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.

The rare DAT coding variant Val559 perturbs DA neuron function, changes behavior, and alters in vivo responses to psychostimulants

“…Our efforts originated from identification of the Val559 variant in two male siblings with an ADHD diagnosis (47). Although our starting point concerned efforts to detect rare, functional gene variation impacting DAT expression or function that could contribute to risk for ADHD, the variant has also been detected in a female subject with BPD (48), and in two unrelated males with an ASD diagnosis (49). ADHD and BPD are found at higher incidence in Lack of genotype differences in relative time spent in the center versus the edge of the activity chambers.…”

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…These authors also observed a decrease in AMPHinduced stimulation of locomotor behavior. Interestingly, AMPH-evoked DA efflux in transfected HEK-293 cells as monitored by amperometry was found to be insensitive to PKCb antagonism, although these inhibitors restored AMPH evoked efflux lost in the ADHD-associated DAT coding variant Val559 (Bowton et al, 2014).…”

Regulation of the Dopamine and Vesicular Monoamine Transporters: Pharmacological Targets and Implications for Disease

“…The following sections include a description of some commonly investigated diseases and the putative role of DAT and VMAT (for examples of other diseases such as autism and bipolar disorder, see Grünhage et al, 2000;Nakamura et al, 2010;Bowton et al, 2014;Nguyen et al, 2014).…”