A network of phosphatidylinositol (4,5)-bisphosphate (PIP2) binding sites on the dopamine transporter regulates amphetamine behavior in Drosophila Melanogaster

Abstract

Reward modulates the saliency of a specific drug exposure and is essential for the transition to addiction. Numerous human PET-fMRI studies establish a link between midbrain dopamine (DA) release, DA transporter (DAT) availability, and reward responses. However, how and whether DAT function and regulation directly participate in reward processes remains elusive. Here, we developed a novel experimental paradigm in Drosophila melanogaster to study the mechanisms underlying the psychomotor and rewarding properties of amphetamine (AMPH). AMPH principally mediates its pharmacological and behavioral effects by increasing DA availability through the reversal of DAT function (DA efflux). We have previously shown that the phospholipid, phosphatidylinositol (4, 5)-bisphosphate (PIP₂), directly interacts with the DAT N-terminus to support DA efflux in response to AMPH. In this study, we demonstrate that the interaction of PIP₂ with the DAT N-terminus is critical for AMPH-induced DAT phosphorylation, a process required for DA efflux. We showed that PIP₂ also interacts with intracellular loop 4 at R443. Further, we identified that R443 electrostatically regulates DA efflux as part of a coordinated interaction with the phosphorylated N-terminus. In Drosophila, we determined that a neutralizing substitution at R443 inhibited the psychomotor actions of AMPH. We associated this inhibition with a decrease in AMPH-induced DA efflux in isolated fly brains. Notably, we showed that the electrostatic interactions of R443 specifically regulate the rewarding properties of AMPH without affecting AMPH aversion. We present the first evidence linking PIP₂, DAT, DA efflux, and phosphorylation processes with AMPH reward.

Fig. 1. PIP₂/N-terminus interactions regulate DAT phosphorylation, a posttranslational modification that supports PIP₂ independent DA efflux.

a hDAT WT, hDAT K/N, or hDAT K/A cells were labeled with ³²P prior to vehicle or AMPH (10 μM, 30 min) incubation. Equal amounts of DAT determined by immunoblotting were immunoprecipitated and subjected to SDS-PAGE/auto-radiography. Left: representative autoradiographs showing hDAT phosphorylation after vehicle or AMPH exposure, along with respective immunoblots of total hDAT. Right: DAT phosphorylation is quantified as ³²P labeling normalized to basal hDAT ³²P labeling. hDAT WT phosphorylation increased post-AMPH treatment relative to vehicle (n = 4; F_{(2, 18)} = 5.05, p = 0.002). In hDAT K/N and hDAT K/A cells, displayed no changes in DAT phosphorylation in response to AMPH compared with vehicle (p > .05). b Left: representative traces of amperometric currents recorded from hDAT WT (black traces) and hDAT S/D (green traces) preincubated in either vehicle or PAO (20 μM, 10 min) and subsequently treated with AMPH (10 μM; indicated by arrow). Right: quantitation of mean peak current amplitudes in hDAT WT and hDAT S/D cells. Preincubation with PAO significantly decreased DA efflux in hDAT WT cells (0.33 ± 0.04 pA) with respect to vehicle (0.16 ± 0.02 pA; p = 0.03; n = 4), but not in hDAT S/D cells (vehicle: 0.38 ± 0.06 pA, PAO: 0.35 ± 0.05 pA; p > 0.05; n = 4). c Representative traces from hDAT S/D cells after whole-cell patch delivery of control peptide (3 μM, pal-HAQKHFEAAA) or PIP₂ sequestering peptide (3 μM, pal-HRQKHFEKRR) to the cytoplasm of the cell prior (10 min) to the application of AMPH (10 μM, indicated by arrow). Delivery of control or PIP₂ sequestering peptides resulted in comparable peak currents in response to AMPH (n = 4; p > 0.05). Data are presented as mean ± SEM. Two-way ANOVA with Bonferroni’s multiple comparison test: a, b; Student’s t test: c.

