Protein Kinase Cβ Is a Critical Regulator of Dopamine Transporter Trafficking and Regulates the Behavioral Response to Amphetamine in Mice

Abstract

The dopamine transporter (DAT) is a key mediator of dopaminergic neurotransmission and a major target for amphetamine. We found previously that protein kinase C (PKC) ␤ regulates amphetamine-mediated dopamine efflux. Here, using PKC␤ wild-type (WT) and knockout (KO) mice, we report a novel role for PKC␤ in amphetamine-induced regulation of DAT trafficking and activity. PKC␤ KO mice have less striatal surface DAT, [ 3 H]dopamine uptake, and amphetamine-stimulated dopamine efflux, yet higher novelty-induced locomotor activity than WT mice. Although a short exposure (Յ90 s) to amphetamine rapidly increases striatal surface DAT and [3 H]dopamine uptake in WT mice, this treatment decreases surface DAT and [3 H]dopamine uptake in KO mice. Increases in surface DAT and [ 3 H]dopamine uptake are not evident in KO mice until a longer exposure (60 min) to amphetamine, by which time WT mice exhibit decreased surface DAT and dopamine uptake. The slowness of amphetamine-induced striatal DAT trafficking in PKC␤ KO mice was mimicked by the use of a specific PKC␤ inhibitor, LY379196, in WT mice. Furthermore, PKC␤ KO mice exhibit reduced locomotor responsiveness to amphetamine compared with WT, which could be explained by reduced surface DAT and delayed amphetamine-induced DAT trafficking in KO mice. Our results indicate that PKC␤ is crucial for proper trafficking of DAT to the surface and for functioning of DAT and amphetamine signaling, providing new insight into the role of PKC␤ as an important regulator of dopaminergic homeostasis.The dopamine transporter (DAT) is an integral membrane protein containing 12 putative transmembrane domains and cytosolic amino and carboxyl termini. It belongs to a superfamily of Na ϩ /Cl Ϫ -dependent neurotransmitter transporters. Dopaminergic transmission in the synapse is primarily terminated by removal of dopamine (DA) from presynaptic nerve terminals via DAT. DAT is also a primary target of psychostimulants such as amphetamine (AMPH). As a substrate, AMPH elicits reverse transport of DA through DAT, a mechanism underlying AMPH addiction. Dysfunctional DAT signaling is also implicated in various other neurodegenerative and psychiatric disorders such as Parkinson's disease, attention deficit/hyperactive disorder, and schizophrenia.DAT trafficking and activity are regulated by a complicated protein network involving multiple protein kinases and DAT-interacting proteins (Torres, 2006), among which protein kinase C (PKC) is the most characterized. Longer term activation of PKC (Ͼ20 min) by phorbol 12-myristate 13-acetate, a general PKC activator, leads to increased DAT endocytosis and reduced DAT recycling, resulting in reduced surface DAT expression and activity in rat synaptosomes and cultured cells (Daniels and Amara, 1999;Melikian and Buckley, 1999;Loder and Melikian, 2003). In accordance, PKC inhibitors block phorbol 12-myristate 13-acetate-induced DAT internalization (Daniels and Amara, 1999;Melikian and Buckley, 1999;Chang et al., 2001). An endocytic motif in the C terminus of DAT t...

Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters

“…Consistent with this idea, PKCb knockout mice demonstrate reduced, though notably not completely eliminated, AMPH-evoked DA efflux (Chen et al, 2009). Similar results were recently observed by the Gnegy group, who found that perfusion of PKCb inhibitors into the nucleus accumbens of rats reduced AMPHevoked DA efflux by approximately 50% (Zestos et al, 2016).…”

Temporal Pattern of Cocaine Intake Determines Tolerance vs Sensitization of Cocaine Effects at the Dopamine Transporter

“…It is possible that allosteric modifications to the DAT protein itself are mediating the decreased potency following LgA and the increased potency following IntA. Indeed, the DAT has been shown to undergo glycosylation and phosphorylation (Foster et al, 2008;Chen et al, 2009). Therefore, it is possible that changes in the interaction of the DAT protein with downstream effectors responsible for these post-translational modifications are altered following self-administration in a way that changes the ability of ligands to bind to the DAT and inhibit the clearance of DA.…”

Amphetamine-induced ERM Proteins Phosphorylation Is through PKCβ Activation in PC12 Cells

“…AMPH also activates PKC in PC12 cells [8] is a critical regulator of DAT trafficking and regulates the behavioral response to AMPH in mice [15]. Thus, we examined AMPH-induced PKCβ activation by using anti-phospho PKCβ antbody.…”