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• W2018678300 abstract "Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na+/Cl--dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPH-induced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane. Dopaminergic neurotransmission is fine-tuned by the rate of removal of dopamine (DA) from the extracellular space via the Na+/Cl--dependent DA transporter (DAT). DAT is a target of psychostimulants such as amphetamine (AMPH) and cocaine. Previously, we reported that AMPH redistributes the human DAT away from the cell surface. This process was associated with a reduction in transport capacity. This loss of transport capacity may result either from a modification of the function of DAT that is independent of its cell surface redistribution and/or from a reduction in the number of active transporters at the plasma membrane that results from DAT trafficking. To discriminate between these possibilities, we stably transfected HEK-293 cells with a yellow fluorescent protein (YFP)-tagged human DAT (hDAT cells). In hDAT cells, acute exposure to AMPH induced a time-dependent loss of hDAT activity. By coupling confocal imaging with patch-clamp whole-cell recordings, we have demonstrated for the first time that the loss of AMPH-induced hDAT activity temporally parallels the accumulation of intracellular hDAT. In addition, presteady-state current analysis revealed a cocaine-sensitive, voltage-dependent capacitance current that correlated with the level of transporter membrane expression and in turn served to monitor the AMPH-induced trafficking of hDAT. We found that the decrease in hDAT cell surface expression induced by AMPH was not paralleled by changes in the ability of the single transporter to carry charges. Quasi-stationary noise analysis of the AMPH-induced hDAT currents revealed that the unitary transporter current remained unaltered during the loss of hDAT membrane expression. Taken together, these data strongly suggest that the AMPH-induced reduction of hDAT transport capacity results from the removal of active hDAT from the plasma membrane. The Na+/Cl--dependent transporter family includes plasmalemmal carriers for monoamines such as DA, 1The abbreviations used are: DA, dopamine; DAT, DA transporter; AMPH, amphetamine; hDAT, human DAT; YFP, yellow fluorescent protein; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; NET, norepinephrine transporter; ANOVA, analysis of variance. serotonin, and norepinephrine (1Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 2Blakely R.D. De Felice L.J. Hartzell H.C. J. Exp. Biol. 1994; 196: 263-281Crossref PubMed Google Scholar). DAT tunes the spatial and temporal characteristics of dopaminergic neurotransmission by regulating extracellular DA concentration (3Giros B. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2076) Google Scholar, 4Jones S.R. Gainetdinov R.R. Wightman R.M. Caron M.G. J. Neurosci. 1998; 18: 1979-1986Crossref PubMed Google Scholar). Although diffusion and enzymatic degradation also reduce the synaptic concentration of this monoamine, the development of a DAT knockout mouse established reuptake as the primary mechanism controlling extracellular DA levels (3Giros B. Nature. 1996; 379: 606-612Crossref PubMed Scopus (2076) Google Scholar). Dopaminergic neurotransmission mediates numerous biological events, including reward, addiction, movement, and lactation (1Giros B. Caron M.G. Trends Pharmacol. Sci. 1993; 14: 43-49Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 5Iversen L.L. Biophys. J. 1971; 41: 571-591Google Scholar). Several therapeutic agents (6Howell L.L. Wilcox K.M. J. Pharmacol. Exp. Ther. 2001; 298: 1-6PubMed Google Scholar), environmental toxins (7Javitch J.A. D'Amato R.J. Strittmatter S.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2173-2177Crossref PubMed Scopus (1154) Google Scholar, 8Pifl C. Giros B. Caron M.G. J. Neurosci. 1993; 13: 4246-4253Crossref PubMed Google Scholar), and psychostimulants (AMPH and cocaine) (9Koob G.F. Bloom F.E. Science. 1988; 242: 715-723Crossref PubMed Scopus (1789) Google Scholar) have been shown to generate their effects by targeting DAT. AMPH rapidly decreases DA clearance and stimulates an DAT-mediated DA efflux (10Sulzer D. Maidment N.T. Rayport S. J. Neurochem. 