The human concentrative (Na(+)-linked) plasma membrane transport proteins hCNT1 and hCNT2 are selective for pyrimidine nucleosides (system cit) and purine nucleosides (system cif), respectively. Both have homologs in other mammalian species and belong to a gene family (CNT) that also includes hfCNT, a newly identified broad specificity pyrimidine and purine Na(+)-nucleoside symporter (system cib) from the ancient marine vertebrate, the Pacific hagfish (Eptatretus stouti). We now report the cDNA cloning and characterization of cib homologs of hfCNT from human mammary gland, differentiated human myeloid HL-60 cells, and mouse liver. The 691- and 703-residue human and mouse proteins, designated hCNT3 and mCNT3, respectively, were 79% identical in amino acid sequence and contained 13 putative transmembrane helices. hCNT3 was 48, 47, and 57% identical to hCNT1, hCNT2, and hfCNT, respectively. When produced in Xenopus oocytes, both proteins exhibited Na(+)-dependent cib-type functional activities. hCNT3 was electrogenic, and a sigmoidal dependence of uridine influx on Na(+) concentration indicated a Na(+):uridine coupling ratio of at least 2:1 for both hCNT3 and mCNT3 (cf 1:1 for hCNT1/2). Phorbol myristate acetate-induced differentiation of HL-60 cells led to the parallel appearance of cib-type activity and hCNT3 mRNA. Tissues containing hCNT3 transcripts included pancreas, bone marrow, trachea, mammary gland, liver, prostate, and regions of intestine, brain, and heart. The hCNT3 gene mapped to chromosome 9q22.2 and included an upstream phorbol myristate acetate response element.

We investigated the mechanism of the transport of ribavirin (1-beta-D-ribofuranosyl-1,2,4-trizole-3-carboxamide) into placental epithelial cells using human choriocarcinoma (BeWo) cells and Xenopus oocytes expressing human nucleoside transporters. In BeWo cells, when a relatively low concentration (123 nM) of ribavirin was used, both Na(+)-dependent uptake and -independent uptake of ribavirin were observed. On the other hand, when a higher concentration (100 microM) of ribavirin was used, Na(+)-independent uptake was observed, but there was only a slight Na(+)-dependent uptake. In Xenopus oocytes, influxes of ribavirin mediated by hCNT2 (concentrative nucleoside transporter 2), hCNT3 (concentrative nucleoside transporter 3), hENT1 (equilibrative nucleoside transporter 1) and hENT2 (equilibrative nucleoside transporter 2) were saturable, and apparent K(m) values were 18.0 microM, 14.2 microM, 3.46 mM and 3.71 mM, respectively. These data indicate that hCNT2 and hCNT3 have higher affinity for ribavirin than do hENT1 and hENT2. Moreover, analysis by RT-PCR showed that BeWo cells express mRNA of hCNT3, hENT1 and hENT2. These results suggest that ribavirin is taken up by BeWo cells via both the high-affinity Na(+)-dependent transporter hCNT3 and the low-affinity Na(+)-independent transporters hENT1 and hENT2.

We characterized the electrophysiology, kinetics, and quantitative structure-activity relationship (QSAR) of the human concentrative nucleoside transporter 3 (hCNT3) expressed in Xenopus laevis oocytes by measuring substrate-induced inward currents using a two-microelectrode voltage-clamp system. At membrane potentials between -30 and -150 mV, sodium activation of gemcitabine transport was sigmoidal, with a K0.5 of 8.5+/-0.3 mM for Na+ and a Hill coefficient of 2.2+/-0.25 independent of membrane potential. We measured the Imax and K0.5 for substrate at -50 mV for the nucleoside analog drugs gemcitabine (638+/-58 nA, 59.7+/-17.5 microM), ribavirin (546+/-37 nA, 61.0+/-13.2 microM), AZT (420+/-4 nA, 310+/-9 microM), and 3-deazauridine (506+/-30 nA, 50.8+/-9.90 microM). K0.5 and Imax for substrate were dependent on membrane potential (both increasing as the membrane became more hyperpolarized) for all four drugs. hCNT3 also exhibited pre-steady-state currents. The quantitative structure-activity relationship (QSAR) was examined using comparative molecular field analysis and comparative molecular similarity indices analysis of the inward currents induced by 27 nucleoside analogs with substitutions at both the ribose and the nucleobase. Two statistically significant QSAR models identified electrostatic interaction as the major force in hCNT3 transport and attributed a critical role to the 3'-hydroxyl position of hCNT3 substrates. Steric hindrance at the 3-position and positive charge at the 5-position of the pyrimidine ring were favorable for transport. Two hCNT3 pharmacophore models revealed the minimal features required for hCNT3 transport as two hydrogen bond acceptors at 3'-OH and 5'-O and the hydrophobic center occupied by the base ring.