SLC6A3 coding variant Ala559Val found in two autism probands alters dopamine transporter function and trafficking

Abstract

Emerging evidence associates dysfunction in the dopamine (DA) transporter (DAT) with the pathophysiology of autism spectrum disorder (ASD). The human DAT (hDAT; SLC6A3) rare variant with an Ala to Val substitution at amino acid 559 (hDAT A559V) was previously reported in individuals with bipolar disorder or attention-deficit hyperactivity disorder (ADHD). We have demonstrated that this variant is hyper-phosphorylated at the amino (N)-terminal serine (Ser) residues and promotes an anomalous DA efflux phenotype. Here, we report the novel identification of hDAT A559V in two unrelated ASD subjects and provide the first mechanistic description of its impaired trafficking phenotype. DAT surface expression is dynamically regulated by DAT substrates including the psychostimulant amphetamine (AMPH), which causes hDAT trafficking away from the plasma membrane. The integrity of DAT trafficking directly impacts DA transport capacity and therefore dopaminergic neurotransmission. Here, we show that hDAT A559V is resistant to AMPH-induced cell surface redistribution. This unique trafficking phenotype is conferred by altered protein kinase C β (PKCβ) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCβ activity, inhibition of which restores the AMPH-induced hDAT A559V membrane redistribution. Mechanistically, we link the inability of hDAT A559V to traffic in response to AMPH to the phosphorylation of the five most distal DAT N-terminal Ser. Mutation of these N-terminal Ser to Ala restores AMPH-induced trafficking. Furthermore, hDAT A559V has a diminished ability to transport AMPH, and therefore lacks AMPH-induced DA efflux. Pharmacological inhibition of PKCβ or Ser to Ala substitution in the hDAT A559V background restores AMPH-induced DA efflux while promoting intracellular AMPH accumulation. Although hDAT A559V is a rare variant, it has been found in multiple probands with neuropsychiatric disorders associated with imbalances in DA neurotransmission, including ADHD, bipolar disorder, and now ASD. These findings provide valuable insight into a new cellular phenotype (altered hDAT trafficking) supporting dysregulated DA function in these disorders. They also provide a novel potential target (PKCβ) for therapeutic interventions in individuals with ASD.

Figure 1

Validation of the hDAT A559V variant in two unrelated ASD probands. (a and b) Sanger sequence validation is shown for the two proband cases of the A559V genotype in the study. Chromatogram data is shown with the corresponding coding sequence flanking amino acid residue 559. Variant-nucleotide sequence is indicated by an asterisk representing heterozygous C/T at this position. Filled symbols indicate individuals with an ASD diagnosis, whereas open symbols reflect individuals without an ASD diagnosis. The circles represent females and the squares males. Individual IDs in the respective pedigrees correspond to NIMH cell line IDs. ASD, autism spectrum disorder.

Figure 2

hDAT A559V does not transport AMPH. (a) Uptake was performed with 10 nM AMPH for 5 min in cells transfected with either empty vector (pcDNA3), hDAT or hDAT A559V. hDAT cells exhibited robust AMPH uptake blocked by pretreatment with 10 μM cocaine (*P<0.05, one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test, n=3). hDAT A559V cells exhibited uptake not significantly different from the empty vector control (P>0.05, one-way ANOVA followed by Dunnett's multiple comparison test, n=3). inset: representative immunoblot for DAT demonstrates comparable levels of transporter expression between hDAT and hDAT A559V cells. (b) hDAT or hDAT A559V cells were treated with AMPH (10 μM) for the indicated time points. Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT') and total cellular lysate (‘total DAT') following AMPH (10 μM) treatment. Bottom, quantification of biotinylated DAT protein fractions following AMPH treatment. The ratio of surface to total DAT protein is plotted normalized to time point 0 (no AMPH treatment). AMPH decreases surface DAT in hDAT cells, but not in hDAT A559V cells (**P<0.01, *P<0.05, one-way ANOVA followed by Dunnett's multiple comparison test, n=6). (c) hDAT or hDAT A559V cells were treated with AMPH (10 nM) for the indicated time points. Top, representative immunoblot of DAT biotinylated fraction (‘surface DAT') and total cellular lysate (‘total DAT'). Bottom, quantification of biotinylated DAT protein fractions following AMPH treatment. The ratio of surface to total DAT protein is plotted normalized to time point 0 (no AMPH treatment). The 10 nM concentration of AMPH decreases surface DAT in hDAT cells, but not in hDAT A559V cells (*P<0.05, Student's t-test, n=4). AMPH, amphetamine; DAT, dopamine transporter; hDAT, human dopamine transporter.

