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, 10 (1), 122-35

Mutations at Tyrosine 88, Lysine 92 and Tyrosine 470 of Human Dopamine Transporter Result in an Attenuation of HIV-1 Tat-induced Inhibition of Dopamine Transport

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Mutations at Tyrosine 88, Lysine 92 and Tyrosine 470 of Human Dopamine Transporter Result in an Attenuation of HIV-1 Tat-induced Inhibition of Dopamine Transport

Narasimha M Midde et al. J Neuroimmune Pharmacol.

Abstract

HIV-1 transactivator of transcription (Tat) protein disrupts the dopamine (DA) neurotransmission by inhibiting DA transporter (DAT) function, leading to increased neurocognitive impairment in HIV-1 infected individuals. Through integrated computational modeling and pharmacological studies, we have demonstrated that mutation of tyrosine470 (Y470H) of human DAT (hDAT) attenuates Tat-induced inhibition of DA uptake by changing the transporter conformational transitions. The present study examined the functional influences of other substitutions at tyrosine470 (Y470F and Y470A) and tyrosine88 (Y88F) and lysine92 (K92M), two other relevant residues for Tat binding to hDAT, in Tat-induced inhibitory effects on DA transport. Y88F, K92M and Y470A attenuated Tat-induced inhibition of DA transport, implicating the functional relevance of these residues for Tat binding to hDAT. Compared to wild type hDAT, Y470A and K92M but not Y88F reduced the maximal velocity of [(3)H]DA uptake without changes in the Km. Y88F and K92M enhanced IC50 values for DA inhibition of [(3)H]DA uptake and [(3)H]WIN35,428 binding but decreased IC50 for cocaine and GBR12909 inhibition of [(3)H]DA uptake, suggesting that these residues are critical for substrate and these inhibitors. Y470F, Y470A, Y88F and K92M attenuated zinc-induced increase of [(3)H]WIN35,428 binding. Moreover, only Y470A and K92M enhanced DA efflux relative to wild type hDAT, suggesting mutations of these residues differentially modulate transporter conformational transitions. These results demonstrate Tyr88 and Lys92 along with Tyr470 as functional recognition residues in hDAT for Tat-induced inhibition of DA transport and provide mechanistic insights into identifying target residues on the DAT for Tat binding.

