Identification of Critical Residues in the Carboxy Terminus of the Dopamine Transporter Involved in the G Protein βγ-Induced Dopamine Efflux

Abstract

The dopamine transporter (DAT) plays a crucial role in the regulation of brain dopamine (DA) homeostasis through the re-uptake of DA back into the presynaptic terminal. In addition to re-uptake, DAT is also able to release DA through a process referred to as DAT-mediated DA efflux. This is the mechanism by which potent and highly addictive psychostimulants, such as amphetamine (AMPH) and its analogues, increase extracellular DA levels in motivational and reward areas of the brain. Recently, we discovered that G protein βγ subunits (Gβγ) binds to the DAT, and that activation of Gβγ results in DAT-mediated efflux - a similar mechanism as AMPH. Previously, we have shown that Gβγ binds directly to a stretch of 15 residues within the intracellular carboxy terminus of DAT (residues 582–596). Additionally, a TAT peptide containing residues 582 to 596 of DAT was able to block the Gβγ-induced DA efflux through DAT. Here, we use a combination of computational biology, mutagenesis, biochemical, and functional assays to identify the amino acid residues within the 582–596 sequence of the DAT carboxy terminus involved in the DAT-Gβγ interaction and Gβγ-induced DA efflux. Our in-silico protein-protein docking analysis predicted the importance of F587 and R588 residues in a network of interactions with residues in Gβγ. In addition, we observed that mutating R588 to alanine residue resulted in a mutant DAT which exhibited attenuated DA efflux induced by Gβγ activation. We demonstrate that R588, and to a lesser extent F5837, located within the carboxy terminus of DAT play a critical role in the DAT-Gβγ physical interaction and promotion of DA efflux. These results identify a potential new pharmacological target for the treatment of neuropsychiatric conditions in which DAT functionality is implicated including ADHD and substance use disorder.

Kappa Opioid Receptor Antagonism Restores Phosphorylation, Trafficking and Behavior induced by a Disease Associated Dopamine Transporter Variant

“…Alternatively, antagonism of KOR could further disrupt the potentially enhanced interaction of DAT Val559 with proteins and lipids that bias DAT towards more efflux-prone states (49,(93)(94)(95)(96). Activated Gbg proteins have also been found to induce DA efflux similar to that seen with DAT Val559 and amphetamines (44,45). Whether the pathways involved with KOR antagonism intersect with this mechanism and its regulators are worthy of study.…”

“…Interestingly, ADE has now been recognized as a functional correlate of multiple disease-associated DAT variants (21,30,34,41,42) and can also be triggered in wildtype (WT) DAT via G protein interactions (44,45), extending the ADE phenotype beyond that seen with rare genetic variants.…”

“…DAT dependence of DAT Val559-mediated ADE was established through a block of efflux by methylphenidate and amphetamine, as well as cocaine (41,42). Interestingly, ADE has now been recognized as a functional correlate of multiple disease-associated DAT variants (21,30,34,41,42) and can also be triggered in wildtype (WT) DAT via G protein interactions (44,45), extending the ADE phenotype beyond that seen with rare genetic variants.…”

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

“…In addition to exogenous agents like amphetamines, certain intracellular signaling mechanisms, such as protein kinase C (PKC) and Ca+2/calmodulin-dependent protein kinase II (CAMKII), can also trigger transporter-mediated efflux [11,12]. Recent studies conducted by our group have demonstrated that G protein βγ subunits can bind to DAT and facilitate DA efflux [13,14]. This effect has been observed in transfected cells, brain slices, and in vivo [13,15].…”

Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2+

“…This phosphorylation can be mediated by CaMKIIα and facilitated by an interaction of the kinase with the DAT C-terminus ( 25 ). AMPH-induced efflux might also be regulated by the interaction of DAT with syntaxin1A ( 30 ), G protein βγ subunits and the neuronal GTPase, Rit2 ( 31 , 32 , 33 ), as well as by lipid messengers ( e.g ., phosphatidylinositol 4,5-bisphosphate) ( 34 ). Taken together, multiple mechanisms have been implicated in AMPH-induced efflux; however, since many studies have been done in heterologous cells, the picture is still blurry as to what mechanisms represent the key drivers of efflux in dopaminergic (DArgic) neurons ( 35 ).…”