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

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.