Materials

[3H]DA (3,4-ethyl-2[N-3H]dihydroxyphenylethylamine; specific activity, 31 Ci/mmol), [3H]WIN 35,428 (specific activity, 85 Ci/mmol) and [3H]MPP+ (Methyl-4-Phenylpyridinium Acetate, N-[Methyl-3H]-) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Recombinant HIV-1 transactivator of transcription (Tat1− 86, REP0002a) protein and its mutant Tat protein at cysteine22 residue (substituted to glycine, C22G) and at lysine19 residue (substituted to alanine, K19A) were purchased from Diatheva (Fano, Italy). Antibodies recognizing hDAT (C-20; goat polyclonal antibody), β-tubulin (H-253; rabbit monoclonal antibody) and MTSET chloride were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Anti-goat IgG horseradish peroxidase and anti-rabbit-IgG horseradish peroxidase were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). PC12 cells (ATCC® CRL-1721TM), HEK293 cells (ATCC® CRL-1573TM) and RPMI-1640 cell culture medium were purchased from American Type Culture Collection (Manassas, VA). Fetal bovine serum was purchased from Atlanta Biologicals (Flowery Branch, GA). Horse serum, penicillin/streptomycin, and trypsin/EDTA were purchased from Fisher Scientific (Waltham, MA). D-Glucose, L-ascorbic acid, cocaine, GBR 12,909, WIN 35,428, nomifensine, desipramine, DMSO, ZnCI2, bovine serum albumin, pyrocatechol, α-D-glucose, HEPES, isopropanol, sucrose, MTSET, 2-bromopalmitate or 2-bromohexadecanoic acid (2-BP) and Tween 20 were purchased from Sigma-Aldrich (St. Louis, MO).

Predicting the Site for hDAT Binding With Tat

The binding structure of hDAT with HIV-1 clade B type TatR1 − 86 Rwas modeled and simulated based on the nuclear magnetic resonance (NMR) structures of Tat (Peloponese et al. 2000) and the previously constructed structure of the hDAT-DA complex (Midde et al. 2015; Yuan et al. 2016a). Protein docking and molecular dynamics (MD) simulation were performed to identify the conformation of hDAT-Tat complex. The energy-minimized complex structure used in this work was extracted from the long-time equilibrated MD trajectories in our previous studies (Midde et al. 2013; Yuan et al. 2015, 2016a; Quizon et al. 2016).

Construction of Plasmids

All mutants on hDAT were generated based on the predictions made using our computational model, as described previously using site-directed mutagenesis based on the wild-type hDAT (WT hDAT) sequence (NCBI, cDNA clone MGC: 164,608 IMAGE: 40,146,999) (Quizon et al. 2016). The combination of hDAT mutations on Asp206 and His547 (Aspartic acid 206 to leucine and histidine 547 to alanine, D206L/H547A) were predicted to eliminate two hydrogen bonds between Tat and hDAT. In addition, the combination of mutations on Tyr88, Asp206, and His547 (tyrosine88 to phenylalanine and aspartic acid 206 to leucine and histidine 547 to alanine, Y88F/D206L/H547A) were predicted to eliminate three hydrogen bonds from the Tat-hDAT complex. Two extracellular cysteine90 and cysteine306 residues on WT hDAT were mutated to alanine as E2C hDAT (C90A/C306A hDAT), rendering the E2C hDAT MTSET-insensitive background (Pedersen et al. 2014). A triple mutation of the two cysteine90 and 306 residues to alanine and aspartic acid151 to cysteine on WT hDAT was generated as E2C-I159C hDAT (C90A/C306A/I159C) for MTSET-sensitive construct. A combination of C90A/C306A/H547A was generated as E2C/H547A hDAT for MTSET-insensitive control for H547A. To determine whether H547A-enhanced DA transport is mediated by changing transporter conformation, two combinations of C90A/C306A/I159C/H547A and C90A/C306A/I159A/H547A were generated as E2C-I159C/H547A and E2C-I159A/H547A for positive and negative controls for MTSET sensitive background, respectively. Synthetic cDNA encoding hDAT subcloned into pcDNA3.1+ (provided by Dr. Haley E Melikian, University of Massachusetts) was used as a template to generate mutants using site-directed mutagenesis performed by GENEWIZ (South Plainfield, NJ). DNA sequencing was also performed by GENEWIZ to confirm the sequences of the mutant constructs. Plasmid DNA were propagated and purified using the Qiagen Hi-speed maxi prep plasmid DNA isolation kit (Qiagen, Valencia, CA, USA).

