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 ↓8.4-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 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 K_t 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 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 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.

Introduction

This laboratory recently identified SLC46A1 as a proton-coupled 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 commonly use a proton gradient to drive uphill transport of substrates (10), there are only eight proton-coupled solute carrier families in humans; pcft is the latest to be identified (1, 11). One or more His residues have been shown to be critical for function of some of these transporters (12–14). Prior to the identification of PCFT, proton-coupled uphill folate transport was demonstrated in membrane vesicles derived from rat hepatocytes (15) and in rabbit jejunal brush border membrane vesicles (16, 17). In the latter case, His residues were implicated, because this process was blocked by diethyl pyrocarbonate (18). However, diethyl pyrocarbonate effects are nonspecific; this reagent can also react with tyrosine, lysine, and cysteine residues (19, 20). The current study was designed to assess directly, by site-directed mutagenesis, the potential role that His residues might play in folate transport mediated by the human PCFT. This study also addresses, for the first time, the direct assessment of cellular acidification, and the relationship between folate and proton transport in wild-type and His mutants, which accompanies PCFT-mediated transport in Xenopus oocytes.

EXPERIMENTAL PROCEDURES

Chemicals

Tritiated folic acid (diammonium salt, 3′,5′,7,9-[³H], cat. no. MT-783) and methotrexate (MTX-disodium salt, 3′,5′,7-[³H](N), cat. no. MT701) were obtained from Moravek Biochemicals Inc. (Brea, CA), purified by liquid chromatography, and maintained as previously described (21). Unlabeled folic acid and tunicamycin (T7765) were purchased from Sigma-Aldrich. MTX, (6S)5-formylTHF, and (6S)5-methylTHF were obtained from Schircks Laboratories (Jona, Switzerland). Protease inhibitor mixture (cat. no. 11836170001) was obtained from Roche Applied Science. All other reagents were of the highest purity available from commercial sources.

Cell Lines, Cell Culture Conditions, Transient Transfection, and Tunicamycin Treatment

HeLa cells, originally obtained from the American Type Tissue Collection (Manassas, VA), have been maintained in this laboratory in RPMI 1640 medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO₂. HeLa R1-11 cells (4) is a more stable subclone of the HeLa R1 cell line (22) that lacks both PCFT and reduced folate carrier expression, the later due to a genomic deletion. Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's protocol for transient transfection of plasmid DNAs into these cells. R1-11 or HeLa cells were transfected with empty pcDNA3.1(+) vector (mock), or the same vector containing wild-type (WT), or mutant pcft cDNAs.

Tunicamycin was dissolved in DMSO and added to growth medium to achieve a concentration of 1 μg/ml, a level previously shown to block the N-linked glycosylation of PCFT with minimal loss of cell viability (23). Cells were exposed to tunicamycin in growth medium for 48 h following which they were prepared for Western blot analysis.

Site-directed Mutagenesis, Epitope Tagging, and Generation of pcft Xenopus Oocyte Expression Constructs

Site-directed mutagenesis was carried out according to the QuikChange II XL protocol from Stratagene (La Jolla, CA). WT-pcft cDNA, cloned into the BamHI site of the mammalian expression vector pcDNA3.1(+), was used as the template (1). Briefly, complementary forward and reverse primers, which carry the targeted nucleotide changes in the middle region, were designed individually for introducing Ala into human pcft primary sequence at positions indicated in supplemental Table S1. Initial DNA denaturation, number of plasmid amplification cycles, and annealing and extension conditions were the same as previously defined (23). DpnI (10 units/μl) restriction enzyme was used to digest the parental double strand DNA. The mixture (3 μl) was transformed into DH5α-competent cells (Invitrogen). Plasmids carrying the desired mutations were identified by DNA-automated sequencing in the Albert Einstein Cancer Center Genomics Shared Resource. The entire coding region of pcft was sequenced to confirm the mutation and the fidelity of DNA. A hemagglutinin (HA) peptide epitope (YPYDVPDYA) was fused to the C terminus of WT and the various pcft constructs by PCR-based site-directed mutagenesis as previously described (23). The C-HA-pcft vector was used as a template for the amino acid substitutions at His²⁴⁷, His²⁸¹, and Ser¹⁷². The forward primers used to introduce these mutations are shown in supplemental Table S1.

