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Papers In Press, published online ahead of print December 29, 2003
J. Biol. Chem, 10.1074/jbc.M311940200
Submitted on October 31, 2003
Revised on December 19, 2003
Accepted on December 22, 2003

Electrophysiological characterization of the human Na+/nucleoside cotransporter 1 (hCNT1) and role of adenosine on hCNT1 function

Ignacio M. Larrayoz, Francisco Javier Casado, Marcal Pastor-Anglada, and M. Pilar Lostao

Fisiologia y Nutricion, Universidad de Navarra, Pamplona, Navarra 31008

Corresponding Author: plostao{at}unav.es

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-FU. 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 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 Ki of 6.5 mu M. The kinetic parameters for uridine, gemcitabine and 5´-DFUR were studied as a function of membrane potential: at -50 mV, K0.5 was 37, 18 and 245 mu M respectively and remained voltage independent. Imax for gemcitabine was voltage-independent and account for ~ 40 % of that for uridine at -50 mV. Maximal current for 5´-DFUR was voltage-dependent and ~ 150 % of that for uridine at all membrane potentials. for Na+ was voltage-independent at hyperpolarized membrane potential (1.25 mM at -50 mV) while resulted 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 1.85 positive inward charges per nucleoside and 1.07 Na+ per positive inward charge, suggesting a stoichiometry of 2 Na+/nucleoside.


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