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

Abstract

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.

Figure 1. Cross-species conservation and in silico mutagenesis of T356

A) Alignment of the DAT amino acid sequence across multiple species. Threonine 356 is represented in red. B) In an equilibrated three-dimensional homology model of hDAT, the T356M mutation is located on transmembrane domain 7 (TM7). Top: Schematic views representing a 180° rotation show T356M with respect to the TM helices. TM7 is shown in dark blue. Bottom: Critical residues that interact with DA^, are shown, as well as the bound Na⁺ and Cl⁻ ions. The methionine is rendered together at position 356 with the wild type threonine (green).

Figure 2. Human dopamine transporter (hDAT) T356M has impaired function

A) Top: Kinetic parameters (V_max and K_m) for hDAT and hDAT T356M (V_max: p ≤ 0.005; by Student’s t-test; n = 3, in triplicate; K_m: p ≥ 0.20; by Student’s t-test; n = 3, in triplicate). Bottom: Representative plot of [³H]DA uptake kinetics in hDAT (filled squares) or hDAT T356M (empty squares) cells (** = p ≤ 0.01, *** = p ≤ 0.001; by two-way ANOVA followed by Bonferroni posttest; n = 3, in triplicate). B) Representative immunoblots for biotinylated (surface) and total protein fractions from hDAT and hDAT T356M cells. Surface fractions were quantitated, normalized to total DAT (Glycosylated), and expressed as a percent of hDAT (p ≥ 0.05; by Student’s t-test; n = 8–11).

Figure 3. hDAT T356M exhibits robust ADE

A) Top: representative amperometric currents recorded from hDAT and hDAT T356M cells. Arrows indicate application of 10 µM cocaine (COC). Bottom: quantitation of the cocaine-induced reduction in the amperometric current (ADE). Data are reported as maximal current (*** = p ≤ 0.001 by Student’s t-test; n = 4–5). B) De novo mutation in the hDAT gene causes DA dysfunction 25 hDAT T356M cells do not display altered resting membrane potential with respect to hDAT cells (p ≥ 0.05 by Student’s t-test, n = 9–25). C) Representative AMPH-induced amperometric currents recorded from hDAT and hDAT T356M cells. Arrows indicate application of 10 µM AMPH. Bottom: quantitation of AMPH-induced DA efflux. Data are represented as maximal current expressed as percent of the current recorded in hDAT cells (** = p ≤ 0.01 by Student’s t-test; n = 5–7).

Figure 4. In LeuT, substitution of Ala289 with a Met supports an outward-open facing conformation

Distance distributions of extracellular and intracellular spin labeled Cys pairs in LeuT reveal changes in the conformational equilibrium caused by mutating Ala289 to a Met. A) Left: extracellular reporter pairs (309–480) tagged on three-dimensional structure of LeuT. Right: distance of the extracellular reporter pair for LeuT (black) and A289M (red), in the Apo conformation (Apo) and in the presence of Na⁺ and Leu (+NaL). B) Left: intracellular reporter pairs (7–86) tagged on the three-dimensional structure of LeuT. Right: distance of the intracellular reporter pair for LeuT (black) and A289M (red), in the Apo conformation (Apo) and in the presence of Na⁺ and Leu (+NaL). The LeuT structure was obtained from PDB 2A65. The structures were generated using PyMOL.

Figure 5. Expression of hDAT T356M in Drosophila leads to hyperactivity

hDAT or hDAT T356M was expressed in DA neurons of dDAT KO flies. A) Locomotor activity was assayed over 32 hours during the light (horizontal white bars) or dark (horizontal black bars) cycle. Flies expressing hDAT T356M (red squares), as well as dDAT KO flies (blue squares), were hyperactive throughout the 32 hour period with respect to flies expressing wild type hDAT (black squares) (n = 32; beam breaks binned in 15 minute intervals). B) Quantitation of total beam crosses over 24 hours for hDAT, hDAT T356M, and dDAT KO flies (**** = p ≤ 0.0001; n = 32).