To express WT and mutant transporters in Xenopus oocytes, WT-pcft was initially cloned into the pGEMHE vector at the EcoRI and XbaI restriction sites (24). The pGEMHE vector carrying WT-pcft was employed as a template for site-directed mutagenesis to create the H281A construct using the same primers as described above (supplemental Table S1).

Membrane Transport Measurements

Influx measurements were performed by rapid determination of membrane transport parameters as previously described (23). R1-11 cells were seeded in 17-mm glass scintillation vials at 4 × 10⁵ cells/ml density. Influx was assessed at mid-log phase growth ∼72 h after transfection. Most transport studies were performed in MES-buffered saline (20 mm MES, 140 mm NaCl, 5 mm KCl, 2 mm MgCl₂, 5 mm dextrose), at pH 5.5. This buffer was titrated to achieve pH levels of 4.5, 5.0, 6.0, and 6.5. For pH levels ≥ 7.0, MES-buffered saline was replaced with HBS and titrated to the appropriate pH. Cells were incubated in HEPES buffer (HBS) (20 mm HEPES, 140 mm NaCl, 5 mm KCl, 2 mm MgCl₂, 5 mm dextrose, at pH 7.4) at 37 °C for 20 min before transfer into the prewarmed uptake buffer containing tritiated folate/antifolate substrate. Uptake was halted after 1 min by the injection of 8 ml of HBS (pH 7.4) at 0 °C, an interval over which the relationship between cell folate and time was linear and the ordinate intercept at zero time was near the point of origin. Cells were washed twice in the 0 °C HBS buffer after which radioactivity and protein levels were determined as reported previously (23). Influx is expressed as picomoles of [³H]MTX or [³H]folic acid/mg of protein/min.

[³H]Folic acid and [³H]MTX influx kinetic parameters (K_t and V_max) were obtained from a non-linear regression of influx as a function of extracellular folate/antifolate concentration according to the Michaelis-Menten equation. Influx K_i values for (6S)5-formylTHF, (6S)5-methylTHF, and folic acid were based upon inhibition of [³H]MTX influx according to the formula: V_inhibited = V_maxS/(1 + i/K_i)K_t + S, where i and S are the inhibitor and substrate concentrations, respectively, and K_t and V_max are the parameters determined for WT-PCFT. The [³H]MTX concentrations used were comparable to the K_t for each PCFT construct, and the concentrations of inhibitors were adjusted so that the level of inhibition achieved was in the 35–65% range.

Western Blot Analysis

As reported previously (23), a rabbit anti-HA antibody (#H6908) obtained from Sigma was used to detect HA-tagged proteins in cell plasma membrane protein fractions prepared from HeLa cells transiently transfected with empty vector (mock), WT-pcft, or mutant constructs at equal amounts. Briefly, to obtain membrane fractions, 200 μl (10⁶ cells) were incubated on ice for 30 min in hypotonic buffer (0.5 mm Na₂HPO₄, 0.1 mm EDTA at pH 7.4) containing protease inhibitor. After centrifugation at 14,000 rpm for 2 min, the pellet was dissolved in 20 mm Tris base, 150 mm NaCl, 1% Triton X-100, 0.1% SDS, 1 mm EDTA buffer. Proteins were dissolved in SDS-PAGE loading buffer (0.225 m Tris.Cl (pH 6.8), 50% glycerol, 5% SDS, 0.05% bromphenol blue) with 0.25 m dithiothreitol). A 12% SDS-polyacrylamide gel was used to resolve the proteins, which were then transferred to polyvinylidene difluoride Transfer Membranes (Amersham Biosciences). After antibody treatment, membranes were processed by the ECL Plus Western Blotting Detection System (Amersham Biosciences). Rabbit β-actin antibody (#4967L, Cell Signaling Technology) was used to determine the loading amounts.

Electrophysiological Analysis

Xenopus oocytes were employed to assess the functional roles of the WT and mutant pcfts based upon currents generated and intracellular acidification. cRNA (23 ng in 50 nl) or water (50 nl) were injected into stage V/VI oocytes, and electrophysiological measurements were made 3–7 days later. Voltage electrodes were made from fiber-capillary borosilicate and filled with 3 m KCl; pH electrodes were pulled similarly, silanized, filled with hydrogen ionophore 1 mixture B (Fluka), and back-filled with phosphate buffer (pH 7.0). These procedures and the electrical configuration were the same as described previously (25, 26). For these experiments, as with the divalent metal transporter, DMT1, oocytes were voltage clamped to −90 mV to maximize folate-induced currents (26, 27). ND90 solution (96 mm NaCl, 1.8 mm CaCl₂, 1 mm MgCl₂) was pH-adjusted using Tris (for pH 7.4) or MES (for pH 4.5–5.5). During these experiments, oocytes were continuously superfused with ND90 solutions (with and without folates as indicated) at 5 ml/min.

