Impedance-Based Phenotypic Readout of Transporter Function: A Case for Glutamate Transporters

Abstract

Excitatory amino acid transporters (EAAT/SLC1) mediate Na+-dependent uptake of extracellular glutamate and are potential drug targets for neurological disorders. Conventional methods to assess glutamate transport in vitro are based on radiolabels, fluorescent dyes or electrophysiology, which potentially compromise the cell’s physiology and are generally less suited for primary drug screens. Here, we describe a novel label-free method to assess human EAAT function in living cells, i.e., without the use of chemical modifications to the substrate or cellular environment. In adherent HEK293 cells overexpressing EAAT1, stimulation with glutamate or aspartate induced cell spreading, which was detected in real-time using an impedance-based biosensor. This change in cell morphology was prevented in the presence of the Na+/K+-ATPase inhibitor ouabain and EAAT inhibitors, which suggests the substrate-induced response was ion-dependent and transporter-specific. A mechanistic explanation for the phenotypic response was substantiated by actin cytoskeleton remodeling and changes in the intracellular levels of the osmolyte taurine, which suggests that the response involves cell swelling. In addition, substrate-induced cellular responses were observed for cells expressing other EAAT subtypes, as well as in a breast cancer cell line (MDA-MB-468) with endogenous EAAT1 expression. These findings allowed the development of a label-free high-throughput screening assay, which could be beneficial in early drug discovery for EAATs and holds potential for the study of other transport proteins that modulate cell shape.

Still in Search for an EAAT Activator: GT949 Does Not Activate EAAT2, nor EAAT3 in Impedance and Radioligand Uptake Assays

“…Although inhibition of both these EAAT subtypes can be robustly shown in this assay, activation appears to not be as trivial. 21 At this moment, there are few known allosteric modulators available, which could act as a positive control on EAAT2 and EAAT3 activation. 13 Therefore, this does not allow us to conclude whether the assay cannot readily pick up activation or whether GT949 is failing in mechanistically activating EAAT2.…”

“…The Jump in T-Rex HEK293 (JumpIn) overexpressing human EAAT2/3 were kindly provided by the RESOLUTE consortium () …”

“…The observed changes are attributed to EAATmediated cell swelling as a consequence of increased intracellular levels Na + and K + ions upon L-glutamate uptake by EAATs. 21 These cell-swelling effects could be blocked with the EAAT inhibitor TFB-TBOA, indicating that the observed changes in cell morphology in the impedance-based assay are EAAT specific. This high-throughput amenable technology is very sensitive and could even be used to assess the activity of EAAT1 mutants and their inhibition with allosteric inhibitor UCPH-101.…”

“…Recently, Sijben and co-workers introduced a novel impedance-based assay that can detect l -glutamate uptake by EAATs in real time on whole cells . This assay relies on the fact that EAAT-mediated l -glutamate uptake leads to changes in cell morphology, which can be detected as changes in cellular impedance using the xCELLigence real-time cell analyzer.…”

“…6 Recently, Sijben and co-workers introduced a novel impedance-based assay that can detect L-glutamate uptake by EAATs in real time on whole cells. 21 This assay relies on the fact that EAAT-mediated L-glutamate uptake leads to changes in cell morphology, which can be detected as changes in cellular impedance using the xCELLigence real-time cell analyzer. The observed changes are attributed to EAATmediated cell swelling as a consequence of increased intracellular levels Na + and K + ions upon L-glutamate uptake by EAATs.…”