Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells.
We examined the molecular and functional characterization of choline uptake in human colon carcinomas using the cell line HT-29. Furthermore, we explored the possible correlation between choline uptake and cell proliferation. Choline uptake was saturable and mediated by a single transport system. Interestingly, removal of Na(+) from the uptake buffer strongly enhanced choline uptake. This increase in component of choline uptake under Na(+)-free conditions was inhibited by a Na(+)/H(+) exchanger 1 (NHE1) inhibitor. Collapse of the plasma-membrane H(+) electrochemical gradient by a protonophore inhibited choline uptake. Choline uptake was inhibited by the choline analogue hemicholinium-3 (HC-3) and various organic cations, and was significantly decreased by acidification of the extracellular medium and by intracellular alkalinization. Real-time PCR revealed that choline transporter-like protein 1 (CTL1), CTL2, CTL4 and NHE1 mRNA are mainly expressed in HT-29 cells. Western blot and immunocytochemical analysis indicated that CTL1 protein was expressed in plasma membrane. The biochemical and pharmacological data indicated that CTL1 is functionally expressed in HT-29 cells and is responsible for choline uptake in these cells. We conclude that choline transporters, especially CTL1, use a directed H(+) gradient as a driving force, and its transport functions in co-operation with NHE1. Finally, cell proliferation was inhibited by HC-3 and tetrahexylammonium chloride (THA), which strongly inhibits choline uptake. Identification of this novel CTL1-mediated choline uptake system provides a potential new target for therapeutic intervention.
Kouji
Hironobu
H
Department of Pharmacology, Tokyo Medical University, Shinjuku-ku, Japan.
Inazu
Masato
M
Yamada
Tomoko
T
Tajima
Hirohisa
H
Aoki
Tatsuya
T
Matsumiya
Teruhiko
T
D001693:Biological Transport, Active / Q000187:drug effects; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002794:Choline / Q000378:metabolism*; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D019073:HT29 Cells; D006426:Hemicholinium 3 / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D018892:Proton-Motive Force; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000235:genetics / Q000378:metabolism
D027682:Cation Transport Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C484524:SLC44A2 protein, human; C510731:SLC5A7 protein, human; C490998:SLC9A1 protein, human; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D027981:Symporters; C065637:choline transporter; D006426:Hemicholinium 3; D002794:Choline