Electrophysiological characterization of a recombinant human Na+-coupled nucleoside transporter (hCNT1) produced in Xenopus oocytes

J Physiol. 2004 Aug 1;558(Pt 3):807-23. doi: 10.1113/jphysiol.2004.068189. Epub 2004 Jun 11.


Human concentrative nucleoside transporter 1 (hCNT1) mediates active transport of nucleosides and anticancer and antiviral nucleoside drugs across cell membranes by coupling influx to the movement of Na(+) down its electrochemical gradient. The two-microelectrode voltage-clamp technique was used to measure steady-state and presteady-state currents of recombinant hCNT1 produced in Xenopus oocytes. Transport was electrogenic, phloridzin sensitive and specific for pyrimidine nucleosides and adenosine. Nucleoside analogues that induced inwardly directed Na(+) currents included the anticancer drugs 5-fluorouridine, 5-fluoro-2'-deoxyuridine, cladribine and cytarabine, the antiviral drugs zidovudine and zalcitabine, and the novel thymidine mimics 1-(2-deoxy-beta-d-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-beta-d-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent K(m) values for 5-fluorouridine, 5-fluoro-2'-deoxyuridine and zidovudine were 18, 15 and 450 microm, respectively. hCNT1 was Na(+) specific, and the kinetics of steady-state uridine-evoked Na(+) currents were consistent with an ordered simultaneous transport model in which Na(+) binds first followed by uridine. Membrane potential influenced both ion binding and carrier translocation. The Na(+)-nucleoside coupling stoichiometry, determined directly by comparing the uridine-induced inward charge movement to [(14)C]uridine uptake was 1: 1. hCNT1 presteady-state currents were used to determine the fraction of the membrane field sensed by Na(+) (61%), the valency of the movable charge (-0.81) and the average number of transporters present in the oocyte plasma membrane (6.8 x 10(10) per cell). The hCNT1 turnover rate at -50 mV was 9.6 molecules of uridine transported per second.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / metabolism
  • Antineoplastic Agents / pharmacology
  • Biological Transport / drug effects
  • Biological Transport / physiology
  • Dose-Response Relationship, Drug
  • Electrophysiology
  • Female
  • Humans
  • Membrane Transport Proteins / metabolism
  • Membrane Transport Proteins / physiology*
  • Nucleosides / chemistry
  • Nucleosides / metabolism
  • Nucleosides / pharmacology
  • Oocytes / metabolism*
  • Protein Binding / drug effects
  • Protein Binding / physiology
  • Recombinant Proteins / metabolism*
  • Xenopus laevis


  • Antineoplastic Agents
  • Membrane Transport Proteins
  • Nucleosides
  • Recombinant Proteins
  • cif nucleoside transporter