Molecular mechanism: the human dopamine transporter histidine 547 regulates basal and HIV-1 Tat protein-inhibited dopamine transport

Abnormal dopaminergic transmission has been implicated as a risk determinant of HIV-1-associated neurocognitive disorders. HIV-1 Tat protein increases synaptic dopamine (DA) levels by directly inhibiting DA transporter (DAT) activity, ultimately leading to dopaminergic neuron damage. Through integrated computational modeling prediction and experimental validation, we identified that histidine547 on human DAT (hDAT) is critical for regulation of basal DA uptake and Tat-induced inhibition of DA transport. Compared to wild type hDAT (WT hDAT), mutation of histidine547 (H547A) displayed a 196% increase in DA uptake. Other substitutions of histidine547 showed that DA uptake was not altered in H547R but decreased by 99% in H547P and 60% in H547D, respectively. These mutants did not alter DAT surface expression or surface DAT binding sites. H547 mutants attenuated Tat-induced inhibition of DA transport observed in WT hDAT. H547A displays a differential sensitivity to PMA- or BIM-induced activation or inhibition of DAT function relative to WT hDAT, indicating a change in basal PKC activity in H547A. These findings demonstrate that histidine547 on hDAT plays a crucial role in stabilizing basal DA transport and Tat-DAT interaction. This study provides mechanistic insights into identifying targets on DAT for Tat binding and improving DAT-mediated dysfunction of DA transmission.


Results
Computational modeling: His547 and functional relevant residues of human DAT. Based on the constructed hDAT-Tat binding model in our previous work 36 , both the side chain and backbone of H547 forms a hydrogen bond with residue R49 of HIV-Tat (Fig. 1B). The hydrogen bond with the H547 side chain is expected to be broken with the H547A mutation, which is consistent with decreased inhibitory activity of Tat on hDAT-H547A. Based on the computational model proposed in our previous work 36 , the Y548-Y470-Y551 interaction motif (denoted as the YYY motif) (Fig. 1C) could stabilize the first part of transmembrane helix 10 (TM10a) by tying down residue Y470 with extracellular loop 6. Instability of TM10a could promote the formation of a salt bridge between D476 (in TM10a) and R85 (in TM1b); however, the D476-R85 salt bridge would close the "entrance gate" of DA 35,36,[44][45][46][47] . As a result, instability of TM10a is expected to block the DA entrance in the outward-open state, which may decrease the DA uptake efficiency of hDAT. It could be observed that the three phenol rings in the YYY motif are packed in a specific face to face style, which indicates that the EL6 should also take a specific conformation for the forming of the YYY motif. Therefore, any conformation change on EL6 that occurs close to the YYY motif is expected to regulate the interaction strength of the YYY motif. As one of the nearest residue to the YYY motif on EL6, residue H547 is an appropriate candidate for validating our proposed hypothesis. The H547P, H547A, H547D, and H547R single mutations were tested because proline and alanine mutations are expected to be the most significant change in backbone conformation, while aspartic acid or arginine mutation is expected to introduce significant side-chain conformational disturbances due to their non-neutral electrostatic potentials. According to the molecular dynamics (MD) simulation and the potential of main force (PMF) energy calculation performed in our previous work 36 , we found that the H547A mutation could enhance the strength of the YYY motif. On the contrary, the H547P mutation could weaken the strength of the YYY motif, which is consistent with the increased DA uptake efficiency for the H547A mutant and decreased DA uptake efficiency for the H547P mutant. Furthermore, the relatively weaker influence of the H547R and H547D mutations on DA uptake efficiency also support that the backbone conformation of residue 547 plays a key role in the stability of the YYY motif.