1993; 60: 527-535Crossref PubMed Scopus (253) Google Scholar). The subsequent increase in dopaminergic signaling in limbic areas of the brain is believed to mediate the rewarding and addictive properties of AMPH (9Koob G.F. Bloom F.E. Science. 1988; 242: 715-723Crossref PubMed Scopus (1789) Google Scholar). In 2000, Saunders and colleagues proposed a novel action of AMPH by demonstrating that acute application of AMPH reduces hDAT cell surface expression (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar). Sorkina and colleagues (12Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) recently extended this observation by showing that AMPH induced an intracellular accumulation of hDAT in early and late endosomal vesicles, which coexpress the endosomal proteins Rab5, Rab11, Hrs, and EEA.1. Regulators of DAT activity include AMPH and cocaine (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 12Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Carvelli L. Moron J.A. Kahlig K.M. Ferrer J.V. Sen N. Lechleiter J.D. Leeb-Lundberg F. Merrill G. Lafer E.M. Ballou L.M. Shippenberg T.S. Javitch J.A. Lin R.Z. Galli A. J. Neurochem. 2002; 81: 859-869Crossref PubMed Scopus (181) Google Scholar, 14Daws L.C. Callaghan P.D. Moron J.A. Kahlig K.M. Shippenberg T.S. Javitch J.A. Galli A. Biochem. Biophys. Res. Commun. 2002; 290: 1545-1550Crossref PubMed Scopus (147) Google Scholar, 15Little K.Y. Elmer L.W. Zhong H. Scheys J.O. Zhang L. Mol. Pharmacol. 2002; 61: 436-445Crossref PubMed Scopus (128) Google Scholar, 16Gulley J.M. Doolen S. Zahniser N.R. J. Neurochem. 2002; 83: 400-411Crossref PubMed Scopus (72) Google Scholar), G-protein-coupled receptors such as the D2 and mGluR5 receptors (17Mayfield R.D. Zahniser N.R. Mol. Pharmacol. 2001; 59: 113-121Crossref PubMed Scopus (89) Google Scholar, 18Page G. Peeters M. Najimi M. Maloteaux J.M. Hermans E. J. Neurochem. 2001; 76: 1282-1290Crossref PubMed Scopus (73) Google Scholar), kinases such as protein kinase C, phosphatidylinositol 3-kinase, tyrosine kinase, and Ca2+/calmodulin kinase (12Sorkina T. Doolen S. Galperin E. Zahniser N.R. Sorkin A. J. Biol. Chem. 2003; 278: 28274-28283Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 13Carvelli L. Moron J.A. Kahlig K.M. Ferrer J.V. Sen N. Lechleiter J.D. Leeb-Lundberg F. Merrill G. Lafer E.M. Ballou L.M. Shippenberg T.S. Javitch J.A. Lin R.Z. Galli A. J. Neurochem. 2002; 81: 859-869Crossref PubMed Scopus (181) Google Scholar, 19Melikian H.E. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar, 20Daniels G.M. Amara S.G. J. Biol. Chem. 1999; 274: 35794-35801Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 21Doolen S. Zahniser N.R. J. Pharmacol. Exp. Ther. 2001; 296: 931-938PubMed Google Scholar, 22Derbez A.E. Mody R.M. Werling L.L. J. Pharmacol. Exp. Ther. 2002; 301: 306-314Crossref PubMed Scopus (39) Google Scholar, 23Loder M.K. Melikian H.E. J. Biol. Chem. 2003; 278: 22168-22174Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), and transporter interacting proteins (24Torres G.E. Yao W.D. Mohn A.R. Quan H. Kim K.M. Levey A.I. Staudinger J. Caron M.G. Neuron. 2001; 30: 121-134Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 25Carneiro A.M. Ingram S.L. Beaulieu J.M. Sweeney A. Amara S.G. Thomas S.M. Caron M.G. Torres G.E. J. Neurosci. 2002; 22: 7045-7054Crossref PubMed Google Scholar). In addition, some of these studies suggest that the regulation of DAT activity may originate from a change in DAT cell surface expression (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 13Carvelli L. Moron J.A. Kahlig K.M. Ferrer J.V. Sen N. Lechleiter J.D. Leeb-Lundberg F. Merrill G. Lafer E.M. Ballou L.M. Shippenberg T.S. Javitch J.A. Lin R.Z. Galli A. J. Neurochem. 2002; 81: 859-869Crossref PubMed Scopus (181) Google Scholar, 14Daws L.C. Callaghan P.D. Moron J.A. Kahlig K.M. Shippenberg T.S. Javitch J.A. Galli A. Biochem. Biophys. Res. Commun. 2002; 290: 1545-1550Crossref PubMed Scopus (147) Google Scholar, 15Little K.Y. Elmer L.W. Zhong H. Scheys J.O. Zhang L. Mol. Pharmacol. 2002; 61: 436-445Crossref PubMed Scopus (128) Google Scholar, 16Gulley J.M. Doolen S. Zahniser N.R. J. Neurochem. 2002; 83: 400-411Crossref PubMed Scopus (72) Google Scholar, 17Mayfield R.D. Zahniser N.R. Mol. Pharmacol. 