In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT)

Aggarwal, Shaili; Mortensen, Ole V.

doi:10.1002/cpph.33

Cited by 7 publications

(6 citation statements)

References 20 publications

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“…Transport assays were performed as described previously. 45 Uptake experiments were performed 2 days later. The media was removed, and the cells were washed with PBS-CM.…”

Section: ■ Methodsmentioning

confidence: 99%

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Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter

Aggarwal

Liu

Rice

et al. 2019

ACS Chem. Neurosci.

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The dopamine transporter (DAT) serves a pivotal role in controlling dopamine (DA)-mediated neurotransmission by clearing DA from synaptic and perisynaptic spaces and controlling its action at postsynaptic DA receptors. Major drugs of abuse such as amphetamine and cocaine interact with DAT to mediate their effects by enhancing extracellular DA concentrations. We previously identified a novel allosteric site in the related human serotonin transporter that lies outside the central substrate and inhibitor binding pocket. We used the hybrid structure based (HSB) method to screen for allosteric modulator molecules that target a similar site in DAT. We identified a compound, KM822, that was found to be a selective, noncompetitive inhibitor of DAT. We confirmed the structural determinants of KM822 allosteric binding within the allosteric site by structure/function and substituted cysteine scanning accessibility biotinylation experiments. In the in vitro cell-based assay and ex vivo in both rat striatal synaptosomal and slice preparations, KM822 was found to decrease the affinity of cocaine for DAT. The in vivo effects of KM822 on cocaine were tested on psychostimulant-associated behaviors in a planarian model where KM822 specifically inhibited the locomotion elicited by DAT-interacting stimulants amphetamine and cocaine. Overall, KM822 provides a unique opportunity as a molecular probe to examine allosteric modulation of DAT function.

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“…Transport assays were performed as described previously. 45 Uptake experiments were performed 2 days later. The media was removed, and the cells were washed with PBS-CM.…”

Section: ■ Methodsmentioning

confidence: 99%

“…Transport assays were performed as described previously . Briefly, COS-7 cells were transfected and plated in 96-well plates.…”

Section: Methodsmentioning

confidence: 99%

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter

Aggarwal

Liu

Rice

et al. 2019

ACS Chem. Neurosci.

Self Cite

View full text Add to dashboard Cite

show abstract

“…For the determination of K M , K i , and V max values, the GABA uptake assays were carried out in 96-well plates (Aggarwal & Mortensen, 2017). HEK 293 GnTI À cells were infected with the baculovirus of respective mutant, and cells were plated at a cell density of 50,000 cells per well 36-h post-infection.…”

Section: Gaba Uptake and Inhibition Assay In 96-well Plate Formatmentioning

confidence: 99%

Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter

2022

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c-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.

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“…Radioactive DA uptake assay was performed following previous published protocols. 86 N2a cells and SH-SY5Y cells were seeded in triplicates in the 24-well plate two days before the uptake assay. Cells typically reach 80% confluency on day of testing.…”

Section: Methodsmentioning

confidence: 99%

Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter

et al. 2023

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In Vitro Assays for the Functional Characterization of the Dopamine Transporter (DAT)

Cited by 7 publications

References 20 publications

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter

Identification of a Novel Allosteric Modulator of the Human Dopamine Transporter

Structural insights into GABA transport inhibition using an engineered neurotransmitter transporter

Cocaine-regulated trafficking of dopamine transporters in cultured neurons revealed by a pH sensitive reporter

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