Electrophysiological Characterization of the Human Na +/Nucleoside Cotransporter 1 (hCNT1) and Role of Adenosine on hCNT1 Function

Abstract

We previously reported that the human Na+/nucleoside transporter pyrimidine-preferring 1 (hCNT1) is electrogenic and transports gemcitabine and 5′-deoxy-5-fluorouridine, a precursor of the active drug 5-fluorouracil. Nevertheless, a complete electrophysiological characterization of the basic properties of hCNT1-mediated translocation has not been performed yet, and the exact role of adenosine in hCNT1 function has not been addressed either. In the present work we have used the two-electrode voltage clamp technique to investigate hCNT1 transport mechanism and study the kinetic properties of adenosine as an inhibitor of hCNT1. We show that hCNT1 exhibits presteady-state currents that disappear upon the addition of adenosine or uridine. Adenosine, a purine nucleoside described as a substrate of the pyrimidine-preferring transporters, is not a substrate of hCNT1 but a high affinity blocker able to inhibit uridine-induced inward currents, the Na +-leak currents, and the presteady-state currents, with a K i of 6.5 μM. The kinetic parameters for uridine, gemcitabine, and 5′-deoxy-5-fluorouridine were studied as a function of membrane potential; at -59 mV, K0.5 was 37, 18, and 245 μM, respectively, and remained voltage-independent. Imax for gemcitabine was voltage-independent and accounts for ∼40% that for uridine at -59 mV. Maximal current for 5′-DFUR was voltage-dependent and was ∼150% that for uridine at all membrane potentials. K0.5Na+ for Na+ was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas ImaxNa+ was voltage-dependent, increasing 2-fold from -50 to -150 mV. Direct measurements of 3H-nucleoside or 22Na fluxes with the charge-associated revealed a ratio of two positive inward charges per nucleoside and one Na + per positive inward charge, suggesting a stoichiometry of two Na+/nucleoside.