Phosphorylation of the Amino Terminus of the Dopamine Transporter: Regulatory Mechanisms and Implications for Amphetamine Action

Abstract

Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. Their actions at dopaminergic neurons are thought to mediate their therapeutic efficacy as well as their liability for abuse and dependence. AMPHs target the dopamine transporter (DAT), the plasmalemmal membrane protein that mediates the inactivation of released dopamine (DA) through its reuptake. AMPHs act as substrates for DAT and are known to cause mobilization of dopamine (DA) to the cell exterior via DAT-mediated reverse transport (efflux). It has become increasingly evident that the mechanisms that regulate AMPH-induced DA efflux are distinct from those that regulate DA uptake. Central to these mechanisms is the phosphorylation of the DAT amino (N)-terminus, which has been repeatedly demonstrated to facilitate DAT-mediated DA efflux, without impacting other aspects of DAT physiology. This review aims to summarize the current status of knowledge regarding DAT N-terminal phosphorylation and its regulation by protein modulators and the membrane microenvironment. A better understanding of these mechanisms may lead to the identification of novel therapeutic approaches that interfere selectively with the pharmacological effects of AMPHs without altering the physiological function of DAT.

Keywords: Dopamine efflux; Kinases; Lipid rafts; Phosphatases; Psychostimulants; Vesicular monoamine transporters.

Fig. 1

Regulation of DAT phosphorylation. AMPH acts as a substrate for DAT (orange oval) and is transported into the neuron (dashed arrow pointing intracellularly), which leads to an increase in intracellular Ca²⁺ levels, an increase in CaMKIIα and PKC activity, and an increase in the levels of DAT phosphorylation, all of which have been demonstrated to facilitate AMPH-induced DAT-mediated DA efflux (dashed arrow pointing extracellularly). Activation of PKC by the phorbol ester PMA also increases DAT phosphorylation, as does inhibition of protein phosphatases by okadaic acid (OA). The mechanistic relationship between CaMKII and PKC, with respect to DAT phosphorylation, remains unclear (as indicated by the question mark). Localization of DAT to cholesterol-rich membrane microdomains (purple section of plasma membrane) facilitates its phosphorylation in response to AMPH, and thereby the consequent DA efflux and associated behaviors.