Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved..
Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:299 |
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| Enthalten in: |
The Journal of biological chemistry - 299(2023), 8 vom: 18. Aug., Seite 105063 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Støier, Jonatan Fullerton [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 01.09.2023 Date Revised 06.09.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.jbc.2023.105063 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a Støier, Jonatan Fullerton |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2 |
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| 520 | |a Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved. | ||
| 520 | |a Amphetamines (AMPHs) are substrates of the dopamine transporter (DAT) and reverse the direction of dopamine (DA) transport. This has been suggested to depend on activation of Ca2+-dependent pathways, but the mechanism underlying reverse transport via endogenously expressed DAT is still unclear. Here, to enable concurrent visualization by live imaging of extracellular DA dynamics and cytosolic Ca2+ levels, we employ the fluorescent Ca2+ sensor jRGECO1a expressed in cultured dopaminergic neurons together with the fluorescent DA sensor GRABDA1H expressed in cocultured "sniffer" cells. In the presence of the Na+-channel blocker tetrodotoxin to prevent exocytotic DA release, AMPH induced in the cultured neurons a profound dose-dependent efflux of DA that was blocked both by inhibition of DAT with cocaine and by inhibition of the vesicular monoamine transporter-2 with Ro-4-1284 or reserpine. However, the AMPH-induced DA efflux was not accompanied by an increase in cytosolic Ca2+ and was unaffected by blockade of voltage-gated calcium channels or chelation of cytosolic Ca2+. The independence of cytosolic Ca2+ was further supported by activation of N-methyl-D-aspartate-type ionotropic glutamate receptors leading to a marked increase in cytosolic Ca2+ without affecting AMPH-induced DA efflux. Curiously, AMPH elicited spontaneous Ca2+ spikes upon blockade of the D2 receptor, suggesting that AMPH can regulate intracellular Ca2+ in an autoreceptor-dependent manner regardless of the apparent independence of Ca2+ for AMPH-induced efflux. We conclude that AMPH-induced DA efflux in dopaminergic neurons does not require cytosolic Ca2+ but is strictly dependent on the concerted action of AMPH on both vesicular monoamine transporter-2 and DAT | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a amphetamine | |
| 650 | 4 | |a biosensors | |
| 650 | 4 | |a calcium signaling | |
| 650 | 4 | |a dopamine transporter | |
| 650 | 4 | |a genetically encoded dopamine sensor | |
| 650 | 4 | |a live fluorescent imaging | |
| 650 | 7 | |a Amphetamine |2 NLM | |
| 650 | 7 | |a CK833KGX7E |2 NLM | |
| 650 | 7 | |a Cocaine |2 NLM | |
| 650 | 7 | |a I5Y540LHVR |2 NLM | |
| 650 | 7 | |a Dopamine |2 NLM | |
| 650 | 7 | |a VTD58H1Z2X |2 NLM | |
| 650 | 7 | |a Dopamine Plasma Membrane Transport Proteins |2 NLM | |
| 650 | 7 | |a Vesicular Monoamine Transport Proteins |2 NLM | |
| 700 | 1 | |a Konomi-Pilkati, Ainoa |e verfasserin |4 aut | |
| 700 | 1 | |a Apuschkin, Mia |e verfasserin |4 aut | |
| 700 | 1 | |a Herborg, Freja |e verfasserin |4 aut | |
| 700 | 1 | |a Gether, Ulrik |e verfasserin |4 aut | |
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