Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.
Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK(1) cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.
Mangravite
L M
LM
Department of Biopharmaceutical Sciences, Cardiovascular Research Institute, University of California-San Francisco, San Francisco, CA 94143-0446, USA.
Lipschutz
J H
JH
Mostov
K E
KE
Giacomini
K M
KM
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D010957:Plasmids / Q000235:genetics; D013552:Swine; D014162:Transfection; D014529:Uridine / Q000378:metabolism
D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; C065171:cif nucleoside transporter; D049452:Green Fluorescent Proteins; D014529:Uridine