To determine whether the H547A-induced increase in V max is associated with altered subcellular distribution of DAT, biotinylation and immunoblot assays were performed. Three subcellular fractions were prepared from PC12 cells transfected with WT hDAT and H547A-hDAT. DAT immunoreactivity in both total fraction and cell surface fraction (biotinylated) were examined (Fig. 2B). No differences between WT hDAT and H547A-hDAT were found in the ratio of surface DAT (biotinylated DAT) to total DAT (biotinylated/total: WT hDAT, 0.75 ± 0.10; and H547A, 0.97 ± 0.17, t (17) = 1.18, p = 0.25), indicating that increased V max in H547A-hDAT is not due to alteration of the available DAT on the cell surface. With regard to H547P, neither surface nor total DAT signal was detectable (data not shown). The WIN35,428 binding site shares pharmacological identity with the DA uptake carrier 48   hDAT and Tat are represented as cyan surface and golden ribbon, respectively. (B) A local view of DAT residue H547 and its direct interaction with Tat residue R49, hDAT is represented as a cyan ribbon. The residues are represented in sticks, with hydrogen bonds between the two residues represented with dashed lines labeled with their corresponding coordination distances. (C) Structural details of the residues Y470, Y551, H547, D476, and R85 on hDAT. hDAT is represented as a cyan ribbon, while the first part of transmembrane helix 10 (TM10a) and extracellular loop 6 (EL6) are colored in green and orange, respectively. Dopamine is represented as a ball-and-stick molecule in purple. Hydrogen bonds between D476 and R85 are indicated by dashed lines with coordinating distances labeled.      binding. In general, the conformational changes in DA transport processes involve conversions between outwardand inward-facing conformations 50 . Occupancy of the endogenous Zn 2+ binding site in WT hDAT stabilizes the transporter in an outward-facing conformation, which allows DA to bind but inhibits its translocation, thereby decreasing DA uptake 51 but increasing [ 3 H]WIN35,428 binding 52 . Addition of Zn 2+ is able to partially reverse an inward-facing state to an outward-facing state 51,52 . On the basis of this principle, the addition of Zn 2+ to WT hDAT would inhibit DA uptake, whereas in a functional mutation in DAT Zn 2+ might diminish the preference for the inward-facing conformation and thus enhance DA uptake. As shown in Fig. 5, two-way ANOVA on the specific [ 3 H]DA uptake in WT and H547A-hDAT and H547D-hDAT revealed a significant main effect of zinc [F (3,48)

Discussion
Our recent computational-experimental study demonstrated that an alanine mutation to hDAT His547 (H547A) plays a crucial role in the hDAT-Tat binding and DA uptake by hDAT 38 . The present study aims to pharmacologically characterize His547 and its functional influence in Tat-induced inhibition of DA uptake. There were two main findings. First, H547A enhances DA transport in a PKC-dependent manner, and other substitutions of His547 (H547P, H547R, H547D) differentially altered DA uptake. Second, Tat inhibited DA uptake in WT hDAT, which was attenuated in His547 mutants. In addition, H547A attenuated zinc modulation of and Y551H mutations are expected to partially reproduce the effect of Y470H, as they only impair one of the Y470-Y548 and Y470-Y551 interactions. The Y470H mutant was reported to retain only ~18% (17.8% 37 ,17.7% 38 in our previous reports) V max comparing to WT hDAT, whereas the present study shows that Y548H and Y551H mutants retain 24% and 74% V max compared to WT hDAT, respectively (Table 1). Interestingly, based on the YYY motif model, the combined effect of the Y548H and Y551H mutations may be expected to retain 18% (24% × 74%) V max compared to WT hDAT, which is consistent with the effect of the Y470H single mutation on V max . This observation confirms our computational prediction that YYY motif could stabilize the critical transmembrane 10 (TM10) by tying down Tyr470 with EL6 for enhanced transporter stability 38 . Disrupting this motif will result in perturbation to the transport function, as evidenced by the decrease in V max in the mutants Y548H, Y470H, and Y551H. According to our previous work [35][36][37] , the center residue of YYY motif, i.e., residue Y470, is critical for Tat-induced inhibition of DA uptake, which indicates that stability of YYY motif is also involved in hDAT/Tat binding. Thus, mutating tyrosine551 to histidine may not only change the transporter's capacity for uptake but also inflict a structural change of hDAT/Tat binding. Our results show that the Y551H mutation attenuates Tat-induced inhibition of DA uptake (Fig. 4), which is also consistent with our computational model.
