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 comparative structure
models, we cross-linked hCNT3 protomers in a cell-based assay, thus
showing the existence of hCNT3 homotrimers in human cells. The presence
and absence of cross-links at specific locations along TM9 informs
us of important structural differences between vcCNT and hCNT3. Comparative
modeling of the trimerization domain and sequence coevolution analysis
both indicate that oligomerization is critical to the stability and
function of hCNT3. In particular, trimerization appears to shorten
the translocation path for nucleosides across the plasma membrane
and may allow modulation of the transport function via allostery.