2001; 59: 113-121Crossref PubMed Scopus (89) Google Scholar, 19Melikian H.E. Buckley K.M. J. Neurosci. 1999; 19: 7699-7710Crossref PubMed Google Scholar, 21Doolen S. Zahniser N.R. J. Pharmacol. Exp. Ther. 2001; 296: 931-938PubMed Google Scholar, 23Loder M.K. Melikian H.E. J. Biol. Chem. 2003; 278: 22168-22174Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 24Torres G.E. Yao W.D. Mohn A.R. Quan H. Kim K.M. Levey A.I. Staudinger J. Caron M.G. Neuron. 2001; 30: 121-134Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 25Carneiro A.M. Ingram S.L. Beaulieu J.M. Sweeney A. Amara S.G. Thomas S.M. Caron M.G. Torres G.E. J. Neurosci. 2002; 22: 7045-7054Crossref PubMed Google Scholar). Although these results suggest that trafficking of the transporter modulates the transport capacity of the system, DAT could be modified and functionally inactivated by AMPH prior to trafficking from the plasma membrane. Therefore, the extent to which hDAT trafficking determines the AMPH-induced down-regulation of hDAT activity is unknown. The present work combines confocal imaging, whole cell steady-state and transient current recordings, as well as noise analysis, to simultaneously monitor DAT cell surface expression and activity. Our data suggest that upon AMPH application hDAT redistributes from the plasma membrane as an active carrier. Cell Culture—A fluorescently tagged hDAT was constructed by fusing the C terminus of the coding region of enhanced yellow fluorescent protein (YFP) from pEYFP-N1 (Clontech) to the N terminus of the human synthetic DAT cDNA, thereby creating the fusion construct YFP-hDAT. This construct was subcloned into a bicistronic expression vector (26Rees S. Coote J. Stables J. Goodson S. Harris S. Lee M.G. BioTechniques. 1996; 20 (106, 108-110): 102-104Crossref PubMed Scopus (263) Google Scholar) modified to express the synthetic hDAT from a cytomegalovirus promoter and the hygromycin resistance gene from an internal ribosomal entry site (pciHyg), as described previously (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar). EM4 cells, an HEK 293 cell line stably transfected with macrophage scavenger receptor (27Robbins A.K. Horlick R.A. BioTechniques. 1998; 25: 240-244Crossref PubMed Scopus (57) Google Scholar) (R. Horlick, Pharmacopeia, Cranberry, NJ), were transfected with the YFP-DAT using LipofectAMINE (Invitrogen), and a stably transfected pool (hDAT cells) was selected in 250 μg/ml hygromycin as described previously (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 28Ferrer J.V. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9238-9243Crossref PubMed Scopus (111) Google Scholar). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Reagents were purchased from Sigma (St. Louis, MO) unless otherwise noted. Electrophysiology and Confocal Microscopy—Parental or stably transfected cells were plated at a density of 105 per 35-mm culture dish. Before electrical recordings, attached cells were washed twice with the bath solution containing the following (in mm): 130 NaCl, 10 HEPES, 1.5 CaCl2, 0.5 MgSO4, 1.3 KH2PO4, and 34 dextrose adjusted to pH 7.35. In experiments investigating the Na+ dependence of the hDAT transient charge movement, Na+ was iso-osmotically replaced with choline. The hDAT cells were sequentially perfused with bath solutions containing the following Na+ concentrations (in mm): 3, 24, 53, 94, 122, and 130. The recording pipette was filled with a solution containing the following (in mm); 120 KCl, 2.0 MgCl2, 0.1 CaCl2, 1.1 EGTA, 10 HEPES, and 30 dextrose adjusted to pH 7.35. Free Ca+2 was calculated to be 0.1 mm. A programmable puller (model P-2000, Sutter Instruments, Novato, CA) was used to fabricate quartz-recording pipettes with a resistance of 5 mΩ. Whole cell currents were recorded using an Axopatch 200B with a low pass Bessel filter set at 5,000 Hz. Current-voltage relationships were generated by stepping the membrane voltage from a holding potential of -20 mV to voltages between -160 and 100 mV in 20-mV increments for 1 s. A waveform generator (Challenger VM-2C) was used to vary the membrane potential. Data were stored on a VCR and analyzed with a Nicolet Integra Model 20 oscilloscope and a DELL computer using programs written by W. N. Goolsby (Emory