Prediction of Dopamine Transporter Binding Availability by Genotype: A Preliminary Report
Leslie K. JacobsenJulie K. StaleySami S. ZoghbiJohn SeibylThomas R. KostenRobert B. InnisJoel Gelernter
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OBJECTIVE: Evidence of a relationship between genotype and binding availability was assessed for the dopamine and serotonin transporter genes. METHOD: The authors assessed dopamine transporter genotype at the SLC6A3 3′ variable number of tandem repeats (VNTR) polymorphism and serotonin transporter genotype at the SLC6A4 promotor VNTR polymorphism in 30 healthy subjects who also underwent single photon emission computed tomography with [123I]β-CIT. RESULTS: Subjects homozygous for the 10-repeat allele at the SLC6A3 locus demonstrated significantly lower dopamine transporter binding than carriers of the nine-repeat allele. There was no effect of SLC6A4 genotype upon serotonin transporter binding. CONCLUSIONS: These findings suggest that genetic variation at the SLC6A3 3′ VNTR polymorphism may modify dopamine transporter function.Cite
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Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn2+. Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn2+. Without Zn2+, E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn2+ (10 μm), however, MTSET (0.5 mm) caused up to ∼60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn2+-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT. Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn2+. Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn2+. Without Zn2+, E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn2+ (10 μm), however, MTSET (0.5 mm) caused up to ∼60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn2+-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT. The dopamine transporter (DAT) 1The abbreviations used are: DATdopamine transporterhDAThuman dopamine transporterNETnorepinephrine transporterSERTserotonin transporterCFT2β-carbomethoxy-3β-(4-fluorophenyl)tropaneMTSmethanethiosulfonateMTSET[2-(trimethylammonium)ethyl]-methanethiosulfonateTMtransmembrane segmentWTwild typeICLintracellular loop.1The abbreviations used are: DATdopamine transporterhDAThuman dopamine transporterNETnorepinephrine transporterSERTserotonin transporterCFT2β-carbomethoxy-3β-(4-fluorophenyl)tropaneMTSmethanethiosulfonateMTSET[2-(trimethylammonium)ethyl]-methanethiosulfonateTMtransmembrane segmentWTwild typeICLintracellular loop. is responsible for the rapid re-uptake of dopamine released upon neuronal stimulation (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Google Scholar, 2Blakely R.D. Bauman A.L. Curr. Opin. Neurobiol. 2000; 10: 328-336Google Scholar, 3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar). In this way, the transporter controls the availability of dopamine in the synaptic cleft and thereby plays a key role in regulating the broad spectrum of physiological function mediated by dopamine (1Amara S.G. Kuhar M.J. Annu. Rev. Neurosci. 1993; 16: 73-93Google Scholar, 2Blakely R.D. Bauman A.L. Curr. Opin. Neurobiol. 2000; 10: 328-336Google Scholar, 3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar). The critical physiological role of DAT has been illustrated by targeted disruption of the DAT gene in mice, which resulted in multiple deficits and profoundly altered dopaminergic neurotransmission (4Giros B. Jaber M. Jones S.R. Wightman R.M. Caron M.G. Nature. 1996; 379: 606-612Google Scholar, 5Gainetdinov R.R. Caron M.G. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 261-284Google Scholar). Furthermore, the DAT has been the focus of much attention because it represents the principle target for the action of widely abused psychostimulants such as cocaine and amphetamine (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar, 6Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Google Scholar). dopamine transporter human dopamine transporter norepinephrine transporter serotonin transporter 2β-carbomethoxy-3β-(4-fluorophenyl)tropane methanethiosulfonate [2-(trimethylammonium)ethyl]-methanethiosulfonate transmembrane segment wild type intracellular loop. dopamine transporter human dopamine transporter norepinephrine transporter serotonin transporter 2β-carbomethoxy-3β-(4-fluorophenyl)tropane