Abstract
Uptake through the Dopamine Transporter (DAT) is the primary mechanism of terminating dopamine signaling within the brain, thus playing an essential role in neuronal homeostasis. Deregulation of DAT function has been linked to several neurological and psychiatric disorders including ADHD, schizophrenia, Parkinson’s disease, and drug addiction. Over the last 15 years, several studies have revealed a plethora of mechanisms influencing the activity and cellular distribution of DAT; suggesting that fine-tuning of dopamine homeostasis occurs via an elaborate interplay of multiple pathways. Here, we show for the first time that the βγ subunits of G proteins regulate DAT activity. In heterologous cells and brain tissue, a physical association between Gβγ subunits and DAT was demonstrated by co-immunoprecipitation. Furthermore, in vitro pull-down assays using purified proteins established that this association occurs via a direct interaction between the intracellular carboxy-terminus of DAT and Gβγ. Functional assays performed in the presence of the non-hydrolyzable GTP analog GTP-γ-S, Gβγ subunit overexpression, or the Gβγ activator mSIRK all resulted in rapid inhibition of DAT activity in heterologous systems. Gβγ activation by mSIRK also inhibited dopamine uptake in brain synaptosomes and dopamine clearance from mouse striatum as measured by high-speed chronoamperometry in vivo. Gβγ subunits are intracellular signaling molecules that regulate a multitude of physiological processes through interactions with enzymes and ion channels. Our findings add neurotransmitter transporters to the growing list of molecules regulated by G-proteins and suggest a novel role for Gβγ signaling in the control of dopamine homeostasis.
Highlights
Termination of dopamine (DA) neurotransmission is accomplished primarily through a plasma membrane sodium-dependent re-uptake process mediated by the dopamine transporter (DAT)
Immunoprecipitations with two antibodies directed against different DAT epitopes resulted in co-precipitation of Gbc subunits from mouse striatum, while no bands were detected in samples precipitated using the corresponding control IgGs (Figure 1A)
While these results cannot rule out the possibility of an interaction between DAT and Ga subunits, they strongly support a physical association between the transporter and Gbc subunits
Summary
Termination of dopamine (DA) neurotransmission is accomplished primarily through a plasma membrane sodium-dependent re-uptake process mediated by the dopamine transporter (DAT). Depletion of DA stores in DAT knock-out mice was observed despite the fact that the DA synthesis rate was elevated by twofold, suggesting a major role for the transporter in both the termination of DA signaling and vesicular DA replenishment. Signaling pathways involved in rapid regulation of DAT include the activation of G protein-coupled receptors, intracellular second messenger systems, and the effect of protein-protein interactions [3,4,5,6]. As a consequence, these multiples modes of regulation result in dynamic changes in DAT function, which are expected to have profound consequences in DA transmission and DA-related behaviors
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