The functional roles of the His247 and His281 residues in folate and proton translocation mediated by the human proton-coupled folate transporter SLC46A1

J Biol Chem. 2009 Jun 26;284(26):17846-57. doi: 10.1074/jbc.M109.008060. Epub 2009 Apr 23.


This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was downward arrow 8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to approximately 12-fold upward arrow in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Biological Transport
  • Blotting, Western
  • Electrophysiology
  • Folic Acid / pharmacokinetics*
  • HeLa Cells
  • Histidine / chemistry
  • Histidine / genetics
  • Histidine / metabolism*
  • Humans
  • Kinetics
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism*
  • Mutagenesis, Site-Directed
  • Oocytes / cytology
  • Oocytes / drug effects
  • Oocytes / metabolism
  • Proton-Coupled Folate Transporter
  • Protons*
  • Substrate Specificity
  • Tunicamycin / pharmacology
  • Xenopus laevis / metabolism


  • Membrane Transport Proteins
  • Proton-Coupled Folate Transporter
  • Protons
  • SLC46A1 protein, human
  • Tunicamycin
  • Histidine
  • Folic Acid