Conflict of interest statement

Conflicts of Interest

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Modeled hDAT-Tat binding structure. (A) HIV-1 Tat protein is shown as ribbon and colored in gold, and hDAT is represented as semi-transparent surface and colored in cyan. Dopamine (DA) is shown as blue spheres. (B) Atomic interactions between HIV-1 Tat and hDAT as observed from the binding structure. Key residues for the intermolecular interactions between HIV-1 Tat and hDAT are shown in stick style. Residues T-M1, T-P18, and T-K19 of HIV-1 Tat are colored in purple, whereas residues D-Y470, D-K92, and D-Y88 are colored in green. Two dashed lines on the left side represent intermolecular hydrogen bonds with key distances labeled. The orange ball indicates the center of aromatic ring, and the dashed line pointing to the orange ball represents the cation-π interaction with the key distance labeled.
Figure 2
Figure 2
Structural details of residues Y470, K92, and Y88 in the MD-simulated hDAT-Tat binding structure. Protein hDAT is represented as cyan ribbon, and its residues Y470, K92, Y88, and D313 are shown in stick colored in green. The hydrogen bond between K92 and D313 is represented as a dashed line with the key distance given; the hydrogen bond favorably connects M1b with TM6a in hDAT.
Figure 3
Figure 3
[3H]DA uptake and DAT surface expression in WT hDAT and mutants. (A) Kinetic analysis of [3H]DA uptake in WT hDAT, Y470F-hDAT and Y470A-hDAT. PC12 cells transfected with WT hDAT, Y470F-hDAT or Y470A-hDAT were incubated with one of 6 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 expression of WT hDAT, Y470F-hDAT and Y470A-hDAT was analyzed by biotinylation assay. Top panel: representative immunoblots in CHO cells expressing WT hDAT (WT), Y470F-hDAT (Y470F) or Y470A-hDAT (Y470A). Bottom panel: DAT immunoreactivity is expressed as mean ± S.E.M. densitometry units from three independent experiments (n = 3).
Figure 4
Figure 4
[3H]DA uptake and DAT surface expression in WT hDAT and mutants. (A) Kinetic analysis of [3H]DA uptake in WT hDAT, Y88F-hDAT and K92M-hDAT. CHO cells transfected with WT hDAT, Y88F-hDAT or K92M-hDAT were incubated with one of 6 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 expression of WT hDAT, Y88F-hDAT and K92M-hDAT was analyzed by biotinylation assay. Top panel: representative immunoblots in CHO cells expressing WT hDAT (WT), Y88F-hDAT (Y88F) or K92M-hDAT (K92M). 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 5
Figure 5
Effects of Tat or Tat Cys22 on kinetic analysis of [3H]DA uptake in WT hDAT and Tyr470 mutants. (A) PC12 cells transfected with WT hDAT (WT), Y470F-hDAT (Y470F), Y470H-hDAT (Y470H) or Y470A-hDAT (Y470A) were preincubated with or without recombinant Tat1–86 (rTat1–86) or Tat Cys22 (500 nM, final concentration) at room temperature for 20 min followed by the addition of 0.05 µM final concentration of the [3H]DA. Nonspecific uptake was determined in the presence of 10 µM final concentration of nomifensine. (B) [3H]DA uptake in cells transfected with WT hDAT (WT), Y88F-hDAT (Y88F) and K92M-hDAT (K92M) was determined in the presence or absence of Tat Cys22 or rTat1–86 (500 nM, final concentration). Data are expressed as means from seven independent experiments ± S.E.M. * p < 0.05 compared with the respective control values. # p < 0.05 compared to WT hDAT.
Figure 6
Figure 6
Effects of Y470F, Y470A and Y470H mutants on transporter conformational transitions. Mutations of Tyr470 affect zinc regulation of [3H]DA uptake (A) and [3H]WIN 35,428 binding (B). CHO cells transfected with WT hDAT (WT), Y470F-hDAT (Y470F) and Y470A-hDAT (Y470A) were incubated with KRH buffer alone (control) or ZnCl2 (10 µM, final concentration) followed by [3H]DA uptake or [3H]WIN 35,428 binding (n = 6). 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 ZnCl2. (C) Functional DA efflux of DA properties of WT hDAT and mutants. CHO cells transfected with WT hDAT or mutants were preincubated with KRH buffer containing [3H]DA (0.05 µM, final concentration) at room temperature for 20 min. After incubation, cells were washed and incubated with fresh buffer as indicated time points. Subsequently, the buffer was removed from cells, and radioactivity in the buffer and remaining in the cells was counted. Each fractional efflux of [3H]DA in WT hDAT (WT), Y470F-hDAT (Y470F), Y470A-hDAT (Y470A) or Y470H-hDAT was expressed as percentage of total [3H]DA in the cells at the start of the experiment. Fractional [3H]DA efflux levels at 1, 10, 20, 30, 40 and 50 min are expressed as the percentage of total [3H]DA with preloading with 0.05 µM (WT hDAT: 26837 ± 5089 dpm, Y470F-hDAT: 20908 ± 4209 dpm, Y470A-hDAT: 1158 ± 123 dpm and Y470H-hDAT: 2488 ± 150 dpm) presented in the cells at the start of the experiment (n = 4). ××p < 0.05 compared to WT hDAT (Bonferroni t-test). (D) Functional MPP+ efflux properties of WT hDAT and mutants. CHO cells transfected with WT hDAT or mutants were preincubated with KRH buffer containing [3H]MPP+ (5 nM, final concentration) at room temperature for 30 min. Fractional [3H]MPP+ efflux levels at each time point are expressed as the percentage of total [3H]MPP+ with preloading with 0.05 µM (WT hDAT: 12120 ± 397 dpm, Y470F-hDAT: 7399 ± 359 dpm, Y470A-hDAT: 460 ± 46 dpm and Y470H-hDAT: 602 ± 28 dpm) presented in the cells at the start of the experiment (n = 4). ××p < 0.05 compared to WT hDAT (Bonferroni t-test).
Figure 7
Figure 7
Effects of Y88F and K92M mutants on transporter conformational transitions. Mutations of Tyr88 and Lys92 affect zinc regulation of [3H]DA uptake (A) and [3H]WIN 35,428 binding (B). CHO cells transfected with WT hDAT (WT), Y88F-hDAT (Y88F) and K92M-hDAT (K92M) were incubated with KRH buffer alone (control) or ZnCl2 (10 µM, final concentration) followed by [3H]DA uptake or [3H]WIN 35,428 binding (n = 6). 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 ZnCl2. (C) Functional DA efflux of DA properties of WT hDAT and mutants. CHO cells transfected with WT hDAT or mutants were preincubated with KRH buffer containing [3H]DA (0.05 µM, final concentration) at room temperature for 20 min. After incubation, cells were washed and incubated with fresh buffer as indicated time points. Subsequently, the buffer was separated from cells, and radioactivity in the buffer and remaining in the cells was counted. Each fractional efflux of [3H]DA in WT hDAT (WT), Y88F-hDAT and K92M-hDAT was expressed as percentage of total [3H]DA in the cells at the start of the experiment. Fractional [3H]DA efflux levels at 1, 10, 20, 30, 40 and 50 min are expressed as the percentage of total [3H]DA with preloading with 0.05 µM (WT hDAT: 70082 ± 8256 dpm, Y88F-hDAT: 41805 ± 6887 dpm and K92M-hDAT: 9655 ± 2160 dpm) presented in the cells at the start of the experiment (n = 3). ××p < 0.05 compared to WT hDAT (Bonferroni t-test). (D) Functional MPP+ efflux properties of WT hDAT and mutants. CHO cells transfected with WT hDAT or mutants were preincubated with KRH buffer containing [3H]MPP+ (5 nM, final concentration) at room temperature for 30 min. Fractional [3H]MPP+ efflux levels at each time point are expressed as the percentage of total [3H]MPP+ with preloading with 0.05 µM (WT hDAT: 15516 ± 920 dpm, Y88F-hDAT: 5695 ± 450 dpm and K92M-hDAT: 967 ± 121 dpm) presented in the cells at the start of the experiment (n = 4).

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