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 K t at pH 5.5 for H247A was 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 ϳ12-fold1 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 (slippage). 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.This laboratory recently identified SLC46A1 as a protoncoupled folate transporter (PCFT) 2 that mediates transport of folates across the apical (brush border) membrane of enterocytes within the acidic microclimate at the absorptive surface of the proximal jejunum (1). The critical role that PCFT plays at this epithelium was confirmed with the demonstration by this laboratory that there are loss-of-function mutations in this gene in patients with the autosomal recessive disorder, hereditary folate malabsorption (Online Mendelian Inheritance in Man, 229050) (1, 2). Hereditary folate malabsorption is characterized by impaired intestinal folate absorption and impaired transport of folates into the central nervous system (3), the latter likely due to a defect in transport across the blood-choroid plexus-cerebrospinal fluid barrier. While operating most efficiently at low pH, PCFT contributes to the transport of folates and the pharmacological activity of the new generation antifolate, pemetrexed, even at neutral pH (4). PCFT may be even more important to the delivery of this, and possibly other antifolates, within the acidic interstitium of solid tumors (5, 6). PCFT may also play a role in the export of folates from acidified endosomes (7) during folate receptor-mediated endocytosis (8, 9). Although facilitative carriers in lower organisms com...

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

“…PCFT consists of 12 transmembrane segments as determined by the substituted cysteine accessibility method [27]. Three-dimensional homology models of PCFT were built using high-resolution crystal structures of the GlpT or the EmrD, the closest homologues with a known crystal structure [2,28]. Secondary structure content in the form of computed lengths of a-helical segments based on a GlpT homology model is included in our topological model (Fig.…”

Identification and Functional Impact of Homo-oligomers of the Human Proton-coupled Folate Transporter

“…Based on results with substituted cysteine-scanning mutagenesis of a Cys-less hPCFT and accessibilities to thiol-reactive agents (27) and on studies of hemagglutinin (HA) epitope accessibilities to epitope-specific antibody (28,29), hPCFT has 12 TMDs with N and C termini directed to the cytosol. Structurally and/or functionally important residues have been identified in hPCFT and include (with possible roles) Glu-185 (which is required for proton coupling) (30), His-247 and Ser-172 (which modulate folate and proton access to a high affinity binding site) (31), His-281 (which is important for substrate binding) (31), Arg-376 (which impacts proton and substrate binding) (12), and Asp-156 (which contributes to PCFT protein stability) (13) (Fig. 1).…”

Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

“…Among the mutants that were expressed and trafficked to the plasma membrane, E232G appeared to have a defect in the ability of the carrier to undergo the required conformational changes associated with substrate transport. In fact, the affinity of this mutant for MTX was markedly increased, a pattern that has been observed with other mutant PCFTs usually accompanied by a fall in V max (20). The I304F mutant had a defect in MTX binding (slightly decreased influx V max and a larger increase in influx K t ).…”

“…Functional Analysis of the E232G, L161R, and I304F PCFT Mutants-Because the E232G, L161R, and I304F mutants were expressed, studies were conducted to examine their function and to explore the kinetic basis for their decrease in transport activity. Although the H247P mutant was expressed well at the plasma membrane, it was not included because the role of His-247 was characterized in detail previously (20). MTX influx at neutral pH was markedly decreased in these mutants as compared with the wild-type PCFT excluding the possibility that the mutations disrupt proton coupling as observed for the E185A mutation (18) (data not shown).…”