Role of the glutamate 185 residue in proton translocation mediated by the proton-coupled folate transporter SLC46A1

• Published in: American Journal of Physiology: Cell Physiology Vol. 297; no. 1; pp. C66 - C74
• Main Authors: Unal, Ersin Selcuk, Zhao, Rongbao, Goldman, I. David
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.07.2009
• Subjects: Amino Acid Sequence; Cell Membrane - metabolism; Conserved Sequence; Evolution, Molecular; Folic Acid - metabolism; Genes; Glutamic Acid; HeLa Cells; Humans; Hydrogen-Ion Concentration; Kinetics; Leucovorin - metabolism; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Membrane Transporters, Ion Channels, and Pumps; Methotrexate - metabolism; Models, Biological; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Conformation; Proton-Coupled Folate Transporter; Protons; Salt; Small intestine; Sorption; Structure-Activity Relationship; Transfection; Vitamin B
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00096.2009

Departments of 1 Molecular Pharmacology and 2 Medicine, Albert Einstein College of Medicine, Bronx, New York Submitted 3 March 2009 ; accepted in final form 26 April 2009 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 µM). 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 ( 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. heme carrier protein-1; proton-coupled folate transporter/heme carrier protein-1; folate transport; hereditary folate malabsorption; methotrexate; pemetrexed; proton-coupled transporters Address for reprint requests and other correspondence: I. D. Goldman, Albert Einstein Cancer Center, Chanin 2, 1300 Morris Park Ave., Bronx, NY 10461 (e-mail: igoldman{at}aecom.yu.edu )