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Stereoselective transport of human His27- and Arg27-reduced folate carrier

Tomoya Narawa1, Hideaki Yanagisawa, Tomoo Itoh

  • 1School of Pharmacy, Kitasato University.

Biological & Pharmaceutical Bulletin
|March 4, 2014
PubMed

Insights

The human reduced folate carrier (hRFC) transports methotrexate enantiomers stereoselectively. Differences in binding affinity explain the differential uptake of L-methotrexate and D-methotrexate by hRFC.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Methotrexate (MTX) is a crucial antifolate drug used in chemotherapy.
  • Understanding the transport mechanisms of MTX enantiomers is vital for optimizing drug efficacy.
  • The human reduced folate carrier (hRFC) plays a significant role in cellular folate uptake.

Purpose of the Study:

  • To investigate the stereoselective transport of L-methotrexate (L-MTX) and D-methotrexate (D-MTX) by hRFC.
  • To characterize the kinetic parameters and pH dependency of hRFC-mediated MTX enantiomer transport.
  • To determine the inhibitory effects of MTX enantiomers on each other's transport.

Main Methods:

  • HEK293 cells expressing specific hRFC variants (H27-hRFC and R27-hRFC) were utilized.
  • Uptake assays were performed at varying extracellular pH levels (6.0 to 7.4).
  • Kinetic analysis (Michaelis-Menten, Dixon plots) and competitive inhibition studies were conducted.

Main Results:

  • Extracellular pH significantly influenced the uptake of both L-MTX and D-MTX.
  • hRFC-mediated uptake of L-MTX followed Michaelis-Menten kinetics with distinct Km values for H27- and R27-hRFC variants.
  • D-MTX competitively inhibited L-MTX uptake, indicating shared binding sites but with lower affinity compared to L-MTX.

Conclusions:

  • hRFC exhibits stereoselective transport for methotrexate enantiomers.
  • Significant differences in the affinities of L-MTX and D-MTX to hRFC underlie this stereoselectivity.
  • These findings provide insights into the molecular basis of folate carrier function and MTX pharmacokinetics.

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