title: Inhibition of N-linked glycosylation affects organic cation transport across the brush border membrane of opossum kidney (OK) cells.
abstract: Many organic cations are transported across the apical membrane of the proximal tubule by specific saturable mechanisms. The goal of this study was to determine if the transporter for tetraethylammonium (TEA) in the brush border membrane of an established opossum kidney (OK) cell line is glycosylated and to elucidate the function of this glycosylation. The uptake of TEA was determined in OK cell monolayers treated with tunicamycin (TM), a compound that prevents synthesis of the core oligosaccharide precursor molecules. TM exposure significantly decreased the incorporation of [3H]mannose in OK cell proteins and significantly reduced TEA uptake in a time and a concentration dependent manner. No effect of TM exposure on cellular protein synthesis, DNA content, cell viability, or on [3H]proline uptake was observed. The transport of TEA in control cells was characterized by a Km of 26.9 +/- 16.4 microM and a Vmax of 378 +/- 39 pmol/mg of protein/min. TM treatment (1 microgram/ml for 21 h) significantly increased the Km by over 4-fold to 111.5 +/- 18.4 microM while not affecting the Vmax. The apparent KI values of other organic cations known to interact with this transport system were also significantly increased by TM exposure. Estimated KI values of N1-methylnicotinamide, cimetidine, and mepiperphenidol increased by 6-fold, 4-fold, and 2-fold, respectively, after exposure of OK cells to TM. An increased KI for protons was also observed. Additional inhibitors of the N-linked glycosylation pathway, castanospermine, deoxynojirimycin, and deoxymannojirimycin significantly decreased TEA transport, whereas swainsonine had no effect. Our results suggest that the organic cation transporter is glycosylated. The N-linked oligosaccharide side chain appears to be of the hybrid type, and it either directly or indirectly affects the binding site of the transporter for both organic cations and protons. This is the first report describing the importance of glycosylation in the function of the organic cation transporter in the apical membrane of OK cells.
authors.affiliation: School of Pharmacy, University of California, San Francisco 94143.
authors.forename: R J
authors.initials: RJ
authors.lastname: Ott
authors.forename: A C
authors.initials: AC
authors.lastname: Hui
authors.forename: K M
authors.initials: KM
authors.lastname: Giacomini
mesh_terms: D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002412:Cations / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism; D009893:Opossums; D019789:Tetraethylammonium; D013757:Tetraethylammonium Compounds / Q000378:metabolism; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D002412:Cations; D006023:Glycoproteins; D013757:Tetraethylammonium Compounds; D014415:Tunicamycin; D019789:Tetraethylammonium