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Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release*

2013; Elsevier BV; Volume: 288; Issue: 38 Linguagem: Inglês

10.1074/jbc.m112.441295

1083-351X

Mattias Rickhag, William A. Owens, Marie-Therese Winkler, Kristine Nørgaard Strandfelt, Mette Rathje, G. Sørensen, Bjørn Andresen, Kenneth L. Madsen, Trine N. Jørgensen, Gitta Wörtwein, David P.D. Woldbye, Harald H. Sitte, Lynette C. Daws, Ulrik Gether,

Neuroscience and Neuropharmacology Research

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca 2+ -calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects. Background: The significance of dopamine transporter (DAT) C-terminal protein-protein interactions for amphetamine-induced dopamine efflux is unsettled. Results: Cell-permeable C-terminal DAT peptides attenuate amphetamine-induced dopamine efflux and locomotor activity in mice. Conclusion: DAT C-terminal protein-protein interactions are critical for the effects of amphetamine in vivo . Significance: Targeting protein-protein interactions might be a way of inhibiting the effects of psychostimulants.