methanethiosulfonate [2-(trimethylammonium)ethyl]-methanethiosulfonate transmembrane segment wild type intracellular loop. The DAT is a prototypic member of the class of Na+/Cl–-dependent transporters, along with neurotransmitter transporters, including the norepinephrine (NET), serotonin (SERT), γ-aminobutyric acid, and glycine transporters (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar, 6Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Google Scholar). This family of transporters is believed to contain 12 putative transmembrane segments (TMs) connected by alternating extracellular and intracellular loops (ICLs) with an intracellular location of the N and C termini (Fig. 1) (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar, 6Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Google Scholar). A high-resolution structure is not yet available for DAT or any related transporter, and our insight into the packing of the 12 helices remains limited (3Norregaard L. Gether U. Curr. Opin. Drug Discov. Dev. 2001; 4: 591-601Google Scholar, 6Chen N. Reith M.E. Eur. J. Pharmacol. 2000; 405: 329-339Google Scholar). The first series of proximity relationships have only quite recently been described in the tertiary structure of the human DAT (hDAT) (7Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Google Scholar, 8Loland C.J. Norregaard L. Gether U. J. Biol. Chem. 1999; 274: 36928-36934Google Scholar, 9Norregaard L. Visiers I. Loland C.J. Ballesteros J. Weinstein H. Gether U. Biochemistry. 2000; 39: 15836-15846Google Scholar). Interestingly, an increasing amount of evidence suggest that the DAT exists in the membrane as an oligomeric structure, but the functional significance of this still needs to be clarified (10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Google Scholar, 11Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Google Scholar, 12Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Google Scholar, 13Norgaard-Nielsen K. Norregaard L. Hastrup H. Javitch J.A. Gether U. FEBS Lett. 2002; 524: 87-91Google Scholar). Transport of substrate mediated by Na+/Cl–-dependent transporters is energetically coupled to the transmembrane sodium gradient maintained by the Na+/K+-ATPase (14Rudnick G. Reith, M.E.A Neurotransmitter Transporters: Structure, Function, and Regulation. 1st Ed. Humana Press, Totowa, NJ1997: 73-100Google Scholar). Accordingly, binding of Na+ (two ions in case of DAT) together with substrate is assumed to trigger a critical conformational change that leads to transition of the transporter from an "outward" facing conformation in which the substrate binding site is exposed to the extracellular medium to an "inward" facing conformation in which the substrate binding site is exposed to the intracellular environment (14Rudnick G. Reith, M.E.A Neurotransmitter Transporters: Structure, Function, and Regulation. 1st Ed. Humana Press, Totowa, NJ1997: 73-100Google Scholar). This allows for the release of substrate and sodium and potential return of the empty transporter to an outward facing conformation (14Rudnick G. Reith, M.E.A Neurotransmitter Transporters: Structure, Function, and Regulation. 1st Ed. Humana Press, Totowa, NJ1997: 73-100Google Scholar). A prerequisite for such an alternating access model is the existence of both external and internal "gates," that is protein domains that undergo significant conformational changes during the transport cycle and are capable of occluding access to the substrate binding site from the extracellular or intracellular environment, respectively. Currently we know rather little about these putative gating domains, although the application of the substituted cysteine accessibility method has identified several conformationally active regions of the transporter (15Ferrer J. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Google Scholar, 16Chen N. Ferrer J.V. Javitch J.A. Justice Jr., J.B. J. Biol. Chem. 2000; 275: 1608-1614Google Scholar, 17Chen J.G. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1044-1049Google Scholar, 18Androutsellis-Theotokis A. Ghassemi F. Rudnick G. J. Biol. Chem. 2001; 276: 45933-45938Google Scholar, 19Androutsellis-Theotokis A. Rudnick G. J. Neurosci. 