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

Karl Mattias Rickhag; WA Owens; Marie-Therese Winkler; Kristine Nørgaard Strandfelt; Mette Rathje; Gunnar Sørensen; Bjørn Andresen; Kenneth Lindegaard Madsen; Trine Nygaard Jørgensen; Gitta Wörtwein; David Paul Drucker Woldbye; Harald H Sitte; Lynette C. Daws; Ulrik Gether

doi:10.1074/jbc.M112.441295

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

Karl Mattias Rickhag, WA Owens, Marie-Therese Winkler, Kristine Nørgaard Strandfelt, Mette Rathje, Gunnar Sørensen, Bjørn Andresen, Kenneth Lindegaard Madsen, Trine Nygaard Jørgensen, Gitta Wörtwein, David Paul Drucker Woldbye, Harald H Sitte, Lynette C. Daws, Ulrik Gether

21 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 language	English
Journal	Journal of Biological Chemistry
Volume	288
Pages (from-to)	27534-44
Number of pages	11
ISSN	0021-9258
DOIs	https://doi.org/10.1074/jbc.M112.441295
Publication status	Published - 20 Sept 2013

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Rickhag, K. M., Owens, WA., Winkler, M.-T., Strandfelt, K. N., Rathje, M., Sørensen, G., Andresen, B., Madsen, K. L., Nygaard Jørgensen, T., Wörtwein, G., Woldbye, D. P. D., Sitte, H. H., Daws, L. C., & Gether, U. (2013). Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release. Journal of Biological Chemistry, 288, 27534-44. https://doi.org/10.1074/jbc.M112.441295

Rickhag, KM, Owens, WA, Winkler, M-T, Strandfelt, KN, Rathje, M, Sørensen, G, Andresen, B, Madsen, KL, Nygaard Jørgensen, T, Wörtwein, G , Woldbye, DPD, Sitte, HH, Daws, LC & Gether, U 2013, 'Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release.', Journal of Biological Chemistry, vol. 288, pp. 27534-44. https://doi.org/10.1074/jbc.M112.441295

@article{8b49901d870241f49f8088318fc9d815,

title = "Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release.",

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.",

author = "Rickhag, {Karl Mattias} and WA Owens and Marie-Therese Winkler and Strandfelt, {Kristine N{\o}rgaard} and Mette Rathje and Gunnar S{\o}rensen and Bj{\o}rn Andresen and Madsen, {Kenneth Lindegaard} and {Nygaard J{\o}rgensen}, Trine and Gitta W{\"o}rtwein and Woldbye, {David Paul Drucker} and Sitte, {Harald H} and Daws, {Lynette C.} and Ulrik Gether",

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doi = "10.1074/jbc.M112.441295",

language = "English",

volume = "288",

pages = "27534--44",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology, Inc.",

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T1 - Membrane permeable C-terminal dopamine transporter peptides attenuate amphetamine-evoked dopamine release.

AU - Rickhag, Karl Mattias

AU - Owens, WA

AU - Winkler, Marie-Therese

AU - Strandfelt, Kristine Nørgaard

AU - Rathje, Mette

AU - Sørensen, Gunnar

AU - Andresen, Bjørn

AU - Madsen, Kenneth Lindegaard

AU - Nygaard Jørgensen, Trine

AU - Wörtwein, Gitta

AU - Woldbye, David Paul Drucker

AU - Sitte, Harald H

AU - Daws, Lynette C.

AU - Gether, Ulrik

PY - 2013/9/20

Y1 - 2013/9/20

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.

U2 - 10.1074/jbc.M112.441295

DO - 10.1074/jbc.M112.441295

M3 - Journal article

SN - 0021-9258

VL - 288

SP - 27534

EP - 27544

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

ER -

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

Abstract

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