title: Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1.
abstract: The proton-coupled folate transporter (PCFT) SLC46A1 mediates uphill folate transport into enterocytes in proximal small intestine coupled to the inwardly directed proton gradient. Hereditary folate malabsorption is due to loss-of-function mutations in the PCFT gene. This study addresses the functional role of conserved charged amino acid residues within PCFT transmembrane domains with a detailed analysis of the PCFT E185 residue. D156A-, E185A-, E232A-, R148A-, and R376A-PCFT mutants lost function at pH 5.5, as assessed by transient transfection in folate transport-deficient HeLa cells. At pH 7.4, function was preserved only for E185A-PCFT. Loss of function for E185A-PCFT at pH 5.5 was due to an eightfold decrease in the [(3)H]methotrexate (MTX) influx V(max); the MTX influx K(t) was identical to that of wild-type (WT)-PCFT (1.5 microM). Consistent with the intrinsic functionality of E185A-PCFT, [(3)H]MTX influx at pH 5.5 or 7.4 was trans-stimulated in cells preloaded with nonlabeled MTX or 5-formyltetrahydrofolate. Replacement of E185 with Leu, Cys, His, or Gln resulted in a phenotype similar to E185A-PCFT. However, there was greater preservation of activity (approximately 38% of WT) for the similarly charged E185D-PCFT at pH 5.5. All E185 substitution mutants were biotin accessible at the plasma membrane at a level comparable to WT-PCFT. These observations suggest that the E185 residue plays an important role in the coupled flows of protons and folate mediated by PCFT. Coupling appears to have a profound effect on the maximum rate of transport, consistent with augmentation of a rate-limiting step in the PCFT transport cycle.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
authors.forename: Ersin Selcuk
authors.initials: ES
authors.lastname: Unal
authors.forename: Rongbao
authors.initials: R
authors.lastname: Zhao
authors.forename: I David
authors.initials: ID
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D017124:Conserved Sequence; D019143:Evolution, Molecular; D005492:Folic Acid / Q000378:metabolism*; D018698:Glutamic Acid; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D002955:Leucovorin / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008727:Methotrexate / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D058979:Proton-Coupled Folate Transporter; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; C505058:SLC46A1 protein, human; D018698:Glutamic Acid; D005492:Folic Acid; D002955:Leucovorin; D008727:Methotrexate