Knowledge of factors that regulate the plasmalemma dopamine transporter (DAT) is crucial to a complete understanding of the actions of several drugs of abuse. The motor stimulant and reinforcing effects of amphetamine (AMPH) and methamphetamine (METH), substrates of DAT, are due to their ability to increase the release of dopamine (DA). AMPHs appear to elicit reverse transport through DAT by an exchange diffusion mechanism. Our data reveal an unexpected component in AMPH-mediated reverse transport of DA. We find that AMPH-mediated outward transport of DA requires protein kinase C (PKC) activity and intracellular Ca2+. Direct PKC activation mimics the releasing activity of AMPH. The substrate for the PKC phosphorylation could be the DA transporter itself since both AMPH and PKC activators elicit phosphorylation of DAT. The focus of this proposal is to elucidate the mechanism by which PKC exerts its effects on DA outward transport. The hypothesis to be tested is that AMPH transport activates PKC which elicits a phosphorylation of DAT that, in turn, enhances outward transport.
The specific aims are: 1. Determine whether DAT agonists other than AMPH increase the phosphorylation of DAT. 2. Test the hypothesis that an AMPH-mediated phosphorylation of DAT is responsible for enhanced AMPH-mediated DA release. 3. Determine whether TPA and the AMPH-induced increase in phosphorylation alter insertion of DAT into the membranes of rat synaptosomes and of hDAT-transfected cos-7 cells. 4. Test the hypothesis that AMPH increases PKC activity by increasing translocation of a PKC isozyme proximal to DAT. Smokable forms of METH are becoming more popular in America, signalling the start of a new wave of AMPH abuse. Since AMPH is a substrate for DAT, its mechanism of action is different from that of cocaine, an antagonist. AMPH does not act at a classical receptor, and the DAT antagonist, cocaine, is also widely abused. Thus, an ideal inhibitor of AMPH would block its ability to release DA. A more complete understanding of the mechanism of AMPH action can lead to a more rational design of inhibitors of the amphetamines.
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