To investigate the effect of the H547 mutation on influencing the YYY motif and hDAT transport, experiments were performed to obtain the kinetics data for four mutations, including H547A, H547P, H547R, and H547D. Alanine has the highest helix propensity; on the contrary, proline and glycine are generally known as "helix breakers" 53,54 . Therefore, the H547A and H547P mutations suggest two distinct directions of disturbing the backbone conformation. Additionally, mutation to other residues such as arginine or aspartic acid would introduce the most significant side-chain disturbance. Consequently, the H547R and H547D mutations would be reasonable probes for investigating the role of the side chain of residue 547. The present study shows that an alanine mutation on His547 increases the V max by ~3-fold compared to WT hDAT, indicating that H547A is a critical residue that mediates the enhancement of DA transport, which supports our computational prediction 38 . Furthermore, the H547A-induced enhancement of DA uptake was not observed in other substitutions of H547, in which V max was significantly decreased in H547P but not altered in H547R, whereas the effect of the H547D mutation on V max decrease is relatively weaker than that of the H547P mutation. The hDAT activity shows higher sensitivity to backbone disturbances induced by H547A or H547P rather than side chain disturbances induced by H547D or H547R. Furthermore, the distinct helix propensity of H547A and H547P corresponds to the up-regulation and down-regulation of hDAT activity. Thus, the backbone conformation of residue His547 is expected to play an important role in hDAT function, which also supports our proposed hypothesis based on computational prediction 38 . Notably, the baseline K m values for WT hDAT vary in the current study, which may be affected by the experimental conditions and individual experiments performed across different times. For example, the K m values are relatively higher in either WT or H547A in Fig. 6B than others; this is because the PMA-mediated DA uptake assay was conducted at 37 °C. Previous studies have reported that DAT expression in plasma membrane is increased in a temperature-dependent manner 55 , which may cause increased V max and decreased K m for DA uptake. However, the K m value of H547A hDAT was always compared to its respective WT hDAT control within each individual experiment. Importantly, our results demonstrate that H547A-hDAT displays increased K m value for DA uptake relative to WT hDAT, indicating that mutating the His547 residue may alter the binding site of substrate DA. Given that DA transport efficiency by DAT is largely governed by surface DAT expression 56 , our results show that mutations of these residues do not alter either cell surface DAT expression or [ 3 H]WIN35,428 binding sites in intact cells expressing these mutants, indicating that the altered V max in these mutants is not due to changing surface DAT expression. Interestingly, mutant H547P displays a dramatic decrease in DA uptake, which is accompanied by decreased total DAT expression (data not shown). Considering that site-directed mutagenesis studies on DAT typically result in a decrease in DA uptake 35,37,57,58 , the enhancement of DA transporter as evidenced by the H547A mutant suggests that targeting this residue may provide an exciting knowledge basis for the development of novel concepts for the therapeutic treatment of the HAND.
Consistent with our previous reports 35,37 , the current results show that mutations of His547 completely eliminate the inhibitory effect of Tat on DA transport, further suggesting that the Tat molecule is associated with DAT through intermolecular electrostatic attractions and complementary hydrophobic interactions. Our computational study predicts that the side chain of H547 forms a hydrogen bond with residue R49 of HIV-Tat and that the alanine mutation of H547 would be expected to change the local backbone conformation of hDAT-H547 and eliminate the hydrogen bond between hDAT-H547A/Tat-R49 (Fig. 1). These findings indicate the important role of His547 in Tat-DAT interaction. The allosteric modulation of DAT is responsible for conformational transitions via substrate-and ligand-binding sites on DAT 50,59 . Given that Tat protein regulates DAT function allosterically 34,49 , we tested whether mutated His547 attenuates the Tat effect through alteration to a conformational transporter transition. First, our results show that H547A and H547D attenuate zinc-mediated decrease in DA uptake and increase in WIN binding. Second, H547D but not H547A enhances basal DA efflux, which further supports our previous report showing Tat protein induced enhancement of DA efflux in WT hDAT 60 . These results are consistent with our previous reports 35,37 , suggesting that disrupting the intermolecular interaction of Tat and DAT influences Tat-induced inhibition of DA uptake. Further understanding the functional relevance of additional residues in Tat on DAT modulation will provide useful feedback for further refining the computationally predicted binding model of DAT with Tat.
An important finding from the current study is that promoting PKC phosphorylation of DAT with PMA resulted in 40% and 60% reduction of DA uptake in WT hDAT and H547A, respectively. Similarly, preventing PKC phosphorylation of DAT with BIM produces a 98% and 42% increase in DA uptake in WT hDAT and H547A, respectively. This suggests a differential sensitivity to PMA-or BIM-induced activation or inhibition of DAT function between WT and H547A. It has been well characterized that PKC-dependent phosphorylation of DAT regulates DA uptake velocity 41,61,62 . One possibility is that mutation of His547 alters basal levels of PKC-mediated phosphorylation of DAT, thereby resulting in the enhanced DA uptake. Recent studies demonstrate that the serine-7 DAT residue is critical for PKC-dependent DAT phosphorylation 63 , and the alanine mutation of serine-7 results in an increase in DA uptake relative to WT DAT 64 . As the PKC phosphorylation sites on cytoplasmic domain (intracellular side) of hDAT are structurally far away from the residue H547 on the extracellular side of hDAT 37,38 , the H547A mutation is likely to regulate the PKC-mediated phosphorylation by allosteric effect. Therefore, future studies will be necessary to investigate the double mutant serine-7/His547 as an approach to identify whether the site of reduced PKC phosphorylation is on H547A-hDAT.