2002; 22: 8370-8378Google Scholar). We also know very little about the molecular mechanisms governing the equilibrium between the distinct conformational states in the transport cycle, although this must be tightly regulated for proper transporter function. Interestingly, we have recently identified a tyrosine (Tyr-335) in the third ICL of hDAT that may play a key role in regulating this conformational equilibrium (20Loland C.J. Norregaard L. Litman T. Gether U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1683-1688Google Scholar). This inference was primarily based on the observation that mutation of the tyrosine alters completely the effect of Zn2+ at the previously identified endogenous Zn2+ binding site in the hDAT (Refs. 7Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Google Scholar and 8Loland C.J. Norregaard L. Gether U. J. Biol. Chem. 1999; 274: 36928-36934Google Scholar and Fig. 1). In the WT, Zn2+ acts as a potent non-competitive inhibitor of transport via interaction with three residues on the extracellular face of the transporter (Fig. 1) (7Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Google Scholar). In marked contrast, in the Tyr-335 mutant (Y335A) this inhibitory Zn2+ switch is converted into a stimulatory Zn2+ switch, i.e. the transporter only displays efficient uptake in the presence of Zn2+ (20Loland C.J. Norregaard L. Litman T. Gether U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1683-1688Google Scholar). We inferred that mutation of Tyr-335 produced a constitutive shift in the distribution of conformational states in the transport cycle and that this shift could be reversed in part by Zn2+. A major alteration of the conformational equilibrium in Y335A was further supported by a substantial decrease of up to 150-fold in the apparent affinity for cocaine and related inhibitors and a parallel increase of up to 20-fold in the apparent affinity for substrates (20Loland C.J. Norregaard L. Litman T. Gether U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1683-1688Google Scholar). We proposed that Tyr-335 is part of a network of intramolecular interactions, possibly in the gating domains themselves, that is important for stabilizing the transporter in a conformational state that maintains the structural integrity of the inhibitor binding site and to which extracellular substrate can bind and initiate transport (20Loland C.J. Norregaard L. Litman T. Gether U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1683-1688Google Scholar). If this hypothesis is correct, mutation of other residues might display a similar phenotype as the Y335A mutation, due to their participation in the same network of regulatory intramolecular interactions. In this study, we have sought to test this hypothesis and accordingly to identify additional intracellular residues with a similar phenotype as Y335A. In agreement with our prediction, we identify three residues of 16 mutated charged or bulky residues on the predicted intracellular face of the transporter with such a phenotype. These residues are either in the same loop as Tyr-335, ICL 3, or in the "adjacent" ICL 2 and ICL 4. Furthermore, we establish a structural read-out of the conformational state of the mutant transporters by using the reactivity of a cysteine engineered into position 159 in TM 3. This position was chosen based on recent observations by Rudnick and co-workers (17Chen J.G. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1044-1049Google Scholar) in the homologous SERT and NET indicating that the accessibility of the corresponding residue (position 155 in NET and 179 in SERT) to the positively charged sulfhydryl-reactive compound MTSET ([2-(trimethylammonium)-ethyl]-methanethiosulfonate) is dependent on whether the transporter assumes an outward facing conformation or an inward facing conformation. Consistent with our hypothesis, the accessibility data provide direct structural support for an alteration in the conformational equilibria of the mutant transporters that can be reversed in part by Zn2+ Site-directed Mutagenesis—We generated a synthetic hDAT (syn-DAT) gene that encodes a protein with an amino acid sequence identical to that of hDAT WT, but the nucleotide sequence was altered to increase the number of unique restriction sites and to optimize codon utilization. The nucleotide sequence of this construct and its generation will be described elsewhere. 