Tamoxifen Directly Interacts with the Dopamine Transporter

Abstract

The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and acts as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [³H]WIN 35,428 [(-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane] binding in rat striatal tissue. Tamoxifen dose-dependently blocked dopamine uptake (54% reduction at 10 μM) and amphetamine-stimulated efflux (59% reduction at 10 μM) in synaptosomes. It also produced a small but significant reduction in [³H]WIN 35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicated that tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggested a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies revealed that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its central nervous system permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.

Fig. 1.

Tamoxifen impairs dopamine uptake. (A and B) Synaptosomes from the striatum of male Sprague-Dawley rats were incubated for 1 hour at 37°C with vehicle or indicated concentrations of tamoxifen, then treated with 310 nM (A) or indicated concentrations of [³H]DA and incubated for an additional 3 minutes (A) or 30 seconds (B). (A) Data are represented as the mean ± S.E.M. (n = 5). *P < 0.05; **P < 0.01 compared with vehicle (post hoc Dunnett multiple-comparison tests). (B) Data are represented as the mean ± S.E.M. (n = 5). **P < 0.01; ***P < 0.001 (post hoc Sidak multiple-comparisons test). DA, dopamine; TMX, tamoxifen.

Fig. 2.

Tamoxifen attenuates amphetamine-stimulated dopamine efflux. Striatal synaptosomes were perfused for 1 hour at 37°C with vehicle or various concentrations of tamoxifen as described in the Materials and Methods. Amphetamine (10 μM) was included in the perfusate during fractions 7 and 8. Data were calculated as the area under the curve after treatment with amphetamine. Data are represented as the mean ± S.E.M. (n = 3–5). *P < 0.05; ***P < 0.001 compared with vehicle control (post hoc Dunnett multiple-comparisons test). Baseline release of dopamine in picomoles of dopamine/total dopamine was as follows: vehicle, 6.8 ± 2.0; 1 µM tamoxifen, 7.1 ± 1.8; 3 µM tamoxifen, 8.9 ± 1.2; and 10 µM tamoxifen, 4.9 ± 0.3. AMPH, amphetamine; DA, dopamine.

Fig. 3.

Tamoxifen does not affect surface expression of the DAT. Rat striatal synaptosomes were incubated for 1 hour with 10 μM tamoxifen or vehicle prior to biotinylation of surface proteins with sulfo-NHS-biotin. After avidin-biotin pulldown, DAT content in biotinylated fractions and lysates was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. (B) Representative Western blots showing the biotinylated fraction blotted for DAT protein and Na⁺/K⁺-ATPase, and the corresponding total lysate. Calculations of the ratio (± S.E.M.) of the optical densities of biotinylated transporter–Na⁺/K⁺-ATPase were 1.09 ± 0.2 for vehicle and 0.96 ± 0.1 for tamoxifen (data not shown) (n = 3). TMX, tamoxifen; Veh, vehicle.

Fig. 4.

Tamoxifen inhibits [³H]WIN 35,428 binding to the DAT in rat striatal membranes. Rat striatal membranes were incubated with [³H]WIN 35,428 with or without tamoxifen or vehicle for 3 hours at 4°C. Nonspecific binding was determined with 30 μM nomifensine. (A) Membranes were incubated with 4 nM [³H]WIN 35,428 without and with various concentrations of tamoxifen (n = 8). **P < 0.01; ***P < 0.001 (post hoc Dunnett multiple-comparisons test). (B) Membranes were incubated with 10 μM tamoxifen and various concentrations of [³H]WIN 35,428 to equilibrium. Data are represented as the mean ± S.E.M. (n = 6). **P < 0.01; ****P < 0.001 (post hoc Sidak multiple-comparisons test). TMX, tamoxifen.

Fig. 5.

Tamoxifen stabilizes the outward-facing conformation of the DAT in a cocaine-like manner. Rat striatal synaptosomes were incubated for 1 hour with 100 μM cocaine, 10 μM tamoxifen, or vehicle prior to biotinylation of surface cysteines with maleimide-PEG₂-biotin. DAT content in biotinylated fractions was quantified by Western blotting. (A) Biotinylated transporter/total transporter in lysate. *P < 0.05 compared with vehicle control (post hoc Dunnett multiple-comparisons test). (B) Representative Western blot showing the biotinylated DAT protein, its corresponding total lysate, and the content of GAPDH in the lysate (n = 3). Coc, cocaine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; OD, optical density; TMX, tamoxifen; Veh, vehicle.

Fig. 6.

Tamoxifen does not affect normal locomotor activity in rats. Locomotor activity of male Sprague-Dawley rats was monitored for 2 hours before and 2 hours after intraperitoneal injection. On days 1 and 2, animals received either 5 mg/kg tamoxifen citrate or an equivalent volume of vehicle. On day 3, all animals received saline. Data are represented as the mean ± S.E.M. (n = 8). Hab, XXX; Sal, saline; TMX, tamoxifen; Veh, vehicle.

Fig. 7.

Tamoxifen pretreatment attenuates amphetamine-stimulated hyperactivity. Male Sprague-Dawley rats were pretreated with 5 mg/kg tamoxifen citrate or vehicle 48 and 24 hours prior to administration of amphetamine (1 mg/kg). Saline was administered at 40 minutes and amphetamine (1 mg/kg i.p.) was administered at time point 100. Data are represented as the mean ± S.E.M. (n = 8). *P < 0.05; **P < 0.01; ***P < 0.001 (post hoc Sidak multiple-comparisons test). AMPH, amphetamine.

Fig. 8.

Tamoxifen readily docks in the S2 pocket of hDAT modeled after LeuT. The in silico hDAT model based on LeuT was constructed as described in the Materials and Methods. (A) Final energy-minimized pose of DAT/tamoxifen complex after flexible docking of tamoxifen at the DAT secondary (S2) substrate binding site. Selected binding pocket residues are labeled and rendered as sticks; bound tamoxifen (also shown in sticks) is highlighted in yellow. Cotransported sodium and chloride atoms are labeled and rendered as orange and green spheres, respectively. (B) Two-dimensional interaction diagram of tamoxifen bound at the S2 site of DAT. The interaction map depicts respective DAT residues located within 4.5 Å of the bound tamoxifen molecule (hydrophobic residues are colored green and polar residues are purple). The most significant non–van der Waals DAT/ligand interactions are indicated with dotted lines and a symbol depicting the chemistry of the interaction formed: side-chain hydrogen bond between D476 and amine nitrogen of tamoxifen (green), and an aromatic H/π-bond interaction between one of the tamoxifen rings and W84 (green, hexagon H).