Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding

Biochim Biophys Acta Biomembr. 2017 Nov;1859(11):2193-2202. doi: 10.1016/j.bbamem.2017.08.006. Epub 2017 Aug 9.


The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and folate transport across the choroid plexus. This report addresses the structure/function of the 8th transmembrane helix. Based upon biotinylation of cysteine-substituted residues by MTSEA-biotin, 14 contiguous exofacial residues to Leu316 were accessible to the extracellular compartment of the 23 residues in this helix (Leu303-Leu325). Pemetrexed blocked biotinylation of six Cys-substituted residues deep within the helix implicating an important role for this region in folate binding. Accessibility decreased at 4°C vs RT. The influx Kt, Ki and Vmax were markedly increased for the P314C mutant, similar to what was observed for Y315A and Y315P mutants. However, the Kt, alone, was increased for the P314Y mutant. To correlate these observations with PCFT structural changes during the transport cycle, homology models were built for PCFT based upon the recently reported structures of bovine and rodent GLUT5 fructose transporters in the inward-open and outward- open conformations, respectively. The models predict substantial structural alterations in the exofacial region of the eighth transmembrane helix as it cycles between its conformational states that can account for the extended and contiguous aqueous accessibility of this region of the helix. Further, a helix break in one of the two conformations can account for the critical roles Pro314 and Tyr315, located in this region, play in PCFT function. The data indicates that the 8th transmembrane helix of PCFT plays an important role in defining the aqueous channel and the folate binding pocket.

Keywords: Folate; Homology modeling; Membrane transport; Slc46A1; Transmembrane domain; Transporter.

MeSH terms

  • Amino Acid Substitution
  • Binding Sites / genetics
  • Cell Membrane / chemistry*
  • Cell Membrane / metabolism
  • Folic Acid / metabolism*
  • HeLa Cells
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Protein Binding / genetics
  • Protein Domains / genetics
  • Protein Interaction Domains and Motifs* / genetics
  • Protein Transport / genetics
  • Proton-Coupled Folate Transporter / chemistry*
  • Proton-Coupled Folate Transporter / genetics
  • Proton-Coupled Folate Transporter / metabolism*
  • Water / chemistry


  • Membrane Proteins
  • Mutant Proteins
  • Proton-Coupled Folate Transporter
  • SLC46A1 protein, human
  • Water
  • Folic Acid