Comparative Protein Structure Modeling and Electrostatic Calculations of Human PCFT

Related, experimentally solved three-dimensional structures were identified based upon the sequence of human PCFT with the PSIPRED fold recognition program (28). The most suitable template was that of glycerol-3-phosphate (GlpT) transporter from Escherichia coli (Protein Data Bank code 1pw4) sharing 14% sequence identity to PCFT (29). A three-dimensional comparative protein structure model for PCFT was built using the Multiple Mapping Method (30, 31). This method employs an alignment optimization algorithm to produce accurate input alignment between the target and template for the MODELLER program (32, 33) that generates a comparative model. The quality of the resulting model was checked with PROSA energy function (34). Electrostatic potential around the molecule was calculated with the DelPhi macromolecular electrostatics modeling package (35) and visualized in GRASP2 (36). pK_a values for charged residues were obtained from multiconformation continuum electrostatics calculations (37, 38). Multiconformation continuum electrostatics combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side-chain ionization and conformation.

Statistical Analysis of Membrane Transport Data

Data presented are the means ± S.E. from at least three independent experiments. Statistical comparisons were performed by the two-tailed Student's paired t test. All statistical analyses utilized GraphPad Prism (version 3.0 for Windows, GraphPad Software).

RESULTS

Membrane Localization and Evolutionary Conservation of PCFT His Residues

The human PCFT protein (SLC46A1, NP_542400) consists of 459 amino acids with 10 His residues in the primary sequence. Transmembrane topology prediction programs identified 12 integral transmembrane helices with N and C termini located within the cytoplasm (23, 39). Localization of the termini was subsequently confirmed experimentally (23, 39). Accordingly, His¹⁴⁰ is located in the second extracellular loop, His²⁴⁷ and His²⁴⁸ in the large intracellular loop between transmembrane domains (TMDs) VI and VII, His²⁸¹ in TMD VII, and His³¹² in TMD VIII. Five His residues (His⁴⁹, His⁸⁴, His²⁰¹, His²⁶⁶, and His⁴⁴⁹) are at membrane-aqueous interfaces (Fig. 1). Although all His residues are partially conserved, His⁸⁴, His²⁴⁷, and His²⁸¹ are identical in all species (supplemental Table S2).

FIGURE 1.

Membrane topology model for PCFT predicted by various methods available online indicating locations of His residues (red-filled circles) and Ser¹⁷² (blue-filled circle).

The Effect of His to Ala Substitutions on PCFT Function

All ten His residues were individually replaced with Ala. The mutant pcft cDNA constructs, along with WT-pcft and the empty vector (mock), were transiently transfected, individually, into HeLa-R1-11 cells. As indicated in Fig. 2, influx of 0.5 μm [³H]MTX over 1 min at pH 5.5 in cells transiently transfected with the H49A, H84A, H140A, and H201A mutants was equal to that of the WT carrier with only a modest reduction in function of the H248A and H449A mutants. There were substantial reductions in function for the H247A and H281A mutants, both fully conserved, and, to a much lesser extent, the H312A mutant that is not fully conserved. The pattern of transport changes was the same when influx of 0.5 μm [³H]MTX was assessed in the absence of a proton driving force at pH 7.0, although the overall rate was substantially lower (∼1/25th the rate at pH 5.5, data not shown). These results indicated that His²⁴⁷ and His²⁸¹ are residues that play important roles in PCFT function.

FIGURE 2.

Determination of [³H]MTX influx in R1-11 cells transiently transfected with WT-pcft and mutant constructs in which His residues were substituted with Ala. Influx of [³H]MTX (0.5 μm) was assessed at pH 5.5 and 37 °C over 1 min. Data are the mean ± S.E. from three independent experiments.

