title: Characterization of a Madin-Darby canine kidney cell line stably expressing TRPV5.
abstract: 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.
authors.affiliation: 160 Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500, HB Nijmegen, The Netherlands.
authors.forename: Els
authors.initials: E
authors.lastname: den Dekker
authors.forename: Joost
authors.initials: J
authors.lastname: Schoeber
authors.forename: Catalin N
authors.initials: CN
authors.lastname: Topala
authors.forename: Stan F J
authors.initials: SF
authors.lastname: van de Graaf
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D014162:Transfection
chemical_list: D015220:Calcium Channels; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium