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Am J Physiol Cell Physiol. 2015 Apr 15;308(8):C631-41. doi: 10.1152/ajpcell.00238.2014. Epub 2015 Jan 21.

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

Author information

1
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York;
2
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York;
3
Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, New York; and Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York.
4
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York;
5
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York; Department of Medicine, Albert Einstein College of Medicine, Bronx, New York; i.david.goldman@einstein.yu.edu.

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 [(3)H]methotrexate and [(3)H]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 [(3)H]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.

KEYWORDS:

PCFT, SLC46A1; hereditary folate malabsorption, HFM; methotrexate; pemetrexed; proton-coupled folate transporter; solute transporter

PMID:
25608532
PMCID:
PMC4398847
DOI:
10.1152/ajpcell.00238.2014
[Indexed for MEDLINE]
Free PMC Article

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