Western blot analyses of crude cell plasma membrane protein fractions were evaluated to assess levels of protein expression of the two fully conserved mutants along with the H312A mutant. Immunoblots of crude membrane protein fractions from HeLa cells transiently transfected with empty vector (mock) or C-HA-tagged WT-pcft, H247A, H281A, and H312A-pcft mutants are shown in Fig. 3A. At equivalent protein loading, H281A and H312A expression was similar to WT-pcft. The H247A mutant protein migrated at ∼47 kDa as a narrower band, faster than the other proteins that ran as a broad band at ∼55 kDa. To determine the role of glycosylation in this pattern along with the structural integrity of the H247A mutant, HeLa cells were transiently transfected with WT-PCFT or the H247A mutant and incubated with 1.0 μg/ml tunicamycin for 48 h. Cell membrane protein fractions were prepared and subjected to Western blot analysis along with membranes from cells transfected with the N58Q/N68Q double mutant (deglycosylated PCFT) (23). As indicated in Fig. 3B, high molecular weight broad PCFT bands disappeared for the WT carrier and the protein migrated at the same size (∼35 kDa) as the deglycosylated (C-HA-N58Q/N68Q) carrier. Similarly, the H247A mutant protein showed a size shift from ∼47 kDa to ∼35 kDa in cells exposed to tunicamycin. Hence, H247A PCFT is expressed to the same extent as WT-PCFT, and the protein is not degraded. Rather, the alteration in protein mobility for the H247A mutant appears to be due to a change in the extent of glycosylation or a protein conformational change.

FIGURE 3.

A, Western blot analysis of C-HA-tagged WT-PCFT, H247A, H281A, and H312A mutants. B, Western blot analysis of WT- and the N58Q/N68Q- PCFT [deglycosylated], PCFT along with WT- and H247A-PCFT proteins after tunicamycin treatment (1 μg/ml) of HeLa cells for 48 h. In both panels, equal amounts of crude cell plasma membrane protein fractions (30 μg) were loaded, and the blots were probed with an anti-HA antibody. The numbers on the left indicate the molecular sizes in the protein ladders. The lower bands in each panel indicate the β-actin loading control. The blot is representative of three (A) and two (B) separate experiments.

Analysis of Influx Kinetics and Substrate Specificity

There were profound and distinct changes in influx kinetics mediated by the H247A mutant as compared with WT-PCFT (Fig. 4A). The influx K_t for folic acid decreased by a factor of ∼8.4 (from 1.43 ± 0.64 to 0.17 ± 0.03 μm). This was associated with a ∼11-fold decrease in influx V_max (from 247 ± 37 to 23 ± 1.5 pmol/mg/min). Hence, this amino acid substitution resulted in a marked increase in the affinity of carrier for folic acid. The change in folic acid influx kinetics for the H281A mutant was quite different (Fig. 4B). The influx K_t for folic acid increased ∼12-fold (from 1.88 ± 0.58 to 21.4 ± 2.8 μm). The V_max values for the WT-PCFT and H281A mutant were similar (743 ± 84 and 715 ± 49 pmol/mg/min, respectively, p > 0.05). Hence, in the case of this mutant, the major change was a marked decrease in carrier affinity for folic acid. The inter-experimental differences between V_max values are due to differences in transient transfection efficiency which affect V_max, but not K_t, determinations.

FIGURE 4.

A, [³H]folic acid influx kinetics in R1-11 cells transiently transfected with WT-pcft or the H247A mutant at pH 5.5. B, [³H]folic acid influx kinetics in R1-11 cells transiently transfected with WT-pcft or the H281A mutant at pH 5.5. Data are the mean ± S.E. from three independent experiments. Insets: concentrations of substrates were adjusted to bracket the influx K_t. Influx was determined over 1 min.

The loss of function for the H312A mutant was small, and this was, as expected, associated with small changes in folic acid influx kinetics. The folic acid influx K_t for H312A was only 2-fold higher than WT-PCFT (2.43 ± 0.3 versus 1.22 ± 0.12 μm, respectively). This was accompanied by a 2-fold decrease in influx V_max of 127 ± 4.5 versus 256 ± 5.7 pmol/mg/min, respectively (data not shown).