University, Atlanta, available on request). Application of dextro-AMPH to hDAT cells generated an hDAT current, which after several minutes of stability began to decrease. Because of cell-to-cell variability, we defined “time zero” as the onset of the decreasing phase of the AMPH-induced current. We subsequently defined the initial hDAT current trace (time zero) as the AMPH-induced current recorded immediately before the onset of the decreasing phase of the hDAT-mediated current. The final current was defined as the last current trace recorded before adding cocaine. The AMPH-induced, hDAT-mediated steady-state current and transient charge movement was obtained by subtracting the current trace recorded in the presence of AMPH plus cocaine from the current trace recorded in the presence of AMPH. The hDAT transient charge (Q) movement in response to a voltage step, was obtained by integrating either the “on” or the “off” of the relaxation component of the AMPH-induced current (Fig. 2A). Time-dependent changes in Q were used to evaluate hDAT cell surface expression as described previously (29Mager S. Naeve J. Quick M. Labarca C. Davidson N. Lester H.A. Neuron. 1993; 10: 177-188Abstract Full Text PDF PubMed Scopus (280) Google Scholar, 30Mager S. Min C. Henry D.J. Chavkin C. Hoffman B.J. Davidson N. Lester H.A. Neuron. 1994; 12: 845-859Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 31Galli A. DeFelice L.J. Duke B.J. Moore K.R. Blakely R.D. J. Exp. 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The charge-voltage relationships were fit to the Boltzmann function, Q = (Qdep - Qhyp)/[1 + exp(-(V - V½)qzδ/kT)] + Qhyp; where Qmax = Qdep - Qhyp (Qdep and Qhyp are the charge movements at the depolarizing and hyperpolarizing limits), V is the membrane voltage, V½ is the potential at which half of the charge has moved, q is the elementary charge, zδ is the product of the valence of the moving charge and the fraction of the membrane field through which it moves, k is the Boltzmann constant, and T is the absolute temperature. For simplicity, the slope factor of the fitting was kT/qzδ. Charge-voltage relationships from each experiment were fit, and the results were reported as mean ± S.E. Unless otherwise noted, the steady-state current (I) at a particular voltage was calculated as the average current during the final 100 ms of the voltage step. The time course for the AMPH-induced decrease in hDAT steady-state current (Fig. 5A) was fit with the Boltzmann equation, I = (A1 - A2)/(1 + exp(t - t50)/α) + A2), where A1 is the initial current, A2 is the final current, t50 is the time at which half of A1 remains, and α is the slope of the fit. For all the experiments, the t50 time was reported as mean ± S.E. To synchronize the acquisition of hDAT-mediated currents with the confocal imaging of the cellular distribution of hDAT, we simultaneously triggered the voltage steps and the confocal imaging software (EZ 2000, Coord Automatisering, The Netherlands) with the Challenger VM-2C. Cells were visualized with a Nikon eclipse TE300 inverted microscope. Images were acquired using 488-nm excitation with a 510-nm long pass filter. The image acquired during the initial hDAT current trace (time zero as described above) was defined as the initial confocal image. To minimize the fluorophore bleaching and cytotoxic effects of confocal microscopy, images were acquired every 1.5 min prior to the start of the hDAT current decreasing phase and every 30 s during the decreasing phase. Because membrane proteins such as GLUT4 can be localized to the plasma membrane without being exposed to the extracellular space (37Inoue M. Chang L. Hwang J. Chiang S.H. Saltiel A.R. Nature. 