Molecular and functional characterization of choline transporter in human colon carcinoma HT-29 cells

Arch Biochem Biophys. 2009 Mar 1;483(1):90-8. doi: 10.1016/j.abb.2008.12.008. Epub 2008 Dec 25.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport, Active / drug effects
  • Cation Transport Proteins / antagonists & inhibitors
  • Cation Transport Proteins / genetics
  • Cation Transport Proteins / metabolism
  • Choline / metabolism*
  • Colonic Neoplasms / genetics
  • Colonic Neoplasms / metabolism*
  • HT29 Cells
  • Hemicholinium 3 / pharmacology
  • Humans
  • Kinetics
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Proton-Motive Force
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Sodium-Hydrogen Exchanger 1
  • Sodium-Hydrogen Exchangers / antagonists & inhibitors
  • Sodium-Hydrogen Exchangers / genetics
  • Sodium-Hydrogen Exchangers / metabolism
  • Symporters / genetics
  • Symporters / metabolism

Substances

  • Cation Transport Proteins
  • Membrane Glycoproteins
  • Membrane Transport Proteins
  • RNA, Messenger
  • SLC44A2 protein, human
  • SLC5A7 protein, human
  • SLC9A1 protein, human
  • Sodium-Hydrogen Exchanger 1
  • Sodium-Hydrogen Exchangers
  • Symporters
  • choline transporter
  • Hemicholinium 3
  • Choline