A) Top, representative oxidative currents, filtered at 1 Hz, in hDAT A559V cells following pretreatment with vehicle (left), or 1µM raclopride (right). Arrows indicate application of cocaine. Bottom, data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percent of current recorded in hDAT cells, for both vehicle control conditions (n=7) and with raclopride (n=6) (t=5.6, df=11, p=0.0002, Student’s t-test). B) Top, qPCR demonstrating the presence of endogenous D₂R in HEK 293T cells. Traces from real-time PCR (top, left) and representative PCR gel (top, right) demonstrating that when RNA isolated from HEK 293T cells is reverse transcribed (+RT), PCR product crosses threshold near cycle 30 (arrow) (cT=31.2 ± 0.1, n=8), whereas if no reverse transcription is performed (−RT), no product reaches threshold (arrow). Middle, representative immunoblot for D₂R (top) and actin (bottom) in HEK 293T cells transfected with the indicated cDNA, confirming D₂R expression. Mouse striatum, heavily enriched with endogenous D₂R, was used as a positive control (n=3). Bottom, representative confocal images of single sections demonstrating that endogenous D₂R and transfected hDAT are expressed in HEK 293T cells (upper row) and that cotransfection of hDAT and GFP leads to their coexpression (lower row). C) Top, representative oxidative currents recorded from hDAT A559V cells pretreated for 4 hours with vehicle control (left) or with 200ng/mL pertussis toxin (PTX) (right). Bottom, data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percent of current in hDAT cells, in both vehicle control conditions (n=3) and with PTX (n=5) (t=5.2, df=6, p=0.0019, Student’s t-test). D) Top, representative oxidative currents in hDAT S/D cells (five most distal hDAT N-terminal serines (Ser2, Ser4, Ser7, Ser12, and Ser13) substituted by aspartates to mimic phosphorylated state) following pretreatment with vehicle (left) or 1µM raclopride (right). Bottom, data reported as mean amperometric current ± SEM in hDAT S/D cells, expressed as a percent of current in hDAT cells in both control conditions (hDAT, n=6; hDAT S/D, n=6) and with raclopride (hDAT, n=6; hDAT S/D n=4) (t=0.8, df=8, p=0.44, Student’s t-test).

A) Representative immunoblot for autophosphorylated CaMKII, total CaMKII, and total DAT in hDAT and hDAT A559V cells. B) hDAT (n=5) and hDAT A559V (n=5) cell extracts were immunoblotted for CaMKII autophosphorylated at Thr286 to measure CaMKII activity under basal conditions. The immunoprecipitated band densities were quantified, normalized to total CaMKII, and expressed as a percent of autophosphorylated CaMKII in hDAT cells (t=2.1, df=8, p=0.036, Student’s t-test comparing hDAT A559V to hDAT). C) Representative immunoblot for autophosphorylated CaMKII and total CaMKII following 20 minute treatment with 1 µM of the D₂R antagonist raclopride or vehicle control. D) hDAT A559V (n=5) cell extracts were immunoblotted for autophosphorylated CaMKII with and without raclopride treatment. The immunoprecipitated band densities were quantified and normalized to the corresponding density of total CaMKII. Data reported as a percent of vehicle control (t=2.3, df=8, p=0.047, Student’s t-test). E) Top, representative oxidative currents in hDAT A559V cells following 20 minute pretreatment with vehicle (left), or 5 µM of the cell permeable CaMKII peptide inhibitor, antCaMKIINTide (right). Arrows indicate addition of cocaine. Bottom, data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percent of current recorded in hDAT cells for both control conditions (n=6) and with antCaMKIINTide (n=8) (t=6.8, df=12, p<0.0001, Student’s t-test). Control peptide, ant-Tirap-R, has no effect on basal DA efflux in either hDAT A559V (n=4) or hDAT cells (n=4) (data not shown). F) Top, representative oxidative currents in hDAT S/D cells following pretreatment with vehicle (left), or 5 µM antCaMKIINTide (right). Bottom, data reported as mean amperometric current ± SEM in hDAT S/D cells expressed as a percent of current in hDAT cells in both control conditions (n=6) and with antCaMKIINTide (n=5) (t=0.7, df=9, p=0.48, Student’s t-test).

A) Representative oxidative currents recorded from hDAT, hDAT A559V, and hDAT A559V S/A cells (five most distal N-terminal serines (Ser2, Ser4, Ser7, Ser12, and Ser13) substituted by alanine in A559V background to prevent phosphorylation). Arrows indicate addition of cocaine. B) Data reported as mean amperometric current ± SEM from hDAT (n=5), hDAT A559V (n=6), and hDAT A559V S/A (n=7) cells (F_{(2, 15)}=18.2, P=0.0005, **p<0.01, one-way ANOVA followed by Bonferroni’s multiple-comparison test). C) Representative immunoblot in hDAT and hDAT A559V cells for phosphorylated Serines 7, 12, and 13 (pSer7, pSer12, and pSer13 respectively) and for total DAT. Inset, immunoblot for D₂R (top) and CaMKII (bottom) confirming their presence in Flp-In CHO cells;lanes are shown in duplicate (n=6). D) Quantification of pSer DAT intensities normalized to total DAT. Data are reported as percent of phosphorylation of pSer7, pSer12, and pSer13 in hDAT A559V with respect to hDAT (n=5–7) (F_(3,22)=4.1, P=0.018, *p<0.05 **p<0.01, one-way ANOVA followed by Dunnett’s multiple-comparison test).