The current finding showing an unusual hDAT mutant capable of both enhancing DA transport and preventing Tat inhibitory effect on DAT is of general interest in therapeutic treatment of drug addiction. The interplay of Tat and cocaine augments synaptic DA levels and Tat release by inhibiting DAT activity 33,34 , which may contribute to the progression of HAND underlying the cognitive deficits in HIV-1 positive cocaine-using individuals 5,6 . Similarly, conditioned expression of Tat in the mouse brain further potentiates cocaine rewarding in vivo 65 . Together, these results suggest a synergistic effect of cocaine and Tat on DA transmission contributing to cognitive dysfunction and elevating the cocaine addictive effects. The current findings might provide novel insight into developing small molecule compounds that could bind to the unique residue on hDAT (His547), thereby not only preventing Tat interaction with DAT but also enhancing DA transport function. Ideally, the effectiveness of early intervention for HAND may combine such compound(s) with anti-retroviral therapy, which would be beneficial to the preservation of neurocognitive function in HIV-infected individuals.
Scientific RepoRts | 6:39048 | DOI: 10.1038/srep39048 Methods Predicting the site for hDAT binding with Tat. The binding structure of hDAT with HIV-1 clade B type Tat was modeled and simulated based on the nuclear magnetic resonance (NMR) structures of Tat 66 and the constructed structure of hDAT-DA complex. The protein-protein docking program ZDOCK 67 was used to determine the initial binding structure of the hDAT-Tat complex. A total of 220,000 potential conformations were generated based on 11 NMR structures of Tat, then all of these conformations were evaluated and ranked by ZRANK 68 . Top-3,000 conformations selected from the protein-protein docking process were submitted to energy minimizations, and the docked structures were ranked according to the binding affinity estimated by using the Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) method 69 . Then the top-256 conformations were selected for further evaluation by performing molecular dynamics (MD) simulations. Based on the MD simulations, the most favorable hDAT-Tat binding mode (with the best geometric matching quality and reasonable interaction between hDAT and Tat) was identified, and the final hDAT-Tat binding structure was energy-minimized for analysis.

Construction of plasmids.
All point mutations of His547, Tyrosine548 (Tyr548), and Tyrosine551 (Tyr551) in hDAT were selected based on the predictions of the three-dimensional computational modeling and simulations. Based on the favorable hDAT-Tat binding mode (Fig. 1), it could be expected that mutations of His547 would eliminate a hydrogen bond between D-H547 and T-R49, which impair the binding affinity of Tat with hDAT, thereby diminishing Tat-induced inhibition of DA uptake. Mutations in hDAT at His547 [Histidine to Alanine (H547A), Proline (H547P), Arginine (H547R), or Aspartic acid (H547D)], Tyr548 (tyrosine to histidine, Y548H), and Tyr551 (tyrosine to histidine, Y551H) were generated based on wild type hDAT (WT hDAT) sequence (NCBI, cDNA clone MGC: 164608 IMAGE: 40146999) by site-directed mutagenesis. 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 QuikChange TM site-directed mutagenesis Kit (Agilent Tech, Santa Clara CA). The sequence of the mutant construct was confirmed by DNA sequencing at University of South Carolina EnGenCore facility. Plasmid DNA were propagated and purified using a plasmid isolation kit (Qiagen, Valencia, CA, USA).
Cell culture and DNA transfection. PC12 cells (ATCC ® CRL-1721 TM , American Type Culture Collection, Manassas, VA) were maintained at 37 °C in a 5% CO 2 incubator in Dulbecco's modified eagle medium (DMEM, Life Technologies, Carlsbad, CA) supplemented with 15% horse serum, 2.5% bovine calf serum, 2 mM glutamine, and antibiotics (100 U/ml penicillin and 100 μ g/mL streptomycin). Twenty four hours prior to transfection, cells were seeded into 24 well plates at a density of 1 × 10 5 cells/cm 2 , or allowed to reach 100% confluence on plates. Cells were transfected with plasmids of WT hDAT or its mutants using Lipofectamine 2000 (Life Technologies, Carlsbad, CA). Twenty four hours after transfection, intact cells or cell suspensions were used for experiments. To determine whether H547A-induced increase in V max was mediated by a phosphorylation-dependent mechanism, kinetic analysis of [ 3 H]DA uptake was measured in the presence or absence of PKC activator PMA (Tocris, Bristol, UK) or PKC inhibitor BIM (Sigma-Aldrich, St. Louis, MO). The concentrations were chosen based on previous reports 70,71 . In brief, intact PC12 cells transfected with WT or H547A-hDAT were preincubated with or without PMA or BIM (1 μ M, final concentration) for 30 min at 37 °C or room temperature and then incubated with six concentrations of mixed [ 3 H]DA for 8 min as described above. Nonspecific uptake of each concentration of [ 3 H]DA (in the presence of 10 μ M nomifensine) was subtracted from total uptake to calculate specific DAT-mediated uptake.
