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Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1
Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760145/ https://www.ncbi.nlm.nih.gov/pubmed/31551431 http://dx.doi.org/10.1038/s41598-019-48829-3 |
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author | Vlachodimou, Anna IJzerman, Adriaan P. Heitman, Laura H. |
author_facet | Vlachodimou, Anna IJzerman, Adriaan P. Heitman, Laura H. |
author_sort | Vlachodimou, Anna |
collection | PubMed |
description | Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs), transporter inhibition may affect GPCR signalling due to a change in extracellular concentration of the substrate/ligand, which can be monitored by an impedance-based label-free assay. For this study, a prototypical SLC/GPCR pair was selected, i.e. the equilibrative nucleoside transporter-1 (SLC29A1/ENT1) and an adenosine receptor (AR), for which adenosine is the substrate/ligand. ENT1 inhibition with three reference compounds was monitored sensitively via AR activation on human osteosarcoma cells. Firstly, the inhibitor addition resulted in an increased apparent potency of adenosine. Secondly, all inhibitors concentration-dependently increased the extracellular adenosine concentration, resulting in an indirect quantitative assessment of their potencies. Additionally, AR activation was abolished by AR antagonists, confirming that the monitored impedance was AR-mediated. In summary, we developed a novel assay as an in vitro model system that reliably assessed the potency of SLC29A1 inhibitors via AR signalling. As such, the method may be applied broadly as it has the potential to study a multitude of SLCs via concomitant GPCR signalling. |
format | Online Article Text |
id | pubmed-6760145 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-67601452019-11-12 Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 Vlachodimou, Anna IJzerman, Adriaan P. Heitman, Laura H. Sci Rep Article Transporters are important therapeutic but yet understudied targets due to lack of available assays. Here we describe a novel label-free, whole-cell method for the functional assessment of Solute Carrier (SLC) inhibitors. As many SLC substrates are also ligands for G protein-coupled receptors (GPCRs), transporter inhibition may affect GPCR signalling due to a change in extracellular concentration of the substrate/ligand, which can be monitored by an impedance-based label-free assay. For this study, a prototypical SLC/GPCR pair was selected, i.e. the equilibrative nucleoside transporter-1 (SLC29A1/ENT1) and an adenosine receptor (AR), for which adenosine is the substrate/ligand. ENT1 inhibition with three reference compounds was monitored sensitively via AR activation on human osteosarcoma cells. Firstly, the inhibitor addition resulted in an increased apparent potency of adenosine. Secondly, all inhibitors concentration-dependently increased the extracellular adenosine concentration, resulting in an indirect quantitative assessment of their potencies. Additionally, AR activation was abolished by AR antagonists, confirming that the monitored impedance was AR-mediated. In summary, we developed a novel assay as an in vitro model system that reliably assessed the potency of SLC29A1 inhibitors via AR signalling. As such, the method may be applied broadly as it has the potential to study a multitude of SLCs via concomitant GPCR signalling. Nature Publishing Group UK 2019-09-24 /pmc/articles/PMC6760145/ /pubmed/31551431 http://dx.doi.org/10.1038/s41598-019-48829-3 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Vlachodimou, Anna IJzerman, Adriaan P. Heitman, Laura H. Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title | Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title_full | Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title_fullStr | Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title_full_unstemmed | Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title_short | Label-free detection of transporter activity via GPCR signalling in living cells: A case for SLC29A1, the equilibrative nucleoside transporter 1 |
title_sort | label-free detection of transporter activity via gpcr signalling in living cells: a case for slc29a1, the equilibrative nucleoside transporter 1 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760145/ https://www.ncbi.nlm.nih.gov/pubmed/31551431 http://dx.doi.org/10.1038/s41598-019-48829-3 |
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