Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

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

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 Ca2+-calmodulindependent 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, butnotTAT-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 CaMKIIa 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 CaMKIIa 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.

Original languageEnglish (US)
Pages (from-to)27534-27544
Number of pages11
JournalJournal of Biological Chemistry
Volume288
Issue number38
DOIs
StatePublished - Sep 20 2013

Fingerprint

Dopamine Plasma Membrane Transport Proteins
Amphetamine
Dopamine
Membranes
Peptides
Protein Kinases
Proteins
Human Immunodeficiency Virus tat Gene Products
1-Methyl-4-phenylpyridinium
tat Gene Products
Cell membranes
Protein C
Immunoprecipitation
HIV-1
Carrier Proteins
Cell Membrane

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Rickhag, M., Owens, W. A., Winkler, M. T., Strandfelt, K. N., Rathje, M., Sørensen, G., ... Gether, U. (2013). Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. Journal of Biological Chemistry, 288(38), 27534-27544. https://doi.org/10.1074/jbc.M112.441295

Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. / Rickhag, Mattias; Owens, William A.; Winkler, Marie Therese; Strandfelt, Kristine Nørgaard; Rathje, Mette; Sørensen, Gunnar; Andresen, Bjørn; Madsen, Kenneth L.; Jørgensen, Trine Nygaard; Wörtwein, Gitta; Woldbye, David P D; Sitte, Harald; Daws, Lynette C; Gether, Ulrik.

In: Journal of Biological Chemistry, Vol. 288, No. 38, 20.09.2013, p. 27534-27544.

Research output: Contribution to journalArticle

Rickhag, M, Owens, WA, Winkler, MT, Strandfelt, KN, Rathje, M, Sørensen, G, Andresen, B, Madsen, KL, Jørgensen, TN, Wörtwein, G, Woldbye, DPD, Sitte, H, Daws, LC & Gether, U 2013, 'Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release', Journal of Biological Chemistry, vol. 288, no. 38, pp. 27534-27544. https://doi.org/10.1074/jbc.M112.441295
Rickhag, Mattias ; Owens, William A. ; Winkler, Marie Therese ; Strandfelt, Kristine Nørgaard ; Rathje, Mette ; Sørensen, Gunnar ; Andresen, Bjørn ; Madsen, Kenneth L. ; Jørgensen, Trine Nygaard ; Wörtwein, Gitta ; Woldbye, David P D ; Sitte, Harald ; Daws, Lynette C ; Gether, Ulrik. / Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. In: Journal of Biological Chemistry. 2013 ; Vol. 288, No. 38. pp. 27534-27544.
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T1 - Membrane-permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release

AU - Rickhag, Mattias

AU - Owens, William A.

AU - Winkler, Marie Therese

AU - Strandfelt, Kristine Nørgaard

AU - Rathje, Mette

AU - Sørensen, Gunnar

AU - Andresen, Bjørn

AU - Madsen, Kenneth L.

AU - Jørgensen, Trine Nygaard

AU - Wörtwein, Gitta

AU - Woldbye, David P D

AU - Sitte, Harald

AU - Daws, Lynette C

AU - Gether, Ulrik

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N2 - 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 Ca2+-calmodulindependent 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, butnotTAT-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 CaMKIIa 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 CaMKIIa 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.

AB - 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 Ca2+-calmodulindependent 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, butnotTAT-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 CaMKIIa 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 CaMKIIa 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.

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