Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3

Abstract

The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.

Keywords: amphetamine; autism; dopamine transporter; efflux; leucine transporter.

Fig. 1.

In LeuT, deletion of V269 supports a HOIF conformation of the intracellular gate. Distance distributions of extracellular and intracellular spin-labeled Cys pairs reveal changes in the conformational equilibrium induced by ∆V269 in LeuT. (A) Extracellular reporter pairs (309–480) tagged on a 3D structure of LeuT. (D) Intracellular reporter pairs (7–86) tagged on a 3D structure of LeuT. (B and E) Distance distributions of the extracellular and intracellular reporter pair for LeuT in the apo conformation (black), in the presence of Na⁺ (red), or in the presence of Na⁺ plus Leu (blue). (C and F) Distance distributions of the extracellular and intracellular reporter pair for LeuT ∆V269 in the apo conformation (black), in the presence of Na⁺ (red), or in the presence of Na⁺ and Leu (blue).

Fig. 2.

Structure of LeuT ∆V269. (A) Superposition of LeuT ∆V269 with PDB ID code 2A65. The primary differences in the intracellular region are highlighted in red (PDB ID code 2A65) and blue (∆V269). (B) Reorientation of the residues immediately following the ∆V269 is displayed. The movement of the first helical turn of the TM1 region is displayed in cartoon representation in the background. (C) Residues responsible for movements in the first turn of TM1 are highlighted (also SI Appendix, Fig. S2).

Fig. 3.

Modeling of hDAT ∆N336 predicts the formation of a new K337-D345 hydrogen bond. (A) Modeling using Rosetta shows that hDAT (blue) positions a free residue K337 toward the intracellular space. Modeling hDAT ∆N336 (red) reveals a repositioning of K337 that, in turn, forms a new K337-D345 hydrogen bond that is absent in hDAT. This K337-D345 bond may stabilize IL3 by linking it to TM7. (B) Cartoon representation of a homology model of hDAT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid bilayer. The chloride (cyan) and Na⁺ (blue) ions; DA (orange); and side chains of K66 (green), K337 (yellow), and D345 (red) are highlighted. (C) hDAT at 50 ns: salt bridge between K66 and D345. (D) hDAT ∆N336 at 50 ns: salt bridge between K66 and D345 (replica 2). (E) hDAT ∆N336 at 100 ns: residue D345 forms two salt bridges, one to K66 and a second to K337 (replica 2) (also SI Appendix, Fig. S3).

Fig. 4.

hDAT ∆N336 displays impaired DA transport, reduced AMPH-induced DA efflux, and diminished AMPH-induced currents. (A) Representative plot of [³H]DA uptake kinetics in hDAT (■) or hDAT ∆N336 (□) cells (***P ≤ 0.0001 by two-way ANOVA, followed by Bonferroni posttest; n = 6, performed in triplicate). (B) Representative AMPH-induced amperometric currents recorded from hDAT and hDAT ∆N336 cells. Cells were patch-loaded with DA. Arrows indicate application of 10 μM AMPH. (C) Quantification of AMPH-induced DA efflux. Data are represented as the peak amperometric current ± SEM (*P ≤ 0.01 by t test; n = 5). (D and E) Mean AMPH (10 μM)-induced whole-cell current of 10 consecutive sweeps obtained from hDAT and hDAT ∆N336 cells is plotted. The hDAT and hDAT ∆N336 cells were kept under voltage clamp (−60 mV), and AMPH was rapidly applied to the cells for 1 s by a piezoelectric translator (Methods). In contrast to hDAT ∆N336 cells, hDAT cells display a peak and a steady-state current. (F and G) Quantification of AMPH-induced peak current and steady-state current for hDAT- and hDAT ∆N336-expressing cells. Data are represented as amperometric current ± SEM (*P ≤ 0.05 by t test; n = 9) (also SI Appendix, Figs. S4 and S5).

Fig. 5.

In Drosophila brain, hDAT ∆N336 cells display reduced DA uptake and AMPH-induced efflux. (A) Flies expressing hDAT ∆N336 display reduced DA uptake compared with hDAT-expressing brains. Data are represented as DA uptake (200 nM DA) using four Drosophila brains per well ± SEM (*P ≤ 0.01 by t test; n = 4). (B) Representative AMPH-induced (20 μM) amperometric currents recorded from fly brains expressing hDAT or hDAT ∆N336. Arrows indicate application of AMPH. (C) Quantitation of DA efflux. Data are presented as the peak amperometric current ± SEM (*P ≤ 0.01 by t test; n = 4). (D) Flies expressing hDAT ∆N336, when fed an l-DOPA (5 mM) diet for 24 h, display increased DA efflux compared with vehicle-fed flies. Representative AMPH-induced (20 μM) amperometric currents recorded from hDAT ∆N336 fly brains. Arrows indicate application of AMPH. (E) Quantitation of DA efflux. Data are presented as the peak amperometric current ± SEM (*P ≤ 0.05 by t test; n = 4).

Fig. 6.

hDAT ∆N336 flies are hyperactive and show prolonged freezing and reduced fleeing. (A) Locomotor activity was assayed over 30 h during the light (horizontal white bars) and dark (horizontal dark bars) cycle. Flies expressing hDAT ∆N336 (○) were hyperactive compared with flies expressing hDAT (■) (*P < 0.05 by two-way ANOVA with Sidak’s post hoc test; n = 31–32). Beam crossings were binned into 20-min intervals. (B) Quantitation of total beam crossings over 24 h. Data are presented as the total beam crossing ± SEM (*P ≤ 0.0001 by Mann–Whitney test; n = 30–31). (C) Average velocity (μm⋅ms⁻¹) was assayed over 600 ms following an auditory stimulus for flies expressing hDAT (■) or hDAT ∆N336 (○). The hashed line indicates preaudible baseline velocity. (D) AUC is calculated as the cumulative velocity from the onset to the end of the predatory sound. Quantitation of AUC over 600 ms for hDAT and hDAT ∆N336 flies. Data are presented as the total AUC ± SEM (*P ≤ 0.05 by t test; n = 8).

Fig. 7.

hDAT ∆N336 flies show social impairments as measured by proximity to their neighbors during the escape response. The sum of distances (μm) across four flies (social space) was calculated and assayed over 1,000 ms and normalized to the respective social space calculated at time 0. Over time, hDAT flies (■) display increased flock size, as determined by the slope of the fitting of the sum of distances over time (black dotted line: 2.30 ± 0.05 μm⋅ms⁻¹). In contrast, hDAT ∆N336 flies (○) reveal decreased flock size over time (gray dotted line: −9.36 ± 0.08 μm⋅ms⁻¹).