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First published online on June 11, 2004.
 Copyright © 2004 by The Physiological Society
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jphysiol.2004.068189v1
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Received May 19, 2004
Revised May 24, 2004
Accepted after revision June 4, 2004

Electrophysiological Characterization of a Recombinant Human Na+-coupled Nucleoside Transporter (hCNT1) Produced in Xenopus Oocytes

Kyla M Smith1, Amy M.L. Ng1, Sylvia Y.M. Yao1, Kathy A Labedz2, Edward E Knaus1, Leonard I Wiebe1, Carol E Cass3, Stephen A Baldwin4, Xing-Zhen Chen1, Edward Karpinski5, and James D Young1*

1 University of Alberta
2 Univeristy of Alberta
3 University of Alberta/ Cross Cancer Institute
4 School of Biochemistry and Microbiology, University of Leeds
5 Unversity of Alberta

* To whom correspondence should be addressed. E-mail: james.young{at}ualberta.ca.

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 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 analogs 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. 5-Fluorouridine, 5-fluoro-2'-deoxyuridine and zidovudine apparent Km values 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 valence 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 s.


Key words: Electrophysiology • Oocyte • transporter




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