The Functional Roles of the His247 and His281 Residues in Folate and Proton Translocation Mediated by the Human Proton-coupled Folate Transporter SLC46A1

• Published in: The Journal of biological chemistry Vol. 284; no. 26; pp. 17846 - 17857
• Main Authors: Unal, Ersin Selcuk, Zhao, Rongbao, Chang, Min-Hwang, Fiser, Andras, Romero, Michael F., Goldman, I. David
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.06.2009; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Substitution; Animals; Biological Transport; Blotting, Western; Electrophysiology; Folic Acid - pharmacokinetics; HeLa Cells; Histidine - chemistry; Histidine - genetics; Histidine - metabolism; Humans; Kinetics; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Membrane Transport, Structure, Function, and Biogenesis; Mutagenesis, Site-Directed; Oocytes - cytology; Oocytes - drug effects; Oocytes - metabolism; Proton-Coupled Folate Transporter; Protons; Substrate Specificity; Tunicamycin - pharmacology; Xenopus laevis - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.008060

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 ↓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 ∼12-fold↑ 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.