A) Representative oxidative currents in hDAT A559V cells following whole cell perfusion with vehicle (left), or a peptide corresponding to the first 27 residues of hDAT with the N-terminal serines substituted for aspartates (hDAT S/D peptide, right). Arrows indicate addition of cocaine. Cells were whole cell patch clamped and perfused for 10 minutes to allow for loading of 2 mM DA and either 3 µM hDAT S/D peptide or vehicle control, and measured for ADE as described in Methods. B) Data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percent of current recorded in hDAT cells in both control conditions (n=3) and with hDAT S/D peptide (n=5) (t=5.9, df=6, p=0.001, Student’s t-test). C) Representative oxidative currents in hDAT A559V cells following whole cell perfusion with vehicle (left), or a peptide corresponding to the first 27 residues of hDAT with the N-terminal serines substituted for alanines (hDAT S/A peptide, right). Cells were whole cell patch clamped and perfused as described above. D) Data reported as mean amperometric current ± SEM in hDAT A559V cells expressed as a percent of current recorded in hDAT cells in both control conditions (n=3) and with hDAT S/A peptide (n=3) (t=0, df=4, p=1.0, Student’s t-test). E) Representative oxidative currents in hDAT S/D cells following whole cell perfusion with vehicle (left), or hDAT S/D peptide (right). Cells were whole cell patch clamped and perfused as described above. F) Data reported as mean amperometric current ± SEM in hDAT S/D cells expressed as a percent of current recorded in hDAT cells in both control conditions (n=3) and with hDAT S/D peptide (n=3) (t=3.5, df=4, p=0.02, Student’s t-test).

A) Representative traces of DAT-mediated channel-like activity in hDAT A559V, hDAT, hDAT A559V S/A and hDAT S/D cells. hDAT A559V cells were also treated with raclopride (1 µM) and antCaMKIINTide (5 µM) as described above. Traces shown for both control condition (CTR), and after bath application of 10 µM cocaine (COC) to confirm DAT-specificity. Isolated membrane patches were excised using the outside-out patch clamp technique and held at −20mV. B) Data was quantified using amplitude histogram analysis of 30 sec of data to calculate the open probability (NPo) of the DAT channel-like mode. For both CTR and COC conditions, data is reported as mean NPo ± SEM calculated forhDAT A559V cells (filled bars; n=4), hDAT cells (open bars; n=5), hDAT A559V S/A cells (sparse diagonal striped bars; n=6), hDAT A559V cells pretreated for 20 minutes with 1 µM raclopride (dense diagonal striped bars; n=4), hDAT A559V cells pretreated for 20 minutes with 5 µM antCaMKIINTide (sparse horizontal striped bars; n=4), and hDAT S/D cells (dense horizontal striped bars; n=5) (F_{(5, 20)}=5.9, p=0.0016, **p<0.01, one-way ANOVA followed by Dunnett’s test compared to hDAT A559V).

A) Inset: mouse midbrain neuron expressing both red fluorescence (selection for DA neuron), and green fluorescence (successful transfection with DAT). Confocal image represents the merging (yellow) of both green and red fluorescence, indicating areas where both red and green fluorescence are dually expressed. Top, representative oxidative currents in unloaded hDAT A559V transfected neurons following 20 minute pretreatment with vehicle (left), or 1 µM raclopride (right). Arrows indicate addition of cocaine. Bottom, data reported as mean amperometric current ± SEM in hDAT A559V neurons expressed as a percent of current recorded in hDAT neurons for both control conditions (n=4) and with raclopride (n=4) (t=2.8, df=6, p=0.0327, Student’s t-test). B) Top, representative oxidative currents in unloaded hDAT S/D transfected neurons following 20 minute pretreatment with vehicle (left), or 1 µM raclopride (right). Bottom, data reported as mean amperometric current ± SEM in hDAT S/D neurons expressed as a percent of current recorded in hDAT neurons for both control conditions (hDAT, n=4; hDAT S/D, n=3) and with raclopride (hDAT, n=4; hDAT S/D, n=3) (t=1, df=4, p=0.37, Student’s t-test). C) Top, representative oxidative currents in unloaded hDAT A559V transfected neurons following 20 minute pretreatment with vehicle (left), or 5 µM antCaMKIINTide (right). Bottom, data reported as mean amperometric current ± SEM in hDAT A559V neurons expressed as a percent of current recorded in hDAT neurons for both control conditions (n=4) and with antCaMKIINTide (hDAT, n=3; hDAT A559V, n=4) (t=2.9, df=6, p=0.0248, Student’s t-test).