Substrate specificity of the three His mutants was evaluated. The pcft cDNA constructs were transiently transfected into HeLa-R1-11 cells and inhibition of 0.5 μm [³H]MTX influx by several other folates (at 2 μm) was assessed. As indicated in Fig. 5A, the pattern of [³H]MTX influx inhibition was 5-methyl-THF > 5-formylTHF > folic acid for WT-PCFT. To facilitate comparison of the degree of inhibition by folates, the data were plotted as a percentage of control influx in the absence of inhibitors for each pcft construct (Fig. 5B). The pattern of inhibition of MTX influx into H281A and H312A transfectants by the various folates was similar to that of WT-pcft transfectants. However, the magnitude and pattern of inhibition was remarkably different for the H247A mutant. The degree of inhibition was markedly increased and was comparable for all the folates. This was more evident when the inhibitor concentration was decreased to a level (0.25 μm) so low that there was no inhibition at all of MTX influx mediated by WT-PCFT. Under these conditions there still was marked (∼40%), and comparable, inhibition of MTX influx into the H247A transfectant by the three folate substrates (supplemental Fig. S1). K_i values were computed based upon inhibition of 0.5 μm [³H]MTX influx by 5-methylTHF, 5-formylTHF, or folic acid, each at 2 μm for the WT carrier, and each at 0.25 μm for the H247A mutant (Table 1). Influx K_i values for the H247A mutant were similar for all the substrates and were decreased by factors of ∼3.4, ∼7, and ∼8 for 5-methylTHF, 5-formylTHF, and folic acid, respectively, as compared with the WT-PCFT. Hence, the H247A mutation resulted in both increased affinity for all its folate substrates and a loss of substrate selectivity.

FIGURE 5.

A, inhibition of 0. 5 μm [³H]MTX influx by 2 μm 5-methyl-THF, 5-formyTHF, or folic acid in R1-11 cells transiently transfect with WT-pcft, or His mutants. Data are the mean ± S.E. from three independent experiments. B, the data in A presented as percentage of the control rate for each construct in the absence of inhibitor. Data are the mean ± S.E. from three independent experiments. Influx was determined at pH 5.5 over 1 min.

TABLE 1.

K_i values for 5-methylTHF, 5-formylTHF, and folic acid based upon inhibition of 0.5 μm [³H]MTX influx

K_i values are the mean ± S.E. from three independent experiments. The [³H]MTX concentrations were comparable to the K_t for each carrier, and the concentrations of inhibitors were adjusted so that inhibition achieved was in the 35–65% range.

	ki
	5-formylTHF	5-methylTHF	Folic acid
	μm
WT-PCFT	1.17 ± 0.12	0.67 ± 0.21	1.63 ± 0.18
H247A	0.17 ± 0.02	0.20 ± 0.01	0.20 ± 0.02

Impact of Other Amino Acid Substitutions at the His²⁴⁷ Residue

To further explore the role of the His²⁴⁷ residue in PCFT function, other mutants were constructed with a variety of amino acid substitutions. When His was replaced with Arg (positively charged) there was a marked loss of function (Fig. 6A). On the other hand, substitution with the uncharged Gln produced transport activity higher than that of H247A; substitution with the negatively charged Glu preserved the majority of function. For all these pcft constructs, folic acid produced comparable inhibition of [³H]MTX influx and at a level comparable to what was observed with the H247A mutant (Table 2). These data indicate that substitutions at the His²⁴⁷ residue produce a comparable increase in affinity for folic acid irrespective of charge, polarity, or size of the substituted amino acid.

FIGURE 6.

A, [³H]MTX (0. 5 μm) influx and inhibition by 1 μm folic acid at pH 5.5 and 37 °C over 1 min in R1-11 cells transiently transfected with WT-pcft and His²⁴⁷ substituted with Ala, Arg, Gln, or Glu. Data are the mean ± S.E. from three independent experiments. B, Western blot analysis of crude cell plasma membrane protein fractions from HeLa cells transiently transfected with C-HA-WT-pcft, C-HA-deglycosylated-pcft, and C-HA-tagged His²⁴⁷ Ala, Arg, Gln, and Glu substitution mutants. Equal amounts of crude cell membrane protein fractions (30 μg) were loaded, and the blots were probed with an anti-HA antibody. The numbers on the left indicate the molecular sizes in the protein ladders. β-Actin was the loading control. The blots are representative of three separate experiments.

TABLE 2.

Percentage inhibition of 0.5 μm [³H]MTX influx by 1 μm folic acid

Influx was assessed at pH 5.5 and 37 °C over 1 min. Data are the mean ± S.E. from three independent experiments.

