Simultaneous expression of hCNT1-CFP and hENT1-YFP in Madin-Darby canine kidney cells. Localization and vectorial transport studies

• Published in: The Journal of biological chemistry 2002-10, Vol.277 (40), p.37711-37717
• Main Authors: Lai, Yurong, Bakken, Aimee H, Unadkat, Jashvant D
• Format: Article
• Language: English
• Subjects: Adenosine - metabolism; Alkaline Phosphatase - metabolism; Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological Transport; Cell Differentiation - physiology; Cell Line; Cell Membrane - metabolism; Deoxyadenosines - metabolism; Dogs; Equilibrative Nucleoside Transporter 1 - genetics; Equilibrative Nucleoside Transporter 1 - metabolism; Green Fluorescent Proteins; Humans; Kidney; Kinetics; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Microscopy, Confocal; Purine Nucleosides - metabolism; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - metabolism; Transfection; Uridine - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M204986200

To test the hypothesis that human concentrative and equilibrative nucleoside transporters (hCNT1 and hENT1) are present on the apical and basolateral membrane, respectively, we constructed a Madin-Darby canine kidney (MDCK) cell line that simultaneously and stably expresses recombinant hCNT1 and hENT1 gene products tagged with CFP and YFP fluorescent proteins, respectively. Using a confocal microscope, both hCNT1-CFP and hENT1-YFP were found to be distributed uniformly on the plasma membrane of undifferentiated MDCK cells. Upon differentiation of the MDCK cells on Transwell filter inserts, hCNT1-CFP was visualized exclusively on the apical membrane, whereas hENT1-YFP appeared predominantly on the basolateral membrane. As differentiation proceeded, there was an increase in alkaline phosphatase activity, and activity of hENT1 in the apical compartment decreased while hCNT1 activity remained constant. These results suggest that, on differentiation, hENT1 is sorted to the basolateral membrane. This was confirmed when the hCNT1-mediated uptake of [(3)H]uridine from the apical compartment of the differentiated cells was found to be approximately 20-fold higher and that for hENT1 was approximately 4-fold lower than the corresponding uptake from the basal compartment. As observed in vivo, the net transport of [(3)H]adenosine was from the apical to the basal compartment, whereas that for (14)C-deoxyadenosine was from the basal to the apical compartment. In summary, we have shown for the first time that hCNT1 and hENT1 are expressed in polarized MDCK cells on the apical and basolateral membrane, respectively, allowing vectorial transport in both directions depending on the relative activity (ratio of maximal transporter activity to affinity) of each transporter for their substrates.