Fig 2: pS14 is critical for CDE.(A) Representative traces (top) and quantitative analysis (bottom) of the amplitude of the peak current and CDE (CDE amplitude is defined as the maximum deflection of the current from baseline) of hDAT SD cells treated with CK2i (100 nM) or VEH. CK2i treatment prevented CDE (t = 3.678, n = 5 to 7). (B) Representative traces (top) and quantitative analysis (bottom) of hDAT SD cells expressing either Stx1 S14D or S14A. Stx1 S14A prevented CDE (U = 0, n = 5 to 7). (C) Saturation curves of 3[H]DA uptake measured in hDAT SD cells expressing either Stx1 S14D (red) or S14A (blue). Curves were fit to Michaelis-Menten kinetics to derive Km and Vmax. DA uptake for hDAT SD cells expressing Stx1 S14A was increased compared to hDAT SD cells expressing Stx1 S14D at the two highest DA concentrations used [F(5,22) = 4.197, n = 3 in triplicate, P = 0.0079]. The black dotted line represents hDAT WT cell uptake. (D) Snapshots of MD simulations of the hDAT SD/Stx1 S14A complex at 20, 50, and 100 ns. Red and blue VDW spheres represent acidic and basic residues. Residues from DAT and Stx1 are labeled in boldface and plain face, respectively. Stx1 D25, R26, D27, and R28 were observed to form intermittent salt bridges with hDAT K65/K337, D12/D13, K65/K337, and E61/D68, respectively. For clarification, some residues, e.g., Stx1 D25, hDAT D68, and K337, are not displayed. We observed this in one independent run. In each figure, the binding PIP2 lipids are colored in licorice format. The absence of bound PIP2 near R344 and K66 holds the association of D15 to D19 Stx1 acidic cluster with the basic cluster near hDAT R344/K66. Data are presented as means ± SEM. Student’s unpaired t-test with Welch’s correction (A); Mann-Whitney test (B); two-way ANOVA with Bonferroni’s multiple comparisons test (C).

Fig 3: Stx1 and hDAT phosphorylation status regulate DA efflux in Drosophila brains.(A) Schematic of the amperometric recordings. The amperometric electrode was positioned in the PPL1 region that is enriched in DA neurons. (B) Representative AMPH-induced amperometric traces (top) recorded from the PPL1 region of hDAT WT–expressing Drosophila brains from flies that received oral administration of VEH or CK2i (100 nM) for 24 hours and quantitative analysis of the peak currents (bottom). CK2i significantly reduced DA efflux in hDAT WT brains (t = 2.505, n = 5). (C) Representative AMPH-induced amperometric traces (top) recorded from hDAT SD–expressing Drosophila brains from flies that received oral administration of VEH or CK2i (100 nM) for 24 hours and quantitative analysis of the peak currents (bottom). CK2i significantly reduced CDE in hDAT SD brains (t = 3.359, n = 6 to 7). (D) Representative amperometric traces (top) recorded from the PPL1 region of hDAT WT or hDAT SD Drosophila brains and quantitative analysis of the peak currents (bottom). Brains were exposed acutely to COC (1 μM). COC reveals a CDE in hDAT SD that is not present in hDAT WT brains (t = 2.528, n = 8). Data are presented as means ± SEM. Student’s unpaired t-test (B); Student’s unpaired t-test with Welch’s correction (C and D).

Fig 4: Pedigree and cross-species conservation of hDAT R/W and STX1 R/Q.(A) Variant inheritance is shown for the families harboring the hDAT R/W and the STX1 R/Q genotypes. Filled symbols indicate individuals with an ASD diagnosis, while open symbols reflect individuals without an ASD diagnosis. (B) Alignment of the DAT amino acid sequence across multiple species. Arginine 51 is represented in red. (C) Alignment of the STX1 amino acid sequence across isoforms and multiple species. Arginine 25 is represented in red.

Fig 5: STX1 phosphorylation promotes reverse transport of DA: (A) Top: stably transfected hDAT cells expressing STX1 were treated with either vehicle or 10 μM TBB for 20 min. This was followed by an additional treatment with vehicle or 10 μM AMPH for 15 min. STX1 immunoprecipitates were immunoblotted for either p-STX1 (Ser14) or STX1. Bottom: the immunoprecipitated band densities were quantified, normalized to the corresponding density of total precipitated STX1, and expressed as a percentage of vehicle control (* = p < 0.05 by one-way ANOVA followed by Newman–Keuls Multiple Comparison Test; n = 3). (B) Representative AMPH-induced DA efflux recorded from stably transfected hDAT cells expressing STX1 treated with either vehicle or 10 μM TBB for 20 min just before the amperometric recordings. Arrows indicate application of 10 μM AMPH. Bottom: quantitation of AMPH-induced DA efflux. Data are represented as maximal oxidative current (* = p < 0.05 by Student's t-test; n = 5). (C) Representative AMPH-induced amperometric currents recorded from stably transfected hDAT cells expressing STX1 S14A treated with either vehicle or 10 μM TBB for 20 min. Arrows indicate application of 10 μM AMPH. Bottom: quantitation of maximal oxidative current (p > 0.05 by Student's t-test; n = 4–7). (D) Representative AMPH-induced amperometric currents recorded from stably transfected hDAT cells expressing STX1 S14D treated with either vehicle or 10 μM TBB for 20 min. Arrows indicate application of 10 μM AMPH. Bottom: quantitation of AMPH-induced DA efflux. Data are represented as maximal oxidative current (p > 0.05 by Student's t-test; n = 6).