	Inhibition
	%
WT-PCFT	33.0 ± 2.2
H247A	76.4 ± 5.2
H247R	77.8 ± 4.3
H247Q	73.4 ± 5.1
H247E	75.3 ± 3.8

Western blot analyses were performed on cell plasma membrane protein fractions prepared from HeLa cells transiently transfected with C-HA-WT-, C-HA-deglycosylated-, and C-HA-tagged H247A, H247R, H247Q, and H247E mutants (Fig. 6B). Similar to H247A, other substitutions at this position resulted in a protein that migrated as a narrow band at ∼47 kDa, faster than WT protein. Although these mutations produced a protein with an anomalous migration pattern on Western blot, the data indicate that their levels of expression are similar.

A Homology Model for PCFT; Role of Serine 172 in PCFT Function

A comparative protein structure model was built for PCFT, and electrostatic calculations were performed to obtain charge distribution preferences on the molecular surface and to obtain pK_a values for individual His residues (Fig. 7). Charge distribution provided indirect support for the homology model, because the external, membrane-spanning surface turned out to be rather hydrophobic and most charges appeared at the predicted extra- and intracellular ends of the transporter (Fig. 7, B and D). Continuum electrostatic calculations on the three-dimensional model of PCFT predicted that most His residues would have a pK_a value between 6.0 and 7.0, except for His²⁴⁷ (pK_a = 4.9) and His²⁸¹ (pK_a = 2.72); a pK_a prediction was not possible for His³¹² due to the lack of numerical convergence during the simulation. In the comparative model, as indicated in Fig. 7A, His²⁴⁷ resides at the cytoplasmic end of a solute pathway, at a hydrogen bond distance to Ser¹⁷². Ser¹⁷² is located in the second cytoplasmic loop between the fourth and fifth TMDs and is identical among all species studied in contrast to the two serines (Ser¹⁷³ and Ser¹⁷⁴) that follow it (Fig. 1). The structural environment of His²⁴⁷ and Ser¹⁷² appears to form a substantial part of the cytoplasmic opening of the translocation pathway. To interrupt this predicted interaction, Ser¹⁷² was mutated to Ala and transiently transfected into R1-11 cells. [³H]MTX influx was only slightly lower than transport into cells transfected with WT-pcft; however, 2 μm folic acid markedly inhibited (∼80%) [³H]MTX influx, similar to what was seen with the H247A and other PCFT mutations at this residue (data not shown). The kinetic basis for this change was assessed and compared with kinetic parameters for the H247A mutant and WT-PCFT in the same experiment. As shown in Table 3, the folic acid influx K_t for the S172A mutant was decreased by a factor of ∼4 as compared with the K_t obtained for the WT-PCFT (0.19 versus 0.68 μm, respectively). Although the V_max for the H247A mutant was markedly decreased, as before, there was only a 2-fold reduction in V_max for the S172A mutant. The S172A mutant was expressed to a similar level, and migrated as WT-PCFT, on Western blot analysis (data not shown).

FIGURE 7.

Homology model of the human PCFT based on the crystal structure of the GlpT. A, close-up of the structure around the His²⁴⁷ and S¹⁷² residues viewed from a cytoplasmic perspective. C, the H²⁸¹ and H³¹² residues viewed from an extracellular perspective. The specified residues are shown in stick representation, the surrounding backbone in ribbon representation as prepared with the VMD program (72). Electrostatic surface representations of PCFT: B, viewed from the cytoplasmic perspective; D, viewed from the side. Blue and red colors indicate positive and negative charges, respectively; hydrophobic regions are shown in gray as prepared with the GRASP program (36).

TABLE 3.

[³H]Folic acid influx kinetic parameters in R1-11 cells transiently transfected with C-HA-WT-PCFT, C-HA-H247A, or C-HA-S172A mutants

Influx was assessed at pH 5.5 and 37 °C over 1 min. Data are the mean ± S.E. from three independent experiments.

	kt	Vmax
	μm	pmol/mg/min
C-HA-WT	0.69 ± 0.09	123.2 ± 4.5
C-HA-S247A	0.067 ± 0.004	7.6 ± 0.6
C-HA-S172A	0.19 ± 0.02	55.7 ± 1.2