Cell Culture and Transfection

All cell cultures and transfection were conducted as described previously (Quizon et al. 2016; Sun et al. 2019). PC12 cells were maintained at 37 °C in a 5 % CO2 incubator in Dulbecco’s modified eagle medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 15 % horse serum, 2.5 % fetal bovine serum, 2 mM glutamine, and antibiotics (100 U/ml penicillin and 100 µg/mL streptomycin). HEK293 cells were used for the MTSET experiments and were maintained in Eagle’s minimum essential medium (MEM) supplemented with 10 % fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/mL streptomycin). For transient transfection, cells were seeded into 24-well plates at a density of 1 × 105 cells/cm2, or allowed to reach 100 % confluence, and were transfected 24 h later with WT hDAT or mutant hDAT plasmid DNA using Lipofectamine 2000. Cells were used for experiments 24 h after transfection.

[3H]DA Uptake Assay

The maximal velocity (Vmax) and the Michaelis-Menten constant (Km) of [3H]DA uptake were examined in intact PC12 cells transiently expressing WT or mutant hDAT as previously described (Midde et al. 2013). In brief, the cells were washed twice in 1× Krebs-Ringer-HEPES (1× KRH) buffer, preincubated for 10 min at room temperature with buffer and/or 10 µM nomifensine (final concentration), and then incubated with the addition of one of six concentrations of unlabeled DA (final DA concentrations, 0.03-5 µM) and a fixed concentration of [3H]DA (500,000 DPM/well, specific activity, 21.2 Ci/mmol) at room temperature for 8 min. Specific uptake was calculated by subtracting nonspecific uptake (in the presence of 10 µM nomifensine and desipramine) from total uptake.

The IC50 of DA uptake in WT or mutant hDAT by DAT substrate and inhibitors was determined in intact PC12 cells seeded into 24-well plates as reported previously (Quizon et al. 2016). Briefly, cells were preincubated in 1× KRH buffer containing either DA (1 nM-1 mM, final concentration), GBR12909 (1 nM-10 µM, final concentration), cocaine (1 nM-1 mM, final concentration), or ZnCl2 (1, 10, 100 µM; final concentration) for 10 min at room temperature and then incubated for 8 min after the addition of [3H]DA (0.05 µM, final concentration). The cells were then washed twice with ice-cold 1× KRH buffer. Cells were lysed in 500 µl of 1 % SDS for an hour.

To determine the effects of Tat inhibition on [3H]DA uptake, cells were harvested and resuspended in culture medium and allowed to incubate at room temperature for 10 min. The dissociated cells were then pelleted by centrifugation at 400 × g for 5 min at 4 °C, washed once and resuspended with phosphate-buffered saline, followed by another centrifugation at 400 × g for 5 min at 4 °C, and finally resuspended in 1× KRH buffer. Specific [3H]DA uptake was determined in the cell suspensions prepared from WT hDAT and its mutants in the presence or absence of recombinant Tat1 − 86 (140 nM, final concentration). Cell suspensions were preincubated with Tat for 20 min at room temperature and then incubated for 8 min after adding [3H]DA (0.05 µM, final concentration). 10 µM nomifensine and desipramine (final concentration) were used to determine non-specific [3H]DA uptake. The incubation was terminated by immediate filtration through Whatman GF/B glass filters (presoaked with 1× KRH buffer containing 1 mM pyrocatechol for at least 3 h). Filters were washed three times with 3 ml of ice-cold KRH buffer containing pyrocatechol using a Brandel cell harvester (model M-48; Brandel Inc., Gaithersburg, MD). Radioactivity was determined as described above.

To determine whether the H547A-induced increase in Vmax was due to a palmitoylation-mediated mechanism, kinetic analysis of [3H]DA uptake in WT hDAT and H547A was performed in the presence or absence of 15 µM 2-BP, a palmitoylation inhibitor. This concentration was chosen based on previous studies as well as pilot experiments conducted to determine the optimal concentration (data not shown). A 1.5 mM 2-BP stock was prepared in 100 % DMSO and was diluted to 150 µM/1 % DMSO final concentration. Intact PC12 cells in 24-well plates transiently expressing WT hDAT or H547A were washed twice with 1× KRH buffer. To determine the effects of 2-BP exposure at the zero time point (0 h), the cells were incubated for 8 min at room temperature with 15 µM 2-BP or DMSO control and six concentrations of mixed unlabeled and labeled [3H]DA as described above. Nonspecific uptake was determined in the presence of 10 µM nomifensine and desipramine. To dete×rmine specific uptake, nonspecific uptake was subtracted from total uptake. To determine the effects of 2-BP exposure at the 2 h time point, the cells were incubated similarly as above, but allowed to incubate at room temperature for 2 h. After each incubation, cells were washed twice with ice-cold 1× KRH buffer to terminate the r×eaction, lysed with 500 µl 1 % SDS, and allowed to shake at room temperature for an hour. Lysates were collected into scintillation vials and radioactivity was measured via liquid scintillation as above. Radioactivity was measured the next day using a liquid scintillation counter (model Tri-Carb 2900TR; PerkinElmer Life and Analytical Sciences, Waltham, MA).