2L. Shi and J. A. Javitch, manuscript in preparation. We have used this synthetic construct in previous work, and its expression and function are identical to DAT encoded by the wild type DAT gene (10Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Google Scholar, 21Saunders C. Ferrer J.V. Shi L. Chen J. Merrill G. Lamb M.E. Leeb-Lundberg L.M. Carvelli L. Javitch J.A. Galli A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6850-6855Google Scholar). The E2C background was generated by mutation in the synDAT background of Cys-90 and Cys-306 to alanine. These two endogenous cysteines were previously demonstrated to be accessible to charged sulfhydryl reagents applied extracellularly (15Ferrer J. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Google Scholar). This construct was designated "E2C," because the two extracellular cysteines were mutated. The synDAT gene and synDAT E2C gene were subcloned into pcDNA3 (Invitrogen) using the restriction sites for KpnI and XbaI. All mutations were generated by two-step PCR mutagenesis using Pfu polymerase (Promega, Madison, WI) with either synDAT WT or synDAT E2C as templates. The mutant PCR fragments were digested with the appropriate enzymes and cloned into the expression vector, purified by agarose gel electrophoresis and ligated into the vector using the TaKaRa ligation kit (Takara Bio Inc., Shiga, Japan). All mutations were confirmed by restriction enzyme mapping and DNA sequencing using an ABI 310 automated sequencer according to the manufacturer's instructions. Indexing of Residues—A generic numbering scheme for amino acid residues in the family of Na+/Cl–-coupled transporters has recently been proposed to facilitate direct comparison of positions between the individual members of the transporter family (22Goldberg N.R. Beuming T. Weinstein H. Javitch J.A. Bräuner-Osborne H. Schousboe A. Structure and Function of Neurotransmitter Transporters. Humana Press, Totowa, NJ2003: 213-234Google Scholar). According to this scheme the most conserved residue in each transmembrane segment has been given the number 50, and each residue is numbered according to its position relative to this conserved residue. For example, 1.55 indicates a residue in TM1 five residues carboxyl-terminal to the most conserved residue in this TM (Trp1.50). For the DAT, the most conserved residues in each transmembrane domain is as follows: TM1, Trp-84; TM2, Pro-112; TM3, Tyr-156; TM4, Cys-243; TM5, Leu-287; TM6, Gln-317; TM7, Phe-356; TM8, Phe-412; TM9, Gly-468; TM10, Gly-500; TM11, Pro-529; TM12, Gly-561. The generic numbers for residues mutated in this study are (in superscript): Arg-601.26, Glu-611.27, Phe-1232.61, Ile-1593.53, Lys-2604.67, Lys-2645.27, Tyr-3356.68, Tyr-3437.28, Arg-3446.29, Asp-3456.30, Asp-4218.59, Glu4288.66, Asp-4369.18, Glu-4379.19, Phe-4389.20, Asp-50710.57, Tyr-51911.40, and Tyr-57812.67. In E2C we have mutated Cys-901.56 and Cys-3066.39. Expression in COS-7 Cells—COS-7 cells were maintained at 37 °C in 10% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mml-glutamine, and 0.01 mg/ml gentamicin (all from Invitrogen). WT and mutant transporters were transiently transfected into COS-7 cells by the calcium phosphate precipitation method as previously described (23Johansen T.E. Scholler M.S. Tolstoy S. Schwartz T.W. FEBS Lett. 1990; 267: 289-294Google Scholar, 24Gether U. Marray T. Schwartz T.W. Johansen T.E. FEBS Lett. 1992; 296: 241-244Google Scholar). [3H]Dopamine Uptake Experiments—Uptake assays were performed as modified from Giros et al. (25Giros B. el Mestikawy S. Godinot N. Zheng K. Han H. Yang-Feng T. Caron M.G. Mol. Pharmacol. 1992; 42: 383-390Google Scholar) using 2,5,6-[3H]dopamine (7–21 Ci/mmol) (Amersham Biosciences). The uptake assays were carried out 2 days after transfection of transiently transfected COS-7 cells. Twenty hours after transfection, the cells were plated in poly-d-lysine-coated 24- or 12-well dishes (1 or 3 × 105 cells/well, respectively) depending on the expression level of the particular mutated transporter to achieve an uptake level of 5–10% of total added [3H]dopamine. Prior to the experiment, the cells were washed once in 500 μl of uptake buffer (25 mm HEPES adjusted to pH 7.4 upon addition of 130 mm NaCl, 5.4 mm KCl, 1.2 mm CaCl2, 1.2 mm