Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer

Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by...