Fig. 2. R443A substitution reduced PIP₂ binding and inhibited AMPH-induced DA efflux in vitro.

a hDAT WT or hDAT R443A (containing a His-eGFP tag on the N-terminus) were purified from cellular extracts. Solubilized hDAT WT or hDAT R443A proteins were incubated with a water-soluble analog of PIP₂ conjugated to an orange fluorophore (BODIPY^® TMR-PIP₂). Specific binding was quantified as a ratio of PIP₂ fluorescence to eGFP. Minimal PIP₂ binding was measured in the presence of His-eGFP only (0.16 ± 0.05 AU) relative to hDAT WT (1.38 ± 0.12 AU) or hDAT R443A (0.90 ± 0.24 AU) (F_(2,9) = 55.11, p < 0.0001; n = 4). hDAT R443A displayed a 34.8 ± 9.9% reduction in PIP₂ binding compared with hDAT WT (p = 0.007). b Top: average ³[H]DA saturation curves of DA uptake measured in hDAT WT (closed squares) or hDAT R443A (open squares) cells (n = 4, in triplicate). Curves were fit to Michaelis–Menten kinetics to derive K_m and V_max. DA uptake for hDAT R443A was comparable to hDAT WT at every DA concentration measured (F_(1,120) = 1.40, p > 0.05), as were the kinetic constants, K_m and V_max (p > 0.05). c Left: representative traces of amperometric currents (DA efflux) recorded from hDAT WT (top) and hDAT R443A (bottom) cells, in response to AMPH application (10 μM, indicated by arrow). Right: quantitation of peak current amplitudes. hDAT R443A display a 50.6 ± 15.1% decrease in AMPH-induced DA efflux relative to hDAT WT (p = 0.003; n = 14). d Left: representative immunoblots of surface hDAT (top), total (glycosylated and nonglycosylated) hDAT (middle), and actin as loading control (bottom). Right: hDAT expression is quantified as a ratio of surface to total glycosylated hDAT normalized to hDAT WT. hDAT R443A and hDAT WT had comparable expression (p > 0.05; n = 6, in triplicate). Data are presented as mean ± SEM. One-way ANOVA with Bonferroni’s multiple comparisons test: a; two-way RM ANOVA with Bonferroni’s multiple comparison test: b; Student’s t test: b, d; Wilcoxon matched-pairs signed rank test: c.

Fig. 3. Disrupting R443 electrostatic interactions inhibits DA efflux independent of DAT N-terminus phosphorylation.

a Left: representative immunoblots of surface hDAT (top), total hDAT (middle), and actin (bottom). Right: hDAT expression is quantified as a ratio of surface to total glycosylated hDAT normalized to hDAT S/D. hDAT S/D R443A had comparable expression to hDAT S/D (p > 0.05; n = 4 in triplicate). Dashed lines indicate separate sets of experiments. b Top: average ³[H]DA uptake kinetics measured in hDAT S/D R443A (gray line open squares) and hDAT S/D (green line closed triangle) cells (n = 4, in triplicate). Curves were fit to Michaelis–Menten equation to derive K_m and V_max. Bottom: the V_max and K_m for hDAT S/D R443A were comparable to hDAT S/D cells (p > 0.05). c Left: representative traces of DA efflux recorded from hDAT S/D (green trace) and hDAT S/D R443A cells (gray trace) in response to AMPH application (10 μM, indicated by arrow). Right: quantitation of peak current amplitudes (n = 9–10). DA efflux from hDAT S/D R443A (0.15 ± 0.02 pA; p = 0.01) was significantly lower compared with hDAT S/D cells (0.42 ± 0.07 pA; p = 0.0004). Data are presented as mean ± SEM. Student’s t test: a–c; two-way RM ANOVA with Bonferroni’s multiple comparison test: b.

