Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption

Kris Mahadeo, Ndeye Diop-Bove, Daniel Shin, Ersin Selcuk Unal, Juliana Teo, Rongbao Zhao, Min Hwang Chang, Andreas Fulterer, Michael F. Romero, I. David Goldman

Research output: Contribution to journalArticle

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Abstract

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5- methyltetrahydrofolate (5-MTHF) due to an increase in K m and decrease in V max in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K m for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K m mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume299
Issue number5
DOIs
StatePublished - Nov 2010

Fingerprint

Proton-Coupled Folate Transporter
Pemetrexed
Leucovorin
Glutamine
Folic Acid
Protons
Intestinal Absorption
Xenopus
Oocytes
Mutation
Hereditary Folate Malabsorption

Keywords

  • Choroid plexus
  • Folate deficiency
  • Folate receptors
  • Folate transport
  • Heme carrier protein-1
  • Intestinal folate absorption
  • Reduced folate carrier

ASJC Scopus subject areas

  • Cell Biology
  • Physiology

Cite this

Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption. / Mahadeo, Kris; Diop-Bove, Ndeye; Shin, Daniel; Unal, Ersin Selcuk; Teo, Juliana; Zhao, Rongbao; Chang, Min Hwang; Fulterer, Andreas; Romero, Michael F.; Goldman, I. David.

In: American Journal of Physiology - Cell Physiology, Vol. 299, No. 5, 11.2010.

Research output: Contribution to journalArticle

Mahadeo, Kris ; Diop-Bove, Ndeye ; Shin, Daniel ; Unal, Ersin Selcuk ; Teo, Juliana ; Zhao, Rongbao ; Chang, Min Hwang ; Fulterer, Andreas ; Romero, Michael F. ; Goldman, I. David. / Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption. In: American Journal of Physiology - Cell Physiology. 2010 ; Vol. 299, No. 5.
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abstract = "The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5- methyltetrahydrofolate (5-MTHF) due to an increase in K m and decrease in V max in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K m for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K m mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.",
keywords = "Choroid plexus, Folate deficiency, Folate receptors, Folate transport, Heme carrier protein-1, Intestinal folate absorption, Reduced folate carrier",
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AU - Diop-Bove, Ndeye

AU - Shin, Daniel

AU - Unal, Ersin Selcuk

AU - Teo, Juliana

AU - Zhao, Rongbao

AU - Chang, Min Hwang

AU - Fulterer, Andreas

AU - Romero, Michael F.

AU - Goldman, I. David

PY - 2010/11

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N2 - The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5- methyltetrahydrofolate (5-MTHF) due to an increase in K m and decrease in V max in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K m for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K m mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

AB - The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5- methyltetrahydrofolate (5-MTHF) due to an increase in K m and decrease in V max in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K m for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K m mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

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KW - Folate deficiency

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KW - Folate transport

KW - Heme carrier protein-1

KW - Intestinal folate absorption

KW - Reduced folate carrier

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