PKC phosphorylates residues in the N-terminal of the DA transporter to regulate amphetamine-induced DA efflux.
The DA transporter (DAT), a phosphoprotein, controls extracellular dopamine (DA) levels in the central nervous system through transport or reverse transport (efflux). Multiple lines of evidence support the claim that PKC significantly contributes to amphetamine-induced DA efflux. Other signaling pathways, involving CaMKII and ERK, have also been shown to regulate DAT mediated efflux. Here we assessed the contribution of putative PKC residues (S4, S7, S13) in the N-terminal of the DAT to amphetamine-induced DA efflux by transfecting DATs containing different serine to alanine (S-A) point mutations into DA pre-loaded HEK-293 cells and incubating these cells in amphetamine (2μM). The effects of a S-A mutation at the non-PKC residue S12 and a threonine to alanine (T-A) mutation at the ERK T53 residue were also assessed for comparison. WT-DATs were used as controls. In an initial experiment, we confirmed that inhibiting PKC with Go6976 (130nM) significantly reduced amphetamine-induced DA efflux. In subsequent experiments, cells transfected with the S4A, S12A, S13A, T53A and S4,7,13A mutants showed a reduction in amphetamine-induced DA efflux similar to that observed with Go6976. Interestingly, cells transfected with the S7A mutant, identified by some as a PKC-PKA residue, showed unperturbed WT-DAT levels of amphetamine-induced DA efflux. These results indicate that phosphorylation by PKC of select residues in the DAT N-terminal can regulate amphetamine-induced efflux. PKC can act either independently or in concert with other kinases such as ERK to produce this effect.
Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, Chicago, IL 60637, USA.
Qiang
Q
Wang
Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, Chicago, IL 60637, USA.
Nancy
N
Bubula
Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, Chicago, IL 60637, USA.
Jason
J
Brown
Department of Neurology, 148th Hospital of PLA, Zibo, Shandong 255300, China.
Yunliang
Y
Wang
Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, Chicago, IL 60637, USA.
Veronika
V
Kondev
Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, Chicago, IL 60637, USA. Electronic address: pvezina@uchicago.edu.
Paul
P
Vezina
D000661:Amphetamine / Q000494:pharmacology*; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D000697:Central Nervous System Stimulants / Q000494:pharmacology*; D004298:Dopamine / Q000378:metabolism*; D050483:Dopamine Plasma Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D010766:Phosphorylation; D017354:Point Mutation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats
D002227:Carbazoles; D000697:Central Nervous System Stimulants; D050483:Dopamine Plasma Membrane Transport Proteins; C081021:Go 6976; D000661:Amphetamine; D011493:Protein Kinase C; D004298:Dopamine
Amphetamine; CaMKII; Dopamine release; Dopamine transporter; ERK; PKC; Phosphorylation