Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption.
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.
Mahadeo
Kris
K
Department of Pediatrics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Diop-Bove
Ndeye
N
Shin
Daniel
D
Unal
Ersin Selcuk
ES
Teo
Juliana
J
Zhao
Rongbao
R
Chang
Min-Hwang
MH
Fulterer
Andreas
A
Romero
Michael F
MF
Goldman
I David
ID
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D005492:Folic Acid / Q000378:metabolism*; D005493:Folic Acid Antagonists; D005971:Glutamates / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008286:Malabsorption Syndromes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D000068437:Pemetrexed; D058979:Proton-Coupled Folate Transporter / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D013763:Tetrahydrofolates / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D005493:Folic Acid Antagonists; D005971:Glutamates; D058979:Proton-Coupled Folate Transporter; D011522:Protons; C505058:SLC46A1 protein, human; D013763:Tetrahydrofolates; D000068437:Pemetrexed; D005973:Glutamine; D006147:Guanine; D005492:Folic Acid; D001120:Arginine; C005984:5-methyltetrahydrofolate