Identification of tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1)

Michele Visentin, Ersin Selcuk Unal, Mitra Najmi, Andras Fiser, Rongbao Zhao, I. David Goldman

Research output: Contribution to journalArticle

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Abstract

The proton-coupled folate transporter (PCFT) mediates intestinal folate absorption and transport of folates across the choroid plexus. This study focuses on the role of Tyr residues in PCFT function. The substituted Cys-accessibility method identified four Tyr residues (Y291, Y362, Y315, and Y414) that are accessible to the extracellular compartment; three of these (Y291, Y362, and Y315) are located within or near the folate binding pocket. When the Tyr residues were replaced with Cys or Ala, these mutants showed similar (up to 6-fold) increases in influx Vmax and Kt/Ki for [3H]methotrexate and [3H]pemetrexed. When the Tyr residues were replaced with Phe, these changes were moderated or absent. When Y315A PCFT was used as representative of the mutants and [3H]pemetrexed as the transport substrate, this substitution did not increase the efflux rate constant. Furthermore, neither influx nor efflux mediated by Y315A PCFT was transstimulated by the presence of substrate in the opposite compartment; however, substantial bidirectional transstimulation of transport was mediated by wild-type PCFT. This resulted in a threefold greater efflux rate constant for cells that express wild-type PCFT than for cells that express Y315 PCFT under exchange conditions. These data suggest that these Tyr residues, possibly through their rigid side chains, secure the carrier in a high-affinity state for its folate substrates. However, this may be achieved at the expense of constraining the carrier’s mobility, thereby decreasing the rate at which the protein oscillates between its conformational states. The Vmax generated by these Tyr mutants may be so rapid that further augmentation during transstimulation may not be possible.

Original languageEnglish (US)
Pages (from-to)C631-C641
JournalAmerican Journal of Physiology - Cell Physiology
Volume308
Issue number8
DOIs
StatePublished - Jan 1 2015

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Keywords

  • Hereditary folate malabsorption
  • Hfm
  • Methotrexate
  • Pcft, slc46a1
  • Pemetrexed
  • Proton-coupled folate transporter
  • Solute transporter

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

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