Caron M.G. Neuron. 2001; 30: 121-134Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). Based on our mutants, we hypothesize that ethanol modulates the interaction between DAT and a putative regulatory protein important for ethanol-induced trafficking of DAT and that this interaction occurs at the second intracellular loop. The ethanol-insensitive mutants described in the present work will aid in testing this hypothesis.