Concentrative Nucleoside Transporter Protein Research Articles

Who Is Who in Adenosine Transport.

Extracellular adenosine concentrations are regulated by a panel of membrane transporters which, in most cases, mediate its uptake into cells. Adenosine transporters belong to two gene families encoding Equilibrative and Concentrative Nucleoside Transporter proteins (ENTs and CNTs, respectively). The lack of appropriate pharmacological tools targeting every transporter subtype has introduced some bias on the current knowledge of the role of these transporters in modulating adenosine levels. In this regard, ENT1, for which pharmacology is relatively well-developed, has often been identified as a major player in purinergic signaling. Nevertheless, other transporters such as CNT2 and CNT3 can also contribute to purinergic modulation based on their high affinity for adenosine and concentrative capacity. Moreover, both transporter proteins have also been shown to be under purinergic regulation via P1 receptors in different cell types, which further supports its relevance in purinergic signaling. Thus, several transporter proteins regulate extracellular adenosine levels. Moreover, CNT and ENT proteins are differentially expressed in tissues but also in particular cell types. Accordingly, transporter-mediated fine tuning of adenosine levels is cell and tissue specific. Future developments focusing on CNT pharmacology are needed to unveil transporter subtype-specific events.

Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket

SLC28 genes, encoding concentrative nucleoside transporter proteins (CNT), show little genetic variability, although a few single nucleotide polymorphisms (SNPs) have been associated with marked functional disturbances. In particular, human CNT1S546P had been reported to result in negligible thymidine uptake. In this study we have characterized the molecular mechanisms responsible for this apparent loss of function. The hCNT1S546P variant showed an appropriate endoplasmic reticulum export and insertion into the plasma membrane, whereas loss of nucleoside translocation ability affected all tested nucleoside and nucleoside-derived drugs. Site-directed mutagenesis analysis revealed that it is the lack of the serine residue itself responsible for the loss of hCNT1 function. This serine residue is highly conserved, and mutation of the analogous serine in hCNT2 (Ser541) and hCNT3 (Ser568) resulted in total and partial loss of function, respectively. Moreover, hCNT3, the only member that shows a 2Na(+)/1 nucleoside stoichiometry, showed altered Na(+) binding properties associated with a shift in the Hill coefficient, consistent with one Na(+) binding site being affected by the mutation. Two-electrode voltage-clamp studies using the hCNT1S546P mutant revealed the occurrence of Na(+) leak, which was dependent on the concentration of extracellular Na(+) indicating that, although the variant is unable to transport nucleosides, there is an uncoupled sodium transport.

Differential Transport of Cytosine-Containing Nucleosides by Recombinant Human Concentrative Nucleoside Transporter Protein hCNT1

The transportability of cytosine-containing nucleosides by recombinant hCNT1 was investigated in transfected mammalian cells. Apparent Km values for hCNT1-mediated transport of uridine, cytidine and deoxycytidine were, respectively, 59, 140 and 150 μM. Uridine transport was inhibited 89, 32 and 11%, respectively, by 500 μM gemcitabine, cytarabine and lamivudine, demonstrating that, unlike gemcitabine (a high-affinity hCNT1 permeant), cytarabine and lamivudine are poor hCNT1 permeants.

Functional characterization of a recombinant sodium-dependent nucleoside transporter with selectivity for pyrimidine nucleosides (cNT1rat) by transient expression in cultured mammalian cells.

We have demonstrated that monkey kidney (COS-1) cells have a single type of nucleoside transport process, which, because it was equilibrative, sodium-independent and could be inhibited by nitrobenzylthioinosine (NBMPR), was identified as the 'equilibrative sensitive' or 'es' transporter. Using NBMPR or dilazep to inhibit the endogenous nucleoside transport activity, we have transiently expressed a cDNA that encodes an inhibitor-insensitive, concentrative nucleoside transporter protein (cNT1rat) of rat intestine in COS-1 cells. The production of recombinant cNT1rat was examined by immunoblotting using an epitope-tagged construct and by analysis of inward fluxes of 3H-labelled nucleosides. Recombinant cNT1rat was sodium-dependent and selective for pyrimidine nucleosides, with approximately Km values of 21 microM, 12.5 microM and 15 microM for uridine, thymidine and adenosine, respectively. Although adenosine exhibited high affinity for the recombinant transporter, its Vmax value was low. A variety of anti-viral and anti-cancer nucleoside drugs inhibited cNT1rat-mediated uptake of uridine by transfected COS-1 cells although to different extents (Floxidine > Idoxuridine > Zidovudine > Zalcitabine > Cytarabine > Gemcitabine), suggesting that the concentrative pyrimidine-selective nucleoside transporters, of which cNT1rat is a representative, may play a role in cellular uptake of these drugs. The cNT1rat/COS-1 expression system is a useful tool for analysis of cNT1rat-mediated transport processes.