2003; 422: 629-633Crossref PubMed Scopus (285) Google Scholar), we quantified intracellular instead of plasma membrane fluorescence as a measure of AMPH-induced hDAT membrane redistribution. Therefore, by excluding plasma membrane fluorescence, we may underestimate the amount of intracellular DAT. The quantitation of intracellular fluorescence was performed by normalizing for fluorophore bleaching and cytosolic volume changes (38Lippincott-Schwartz J. Presley J.F. Zaal K.J. Hirschberg K. Miller C.D. Ellenberg J. Methods Cell Biol. 1999; 58: 261-281Crossref PubMed Google Scholar, 39Piston D.W. Patterson G.H. Knobel S.M. Methods Cell Biol. 1999; 58: 31-48Crossref PubMed Google Scholar). ImageJ software (National Institutes of Health, available online) was utilized for this analysis. Normalized data were analyzed with Prism 3.02 software (GraphPad Software, Inc.) and reported as mean ± S.E. unless otherwise indicated. The reported correlation coefficient (r) was obtained by linear regression analysis. Uptake of [3H]DA—[3H]DA uptake was performed as previously described (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 13Carvelli L. Moron J.A. Kahlig K.M. Ferrer J.V. Sen N. Lechleiter J.D. Leeb-Lundberg F. Merrill G. Lafer E.M. Ballou L.M. Shippenberg T.S. Javitch J.A. Lin R.Z. Galli A. J. Neurochem. 2002; 81: 859-869Crossref PubMed Scopus (181) Google Scholar). hDAT cells were seeded into 24-well plates ∼24 h prior to the experiment (150,000 cells per well). After 5 h of serum starvation, the cells were treated in quadruplicate wells with AMPH in uptake buffer, containing (in mm); 120 NaCl, 4.7 KCl, 10 HEPES, 5 Trizma base, 2.2 CaCl2, and 10 dextrose with 100 μm ascorbic acid at pH 7.4 and 37 °C. The plates were removed from the incubator, and the cells were washed (three washes of 5 min each) with 4 °C uptake buffer to remove the AMPH from each well and inhibit protein trafficking. The plates were then placed into an 18 °C incubator in uptake buffer containing 100 μm pargyline, a monoamine oxidase inhibitor. Then 50 nm [3H]DA (PerkinElmer Life Sciences, Boston, MA) together with 15 μm DA was added to reach a final volume of 250 μl. Cells were incubated for 2 min and then the solution was aspirated to terminate uptake. After three quick washes with ice-cold uptake buffer, the cells were lysed with 300 μl of 1% SDS. Radioactivity was measured in a Beckman scintillation counter with UniverSol mixture. Specific uptake was defined as total uptake minus nonspecific uptake in the presence of 10 μm mazindol. Data were analyzed with Prism 3.02 software and reported as mean ± S.E. Cell Surface Biotinylation—Cell surface biotinylation experiments were performed as previously described (40Apparsundaram S. Schroeter S. Giovanetti E. Blakely R.D. J. Pharmacol. Exp. Ther. 1998; 287: 744-751PubMed Google Scholar) with slight modification. hDAT cells were seeded into 6-well plates (106 cells/well) ∼48 h prior to the experiment. After 1 h of serum starvation, the cells were washed twice with 37 °C uptake buffer and treated with AMPH in uptake buffer at 37 °C. The cells were then washed twice with ice-cold PBS containing 0.1 mm CaCl2 and 1 mm MgCl2 (PBS-Ca-Mg) and treated with Sulfo-NHS-S-S-Biotin (1.5 mg/ml in PBS-Ca-Mg, Pierce Chemical Co., Rockford, IL) on ice for 1 h. The reaction was quenched by washing twice with 4 °C PBS-Ca-Mg containing 100 mm glycine (PBS-Ca-Mg-glycine) followed by an incubation with PBS-Ca-Mg-glycine for 30 min on ice. Cells were then washed twice with 4 °C PBS-Ca-Mg before lysis with 1 ml of radioimmune precipitation assay buffer (20 mm Tris, 20 mm EGTA, 1 mm dithiothreitol, 1 mm benzamidine, 1% Triton X-100: supplemented with protease inhibitors 100 μm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml pepstatin) for 30 min on ice with constant shaking. Lysates were centrifuged at 14,000 × g for 30 min at 4 °C. The supernatants were isolated and biotinylated proteins were separated by incubation with ImmunoPure Immobilized Streptavidin beads (Pierce Chemical Co., Rockford, IL) for 1 h at room temperature with constant mixing. Beads were washed three times with radioimmune precipitation assay buffer, and biotinylated proteins were eluted with Laemmli loading buffer for 30 min at room temperature. Total cell lysates and biotinylated proteins (cell surface) were separated by SDS-polyacrylamide electrophoresis (7.5%) and transferred into polyvinylidene difluoride membranes (Millipore). Polyvinylidene difluoride membranes were incubated for 1.5 h in blocking buffer (5% dry milk, 0.1% Tween 20 in Tris-buffered saline) and immunoblotted with a rat monoclonal antibody directed against the N terminus of the human dopamine transporter (1:2000 in blocking buffer, Chemicon Inc., Temecula, CA). Immunoreactive bands were visualized using horseradish peroxidase-conjugated goat anti-rat