MgSO4,1mml-ascorbic acid, 5 mm d-glucose, and 1 μm of the catechol-O-methyltransferase inhibitor Ro 41-0960 (Sigma) at room temperature. Non-labeled compounds (dopamine, norepinephrine, Zn2+, CFT (2β-carbomethoxy-3β-(4-fluorophenyl)tropane), cocaine, or GBR 12,909) were added to the cells at the indicated concentrations. When the cells were co-incubated with 10 μm Zn2+ and the indicated non-labeled compound, Zn2+ was added to the uptake media prior to the addition of non-labeled compound. Uptake was initiated by addition of 50–90 nm [3H]dopamine in a final volume of 500 μl of uptake buffer. After 10 min of incubation at 37 °C, the cells were washed twice with 500 μl of ice-cold uptake buffer, lysed in 300 μl of 1% SDS, and left for 1 h at 37 °C. All samples were subsequently transferred to 24-well counting plates (PerkinElmer Life Sciences) followed by addition of 600 μl of Opti-phase HiSafe 3 scintillation fluid (PerkinElmer Life Sciences). The samples were counted in a Wallac Tri-Lux β scintillation counter (PerkinElmer Life Sciences). Nonspecific uptake was determined in the presence of 1 mm dopamine (RBI, Natick, MA). All determinations were performed in triplicate. [3H]Norepinephrine Uptake Experiments—Assays were performed as described above for [3H]dopamine uptake experiments, except that the [3H]dopamine was substituted by 40–80 nm [7,8-3H]norepinephrine (6–12 Ci/mmol) (Amersham Biosciences). Surface Biotinylation—Biotinylation of cell surface proteins was performed essentially as described (26Granas C. Ferrer J. Loland C.J. Javitch J.A. Gether U. J. Biol. Chem. 2003; 278: 4990-5000Google Scholar) by reaction with the membrane impermeant amine-specific biotinylating reagent sulfo-NHS-SS-biotin (Pierce). Transfected COS-7 cells were seeded in poly-d-lysine-coated 100-mm cell culture dishes (Corning) at 2.5 × 106 cells/dish and grown for 24 h before the experiment. The cells were washed with ice-cold phosphate-buffered saline/Ca-Mg (pH 7.3), before treatment with sulfo-NHS-SS-biotin (1.5 mg/ml) at 4 °C for 40 min in phosphate-buffered saline/Ca-Mg followed by three washes with TBS (0.05 m Tris, pH 7.4, 0.3 m NaCl) and three washes with phosphate-buffered saline/Ca-Mg. The cells were solubilized in 1 ml of solubilization buffer (25 mm Tris, pH 7.5, with 1.0% Triton X-100, 150 mm NaCl, 1 mm EDTA, 5 mmN-ethylmaleimide, 200 μm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture tablet (Roche Diagnostics)), scraped off and left for 30 min at 4 °C with constant shaking. Lysates were centrifuged at 20,000 × g for 30 min at 4 °C, and the protein concentration in the supernatants was determined using the BCA assay kit (Pierce). Monomeric avidin beads (175 μl) (Pierce) were added to 500 μg of total protein from each sample. The volume was adjusted to 1.0 ml with solubilization buffer and the samples were incubated for 1 h at room temperature. The beads were washed four times with 800 μl of solubilization buffer, before elution with 50 μl of 2× loading buffer (100 mm Tris-HCl, pH 6.8, 20% glycerol, 10% SDS, 0.1 m dithiothreitol, and 0.2% bromphenol blue) for 30 min at 37 °C. The eluates (25 μl) were resolved by SDS-PAGE (10%) and immunoblotted with the rat monoclonal antibody MAB 369 directed against the NH2 terminus of the hDAT (Chemicon) diluted 1:1000. Immunoreactive bands were visualized using goat anti-rat horseradish peroxidase-conjugated secondary antibody (1:10,000) and Pico Luminescence (Pierce). Quantification of bands was performed by densitometry measures using Scion Image (Scion, Frederick, MD) using film exposures that were in the linear range. MTSET Experiments—Two days after transfection, cells seeded in 12- or 24-well plates were washed once with 500 μl of uptake buffer (see above). The cells were subsequently incubated with 0.5 mm MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate) (Toronto Research Chemicals, Toronto, Canada) (unless another concentration is indicated) at 37 °C for 10 min. The stock MTSET solution was freshly prepared in H2O and immediately diluted 10-fold by application to the transfected cells into a final volume of 500 μl of uptake buffer. After incubation, the cells were washed twice in 500 μl of uptake buffer before initiation of [3H]dopamine uptake, performed as described above. The effects of substrates