The competitive inhibition of DA uptake by DAT substrate and inhibitors was examined in intact PC12 cells transfected with WT hDAT or its mutants. Cells were preincubated in 400 μ l KRH buffer containing 50 μ l of one of a series of final concentrations of DA (1 nM-1 mM), GBR12909 (1 nM-10 μ M), cocaine (1 nM-1 mM), or ZnCl 2 (1, 10, 100 μ M) for 10 min at room temperature and then incubated for 8 min after the addition of 50 μ l of [ 3 H]DA (0.05 μ M, final concentration). The reaction for DA uptake in intact cells was terminated by removing reaction reagents and washing the cells twice with ice cold 1x KRH buffer. Cells were lysed in 500 μ l of 1% SDS for an hour and radioactivity was measured using a liquid scintillation counter (model Tri-Carb 2900TR; PerkinElmer Life and Analytical Sciences, Waltham, MA). V max and K m were determined using Prism 5.0 (GraphPad Software Inc., San Diego, CA).
To determine the inhibitory effects of Tat on [ 3 H]DA uptake, cells were dissociated with trypsin/EDTA (0.25%/0.1%, 1 mL for one 10 cm dish) and resuspended in culture medium at room temperature. After a 10-min incubation, the dissociated cells were harvested by centrifugation at 400 × g for 5 min at 4 °C and washed once with phosphate-buffered saline followed by another centrifugation at 400 × g for 5 min at 4 °C. The resulted cell pellets were resuspended in 1x KRH buffer. Specific [ 3 H]DA uptake was determined in the cell suspensions prepared from WT hDAT and its mutants in the presence or absence of recombinant Tat    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.
To determine whether DAT mutations alter DAT surface expression, biotinylation assays were performed as described previously 72 . PC12 cells transiently expressing hDAT or mutants were plated on 6-well plates at a density of 10 5 cells/well. 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 KH 2 PO 4 , 9.6 Na 2 HPO 4 , 1 MgCl 2 , 0.1 CaCl 2 , 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 by addition of 500 ml of Lysis buffer (Triton X-100, 1 μ g/ml aprotinin, 1 μ g/ml leupeptin, 1 μ M pepstatin, 250 μ M phenylmethysulfonyl fluoride), followed by incubation and continuous shaking for 20 min at 4 °C. Cells were transferred to 1.5 ml tubes and centrifuged at 20,000 × g for 20 min. The resulting pellets were discarded, and 100 μ l of the supernatants was stored at − 20 °C for determination of immunoreactive total DAT. Remaining supernatants were incubated with continuous shaking in the presence of monomeric avidin beads in Triton X-100 buffer (100 μ l/tube) 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 μ l of Laemmli sample buffer for 20 min at room temperature and stored at − 20 °C if further assays were not immediately conducted.
Basal efflux assay. Basal DA efflux was performed at room temperature as described previously 35,37 . Intact PC12 cells transfected with WT hDAT or its mutants were preloaded with 0.05 μ M [ 3 H]DA 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 [ 3 H]DA 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 [ 3 H]DA. To collect factional efflux samples, buffer (500 μ l) 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 μ l 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 last fractional efflux, cells were lysed and counted as total amount of [ 3 H]DA remaining in the cells from each well.

Data analysis.
Results are presented as mean ± SEM, and n represents the number of independent experiments for each experiment group. Kinetic parameters (V max, K m , B max , and K d ) were determined from saturation curves by nonlinear regression analysis using a one-site model with variable slope. IC 50 values for substrate and inhibitors inhibiting [ 3 H]DA uptake or [ 3 H]WIN 35,428 were determined from inhibition curves by nonlinear regression analysis using a one-site model with variable slope. For experiments involving comparisons between unpaired samples, unpaired Student's t test was used to assess any difference in the kinetic parameters (V max , K m , B max , K d or IC 50 ) between WT and mutant; log-transformed values of IC 50 , K m or K d were used for the statistical comparisons. Significant differences between samples were analyzed with separate ANOVAs followed by post-hoc tests, as indicated in the results Section of each experiment. All statistical analyses were performed using IBM SPSS Statistics version 20, and differences were considered significant at p < 0.05.