The functional roles of the His247 and His281 residues in folate and proton translocation mediated by the human proton-coupled folate transporter SLC46A1.
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.
Unal
Ersin Selcuk
ES
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Zhao
Rongbao
R
Chang
Min-Hwang
MH
Fiser
Andras
A
Romero
Michael F
MF
Goldman
I David
ID
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
D026901:Membrane Transport Proteins; D058979:Proton-Coupled Folate Transporter; D011522:Protons; C505058:SLC46A1 protein, human; D014415:Tunicamycin; D006639:Histidine; D005492:Folic Acid