TY - JOUR

T1 - Amphetamine-induced reverse transport of dopamine does not require cytosolic Ca2+

AU - Støier, Jonatan Fullerton

AU - Konomi-Pilkati, Ainoa

AU - Apuschkin, Mia

AU - Herborg, Freja

AU - Gether, Ulrik

N1 - Publisher Copyright: © 2023 The Authors

PY - 2023

Y1 - 2023

N2 - 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.

AB - 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.

KW - amphetamine

KW - biosensors

KW - calcium signaling

KW - dopamine transporter

KW - genetically encoded dopamine sensor

KW - live fluorescent imaging

U2 - 10.1016/j.jbc.2023.105063

DO - 10.1016/j.jbc.2023.105063

M3 - Journal article

C2 - 37468107

AN - SCOPUS:85167833202

VL - 299

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 8

M1 - 105063

ER -