Vol. 299, Issue 2, 735-740, November 2001

Differential Kinetics of Transport of 2',3'-Dideoxyinosine and Adenosine via Concentrative Na⁺ Nucleoside Transporter CNT2 Cloned from Rat Blood-Brain Barrier

Jian Yi Li, Ruben J. Boado and William M. Pardridge

Department of Medicine, UCLA School of Medicine, Los Angeles, California

The concentrative Na⁺ nucleoside transporter type 2 (CNT2), cloned from a rat blood-brain barrier cDNA library, yields very high flux ratios for purine nucleosides after expression in frog oocytes. This high activity of the rat CNT2 produced from the blood-brain barrier-derived cDNA, designated clone A-11, enabled a kinetic analysis of 2',3'-dideoxyinosine transport via the rat CNT2. CNT2 transported both adenosine and 2',3'-dideoxyinosine. The 2',3'-dideoxyinosine transport parameters included a K_m of 29.2 ± 8.3 µM, a V_max of 0.40 ± 0.11 pmol/oocyte/min, and a constant of nonsaturable transport (K_D) of 15.7 ± 0.6 nl/oocyte/min. The 2',3'-dideoxyinosine V_max was 27-fold lower than the adenosine V_max and the 2',3'-dideoxyinosine K_D was >15-fold greater than the K_D of adenosine transport. Adenosine inhibited both the saturable component of 2',3'-dideoxyinosine transport with a K_I of 14.8 ± 1.6 µM, and inhibited the nonsaturable component of 2',3'-dideoxyinosine transport. Both the saturable and nonsaturable components of 2',3'-dideoxyinosine transport were sodium-dependent with a sodium K_0.5 of 8.7 ± 0.9 mM, and a Hill coefficient of 1.00 ± 0.10. The transport of 2',3'-dideoxyinosine was strongly inhibited by thymidine, whereas thymidine was a weak inhibitor of adenosine transport via rat CNT2. Thymidine was transported by rat CNT2 with a K_m = 130 ± 44 µM and a V_max = 1.7 ± 0.5 pmol/oocyte/min. These studies provide evidence for asymmetric transport sites on rat CNT2, where 2',3'-dideoxyinosine and thymidine compete selectively at a low V_max site on the transporter, whereas adenosine is transported at a high V_max site.