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

Abstract

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-β-d-ribofuranosyl)-2,4-difluoro-5-methylbenzene and 1-(2-deoxy-β-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 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 × 10¹⁰ per cell). The hCNT1 turnover rate at −50 mV was 9.6 molecules of uridine transported per second.