Abstract

Aberrant dopamine (DA) signaling is associated with several psychiatric disorders such as bipolar disorder, addiction, and Parkinson's disease, and several medications that target the DA transporter (DAT) can induce or treat these disorders. Biochemical and crystallographic studies demonstrate that the competitive actions of DAT antagonists and substrates, such as cocaine and D-amphetamine (AMPH), rely on interactions with the transporter's substrate binding site. Agents that exhibit noncompetitive, allosteric modulation of DAT remain a significant topic of investigation, owing to their potential therapeutic applications. We previously identified a novel allosteric modulator of human DAT, KM822, that can decrease the affinity of cocaine for DAT and attenuate cocaine-elicited behaviors, though whether DAT is the sole mediator of KM822 actions in vivo is unproven given the large number of potential off-target sites. The function of C. elegans DAT (DAT-1) has been well characterized and used in both genetic and pharmacological studies of psychostimulant action and transporter regulation. Here, we provide in silico and in vitro evidence that the allosteric site engaged by KM822 is conserved between human DAT and DAT-1 and that KM822 interacts with DAT-1. Through a combination of genetic and pharmacological in vivo approaches we provide evidence that the ability of KM822 to diminish the behavioral actions of AMPH arises through its allosteric modulation of DAT-1. More broadly, our findings demonstrate allosteric modulation of DAT as a behavior modifying strategy and suggests that C. elegans can be operationalized to identify and investigate the interactions of DAT allosteric modulators.

Significance Statement We previously demonstrated that the DAT allosteric modulator KM822 decreases cocaine affinity for human DAT. Here, using in silico and in vivo genetic approaches, we extend this finding to interactions with amphetamine (AMPH), demonstrating evolutionary conservation of the DAT allosteric site. In C. elegans, we report that KM822 suppresses AMPH behavioral effects via specific interactions with DAT-1. Our findings reveal C. elegans as a new tool to study allosteric modulation of DAT and its behavioral consequences.