Mutations of Human DopamineTransporter at Tyrosine88, Aspartic Acid206, and Histidine547 Influence Basal and HIV-1 Tat-inhibited Dopamine Transport

Abstract

HIV-1 transactivator of transcription (Tat) has a great impact on the development of HIV-1 associated neurocognitive disorders through disrupting dopamine transmission. This study determined the mutational effects of human dopamine transporter (hDAT) on basal and Tat-induced inhibition of dopamine transport. Compared to wild-type hDAT, the maximal velocity (V_max) of [³H]dopamine uptake was decreased in D381L and Y88F/D206L/H547A, increased in D206L/H547A, and unaltered in D206L. Recombinant TatR_{1 - 86} inhibited dopamine uptake in wild-type hDAT, which was attenuated in either DAT mutants (D206L, D206L/H547A, and Y88F/D206L/H547A) or mutated TatR_{1 - 86} (K19A and C22G), demonstrating perturbed Tat-DAT interaction. Mutational effects of hDAT on the transporter conformation were evidenced by attenuation of zinc-induced increased [³H]WIN35,428 binding in D206L/H547A and Y88F/D206A/H547A and enhanced basal MPP⁺ efflux in D206L/H547A. H547A-induced outward-open transport conformational state was further validated by enhanced accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of an inserted cysteine (I159C) on a hDAT background.. Furthermore, H547A displayed an increase in palmitoylation inhibitor-induced inhibition of dopamine uptake relative to wide-type hDAT, indicating a change in basal palmitoylation in H547A. These results demonstrate that Y88F, D206L, and H547A attenuate Tat inhibition while preserving DA uptake, providing insights into identifying targets for improving DAT-mediated dopaminergic dysregulation. HIV-1 Tat inhibits dopamine uptake through human dopamine transporter (hDAT) on the presynaptic terminal through a direct allosteric interaction. Key hDAT residues D-H547, D-Y88, and D-D206 are predicted to be involved in the HIV-1 Tat-DAT binding. Mutating these residues attenuates this inhibitory effect by disrupting the Tat-hDAT interaction.

Keywords: Computational modeling; Dopamine transporter; HIV-1 Tat; Mutation; Uptake.

Fig. 1

Key residues D-H547, D-Y88, and D-D206 involved in the HIV-1 Tat-DAT binding. a The entire complex of wild-type DAT binding with Tat from the MD trajectory. Tat and DAT are represented as gold and cyan ribbons, respectively. The dashed box indicates the binding surface between Tat and DAT. b The detailed interaction of Tat with wild-type DAT. Residues T-K19, T-R49, and T-R57 of HIV-1 Tat are represented in ball-stick style and colored in yellow. Residues D-H547, D-Y88, D-D206,, and D-D381 are represented in ball-stick style and colored in green. Dashed lines represent intermolecular hydrogen bonds with labeled distances; the prefix T- and D- indicate residues of Tat and DAT, respectively. c Tat binding with the D206L/H547A mutant of DAT. The double mutations D-D206L/D-H547A on TAT-hDAT structure. D-H547A mutation eliminates one hydrogen bond with T-R49, and D-D206L mutation eliminates the hydrogen bond with T-R57. d Tat binding with the Y88F/D206L/H547A mutant of DAT. The triple mutations D-Y88F/D-D206L/D-H547A on TAT-hDAT structure. Compared to the above double mutations, the addition of Y88F mutation also eliminates the hydrogen bond with T-K19

Fig. 2

Disruption of the direct interaction between Tat and DAT by mutated Tat_1 − 86. a Mutation of wild-type recombinant Tat_1 − 86 (WT rTat_1 − 86) at Lys19 (K19A) attenuates WT rTat_1 − 86-induced inhibition of DA uptake. PC12 cells transiently transfected with WT hDAT were preincubated with or without 140 nM WT rTat_1 − 86, K19A or heated rTat_1 − 86 at room temperature for 20 min followed by addition of 5 nM [³H]DA. Specific [³H]DA uptake was determine in the presence of 10 µM nomifensine and desipramine. Heated rTat_1 − 86 was used as a negative control. b K19A mutant attenuates WT rTat_1 − 86-induced inhibition of DAT binding site. PC12 cells transiently transfected with WT hDAT were preincubated with 5 nM [³H]WIN35,428 on ice for 2 h in the presence or absence of 140 nM WT rTat_1 − 86, K19A or heated rTat_1 − 86. Specific [³H]WIN35,428 binding was determine in the presence of 30 µM cocaine. Heated rTat_1 − 86 was used as a negative control. Data are expressed as means from four independent experiments ± S.E.M. ** p < 0.01, compared to control (in the absence of Tat)