and blockers on MTSET reactivity were investigated by the addition of 100 μm dopamine or 10 μm cocaine immediately prior to the addition of MTSET. Subsequently, the cells were washed twice in 500 μl of uptake buffer before initiation of [3H]dopamine uptake assay. Data Calculations—Uptake data and binding data were analyzed by nonlinear regression analysis using Prism 3.0 from GraphPad Software, San Diego, CA. The IC50 values used in the estimation of Km for uptake were calculated from means of pIC50 values and the S.E. interval from the pIC50 ± S.E. The KI values were calculated from the IC50 values using the equation KI = IC50/(1 + (L/Km)), L = concentration of [3H]dopamine. One-way analysis of variance followed by Newman-Keuls multiple comparison post-hoc test was used for statistical comparisons of the response to MTSET treatment. To achieve an exact measure for the low specific transporter-mediated [3H]dopamine uptake for the E2C I159C/Y335A mutant with no Zn2+ present, an uptake experiment on mock-transfected COS-7 cells was performed in parallel. The nonspecific uptake in these cells was subtracted from the total uptake in E2C I159C/Y335A expressing cells. The nonspecific uptake was less than 20% of total uptake in E2C I159C/Y335A. Mutation of Selected Intracellular Residues in the hDAT—To identify residues that upon mutation might display the same phenotype as our previously described Tyr-335 to alanine mutation (Y335A), we selected 16 residues distributed throughout the intracellular domain of the hDAT (Fig. 1). First, we selected a series of tyrosines and phenylalanines that were either conserved in all or in many of the mammalian Na+/Cl–-coupled transporters (Fig. 2): Phe-123 in ICL 1; Tyr-343 in ICL 3; Phe-438 in ICL 4; Tyr-519 in ICL 5; and Tyr-578 in the COOH terminus (for the generic number of these residues according to the numbering scheme proposed by Goldberg et al. see indexing under "Materials and Methods"). Next we selected a series of highly conserved charged residues because we hypothesized that such residues might be involved in important intramolecular interactions critical for maintaining the proper conformational equilibrium of the transport cycle. The selected residues included: Arg-60 and Glu-61 in the NH2 terminus; Lys-260 and Lys-264 in ICL 2; Arg-344 and Asp-345 in ICL 3; Asp-421, Glu-428, Asp-436, and Glu-437 in ICL 4; and Asp-507 in ICL5. As is apparent from the alignment in Fig. 2, our selection of residues was representative but was not a comprehensive selection of all the conserved intracellular residues in the DAT with a bulky or charged side chain. The selected residues were mutated to alanine one at a time and functionally characterized after transient expression in COS-7 cells. As shown in Table I, all the mutants were functional as assessed by measurement of [3H]dopamine uptake. Like 335A, several mutants, including K264A, D345A, D436A, F438A, and D507A, displayed a substantially lowered Vmax (<10% of WT). Also similar to Y335A, the K264A, D345A, and D436A mutations displayed lower Km values than the WT (7–9-fold) but this was not the case for F438A and D507A, which had Km values similar to the WT (Table I). Of the mutants, only one (D421A) had a Km value higher than the WT (8 versus 1.7 μm, Table I).Table IUptake characteristics of hDAT and mutant transportershDAT mutants[3H]DA Vmax[3H]DA Km (S.E. interval)nZn2+ IC50 (S.E. interval)nCFT KI (S.E. interval)nfmol/min/105 cells ± SEnmμmnmhDAT wt9700 ± 4001.7 (1.5-1.9)100.7 (0.5-0.9)744 (40-48)6R60A3000 ± 6001.20 (1.15-1.26)30.6 (0.40-0.8)328 (23-33)3E61A4000 ± 7001.2 (1.0-1.4)40.33 (0.28-0.39)325 (24-26)3F123A2300 ± 5000.87 (0.82-0.93)40.6 (0.5-0.7)321 (20-23)3K260A3900 ± 9001.0 (0.6-1.6)40.5 (0.2-0.9)327 (23-32)3K264A260 ± 400.16 (0.13-0.19)6Potentiation5220 (170-290)8Y335AaData taken from Ref. 20.16 ± 30.13 (0.10-0.17)9Potentiation52600 (2300-3100)5Y343A2900 ± 5000.5 (0.4-0.6)30.87 (0.85-0.90)350 (40-65)3R344A2000 ± 3001.3 (1.1-1.5)30.63 (0.59-0.68)321 (16-27)3D345A350 ± 300.18 (0.16-0.19)7Potentiation5250 (230-290)3D421A3100 ± 12008 (6-9)32.2 (1.4-3.4)3400 (250-660)3E428A1240 ± 2700.49 (0.43-0.56)41.1 (0.9-1.4)376 (66-88)3D436A450 ± 1500.20 (0.16-0.25)5Potentiation5130 (90-190)3E437A1940 ± 4400.6 (0.5-0.8)40.2 (0.1-0.4)433.6 (33.0-34.3)3F438A47 ± 131.0 (0.3-4)31.97 (1.90-2.04)3NDbND, not determined.0D507A850 ± 3801.1 (0.7-1.6)30.6 (0.4-0.8)320 (12-32)3Y519A1360 ± 4000.7 (0.5-0.9)40.8 (0.7-1.0)469 (65-74)3Y578A5800 ± 14001.2 (1.0-1.6)50.7 (0.6-0.8)429 (23-34)4D436A/E437A40 ± 100.08 (0.07-0.10)3Potentiation3100 (60-150)3a Data taken from Ref. 20Loland C.J. Norregaard L. Litman T. Gether U. