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

Biochemistry. 2017 Jul 11;56(27):3475-3483. doi: 10.1021/acs.biochem.7b00339. Epub 2017 Jun 29.


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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Absorption, Physiological
  • Animals
  • Cell Line
  • Chromatography, Gel
  • Computer Simulation
  • Cross-Linking Reagents / chemistry
  • Glutaral / chemistry
  • Humans
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism
  • Models, Molecular*
  • Molecular Conformation
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Interaction Domains and Motifs
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Sus scrofa
  • Tritium
  • Uridine / metabolism


  • Cross-Linking Reagents
  • Lipid Bilayers
  • Membrane Transport Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • cif nucleoside transporter
  • Tritium
  • Glutaral
  • Uridine