Fig. 3

Kinetic parameters of [³H]DA uptake and [³H]WIN35,428 binding in WT hDAT and mutants. The specific [³H]DA uptake was determined in intact PC12 cells expressing WT hDAT (WT), D206L, D381L (a) and D206L/H547A, Y88F/D206L/H547A (b) using six concentrations of DA (0.03-5.0 µM) mixed with a fixed concentration of [³H]DA (500,000 dpm/well, specific activity: 21.2 Ci/mmol). In parallel, nonspecific uptake of each concentration of [³H]DA in the presence of 10 µM nomifensine and desipramine were subtracted from total uptake for calculating the specific DAT-mediated uptake. The V_max and K_m values were calculated by fitting the data to the Michaelis-Menten equation and represent the means from five to ten independent experiments ± S.E.M. * p < 0.05 compared to the respective WT hDAT. Saturation binding of [³H]WIN35,428 was tested in intact PC12 cells transfected with WT hDAT, D206L, D381L (c) and WT, D206L/H547A, Y88F/D206L/H547A (d) that were incubated with six concentrations of [³H]WIN35,428 (0.5–30 nM) on ice for 2 h. In parallel, nonspecific binding of each concentration of [³H]WIN35,428 in the presence of 30 µM cocaine was subtracted from total binding for calculating the specific DAT binding sites

Fig. 4

DAT surface expression in WT hDAT and mutants. DAT surface expression of WT hDAT and mutants was determined by biotinylation assay followed by Western blotting. a top panels: representative immunoblots of total (Total Lysate) and cell surface (Biotinylated) fraction of DAT in WT hDAT, D206L, and D381L mutants. β-tubulin was used as control protein for sample loading. Bottom panels: the quantification of total and cell surface expression of DAT was expressed as means ± S.E.M of the ratio of total or biotinylated DAT immunoreactivity to β-tubulin immunoreactivity (n = 4–5). *p < 0.05, compared to WT hDAT. b top panels: representative immunoblots of total (Total Lysate) and cell surface (Biotinylated) fraction of DAT in WT hDAT, D206L/H547A, and Y88F/D206L/H547A mutants. Bottom panels: the quantification of total and cell surface expression of DAT was expressed as means ± S.E.M of the ratio of total or biotinylated DAT immunoreactivity to β-tubulin immunoreactivity (n = 4–5). All raw immunoblots are provided in supplementary material. *p < 0.05, **p < 0.01 compared to WT hDAT

Fig. 5

Mutational effect of hDAT on Tat-induced inhibition of [³H]DA uptake and [³H]WIN35,428 binding. a, b PC12 cells expressing WT hDAT (WT), D206L, D381L, D206L/H547A, or Y88F/D206L/H547A mutants were preincubated with or without recombinant Tat_1 − 86 (rTat_1 − 86, 140 nM) at room temperature for 20 min followed by the addition of 5 nM [³H]DA. In parallel, nonspecific uptake (in the presence of 10 µM nomifensine and desipramine) was subtracted from total uptake to calculate DAT-mediated uptake. c PC12 cells expressing WT, D206L/H547A or Y88F/D206L/H547A mutants were incubated with or without recombinant Tat_1 − 86 (rTat_1 − 86, 140 nM) and [³H]WIN35,428 on ice for 2 h. In parallel, nonspecific binding (in the presence of 30 µM cocaine) was subtracted from total binding to calculate specific binding. Data are presented as a percentage of untreated control per group, expressed as means ± S.E.M (n = 4). *p < 0.05, compared to the percentage of control (in the absence of Tat)

Fig. 6

Mutational effect of hDAT on zinc regulation of [³H]DA uptake and [³H]WIN35,428 binding. PC12 cells transiently expressing WT hDAT (WT) or mutants were incubated with buffer alone (control) or three concentrations of ZnCl₂ (1, 10, or 100 µM) followed by [³H]DA uptake or [³H]WIN 35,428 binding. a Mutants D381L and D206L do not affect zinc-mediated decrease in [³H]DA uptake. Two-way ANOVA analysis on the specific [³H]DA uptake in WT, D381L, and D206L revealed a significant main effect of mutation (F_(2,17) = 14.92, p < 0.001), zinc (F_(1,17) = 125.86, p < 0.05), and a significant mutation × zinc interaction (F_(2,17) = 15.97, p < 0.001). b Mutants D381L and D206L do not affect zinc-mediated decrease in increase in [³H]WIN 35,428 binding. Two-way ANOVA analysis revealed a significant main effect of mutation (F_{(2, 9)} = 48.9, p < 0.001), zinc (F_{(1, 9)} = 167.9, p < 0.05), and a significant mutation × zinc interaction (F_{(2, 9)} = 38.2, p < 0.001). c Both D206L/H547A and Y88F/D206L/H547A do not alter zinc-induced decrease in DA uptake. Two-way ANOVA analysis in WT, D206L, and D381L revealed a significant main effect of mutation (F_{(2, 11)} = 28.81, p < 0.001), zinc (F_{(1, 11)} = 140.30, p < 0.001), and a significant mutation × zinc interaction (F_{(2, 11)} = 23.47, p < 0.001). d The zinc-mediated increase in [³H]WIN binding in WT hDAT but not in D206L/H547A and Y88F/D206L/H547A. There were significant main effects of mutation (F_{(2, 12)} = 4.33, p < 0.05) and a significant mutation × zinc interaction (F_{(2, 12)} = 6.43, p < 0.05). The graphs illustrate specific [³H]DA uptake and [³H]WIN 35,428 binding expressed as mean ± S.E.M. of the respective controls set to 100 % for mutants. *p < 0.05, compared to the respective controls. ^# p < 0.05, compared to WT hDAT within same zinc concentration. (n = 4–7)

