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J. Biol. Chem., Vol. 279, Issue 10, 8999-9007, March 5, 2004

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

Ignacio M. Larráyoz, Francisco Javier Casado¶, Marçal Pastor-Anglada¶, and M. Pilar Lostao||

From the Departamento de Fisiología y Nutrición, Universidad de Navarra, Pamplona 31080, Spain and ^¶Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona E-08071, Spain

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 -50 mV, K_0.5 was 37, 18, and 245 µM, respectively, and remained voltage-independent. I_max for gemcitabine was voltage-independent and accounts for 40% that for uridine at -50 mV. Maximal current for 5'-DFUR was voltage-dependent and was 150% that for uridine at all membrane potentials. for Na⁺ was voltage-independent at hyperpolarized membrane potentials (1.2 mM at -50 mV), whereas 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.

Received for publication, October 31, 2003 , and in revised form, December 19, 2003.

^* This work has been supported by the Plan de Investigacíon Universidad de Navarra, Departamento de Educación of the Navarra Government and Ministerio de Ciencia y Tecnología (Spanish Government) Grants BFI2003-01371 (to M. P. L.) and SAF2002-0717 (to M. P.-A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

A fellow of the Asociación de Amigos (University of Navarra).

^|| To whom correspondence may be addressed: Departamento de Fisiología y Nutrición, Universidad de Navarra, c/Irunlarrea s/n, Pamplona 31080, Spain. Tel.: 948-42-56-00; Fax: 948-42-56-49; E-mail: plostao{at}unav.es.

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