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 K-t at pH 5.5 for H247A was down arrow 28.4-fold. Although wild type (WT)-PCFT K-i values varied among the folates, K-i values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His(247) to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser(172). The folic acid influx K-t for S172A-PCFT was decreased similar to H247A. His(281) faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to similar to 12-fold up arrow in the folic acid influx K-t. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx K-t was unchanged, but the K-t 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 (slip-page). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His(247) to Ser(172) 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 His(281) residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.