| Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5. | |
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MedLine Citation:
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PMID: 15924239 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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To provide a cell model for studying specifically the regulation of Ca2+ entry by the epithelial calcium channel transient receptor potential-vanilloid-5 (TRPV5), green fluorescent protein (GFP)-tagged TRPV5 was expressed stably in Madin-Darby canine kidney type I (MDCK) cells. The localization of GFP-TRPV5 in this cell line showed an intracellular granular distribution. Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on plastic supports was threefold higher than in non-transfected cells. Moreover, apical Ca2+ uptake in GFP-TRPV5-MDCK cells cultured on permeable supports was eightfold higher than basolateral Ca2+ uptake, indicating that GFP-TRPV5 is expressed predominantly in the apical membrane. Patch-clamp analysis showed the presence of typical electrophysiological features of GFP-TRPV5, such as inwardly rectifying currents, inhibition by divalent cations and Ca2+-dependent inactivation. Moreover, the TRPV5 inhibitor ruthenium red completely inhibited Ca2+ uptake in GFP-TRPV5-MDCK cells, whereas Ca2+ uptake in non-transfected cells was not inhibited. The characterized GFP-TRPV5-MDCK cell line was used to assess the regulation of TRPV5. The protein kinase C activator phorbol 12-myristate 13-acetate and the cAMP-elevating compounds forskolin/3-isobutyl-1-methylxanthine, 8-Br-cAMP and PGE2 stimulated TRPV5 activity in GFP-TRPV5-MDCK cells by 121+/-7, 79+/-5, 55+/-4 and 61+/-7%, respectively. These compounds did not affect Ca2+ uptake in non-transfected cells. In conclusion, the GFP-TRPV5-MDCK cell line provides a model to specifically study the regulation of TRPV5 activity. |
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Authors:
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Els den Dekker; Joost Schoeber; Catalin N Topala; Stan F J van de Graaf; Joost G J Hoenderop; René J M Bindels |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2005-05-28 |
Journal Detail:
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Title: Pflügers Archiv : European journal of physiology Volume: 450 ISSN: 0031-6768 ISO Abbreviation: Pflugers Arch. Publication Date: 2005 Jul |
Date Detail:
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Created Date: 2005-07-11 Completed Date: 2005-11-23 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 0154720 Medline TA: Pflugers Arch Country: Germany |
Other Details:
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Languages: eng Pagination: 236-44 Citation Subset: IM |
Affiliation:
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160 Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500, HB Nijmegen, The Netherlands. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Calcium / metabolism* Calcium Channels / biosynthesis*, genetics Cell Line* Dogs Green Fluorescent Proteins / metabolism Humans Kidney Rabbits Recombinant Fusion Proteins Transfection |
| Chemical | |
Reg. No./Substance:
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0/Calcium Channels; 0/Recombinant Fusion Proteins; 147336-22-9/Green Fluorescent Proteins; 7440-70-2/Calcium |
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