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, 8 (4), 975-87

Mutation of Tyrosine 470 of Human Dopamine Transporter Is Critical for HIV-1 Tat-induced Inhibition of Dopamine Transport and Transporter Conformational Transitions

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Mutation of Tyrosine 470 of Human Dopamine Transporter Is Critical for HIV-1 Tat-induced Inhibition of Dopamine Transport and Transporter Conformational Transitions

Narasimha M Midde et al. J Neuroimmune Pharmacol.

Abstract

HIV-1 Tat protein plays a crucial role in perturbations of the dopamine (DA) system. Our previous studies have demonstrated that Tat decreases DA uptake, and allosterically modulates DA transporter (DAT) function. In the present study, we have found that Tat interacts directly with DAT, leading to inhibition of DAT function. Through computational modeling and simulations, a potential recognition binding site of human DAT (hDAT) for Tat was predicted. Mutation of tyrosine470 (Y470H) attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of this residue for Tat binding to hDAT. Y470H reduced the maximal velocity of [³H]DA uptake without changes in the K(m) and IC₅₀ values for DA inhibition of DA uptake but increased DA uptake potency for cocaine and GBR12909, suggesting that this residue does not overlap with the binding sites in hDAT for substrate but is critical for these inhibitors. Furthermore, Y470H also led to transporter conformational transitions by affecting zinc modulation of DA uptake and WIN35,428 binding as well as enhancing basal DA efflux. Collectively, these findings demonstrate Tyr470 as a functional recognition residue in hDAT for Tat-induced inhibition of DA transport and transporter conformational transitions. The consequence of mutation at this residue is to block the functional binding of Tat to hDAT without affecting physiological DA transport.

