title: 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.
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: E
authors.initials: E
authors.lastname: Bowton
authors.affiliation: 1] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [2] Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: C
authors.initials: C
authors.lastname: Saunders
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: I A
authors.initials: IA
authors.lastname: Reddy
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: N G
authors.initials: NG
authors.lastname: Campbell
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: P J
authors.initials: PJ
authors.lastname: Hamilton
authors.affiliation: Department of Basic Sciences, University of North Dakota, Grand Forks, ND, USA.
authors.forename: L K
authors.initials: LK
authors.lastname: Henry
authors.affiliation: Department of Psychiatry, University of Utah, Salt Lake City, UT, USA.
authors.forename: H
authors.initials: H
authors.lastname: Coon
authors.affiliation: 1] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [2] Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: D
authors.initials: D
authors.lastname: Sakrikar
authors.affiliation: 1] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [2] Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Veenstra-VanderWeele
authors.affiliation: 1] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [2] Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: R D
authors.initials: RD
authors.lastname: Blakely
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [3] Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sutcliffe
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [3] N-PISA Neuroscience Program In Substance Abuse, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: H J G
authors.initials: HJ
authors.lastname: Matthies
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [3] N-PISA Neuroscience Program In Substance Abuse, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: K
authors.initials: K
authors.lastname: Erreger
authors.affiliation: 1] Departments of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN, USA [2] Vanderbilt Brain Institute, Vanderbilt University Medical Center, Nashville, TN, USA [3] N-PISA Neuroscience Program In Substance Abuse, Vanderbilt University Medical Center, Nashville, TN, USA.
authors.forename: A
authors.initials: A
authors.lastname: Galli
mesh_terms: D001321:Autistic Disorder / Q000235:genetics*; D002465:Cell Movement / Q000235:genetics; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics*; D035781:Siblings; D009435:Synaptic Transmission / Q000235:genetics*
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; C493214:SLC6A3 protein, human