Characterization of the rat Na+/nucleoside cotransporter 2 and transport of nucleoside-derived drugs using electrophysiological methods.
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.
Larráyoz
Ignacio M
IM
Department of Physiology and Nutrition, University of Navarra, Pamplona, Spain.
Fernández-Nistal
Alonso
A
Garcés
Aitziber
A
Gorraitz
Edurne
E
Lostao
M Pilar
MP
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D015195:Drug Design; D008564:Membrane Potentials / Q000502:physiology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D012964:Sodium / Q000378:metabolism; D014982:Xenopus laevis
D026901:Membrane Transport Proteins; D009705:Nucleosides; C065171:cif nucleoside transporter; D012964:Sodium