Figure 3

Intracellular AMPH delivery induces trafficking in hDAT A559V cells measured by transient charge movement. (a) Cells were voltage clamped in the whole-cell configuration at a baseline of −20 mV and the current recorded following a voltage step to −140 mV. DAT-mediated currents were defined by subtracting the nonspecific current obtained in the presence of the DAT blocker cocaine (10 μM) from those recorded in the absence of cocaine. Top, DAT-mediated control currents (dotted lines) before AMPH treatment are plotted and compared with currents recorded after AMPH treatment (10 μM AMPH for 10 min, solid lines). Extracellular AMPH induces DAT trafficking (compare AMPH solid lines with control dotted lines) in hDAT but not hDAT A559V cells. Bottom, the transient charge (Q) was obtained by integrating the area under transient current as an index of the number of transporters at the cell surface. AMPH significantly decreases Q in hDAT cells (*P<0.05, Q_AMPH vs Q_control Student's t-test, n=5) but not hDAT A559V cells (P>0.05, Q_AMPH vs Q_control Student's t-test, n=3). (b) AMPH was applied intracellularly through perfusion by the whole-cell patch clamp pipette internal solution. Top, DAT-mediated control currents immediately after achieving whole-cell access (dotted lines) are plotted compared with currents after perfusing the cell for 10 min with AMPH (10 μM, solid lines). AMPH does induce DAT trafficking of hDAT A559V cells comparable to that of hDAT cells. Bottom, AMPH significantly decreases Q in both hDAT and hDAT A559V cells (*P<0.05, Q_AMPH vs Q_control Student's t-test, n=3). AMPH, amphetamine; DAT, dopamine transporter; hDAT, human dopamine transporter.

Figure 4

PKCβ inhibition restores AMPH-induced hDAT A559V internalization. (a) Representative immunoblots of phospho-PKCβII in hDAT and hDAT A559V cells. The top bands represent PKCβ in the cytosolic fraction. The middle bands represent PKCβ in the membrane fraction. PMA (100 nM for 20 min) was used as a positive control to demonstrate phosphorylation and translocation of PKCβ to the membrane in hDAT cells. The membrane protein Na/K ATPase (bottom bands) is used as a loading control for the membrane fraction. The bar graph shows that basal levels of phospho-PKCβII in the membrane fraction are higher in hDAT A559V cells than hDAT cells (*P<0.05, Student's t-test, n=6–8). (b) Cells were treated for 20 min with the PKCβ inhibitor 3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione (300 nM) before treatment with vehicle or AMPH (10 μM, 60 min). Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT') and total lysate (‘total DAT'). Bottom, quantification of surface to total DAT ratio. PKCβ inhibition restores AMPH-induced trafficking of hDAT A559V (AMPH vs vehicle, *P<0.05 Student's t-test, n=4). (c) Transient currents were recorded as an index of surface DAT in hDAT A559V cells. Cells were dialyzed under whole-cell patch clamp for 10 min with internal solution containing either the PKCβII inhibitor peptide (N-Myr-SLNPEWNET, 10 μM) or a scramble peptide (10 μM). Top, subsequent DAT-mediated currents following a voltage step from −20 mV to −140 mV are plotted for control (dotted lines) compared with currents after extracellular AMPH treatment (10 μM AMPH for 10 min, solid lines). PKCβII inhibition restores the AMPH-induced decrease in transient current in hDAT A559V cells. Bottom, AMPH decreases transient charge movement Q in hDAT A559V cells treated with PKCβII inhibitor peptide (*P<0.05, Q_AMPH vs Q_control Student's t-test, n=4) but not scamble peptide (P>0.05, Q_AMPH vs Q_control Student's t-test, n=4). (d) hDAT A559V S/A cells were treated with vehicle or AMPH (10 μM, 60 min). Top, representative immunoblot of DAT biotinylated proteins (‘surface DAT') and total cellular lysate (‘total DAT'). Bottom, quantification of surface to total DAT ratio. Mutation of N-terminal Ser to Ala in hDAT A559V restores AMPH-induced trafficking (AMPH vs vehicle, *P<0.05 Student's t-test, n=4). AMPH, amphetamine; DAT, dopamine transporter; hDAT, human dopamine transporter.

Figure 5

AMPH-induced DA efflux is impaired in hDAT A559V cells but restored by inhibiting PKCβ activity or mutating N-terminal Ser to Ala. (a) Representative amperometric traces (top) and quantification of amperometric DA signals (bottom) recorded in hDAT, hDAT A559V, and hDAT A559V S/A cells following 20 min pretreatment with either vehicle control or the PKCβ inhibitor 3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione (300 nM). Arrows indicate addition of AMPH (10 μM) or cocaine (COC, 10 μM) to the extracellular bath. AMPH increases amperometric currents from hDAT cells, indicating DA efflux. However, AMPH reduces amperometric currents in hDAT A559V cells, indicating a block of basal DA efflux from hDAT A559V cells. Either pretreatment with the PKCβ inhibitor or mutation of N-terminal serines in hDAT A559V restores the ability of AMPH to induce DA efflux in hDAT A559V cells (**P<0.01 analysis of variance (ANOVA) followed by Newman–Keuls multiple comparison test, n=3–5). (b) Cells were patch clamped and perfused through the whole-cell patch pipette for 10 min to allow for loading of 2 mM DA and either the PKCβ inhibitor peptide (N-Myr-SLNPEWNET, 10 μM) or a scramble control peptide (10 μM). The patch pipette was then withdrawn and AMPH-induced DA signals recorded by amperometry. The PKCβ inhibitor peptide restores the ability of AMPH to induce DA efflux in hDAT A559V cells (**P<0.01 ANOVA followed by Newman–Keuls multiple comparison test, n=3). AMPH, amphetamine; DA, dopamine; DAT, dopamine transporter; hDAT, human dopamine transporter.