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Membrane Protein Research Group, Departments of
1 Physiology
2 Oncology and Faculty of
3 Pharmacy, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
4 Cross Cancer Institute, Edmonton, Alberta T6G 2H7, Canada
5 School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK
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-ß-D-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-ß-D-ribofuranosyl)-2,4-difluoro-5-iodobenzene. Apparent Km values for 5-fluorouridine, 5-fluoro-2'-deoxyuridine and zidovudine were 18, 15 and 450 µM, 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 [14C]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 1010 per cell). The hCNT1 turnover rate at –50 mV was 9.6 molecules of uridine transported per second.
(Received 19 May 2004;
accepted after revision 4 June 2004;
first published online 11 June 2004)
Corresponding author J. D. Young: 7–55 Medical Sciences Building University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Email: james.young{at}ualberta.ca
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