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

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 ß (PKCß) activity. Cells expressing hDAT A559V exhibit constitutively elevated PKCß 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 PKCß 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 (PKCß) for therapeutic interventions in individuals with ASD.

De novo mutation in the dopamine transporter gene associates dopamine dysfunction with autism spectrum disorder.

De novo genetic variation is an important class of risk factors for autism spectrum disorder (ASD). Recently, whole-exome sequencing of ASD families has identified a novel de novo missense mutation in the human dopamine (DA) transporter (hDAT) gene, which results in a Thr to Met substitution at site 356 (hDAT T356M). The dopamine transporter (DAT) is a presynaptic membrane protein that regulates dopaminergic tone in the central nervous system by mediating the high-affinity reuptake of synaptically released DA, making it a crucial regulator of DA homeostasis. Here, we report the first functional, structural and behavioral characterization of an ASD-associated de novo mutation in the hDAT. We demonstrate that the hDAT T356M displays anomalous function, characterized as a persistent reverse transport of DA (substrate efflux). Importantly, in the bacterial homolog leucine transporter, substitution of A289 (the homologous site to T356) with a Met promotes an outward-facing conformation upon substrate binding. In the substrate-bound state, an outward-facing transporter conformation is required for substrate efflux. In Drosophila melanogaster, the expression of hDAT T356M in DA neurons-lacking Drosophila DAT leads to hyperlocomotion, a trait associated with DA dysfunction and ASD. Taken together, our findings demonstrate that alterations in DA homeostasis, mediated by aberrant DAT function, may confer risk for ASD and related neuropsychiatric conditions.

A closer look at amphetamine-induced reverse transport and trafficking of the dopamine and norepinephrine transporters.

Amphetamine (AMPH) and its derivatives are regularly used in the treatment of a wide array of disorders such as attention-deficit hyperactivity disorder (ADHD), obesity, traumatic brain injury, and narcolepsy (Prog Neurobiol 75:406-433, 2005; J Am Med Assoc 105:2051-2054, 1935; J Am Acad Child Adolesc Psychiatry 41:514-521, 2002; Neuron 43:261-269, 2004; Annu Rev Pharmacol Toxicol 47:681-698, 2007; Drugs Aging 21:67-79, 2004). Despite the important medicinal role for AMPH, it is more widely known for its psychostimulant and addictive properties as a drug of abuse. The primary molecular targets of AMPH are both the vesicular monoamine transporters (VMATs) and plasma membrane monoamine-dopamine (DA), norepinephrine (NE), and serotonin (5-HT)-transporters. The rewarding and addicting properties of AMPH rely on its ability to act as a substrate for these transporters and ultimately increase extracellular levels of monoamines. AMPH achieves this elevation in extracellular levels of neurotransmitter by inducing synaptic vesicle depletion, which increases intracellular monoamine levels, and also by promoting reverse transport (efflux) through plasma membrane monoamine transporters (J Biol Chem 237:2311-2317, 1962; Med Exp Int J Exp Med 6:47-53, 1962; Neuron 19:1271-1283, 1997; J Physiol 144:314-336, 1958; J Neurosci 18:1979-1986, 1998; Science 237:1219-1223, 1987; J Neurosc 15:4102-4108, 1995). This review will focus on two important aspects of AMPH-induced regulation of the plasma membrane monoamine transporters-transporter mediated monoamine efflux and transporter trafficking.