[3H]WIN 35,428 Binding Assay

[3H]WIN35,428 binding assay was conducted as described previously (Quizon et al. 2016). Binding assays were conducted to determine whether mutated hDAT alters the kinetic parameters (Bmax or Kd) of [3H]WIN 35,428 binding in intact PC12 cells transfected with WT hDAT or mutants. Cells were washed with sucrose-phosphate buffer (final concentration in mM: 2.1 NaH2PO4, 7.3 Na2HPO47H2O, and 320 sucrose, pH 7.4) and then incubated with one of the six concentrations of [3H]WIN 35,428 (0.5–30 nM final concentrations) in a final volume of 500 µl on ice for 2 h. In parallel, nonspecific binding at each concentration of [3H]WIN 35,428 (in the presence of 30 µM cocaine, final concentration) was subtracted from total binding to calculate the specific binding. For the competitive inhibition experiment, assays were performed in duplicate in a final volume of 500 µl. Intact cells transfected with WT hDAT or its mutants were incubated in buffer containing 50 µl of [3H]WIN 35,428 (final concentration, 5 nM) and one of seven concentrations of unlabeled substrate DA (1 nM – 100 µM), cocaine (1 nM – 100 µM), GBR12909 (0.01 nM – 1 µM) or ZnCIR2R (1, 10, 100 µM) on ice for 2 h. Assays were terminated by removal of reaction reagents in well and then washed three times with ice-cold assay buffer. Cells were lysed with 1 % SDS for an hour. Radioactivity was determined as described above.

Cell Surface Biotinylation and Western Blots

To determine whether the mutations alter DAT cell surface expression, biotinylation assays were performed as described previously (Zhu et al. 2005). PC12 cells transiently expressing WT hDAT or mutants were grown to 90 % confluence in 6-well plates (at a density of 1 × 105 cells/cm2). Cells were incubated with 1 ml of 1.5 mg/ml sulfo-NHS-SS biotin (Pierce, Rockford, IL) in PBS/Ca/Mg buffer (in mM: 138 NaCl, 2.7 KCl, 1.5 KH2PO4, 9.6 Na2HPO4, 1 MgCl2, 0.1 CaCl2, pH 7.3). After incubation, cells were washed 3 times with 1 ml of ice-cold 100 mM glycine in PBS/Ca/Mg buffer and incubated for 30 min at 4 °C in 100 mM glycine in PBS/Ca/Mg buffer. Cells were then washed 3 times with 1 ml of ice-cold PBS/Ca/Mg buffer and then lysed with 500 ml of Lysis buffer (Triton X-100, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µM pepstatin, 250 µM phenylmethysulfonyl fluoride), followed by incubation and continuous shaking for 30 min at 4 °C. Cells were transferred to 1.5 ml tubes and centrifuged at 17,000 × g for 30 min at 4 °C. The resulting pellets were discarded, and 200 µl of the supernatants was stored at -20 °C as the total DAT fraction. The remaining supernatants were incubated with continuous shaking with ImmunoPure Immobilized Streptavidin beads in lysis buffer for 1 h at room temperature. Samples were centrifuged subsequently at 17,000 × g for 4 min at 4 °C, and supernatants (containing the non-biotinylated, intracellular protein fraction) were stored at -20 °C. Resulting pellets containing the avidin-absorbed biotinylated proteins (cell-surface fraction) were resuspended in 1 ml of 1.0 % Triton X-100 buffer and centrifuged at 17,000 × g for 4 min at 4 °C, and pellets were resuspended and centrifuged twice. Final pellets consisted of the biotinylated proteins adsorbed to monomeric avidin beads. Biotinylated proteins were eluted by incubating with 75 µl of Laemmli sample buffer for 20 min at room temperature and stored at -20 °C until western blot analysis was conducted.

