Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site

Abstract

The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium- and chloride-coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine-like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo-electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non-competitive inhibitor AC-4-248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non-competitive inhibition of DAT and attenuation of cocaine potency by AC-4-248 and provide a basis for the rational design of more efficacious atypical inhibitors.

Keywords: atypical inhibitor; cryo‐electron microscopy; dopamine transporter; neurotransmitter transporter.

Figure 1 –. Characterization of AC-4-248 inhibition.

DA uptake assays showing (a) inhibitory effect of AC-4-248 on hDAT and non-selectivity for hSERT, hNET and hDAT, (b) DA uptake inhibition assay confirming AC-4-248 inhibition of dDAT_mfc, (c) DA transport inhibition assay of cocaine in the presence or absence of AC-4-248 in hDAT transfected COS-7 cells, showing AC-4-248 decreases the potency of the inhibitory effect of cocaine on hDAT, and (d) DA uptake inhibition assay with varying DA concentrations, showing AC-4-248 at 64 μM inhibits uptake of dopamine by hDAT in a non-competitive manner with a decrease in V_max and no significant change in the apparent affinity for DA (K_m) compared to vehicle. All uptake data points were measured in triplicate. The average values of K_m, V_max, and IC₅₀ with standard error means or confidence intervals were obtained from three separate uptake experiments. Statistical analysis was performed using Student’s paired t-test (**, p < 0.01).

Figure 2 –. Overall density and structure of dDAT_TS bound to AC-4-248.

A) Cryo-EM density map of dDAT bound to AC-4-248 (left) and a 90° rotation (right) where the densities for the cholesterol and CHS molecules are shown in light green. Map contour level = 8.15 in ChimeraX. B) Slice view of AC-4-248-bound dDAT in the outward open state, showing the AC-4-248-binding site. AC-4-248 is shown in orange. TM1 and TM6 are shown as cartoon representations. Extracellular gating residues Y124, F319, R52, and D475 together with the intracellular gating residues R27, W30, Y331, Y334, and D435 are shown as sticks. The distances between gating residues are shown with dashed lines. C) Cartoon representation of dDAT with non-protein density seen in the middle. The zoom-in view on the left shows the cryo-EM density map of AC-4-248 (in orange, map contour level = 5.6 in ChimeraX) observed in the central substrate binding site and a presumable Tris molecule (red density, map contour level = 5.6 in ChimeraX) occupying an opportunistic site. The zoom-in view on the right shows Na⁺ and Cl⁻ ions densities contoured at 4.75 level in ChimeraX, and their coordinating residues in the AC-4-248-bound dDAT_TS.

Figure 3 –. AC-4-248 binding pocket.

(a) Residues forming the hydrophobic binding pocket of AC-4-248 are depicted in sticks. Direct interactions are marked with dashes. (b) 2D plot of AC-4-248 interaction with dDAT_TS. (c) DA uptake inhibition assay with varying concentrations of inhibitor AC-4-248 in WT, F155Y, F320Y, and W84C hDAT transfected COS-7 cells. The IC₅₀ of AC-4-248 for WT, F155Y, F320Y, and W84C hDAT is 64.01 μM [CI 95%: 62.43, 65.03], 18.24 μM [CI 95%: 12.76, 20.58], 22.05 μM [CI 95%: 20.65, 23.35], 9.108 μM [7.688, 10.30], respectively. All uptake data points were measured in triplicate. The average values of IC₅₀ with confidence intervals were obtained from three separate uptake experiments (n = 3). Statistical analysis was performed using Student’s paired t-test (**, p < 0.01). (d) The central binding pocket comprising subsites A, B, and C (marked with dashed circles) in the outward open, AC-4-248-bound dDAT_TS structure. Superimposition of dDAT outward open structures bound to inhibitors nortriptyline or cocaine, or substrate DA (PDB IDs 4M48, 4XP4, and 4XP1, respectively) on AC-4-248-bound dDAT_TS, shows the binding pose of the ligands in subsites A, B and C. Selected residues indicating the relative hydrophilic/hydrophobic environment of the subsites A, B, and C are shown as sticks.