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HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport
The Human Embryonic Kidney 293 cell line (HEK‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistiv...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Wiley Periodicals, Inc.
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270233/ https://www.ncbi.nlm.nih.gov/pubmed/25263207 http://dx.doi.org/10.14814/phy2.12158 |
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author | Domingue, Jada C. Ao, Mei Sarathy, Jayashree George, Alvin Alrefai, Waddah A. Nelson, Deborah J. Rao, Mrinalini C. |
author_facet | Domingue, Jada C. Ao, Mei Sarathy, Jayashree George, Alvin Alrefai, Waddah A. Nelson, Deborah J. Rao, Mrinalini C. |
author_sort | Domingue, Jada C. |
collection | PubMed |
description | The Human Embryonic Kidney 293 cell line (HEK‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK‐293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channel. While HEK‐293 cells endogenously express mRNA for the Cl(−) channels, ClC‐2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK‐293 cells with EGFP‐CFTR (HEK‐CFTR) and demonstrated CFTR function by measuring forskolin‐stimulated iodide efflux. This efflux was inhibited by CFTR(inh)172, and the protein kinase A inhibitor H89, but not by Ca(2+) chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK‐CFTR. To investigate the role of an endogenous Gα(S)‐coupled receptor, we examined the bile acid receptor, TGR5. Although HEK‐CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5–500 μmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP](i) in HEK‐CFTR suggesting that endogenous TGR5 may not be functionally linked to Gα(S). However, LCA did increase [Ca(2+)](i) and interestingly, abolished forskolin‐stimulated iodide efflux. Thus, we propose that the stable HEK‐CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR. |
format | Online Article Text |
id | pubmed-4270233 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Wiley Periodicals, Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-42702332014-12-24 HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport Domingue, Jada C. Ao, Mei Sarathy, Jayashree George, Alvin Alrefai, Waddah A. Nelson, Deborah J. Rao, Mrinalini C. Physiol Rep Original Research The Human Embryonic Kidney 293 cell line (HEK‐293) readily lends itself to genetic manipulation and is a common tool for biologists to overexpress proteins of interest and study their function and molecular regulation. Although these cells have some limitations, such as an inability to form resistive monolayers necessary for studying transepithelial ion transport, they are nevertheless valuable in studying individual epithelial ion transporters. We report the use of HEK‐293 cells to study the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(−) channel. While HEK‐293 cells endogenously express mRNA for the Cl(−) channels, ClC‐2 and TMEM16A, they neither express CFTR mRNA nor protein. Therefore, we stably transfected HEK‐293 cells with EGFP‐CFTR (HEK‐CFTR) and demonstrated CFTR function by measuring forskolin‐stimulated iodide efflux. This efflux was inhibited by CFTR(inh)172, and the protein kinase A inhibitor H89, but not by Ca(2+) chelation. In contrast to intestinal epithelia, forskolin stimulation does not increase surface CFTR expression and does not require intact microtubules in HEK‐CFTR. To investigate the role of an endogenous Gα(S)‐coupled receptor, we examined the bile acid receptor, TGR5. Although HEK‐CFTR cells express TGR5, the potent TGR5 agonist lithocholic acid (LCA; 5–500 μmol/L) did not activate CFTR. Furthermore, forskolin, but not LCA, increased [cAMP](i) in HEK‐CFTR suggesting that endogenous TGR5 may not be functionally linked to Gα(S). However, LCA did increase [Ca(2+)](i) and interestingly, abolished forskolin‐stimulated iodide efflux. Thus, we propose that the stable HEK‐CFTR cell line is a useful model to study the multiple signaling pathways that regulate CFTR. Wiley Periodicals, Inc. 2014-09-28 /pmc/articles/PMC4270233/ /pubmed/25263207 http://dx.doi.org/10.14814/phy2.12158 Text en © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society. http://creativecommons.org/licenses/by/3.0/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Original Research Domingue, Jada C. Ao, Mei Sarathy, Jayashree George, Alvin Alrefai, Waddah A. Nelson, Deborah J. Rao, Mrinalini C. HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title | HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title_full | HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title_fullStr | HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title_full_unstemmed | HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title_short | HEK‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (CFTR): a model for studying regulation of Cl(−) transport |
title_sort | hek‐293 cells expressing the cystic fibrosis transmembrane conductance regulator (cftr): a model for studying regulation of cl(−) transport |
topic | Original Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270233/ https://www.ncbi.nlm.nih.gov/pubmed/25263207 http://dx.doi.org/10.14814/phy2.12158 |
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