title: The functional roles of the His247 and His281 residues in folate and proton translocation mediated by the human proton-coupled folate transporter SLC46A1.
abstract: This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was downward arrow 8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to approximately 12-fold upward arrow in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.
authors.affiliation: Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Ersin Selcuk
authors.initials: ES
authors.lastname: Unal
authors.forename: Rongbao
authors.initials: R
authors.lastname: Zhao
authors.forename: Min-Hwang
authors.initials: MH
authors.lastname: Chang
authors.forename: Andras
authors.initials: A
authors.lastname: Fiser
authors.forename: Michael F
authors.initials: MF
authors.lastname: Romero
authors.forename: I David
authors.initials: ID
authors.lastname: Goldman
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D004594:Electrophysiology; D005492:Folic Acid / Q000493:pharmacokinetics*; D006367:HeLa Cells; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D058979:Proton-Coupled Folate Transporter; D011522:Protons; D013379:Substrate Specificity; D014415:Tunicamycin / Q000494:pharmacology; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; D011522:Protons; C505058:SLC46A1 protein, human; D014415:Tunicamycin; D006639:Histidine; D005492:Folic Acid