Interaction between the dopamine transporter and the Gβγ subunits of the heterotrimeric G‐protein modifies transporter function and response to psychostimulants

Abstract

Monoaminergic neurotransmission (dopamine, serotonin, and norepinephrine) is altered in complex psychiatric conditions such as depression, attention‐deficit hyperactivity disorder (ADHD) and drug use disorders. The dopamine transporter (DAT) clears extracellular dopamine (DA) through a sodium‐coupled transport mechanism. The fine‐tuning of DA homeostasis is maintained by multiple mechanisms involving protein‐protein interactions. We recently reported that G‐protein beta‐gamma (Gβγ) subunits bind directly to DAT. The binding of Gβγ results in a net change in the direction of the DA flux, increasing extracellular DA levels. The activation of Gβγ by a binding/activating peptide, mSIRK, increases DA efflux through DAT in heterologous systems and primary dopaminergic neurons in culture. The activation of a Gq‐subtype GPCR like the muscarinic receptor 5 (hM5R), also stimulates efflux that can be blocked with gallein, a small molecule that block the Gbg “hot spot” for binding to different effectors. A permeable TAT‐peptide containing the Gβγ interacting domain of DAT (human DAT 582 to 586) blocked the ability of mSIRK and carbachol to induce DA efflux, consistent with a direct interaction of Gβγ with the transporter. To further understand the mechanism of Gβγ regulation on DAT function, we examined whether the functional effects were linked to conformational changes in the DAT. An extracellularly‐oriented cysteine residue (C306) was used to probe conformational changes induced by the Gβγ activator peptide, mSIRK, and ligands such as cocaine, ibogaine and other modulators of DAT function. The accessibility of C306 to an impermeant thiol‐reactive reagent was significantly reduced in cells treated with mSIRK. It has previously been shown that cocaine increases the accessibility of C306 by stabilizing an outward facing conformation of the DAT. However, pretreatment of cells with mSIRK reduced the cocaine‐induced increase in C306 reactivity, suggesting that Gβγ activation limits the available binding sites for cocaine, perhaps by stabilizing an inward conformation. Finally, we wanted to test if the observed conformational changes were result of the Gβγ binding to DAT or if the binding of Gβγ is conformation‐dependent. For that we used the proximity‐ligation assay, new approach to study protein‐protein interactions, to study how the interaction DAT‐Gβγ is modified in the presence of different conformation‐specific DAT‐ligands. Collectively, our data show that Gβγ interacts directly with DAT and facilitate a shift in DAT conformation towards an inwardly‐oriented efflux mode. This novel mechanism may have important implications in the regulation of brain DA homeostasis.Support or Funding InformationNIMH IRP:ZIA MH002946‐04This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.