Dopamine transporter threonine-53 phosphorylation dictates kappa opioid receptor mediated locomotor suppression and conditioned place aversion via transporter upregulation

Dynorphin (DYN)/kappa opioid receptor (KOR) activation contributes to aversion, dysphoria, sedation, depression, and enhanced psychostimulant-rewarding effects, which have been attributed to the inhibition of dopamine (DA) release. DYN fibers synapse onto DA terminals which express both KOR and dopamine transporter (DAT). DAT activity is critical in the regulation of DA dynamics and dopaminergic neurotransmission. Previously, we demonstrated that KOR agonists upregulate DAT activity via ERK1/2 signaling involving phospho-Thr53 DAT (pT53-DAT). However, whether pT53-DAT is involved in KOR-mediated DAT regulation in-vivo and whether such phenomenon contributes to the behavioral effects of KOR agonism are unknown. Here, we investigated the role of endogenous pT53-DAT in KOR-mediated DAT regulation and the effect of KOR agonists on locomotor suppression and aversive behaviors using DAT-Ala53 knock-in mice expressing DAT carrying non-phosphorylatable Ala at position 53 replacing Thr. Acute systemic administration of KOR agonist, U69593 resulted in KOR antagonist-sensitive increases in DAT activity in parallel to increases in pT53-DAT, and DAT Vmax and surface expression in the ventral and dorsal striatum (containing the nucleus accumbens and caudate putamen respectively) of WT, but not DAT-Ala53 mice. KOR agonists produced conditioned place aversion (CPA) and locomotor suppression in WT but not DAT-Ala53 mice. However, both WT and DAT-Ala53 mice exhibited similar lithium chloride-induced CPA and morphine-induced conditioned place preference (CPP). These findings provide the first evidence that locomotor suppression and aversive responses to KOR agonists manifest due to the modulation of DAT activity via DAT-T53 phosphorylation establishing a causal relationship of pT53-DAT in KOR-mediated DAT regulation and KOR agonist-induced adverse effects.