Fig. 4. hDAT R443A limits central and behavioral responses to AMPH.

a hDAT WT or hDAT R443A was expressed in DA neurons in a dDAT KO (fmn) background. ³[H]DA uptake (200 nM, 10 min) was measured in adult isolated Drosophila brain (n = 5). DA uptake measured in hDAT WT flies (86.9 ± 13.7 fmol/brain) was comparable to that measured in hDAT R443A flies (76.9 ± 9.9 fmol/brain; p > 0.05). b Left: representative traces of amperometric currents recorded from a dense cluster of DA neurons (PPL1, boxed in inset) in response to AMPH application (20 μM; indicated by arrow) in hDAT WT (black trace) and hDAT R443A (red trace) brains. Right: quantitation of peak current amplitudes. hDAT WT flies displayed higher peak currents (0.40 ± 0.11 pA) than hDAT R443A flies (0.08 ± 0.02 pA; n = 6; p = 0.004). c Left: locomotor activity was assayed over a 24-h period including both the light (horizonal white bar) and dark (horizontal black bar) cycle. Circadian activity curves show average beam crosses (20-min interval) during the 24-h period for hDAT WT (black line) and hDAT R443A (red line) Drosophila. Right: cumulative beam crosses were not significantly different for hDAT WT (654 ± 61) versus hDAT R443A Drosophila (879 ± 110; p > 0.05; n = 16). d Locomotor activity was measured after a 30-min exposure to vehicle or 1 mM AMPH. The total beam crosses increase in the AMPH (12.8 ± 1.9; n = 39) compared with vehicle group (5.4 ± 0.9; n = 38) of hDAT WT Drosophila (F_(1,138) = 11.83, p = 0.0005). In hDAT R443A Drosophila, AMPH (10.3 ± 1.2; n = 36; p > 0.05) did not increase the total beam crosses compared with vehicle group (8.5 ± 0.5; n = 29) and with respect to hDAT WT vehicle group (p > 0.05). Data are presented as mean ± SEM. Student’s t test: a, c; Mann–Whitney test: b; two-way ANOVA with Bonferroni’s multiple comparison test: d.

Fig. 5. In a two-choice consumption paradigm, hDAT R443A flies display diminished AMPH preference and continued AMPH aversion.

a Schematic illustrating a two-choice consumption paradigm developed to measure AMPH preference in flies. The paradigm is comprised of three 24-h testing periods: acclimation, baseline, and preference, where capillaries were replaced every 24-h period to measure AMPH consumption. Adult Drosophila were placed in custom chambers containing two volumetric capillaries filled with either clear (100 mM sucrose) or blue food (100 mM sucrose, 500 μM blue). During AMPH preference testing, blue food was supplemented with either AMPH (1 mM or 10 mM) for experimental groups (solid line) or vehicle for control groups (dashed line). Preference is presented as a ratio of blue food to total food consumption. b Left: hDAT WT Drosophila exposed to 1 mM AMPH (solid black line) consumed more AMPH (61.5 ± 4.0%) relative to baseline vehicle (46.3 ± 2.8%; n = 12–13, F_{(1, 49)} = 5.23, p = 0.005). hDAT WT control groups consumed equal amounts of blue food during day 1 (45.8 ± 3.1%) and day 2 (46.0 ± 3.0%; n = 14; p > 0.05). Right: hDAT R443A Drosophila (solid red line) did not consume more AMPH (57.4 ± 2.4%) compared with baseline vehicle (51.1 ± 2.8%; n = 13, F_{(1, 50)} = 0.86, p > 0.05). hDAT R443A control groups consumed comparable amounts of blue food during day 1 (46.7 ± 2.6%) and day 2 (48.3 ± 2.2%; n = 14, p > 0.05). c Left: hDAT WT Drosophila exposed to 10 mM AMPH (solid black line) consumed drastically less AMPH (33.5 ± 5.9%) compared with baseline vehicle (54.9 ± 2.5%; n = 12; F_(1,44) = 9.55, p = 0.0004). Control groups for hDAT WT (black dashed line) consumed comparable food on day 1 and day 2 (n = 12; p > 0.05). Right: hDAT R443A Drosophila consumed significantly less 10 mM AMPH (35.0 ± 5.6%) compared with baseline vehicle (52.9 ± 5.9%; n = 11; F_(1,39) = 5.18, p = 0.03). Control groups for hDAT R443A (red dashed line) consumed comparable food on day 1 and day 2 (n = 10–11; p > 0.05). Each data point represents the mean of 10–14 measurements ± SEM. Two-way ANOVA with Bonferroni’s multiple comparison test: b, c.