Abstract
The human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.
Highlights
The uptake of neurotransmitters in and around the synaptic cleft by dedicated membrane transport proteins is a key process in the regulation of neurotransmitter s ignaling[1]
Several reference norepinephrine transporter (NET) inhibitors were tested for their inhibitory potencies, which showed a strong correlation with potencies from an established fluorescent substrate uptake assay[18]
These results render the through receptor activation’ (TRACT) assay suitable for characterization of NET inhibitors and demonstrate that the assay is amenable to high-throughput screening (HTS)
Summary
The uptake of neurotransmitters in and around the synaptic cleft by dedicated membrane transport proteins is a key process in the regulation of neurotransmitter s ignaling[1]. Our group developed a novel functional ‘transport activity through receptor activation’ (TRACT) assay based on a label-free impedance-based technology for the equilibrative nucleoside transporter 1 (ENT1, SLC29A1)[20,21] and the dopamine transporter (DAT, SLC6A3)[22]. In this bioassay a transporter that shares its substrate (e.g., adenosine, dopamine) with a G protein-coupled receptor (GPCR) is expressed in live cells together with a cognate GPCR. Several reference NET inhibitors were tested for their inhibitory potencies, which showed a strong correlation with potencies from an established fluorescent substrate uptake assay[18] These results render the TRACT assay suitable for characterization of NET inhibitors and demonstrate that the assay is amenable to HTS. The detailed read-out, physiological setting and label-free nature of the method make the TRACT assay a meaningful alternative to conventional label-based assays for SLCs
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