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1683-1688Google Scholar.b ND, not determined. Open table in a new tab Effect of Zn2+on Intracellular Mutants—The effect of Zn2+ on [3H]dopamine uptake mediated by the mutants was subsequently investigated. In the hDAT WT, Zn2+ is a potent non-competitive inhibitor of [3H]dopamine uptake showing a biphasic inhibition curve with an IC50 for the high affinity phase of around 1 μm and >1000 μm for the low affinity phase (Ref. 7Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Google Scholar and Table I, Fig. 3). The high affinity inhibition is because of the interaction of Zn2+ with a tridentate Zn2+ binding site involving His-193, His-375, and Glu-396 (Refs. 7Norregaard L. Frederiksen D. Nielsen E.O. Gether U. EMBO J. 1998; 17: 4266-4273Google Scholar and 8Loland C.J. Norregaard L. Gether U. J. Biol. Chem. 1999; 274: 36928-36934Google Scholar, and Fig. 1). In Y335A, the interaction of Zn2+ with this endogenous site does not inhibit uptake but instead causes a remark
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OBJECTIVE: Evidence of a relationship between genotype and binding availability was assessed for the dopamine and serotonin transporter genes. METHOD: The authors assessed dopamine transporter genotype at the SLC6A3 3′ variable number of tandem repeats (VNTR) polymorphism and serotonin transporter genotype at the SLC6A4 promotor VNTR polymorphism in 30 healthy subjects who also underwent single photon emission computed tomography with [123I]β-CIT. RESULTS: Subjects homozygous for the 10-repeat allele at the SLC6A3 locus demonstrated significantly lower dopamine transporter binding than carriers of the nine-repeat allele. There was no effect of SLC6A4 genotype upon serotonin transporter binding. CONCLUSIONS: These findings suggest that genetic variation at the SLC6A3 3′ VNTR polymorphism may modify dopamine transporter function.
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In recent years, there has been substantial progress in studying the dopamine transporter, a unique component of the functioning dopaminergic nerve terminal. The transporter has been studied by direct binding techniques using a variety of ligands which function as inhibitors of transport. Analogues of these ligands have been used as photoaffinity labels to solubilize and further characterize the transporter. While a variety of drugs bind to the transporter, it is clear that the transporter may serve as an important drug receptor, particularly for the reinforcing properties of some psychostimulants such as cocaine. An extension of the in vitro ligand-binding studies reveals that it is possible to preferentially label the transporter in vivo. The success of in vivo labeling has lead to successful positron emission tomographic scanning studies of the transporter. These studies in turn have revealed the usefulness of imaging the transporter, a measure of the presence of dopaminergic nerve terminals, as a potential diagnostic tool in Parkinson's disease.
Neurotransmitter transporter
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Genetic Association
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The aim of this study was to examine the effect of both promoter and intron polymorphisms of the serotonin transporter (5HTT) gene on posttraumatic stress disorder (PTSD) development. For this purpose, two polymorphisms of the 5-HTT gene, which are found in the promoter (5-HTT gene-linked polymorphic region) and second intron (variable number of tandem repeats) of the gene, were analyzed in 100 patients who were admitted to the Emergency Department after a mild physical trauma. None of the 5-HTT polymorphisms studied have an effect on PTSD development after a mild physical injury, but having L allele for 5-HTT gene-linked polymorphic region may cause milder hyperarousal symptoms in those patients who have developed PTSD.
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