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

Sijben, Hubert J.; Acqua, Laura Dall’; Liu, Ronghui; Jarret, Abigail; Christodoulaki, Eirini; Onstein, Svenja; Wolf, Gernot; Verburgt, Simone J.; Dévédec, Sylvia E. Le; Wiedmer, Tabea; Superti‐Furga, Giulio; IJzerman, Adriaan P.; Heitman, Laura H.

doi:10.3389/fphar.2022.872335

Cited by 5 publications

(16 citation statements)

References 80 publications

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“…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.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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

Section: Methodsmentioning

confidence: 99%

“…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.…”

Section: ■ Introductionmentioning

confidence: 94%

“…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.…”

Section: Introductionmentioning

confidence: 99%

“…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.…”

Section: ■ Introductionmentioning

confidence: 99%

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

van Veggel,

Mocking,

Sijben

et al. 2024

ACS Chem. Neurosci.

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Excitatory amino acid transporters (EAATs) are important regulators of amino acid transport and in particular glutamate. Recently, more interest has arisen in these transporters in the context of neurodegenerative diseases. This calls for ways to modulate these targets to drive glutamate transport, EAAT2 and EAAT3 in particular. Several inhibitors (competitive and noncompetitive) exist to block glutamate transport; however, activators remain scarce. Recently, GT949 was proposed as a selective activator of EAAT2, as tested in a radioligand uptake assay. In the presented research, we aimed to validate the use of GT949 to activate EAAT2-driven glutamate transport by applying an innovative, impedance-based, whole-cell assay (xCELLigence). A broad range of GT949 concentrations in a variety of cellular environments were tested in this assay. As expected, no activation of EAAT3 could be detected. Yet, surprisingly, no biological activation of GT949 on EAAT2 could be observed in this assay either. To validate whether the impedance-based assay was not suited to pick up increased glutamate uptake or if the compound might not induce activation in this setup, we performed radioligand uptake assays. Two setups were utilized; a novel method compared to previously published research, and in a reproducible fashion copying the methods used in the existing literature. Nonetheless, activation of neither EAAT2 nor EAAT3 could be observed in these assays. Furthermore, no evidence of GT949 binding or stabilization of purified EAAT2 could be observed in a thermal shift assay. To conclude, based on experimental evidence in the present study GT949 requires specific assay conditions, which are difficult to reproduce, and the compound cannot simply be classified as an activator of EAAT2 based on the presented evidence. Hence, further research is required to develop the tools needed to identify new EAAT modulators and use their potential as a therapeutic target.

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Section: ■ Results and Discussionmentioning

confidence: 99%

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

Section: Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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

van Veggel,

Mocking,

Sijben

et al. 2024

ACS Chem. Neurosci.

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The solute carrier superfamily interactome

Frommelt,

Ladurner,

Goldmann

et al. 2024

Preprint

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Solute carrier (SLC) transporters form a protein superfamily that enables transmembrane transport of diverse substrates including nutrients, ions and drugs. There are about 450 different SLCs, residing in a variety of subcellular membranes. Loss-of-function of an unusually high proportion of SLC transporters is genetically associated with a plethora of human diseases, making SLCs a rapidly emerging but challenging drug target class. Knowledge of their protein environment may elucidate the molecular basis for their functional integration with metabolic and cellular pathways and help conceive pharmacological interventions based on modulating proteostatic regulation. We aimed at obtaining a global survey of the SLC protein interaction landscape and mapped the protein-protein interactions of 396 SLCs by interaction proteomics. We employed a functional assessment based on RNA interference of interactors in combination with measurement of protein stability and localization. As an example, we detail the role of a SLC16A6 phospho-degron, and the contributions of PDZ-domain proteins LIN7C and MPP1 to the trafficking of SLC43A2. Overall, our work offers a resource for SLC-protein interactions for the scientific community.

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Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

Gorostiola González,

Sijben,

Dall’ Acqua

et al. 2023

Front. Mol. Biosci.

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Glutamate is an essential excitatory neurotransmitter and an intermediate for energy metabolism. Depending on the tumor site, cancer cells have increased or decreased expression of excitatory amino acid transporter 1 or 2 (EAAT1/2, SLC1A3/2) to regulate glutamate uptake for the benefit of tumor growth. Thus, EAAT1/2 may be an attractive target for therapeutic intervention in oncology. Genetic variation of EAAT1 has been associated with rare cases of episodic ataxia, but the occurrence and functional contribution of EAAT1 mutants in other diseases, such as cancer, is poorly understood. Here, 105 unique somatic EAAT1 mutations were identified in cancer patients from the Genomic Data Commons dataset. Using EAAT1 crystal structures and in silico studies, eight mutations were selected based on their close proximity to the orthosteric or allosteric ligand binding sites and the predicted change in ligand binding affinity. In vitro functional assessment in a live-cell, impedance-based phenotypic assay demonstrated that these mutants differentially affect L-glutamate and L-aspartate transport, as well as the inhibitory potency of an orthosteric (TFB-TBOA) and allosteric (UCPH-101) inhibitor. Moreover, two episodic ataxia-related mutants displayed functional responses that were in line with literature, which confirmed the validity of our assay. Of note, ataxia-related mutant M128R displayed inhibitor-induced functional responses never described before. Finally, molecular dynamics (MD) simulations were performed to gain mechanistic insights into the observed functional effects. Taken together, the results in this work demonstrate 1) the suitability of the label-free phenotypic method to assess functional variation of EAAT1 mutants and 2) the opportunity and challenges of using in silico techniques to rationalize the in vitro phenotype of disease-relevant mutants.

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

Cited by 5 publications

References 80 publications

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

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

The solute carrier superfamily interactome

Molecular insights into disease-associated glutamate transporter (EAAT1 / SLC1A3) variants using in silico and in vitro approaches

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