Structural Determinants of the Dopamine Transporter Regulation Mediated by G Proteins

Abstract

Dopamine clearance in the brain is controlled by the dopamine transporter (DAT), a protein residing in the plasma membrane, which drives reuptake of extracellular dopamine into presynaptic neurons. Studies have revealed that the βγ subunits of heterotrimeric G proteins modulate DAT function through a physical association with the C-terminal region of the transporter. Regulation of neurotransmitter transporters by Gβγ subunits is unprecedented in the literature; therefore, it is interesting to investigate the structural details of this particular protein–protein interaction. Here, we refined the crystal structure of the Drosophila melanogaster DAT (dDAT), modeling de novo the N- and C-terminal domains; subsequently, we used the full-length dDAT structure to generate a comparative model of human DAT (hDAT). Both proteins were assembled with Gβ1γ2 subunits employing protein–protein docking, and subsequent molecular dynamics simulations were run to identify the specific interactions governing the formation of the hDAT:Gβγ and dDAT:Gβγ complexes. A [L/F]­R­[Q/E]­R sequence motif containing the residues R588 in hDAT and R587 in dDAT was found as key to bind the Gβγ subunits through electrostatic interactions with a cluster of negatively charged residues located at the top face of the Gβ subunit. Alterations of DAT function have been associated with multiple devastating neuropathological conditions; therefore, this work represents a step toward better understanding DAT regulation by signaling proteins, allowing us to predict therapeutic target regions.

The Norepinephrine Transporter - Gβγ Subunit Interaction Promotes Norepinephrine Efflux through the Transporter: In silico and Functional Studies

“…A hydrogen bond was also formed between E586 (NET) and Y59 (Gβγ) at a distance of 1.92 Å, and W585 (NET) interacted with W99 (Gβγ) through van der Waals (VDW) interactions (Figure S1B). These findings are consistent with Rojas et al's (2020) and Pino et al's (2021) [14,22] study of the DAT/Gβγ interaction.…”

“…Previous studies on the interaction between DAT and Gβγ have demonstrated that Gβγ activation leads to the efflux of DA through DAT. Computational studies have also described specific interactions between the carboxy terminus of DAT and Gβγ [14,22]. However, there is a lack of structural information regarding the potential effects of Gβγ on SERT or NET.…”

“…HADDOCK is a widely used molecular docking program that utilizes both experimental and computational data to generate models of protein-protein complexes. For the docking process, we selected the carboxy-terminal residues of NET and specific residues of Gβγ that have been previously identified as important for the interaction with other effectors, including DAT [13,14,22]. The final NET/Gβγ complex was selected based on the ranked energies calculated by the internal scoring function of HADDOCK, which evaluates the quality and stability of the complex structures.…”

The dopamine transporter gene SLC6A3: multidisease risks

“…2a right panel), explaining their impaired reuptake. The mutations associated with psychiatric conditions affect DA efflux and involve interactions with other proteins such as Gβγ subunits of the G-protein for Glu602Gly in BP and Arg615Cys in ADHD or syntaxin for Arg51Trp in autism [ 66 , 67 ], affecting hDAT networking (see Supplementary Text for Fig. 2a ).…”