Figures

Figure 1
Figure 1
A direct interaction between Tat and DAT and the energy-minimized hDAT(DA) binding complex following the MD simulation. Co-IP of DAT and Tat was performed by immunoprecipitation (IP) with anti-DAT antibody as bait and immunoblot (IB) with anti-Tat antibody. (A) Co-IP of DAT and Tat. Rat synaptosomes from spleen, cerebellum, striatum were preincubated with (+, lanes 1, 2 and 4, from left) or without (−, lane 3) 350 nM recombinant Tat1-86 (rTat1-86). Top panel: DAT immunoreactivity was detected in striatum but not in spleen and cerebellum. Bottom panel: rTat1-86 bound to agarose beads was able to immunoprecipitate DAT in rat striatum but not in spleen and cerebellum. rTat1-86 (10 ng) was loaded in lane 5 as the positive control for Tat immunoreactivity. (B) GST-Tat1-86 bound to WT hDAT protein. Top panel: The GST-Tat1-86 fusion proteins were bound to glutathione-sepharose beads, and then incubated with cell lysates from CHO cells transfected with WT hDAT at room temperature for 1 h following Western Blot using anti-DAT. GST-Tat fusion protein bound to glutathione-sepharose was able to pull down DAT, but GST alone was not. Bottom panel: DAT immunoreactivity in CHO cells expressing hDAT was shown in all lanes. (C) Side view of the complex structure. Tat is shown as the ribbon in cyan color and hDAT(DA) as the ribbon in gold color. Atoms of residue C22 (Cys22) of Tat are shown as overlapped balls in cyan color. Atoms of substrate dopamine (DA) and the Cl ion are shown as overlapped balls in green color. 2 Na+ ions are shown as balls in blue color. The vestibule (colored in purple) is represented as the molecular surface calculated by using the program HOLLOW (Ho and Gruswitz, 2008). (D) Local view of the anchoring residues Lys 19 (K19) and Cys22 (C22) of Tat inside the vestibule of hDAT(DA). Residues K19 and C22 of Tat are shown in ball-and-stick style, and colored by the atom types. Residue Tyr470 (Y470) of hDAT(DA) is also shown in ball-and-stick style and colored by the atom types. The hydrogen bonding between the K19 side chain of Tat and the hydroxyl oxygen atom on Y470 side chain of hDAT(DA) is indicated with the dashed line. Non-popar hydrogen atoms are not shown for clarity. The positions of transmembrane domain11 and 12 (TM11 and TM12) of hDAT(DA) are also labeled.
Figure 2
Figure 2
Inhibition of DA uptake by released Tat from Tat-expressing cells. (A) CHO cells transfected with WT hDAT were preincubated in KRH buffer including 100 μl conditioned media collected at 72 h from cells transfected with plasmid Tat1-72, Tat1-86, Tat1−101 DNAs and vector alone (Control) followed by addition of [3H]DA uptake. * p < 0.05 different from control (Dunnett's Multiple comparison test). (B) Specificity of released Tat in inhibition of [3H]DA uptake. Conditioned media collected at 72 h from cells transfected with Tat1-72 were preincubated with anti-Tat antibody or isotype control IgG at 4°C for 3 h, followed by incubation with protein A/G – Agarose beads 4°C for 2 h. Media collected at same time from cells transfected with vector alone was used as control. Cells transfected with WT hDAT were preincubated in KRH buffer containing supernatants from the agarose-antibody-medium-beads complex, followed by [3H]DA uptake. Released Tat1-72 caused significant decrease in [3H]DA uptake, which was attenuated by immunodepletion with anti-Tat antibody but not isotype control antibody (one-way ANOVA followed by Tukey's multiple comparison test). * p < 0.05 different from control. # p < 0.05 different from Tat1-72 and Tat1-72 + Con IgG. (n = 4).
Figure 3
Figure 3
[3H]DA uptake and DAT surface expression in WT hDAT and mutant. (A) Kinetic analysis of [3H]DA uptake in WT hDAT and Y470H-hDAT. CHO cells transfected with WT hDAT or Y470H-hDAT were incubated with one of six mixed concentrations of the [3H]DA as total rate of DA uptake. In parallel, nonspecific uptake of each concentration of [3H]DA (in the presence of 10 μM nomifensine, final concentration) was subtracted from total uptake to calculate DAT-mediated uptake. *p < 0.05 compared to control value (unpaired Student's t test) (n = 5). (B) Cell surface of WT hDAT (WT) or Y470H-hDAT (Y470H) was analyzed by biotinylation. Top panel: representative immunoblots in CHO cells expressing WT hDAT or Y470H-hDAT. Bottom panel: DAT immunoreactivity is expressed as mean ± S.E.M. densitometry units from three independent experiments (n = 3). *p < 0.05 compared to WT hDAT (unpaired Student's t test).
Figure 4
Figure 4
Effects of Tat on kinetic analysis of [3H]DA uptake in WT hDAT and mutant. (A) CHO cells transfected with WT or Y470H-hDAT were preincubated with or without released Tat1-72 (1.0 ng/ml) at room temperature for 20 min followed by the addition of one of six mixed concentrations of the [3H]DA. In parallel, nonspecific uptake at each concentration of [3H]DA (in the presence of 10 μM nomifensine, final concentration) was subtracted from total uptake to calculate DAT-mediated uptake. (B) [3H]DA uptake in cells transfected with WT or Y470H-hDAT was determined in the presence or absence of recombinant Tat1-86 (rTat1-86, 350 nM, final concentration). (C) [3H]DA uptake in cells transfected with WT (0.8 μg plasmid cDNA) or Y470H-hDAT (2.4 μg plasmid cDNA) was determined in the presence or absence of rTat1-86 (350 nM). Data are expressed as means from five independent experiments ± S.E.M. *p < 0.05 compared with the respective control values. # p < 0.05 compared to WT hDAT. (n = 5)
Figure 5
Figure 5
Mutation of Tyr470 alters transporter conformational transitions. Tyr470 mutation of DAT affects zinc regulation of DA uptake (A) and [3H]WIN 35,428 binding (B). CHO cells transfected with WT or Y470H-hDAT were incubated with KRH buffer alone (control) or with ZnCI2 (10 μM, final concentration) followed by [3H]DA uptake or [3H]WIN 35,428 binding (n = 4). The histogram shows [3H]DA uptake and [3H]WIN 35,428 binding expressed as mean ± S.E.M. of the respective controls set to 100% for the mutant. *p < 0.05 compared to control. #p < 0.05 compared to WT hDAT with ZnCI2. (C) Functional DA efflux properties of WT hDAT and mutant. CHO cells transfected WT or Y470H-hDAT were preincubated with [3H]DA (0.05 μM, final concentration) at room temperature for 20 min. After incubation, cells were washed and incubated with fresh buffer at indicated time points. Subsequently, the buffer was separated from cells, and radioactivity in the buffer and remaining in the cells was counted. Each fractional [3H]DA efflux in WT hDAT and Y470H-hDAT was expressed as percentage of total [3H] in the cells at the start of the experiment. Fractional [3H]DA efflux at 1, 10, 20, 30 and 40 min are expressed as the percentage of total [3H]DA with preloading with 0.05 μM (WT hDAT: 15379 ± 1800 dpm and Y470H-hDAT: 2488 ± 150 dpm) present in the cells at the start of the experiment (n = 4). ×p < 0.05 and ××p < 0.01, compared to WT hDAT (Bonferroni t-test). (D) Functional DA efflux properties of WT hDAT in the presence or absence of Tat1-72. CHO cells transfected with WT hDAT were preincubated with released Tat1-72 (1 ng/mg) followed by DA efflux assay. Fractional [3H]DA efflux at 1, 10, 20, 30, 40 and 50 min are expressed as the percentage of total [3H]DA with preloading with 0.05 μM (control: 14200 ± 1448 dpm and released Tat: 10102 ± 1505 dpm) present in the cells at the start of the experiment (n = 6). ^p < 0.05 and ^^p < 0.01, compared to WT hDAT in the absence of Tat (Bonferroni t-test).

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