antibody (1:5000 in blocking buffer, Santa Cruz Biotechnology, Santa Cruz, CA) with ELC-Plus on Hypersensitive ECL film (Amersham Biosciences, Arlington Heights, IL). Band densities were calculated using Scion-Image software (Scion Corp., Frederick, MD) and normalized to the appropriate total extract to control for protein loading. Data were analyzed with Prism 3.02 software and reported as mean ± S.E. The human EM4 cell line provides an appropriate parental background for studying hDAT function because of the absence of [3H]DA uptake and the lack of either DA- or AMPH-induced whole cell currents (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar). To investigate the extent to which transporter trafficking regulates transport capacity, we created a pool of cells stably expressing fluorescently tagged hDAT. Using confocal imaging, cells with easily visualized plasma membrane fluorescence were selected for analysis (Fig. 1A, inset). Addition of the N-terminal YFP tag to DAT did not significantly alter [3H]DA uptake (data not shown) and did not perturb the ability of the transporter to produce substrate-induced currents (Fig. 1A) (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 41Khoshbouei H. Wang H. Lechleiter J.D. Javitch J.A. Galli A. J. Biol. Chem. 2003; 278: 12070-12077Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Fig. 1A shows current traces recorded from an hDAT cell before substrate application (CTR), after bath application of 10 μm AMPH (AMPH), and after the addition of 10 μm cocaine with AMPH still present (COC). The AMPH-induced hDAT-mediated current was calculated as the current recorded upon bath application of AMPH minus the current recorded after the addition of cocaine, with AMPH still present. Cells without detectable fluorescence did not produce any measurable hDAT-mediated whole cell current upon AMPH application (data not shown). Fig. 1B shows the current-voltage relationship for the AMPH-induced current. The membrane voltage of the cell was held at -20 mV and stepped every 4 s, in 20-mV increments, to voltages between -160 mV and +100 mV. Data were normalized to the current measured at -160 mV (-60.2 ± 16.9 pA, n = 8). The AMPH-induced current reversed at potentials more positive than -35.7 ± 4.0 mV (Fig. 1B). Cocaine application induced a minor decrease of the control current due to its ability to block the DAT-mediated leak conductance (Fig. 1A). This substrate-independent current, which has been described previously for DAT (41Khoshbouei H. Wang H. Lechleiter J.D. Javitch J.A. Galli A. J. Biol. Chem. 2003; 278: 12070-12077Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 42Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar) as well as other neurotransmitter transporters (30Mager S. Min C. Henry D.J. Chavkin C. Hoffman B.J. Davidson N. Lester H.A. Neuron. 1994; 12: 845-859Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 31Galli A. DeFelice L.J. Duke B.J. Moore K.R. Blakely R.D. J. Exp. Biol. 1995; 198: 2197-2212Crossref PubMed Google Scholar) constitutes, in HEK 293 cells expressing hDAT, only a small percentage of the total AMPH-induced current at negative voltages (11Saunders 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: 6850-6855Crossref PubMed Scopus (330) Google Scholar, 43Ingram S.L. Prasad B.M. Amara S.G. Nat. Neurosci. 2002; 5: 971-978Crossref PubMed Scopus (186) Google Scholar). Moreover, DAT substrates (such as DA and AMPH) have also been shown to strongly inhibit this leak current (41Khoshbouei H. Wang H. Lechleiter J.D. Javitch J.A. Galli A. J. Biol. Chem. 2003; 278: 12070-12077Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 42Sonders M.S. Zhu S.J. Zahniser N.R. Kavanaugh M.P. Amara S.G. J. Neurosci. 1997; 17: 960-974Crossref PubMed Google Scholar). In hDAT cells the leak current revealed by bath application of 10 μm cocaine was on average -7.4 ± 1.6 pA at -160 mV (n = 8), ∼10% of the AMPH-induced steady-state current. Following a voltage jump, hDAT cells display current relaxations that persist for several milliseconds (Fig. 1A) after the time required to charge the membrane capacitance. The hDAT component of these current relaxations (hDAT-mediated transient current)" @default.
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