Stable polarized expression of hCAT-1 in an epithelial cell line

• Published in: The Journal of membrane biology Vol. 186; no. 1; pp. 23 - 30
• Main Authors: Cariappa, R, Heath-Monnig, E, Furesz, T C, Kamath, S G, Smith, C H
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.03.2002
• Subjects: Animals; Cationic Amino Acid Transporter 1 - genetics; Cationic Amino Acid Transporter 1 - metabolism; Cell Polarity - physiology; Cells, Cultured; Dogs; Epithelial Cells - metabolism; Green Fluorescent Proteins; Humans; Ion Transport - physiology; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Lysine - metabolism; Microscopy, Confocal; Oocytes - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Subcellular Fractions - metabolism; Transfection; Xenopus
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/s00232-001-0133-y

Our laboratory has recently identified and cloned three cationic amino-acid transporters of human placenta. We have now examined the plasma membrane domain localization and functional expression of one of these transporters, hCAT-1, in a polarized epithelial cell line (MDCK). To facilitate identification of expressed protein we first transferred the hCAT-1 cDNA to a vector with C-terminal green fluorescent protein (GFP). The resultant hCAT-1-CT-GFP fusion protein stimulated L-[3H] lysine uptake in Xenopus oocytes. In confluent monolayers of stably transfected cells grown on porous nitrocellulose filters, saturable uptake of L-[3H] lysine from the basolateral surface was stimulated 7-fold over that of untransfected cells. Concentration-dependence studies in Na+-free medium at pH 7.4 demonstrated a Km of approximately 68 +/- 13 microM and a Vmax of 970 +/-170 pmol/mg protein/min. Uptake from the apical plasma membrane surface was negligible in both transfected and untransfected cells. Consistent with these results, confocal microscopy of confluent monolayers of hCAT-1-CT-GFP-expressing cells revealed localization of the transporter solely on the basolateral domain of the cell. This is apparently the first report of a cultured polarized epithelial cell model for stable expression of a cationic amino-acid transporter. It has the potential to aid in the identification of targeting signals for transport protein localization.