To detect immunoreactive DAT protein in total, intracellular, and biotinylated fractions prepared from biotinylation assay above, samples were subjected to gel electrophoresis and Western blotting. Proteins were separated by 10 % SDS-polyacrylamide gel electrophoresis for 90 min at 150 V and transferred to Immobilon-P transfer membranes (0.45-µm pore size; Millipore, Billerica, MA) in transfer buffer (50 mM Tris, 250 mM glycine, and 3.5 mM SDS) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories) for 110 min at 72 V. Transfer membranes were incubated with blocking buffer (5 % dry milk powder in phosphate-buffered saline containing 0.5 % Tween 20) for 1 h at room temperature, followed by incubation with primary goat anti-DAT antibody (1:200 dilution in blocking buffer) overnight at 4 °C. On next day, the transfer membranes were washed five times with wash buffer (phosphate-buffered saline containing 0.5 % Tween 20) at room temperature and then incubated with secondary anti-goat IgG horseradish peroxidase (1:3000 dilution in blocking buffer) at room temperature for 1 h. Blots on transfer membranes were detected using enhanced chemiluminescence and developed on Hyperfilm ECL-Plus (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). After detection and quantification of DAT protein, each blot was stripped in 10 % ReBlot Plus Mild Antibody Stripping Solution (Millipore Bioscience Research Reagents, Temecula, CA) for 20 min at room temperature and reprobed for detection of β-tubulin. β-tubulin (1:2000 dilution in blocking buffer) was used as a control protein to monitor protein loading between samples and determined. Secondary anti-rabbit-IgG horseradish peroxidase (1:8000 dilution in blocking buffer) was used. Multiple autoradiographs were obtained using different exposure times, and immunoreactive bands within the linear range of detection were quantified by densitometric scanning (Scion Image software; Scion Corporation, Frederick, MD). Band density measurements, expressed as relative optical density, were used to determine levels of DAT immunoreactivity in synaptosomes.

Basal and MPP+ Efflux Assay

Basal DA and MPP+ efflux was performed at room temperature as described previously(Midde et al. 2015). Intact PC12 cells transfected with WT hDAT or its mutants were preloaded with 0.05 µM [3H]DA and 0.005 µM [3H]MPPP+P for 20 min and then washed 3 times with KRH buffer prior to collecting fractional efflux samples. To obtain an estimate of the total amount of [3H]DA or [3H]MPPP+P in the cells at the zero time point, cells from a set of wells (four wells/sample) were lysed rapidly in 1 % SDS after preloading with [3H]DA or [3H]MPP+. To collect fractional efflux samples, buffer (500 µl) was added into a separate set of cell wells and transferred to scintillation vials after 1 min as an initial fractional efflux, and another 500 µl buffer was added to the same wells and collected after 10 min as second fractional efflux. Additional fractional efflux at 20, 30, 40, 50 min, respectively, was repeated under the same procedure. After the last fractional efflux, cells were lysed and counted as total amount of [3H]DA or [3H]MPP+ remaining in the cells from each well.

MTSET Assay

This assay was performed based on the previous report with minor modifications (Loland et al. 2004, 2008). First, we generated five WT hDAT based plasmid construct for MTSET-insensitive or sensitive background as we described above. HEK293 cells were grown in poly-d-lysine coated plates at a density of 1 × 105 cells/cm2 were transfected with different E2C- associated hDAT background constructs: E2C hDAT, E2C-I159C hDAT, E2C/H547A hDAT, E2C-I159C/H547A or E2C-I159A/H547A. Twenty-four hours after the transfection, cells were washed twice with 1× KRH buffer, and preincubated with or without the presence of 10 µM nomifensine (for non-specific uptake) and MTSET for 10 min at room temperature. MTSET at 1 mM final concentration was chosen based on a pilot study on optimizing MTSET concentrations (0.1, 0.5 or 1.0 mM). Previous studies show 0.5 mM MTSET for 10 min exhibiting a 40 % inhibition on E2C-I159C (Loland et al. 2004, 2008). Because MTSET is easily hydrolyzed in solution, the amount needed for a 10 × stock was weighed out and dissolved in 1 × KRH buffer just right before adding to the assigned wells, for a 10-fold dilution (final concentration of 1 mM in final volume of 250 µl). After preincubation, [3H]DA (0.05 µM, final concentration) was added and cells were incubated for 8 min at room temperature. Cells were immediately washed twice with ice-cold 1 × KRH buffer to terminate the reaction and were lysed rapidly with the addition of 1 % SDS. Cell lysates were collected after incubation for 1 h at room temperature and radioactivity was determined the next day.

Data Analysis

Data are presented as means ± SEM, and n corresponds to the number of independent experiments performed for each group. GraphPad Prism version 5.0 was used to calculate kinetic parameters (Vmax, Km, Bmax, and Kd) from saturation curves using nonlinear regression via a Michaelis-Menten fit. IC50 values for substrate and inhibitors inhibiting [3H]DA uptake or [3H]WIN 35,428 binding were determined from a inhibitory curve using a one-site model with variable slope. For data involving comparisons between unpaired samples, the unpaired Student’s t test was used to assess differences in kinetic parameters (Vmax, Km, Bmax, Kd or IC50) between WT and mutant; log-transformed values of IC50, Km or Kd were used for the statistical comparisons. To determine significant differences between samples, data were analyzed with Student’s t tests or separate ANOVAs followed by post-hoc tests, as indicated in the results and figure legends of each experiment. The kinetic parameters (Vmax, Km, Bmax, Kd or IC50) were calculated using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). All statistical analyses were performed using IBM SPSS Statistics version 26, with a significance threshold of p < 0.05.