The purine-preferring Na⁺-nucleoside cotransporter (rCNT2) was cloned from a rat blood-brain barrier cDNA library (Yi Li et al. 2001). In the present work, we have expressed rCNT2 in Xenopus laevis oocytes and used the two-electrode voltage-clamp method (Loo et al. 1993; Lostao et al. 1994; Díez-Sampedro et al. 2001) to study the substrate specificity, kinetics properties of Na⁺ and substrate as a function of voltage and stoichiometry.

The apparent affinity constant (K_0.5), at -50 mV membrane potential (V_m), for adenosine, guanosine and uridine was 13.8 ± 2.5 (S.E.M.), 23.5 ± 2 and 14.8 ± 6.1 µM, respectively, and the maximal current (I_max) was similar for the three nucleosides. Inosine, the cellular precursor of purine nucleosides, showed a K_0.5 of 31.2 ± 1.5 µM and an I_max half of that for adenosine. As expected, the pyrimidine nucleosides thymidine and cytidine did not induce any Na⁺ inward current or inhibited the uptake of 50 µM uridine, indicating that they do not interact with the transporter. Similarly, the pyrimidine-derived anticancer drugs gemcitabine and 5â-deoxy-5-fluorouridine do not induce any inward current. Two purine-derived drugs were also investigated. Fludarabine, currently used in the treatment of lymphoproliferative malignancies, exhibited an I_max 2-fold higher than that for adenosine and an affinity one order of magnitude lower than the adenosine affinity (K_0.5 = 378.1 ± 82.6 µM). Formycin B, used in the treatment of parasitic diseases, showed a K_0.5 of 66 ± 6.5 µM and an I_max also 2-fold higher than the adenosine I_max. For all substrates tested, I_max increased as the membrane potential was hyperpolarized from -10 to -150 mV, whereas K_0.5 was not affected by the membrane potential between -30 and -150 mV. The apparent affinity constant for Na⁺ was also independent of membrane potential (0.7 ± 0.2 mM at -150 mV and 1.3 ± 0.5 mM at -30 mV).

To determine the charge-to-nucleoside stoichiometry, radio-labelled substrate influx was measured under voltage-clamp conditions (V_m = -50 mV) resulting in two inward positive charges (most likely due to Na⁺) per molecule of transported nucleoside.

Finally, experiments performed in Na⁺ buffer in the absence of substrate, after rapid steps in membrane potential, revealed the presence of pre-steady-state currents which indicate charge transfer associated to the movement of the transporter in the membrane. The analysis of these currents will be useful to understand the mechanism of this transporter.

This work was supported by PIUNA (University of Navarra) and ‘Departamento de Educacíon y Cultura’ (Navarra Government).