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Dadun > Depósito Académico > Facultad de Farmacia > Departamento de Ciencias de la Alimentación, Fisiología y Toxicología > DA - Farmacia - CAFT - Artículos de revista >

Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.
Authors: Larrayoz, I.M. (Ignacio M.)
Fernandez-Nistal, A. (Alonso)
Garces, A. (Aitziber)
Gorraiz, E. (Edurne)
Lostao, M.P. (María Pilar)
Keywords: NA+ glucose cotransporter
Chromosomal localization
Voltage clamp
Issue Date: 2006
Publisher: American Physiological Society
Publisher version:
ISSN: 0363-6143
Citation: Larrayoz IM, Fernandez-Nistal A, Garces A, Gorraitz E, Lostao MP. Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods. Am J Physiol Cell Physiol. 2006 Dec;291(6):C1395-404.
The Na(+)-dependent nucleoside transporter 2 (CNT2) mediates active transport of purine nucleosides and uridine as well as therapeutic nucleoside analogs. We used the two-electrode voltage-clamp technique to investigate rat CNT2 (rCNT2) transport mechanism and study the interaction of nucleoside-derived drugs with the transporter expressed in Xenopus laevis oocytes. The kinetic parameters for sodium, natural nucleosides, and nucleoside derivatives were obtained as a function of membrane potential. For natural substrates, apparent affinity (K(0.5)) was in the low micromolar range (12-34) and was voltage independent for hyperpolarizing membrane potentials, whereas maximal current (I(max)) was voltage dependent. Uridine and 2'-deoxyuridine analogs modified at the 5-position were substrates of rCNT2. Lack of the 2'-hydroxyl group decreased affinity but increased I(max). Increase in the size and decrease in the electronegativity of the residue at the 5-position affected the interaction with the transporter by decreasing both affinity and I(max). Fludarabine and formycin B were also transported with higher I(max) than uridine and moderate affinity (102 +/- 10 and 66 +/- 6 microM, respectively). Analysis of the pre-steady-state currents revealed a half-maximal activation voltage of about -39 mV and a valence of about -0.8. K(0.5) for Na(+) was 2.3 mM at -50 mV and decreased at hyperpolarizing membrane potentials. The Hill coefficient was 1 at all voltages. Direct measurements of radiolabeled nucleoside fluxes with the charge associated showed a ratio of two positive inward charges per nucleoside, suggesting a stoichiometry of two Na(+) per nucleoside. This discrepancy in the number of Na(+) molecules that bind rCNT2 may indicate a low degree of cooperativity between the Na(+) binding sites.
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