Fig. 7

Mutational effect of hDAT on functional efflux of MPP⁺. PC12 cells transfected with WT hDAT or mutants were preincubated with assay buffer containing 5 nM [³H]MPP⁺ at room temperature for 20 min. After incubation, cells were washed and incubated with fresh buffer at indicated time points. Subsequently, the buffer was removed, and radioactivity in the buffer and residual radioactivity in the cells was counted. Each fractional efflux of [³H]MPP⁺ in WT hDAT or mutants was expressed as a percentage of total [³H]MPP⁺ in the cells at the start of the experiment. Fractional [³H]MPP⁺ efflux levels at 1, 10, 20, 30, 40 and 50 min are expressed as a percentage of total [³H]MPP⁺ with preloading with 0.005 µM introduced to the cells at the start of the experiment. a Two-way ANOVA on functional MPP⁺ efflux of WT hDAT, D206L and D381L revealed a significant main effect of time (F_{(1, 9)} = 53.10, p < 0.001). b Two-way ANOVA analysis on the fractional efflux levels of [³H]MPP⁺ in WT hDAT, D206L/H547A and Y88F/D206L/H547A revealed a main effect of genotype (F_{(2, 21)} = 6.96, p < 0.01), time (F_{(1, 21)} = 36.26, p < 0.001) and significant interaction of genotype × time (F_{(2, 21)} = 8.33, p < 0.01) * p < 0.05, ** p < 0.01 compared to WT hDAT. (n = 4–8)

Fig. 8

H547A renders an inserted cysteine more reactive to MTSET inactivation and alters basal palmitoylation compared to WT hDAT. HEK293 cells transiently expressing WT hDAT (WT) or E2C hDAT constructs were treated with a range of concentrations (0.1, 0.5, 1.0 mM) of MTSET for 10 min followed by the addition of 5 nM [³H]DA for 8 min. a the concentration-dependent effects of MTSET on DA uptake. Two-way ANOVA analysis revealed no significant main effects of genotype and their interaction on MTSET-mediated DA uptake, a significant main effect of MTSET (F_{(1, 6)} = 35.55, p < 0.01) was found. (*p < 0.05, ***p < 0.001; n = 4). b Effects of 1 mM MTSET on DA uptake in E2C, E2C-I159C, E2C/H547A, E2C-I159C/H547A, and E2C-I159A/H547A. Two-way ANOVA analysis on MTSET-mediated [³H]DA uptake in positive control E2C-I159C/H547A and negative control E2C-I159A/H547A revealed a significant main effects of genotype (F_{(4, 40)} = 139.67, p < 0.001) and treatment (F_{(1, 40)} = 40.03, p < 0.001), and genotype × treatment interaction (F_{(4, 40)} = 2.62, p < 0.05). (* p < 0.05, ** p < 0.01 compared to the respective controls). ^# p < 0.001, compared to E2C; ^× p < 0.05 compared to E2C-I159C; ^×× p < 0.05 compared to E2C-I159C/H547A, (n = 5). Effects of 2-BP on V_max of [³H]DA uptake at zero (c) or two-hour (d) time point in WT hDAT and H547A in the presence or absence of 15 µM 2-BP. Two-way ANOVA on the V_max values with or without 2-BP revealed significant main effects of genotype (F_(1,10) = 10.3, p < 0.01) and treatment (F_(1,10) = 34.2, p < 0.001) as well as a significant interaction of genotype × treatment (F_(1,10) = 9.7, p < 0.05). * p < 0.05 compared to control; ^# p < 0.05 compared to WT hDAT within control group, (n = 3–4)