Role of His²⁸¹ Residue in PCFT Function

The homology model localized His²⁸¹ and His³¹² close to the extracellular opening to the translocation pathway (Fig. 7C). As indicated above, the basis for the loss of function for the H281A mutant was the opposite of what was found for the mutations at the His²⁴⁷ residue, a marked decrease in carrier affinity for folic acid (increased K_t), without a change in V_max (Fig. 4B). This raised the possibility that there might be an alteration in binding of co-transported proton(s) to site(s) that modulate directly, or allosterically, folate substrate binding, or a loss of coupling entirely. To explore this possibility, folic acid influx was assessed as a function of pH. As indicated in Fig. 8A, [³H]MTX influx for the H281A mutant PCFT was far less than for WT carrier at all pH levels, increasing only at the very low pHs; there was a “left shift” in the H281A mutant-mediated influx curve. To determine whether a further decrease in pH might compensate for the marked loss of pH sensitivity for the H281A mutant, transport was measured over a pH range of 4.5–6.0. In Fig. 8B, MTX influx was normalized to the rate at pH 6.0 (set to 1). It can be seen that there was only a small (<2-fold) further increase in MTX influx mediated by WT-PCFT. However, MTX influx mediated by the H281A mutant was increased by a factor of 15 when the pH was decreased to 4.5. Consistent with this, at pH 6.0, influx mediated by the H281A mutant was only 11% that of WT-PCFT; this increased to 43% of WT-PCFT mediated influx at pH 4.5. Hence, a reduction in pH partially compensated for the loss of His at the 281 position. The kinetic basis for this enhanced transport at low pH was explored. For WT-PCFT there was only a negligible decrease in the folic acid influx K_t at pH 4.5 versus 5.5 (1.4 ± 0.4 versus 1.9 ± 0.58 μm, respectively), and a small (∼20%) increase in V_max (742 ± 84.4 versus 858 ± 79.1 pmol/mg/min, respectively). However, there was a 4.4-fold decrease in the folic acid influx K_t for the H281A mutant at pH 4.5 versus 5.5 (21 ± 2.8 to 4.9 ± 0.8 μm) accompanied by a ∼50% increase in V_max (1073 ± 50.6 versus 715 ± 49.4 pmol/mg/min, respectively). Hence, under conditions in which WT-PCFT undergoes little change in influx kinetic parameters, the H281A mutant continues to be very sensitive to the decrease in extracellular pH as manifested by a fall in K_t and increase in V_max. This decrease in pH partially reversed the marked increase in K_t associated with the H281A mutation. It is of interest that the pH sensitivity of the H247A mutant, that manifests an increase in affinity for its folate substrates, was also less than that of the WT-PCFT (data not shown).

FIGURE 8.

The pH dependence of MTX influx for WT PCFT and the H281A mutant. A, pH dependence of [³H]MTX influx in cells transfected with WT-pcft or the H281A mutant. B, [³H]MTX influx over a pH range of 6.0–4.5. Data are plotted as -fold change in [³H]MTX influx at pH 6.0, which is set to 1. Data are the mean ± S.E. from three independent experiments. Influx was assessed over 1 min; the [³H]MTX concentration was 0.5 μm.

Electrophysiological Studies in Xenopus Oocytes

Current and intracellular pH (pH_i) were measured in Xenopus oocytes microinjected with WT-pcft, H281A-pcft, or H247A-pcft cRNA (Fig. 9). Oocytes were voltage clamped at −90 mV while simultaneously measuring pH_i. When the chamber containing oocytes that express WT-PCFT was perfused with ND90 at pH 5.5 there was neither current nor acidification. When MTX was added to the perfusate there was rapid onset of an inward current of ∼−100 nA along with cellular acidification (∼0.05 unit). This returned to baseline when MTX was removed from the buffer (Fig. 9A). When the pH of the perfusate was decreased to 4.5, a small inward current was generated in the absence of MTX with a very small decrease in pH_i. Both the current and the pH_i fall increased when oocytes were perfused with the MTX-containing ND90. When MTX-free buffer was then perfused at pH 4.5, there was a marked reversal in current, but not to the baseline level, with a continued small fall in pH_i. However, when the buffer pH was increased to 7.4 the whole cell current returned to baseline, and the fall in pH_i ceased (Fig. 9A). Fig. 9B illustrates that in water-injected oocytes there was essentially no change in intracellular pH nor current under these conditions. In Fig. 9C oocytes injected with H281A-pcft cRNA were studied. At pH 5.5 there was a small MTX-elicited inward current and very small fall in pH_i. The fall in pH_i (dpH_i/dt) for H281A was −15.8 × 10⁻⁵ pH units*s⁻¹ (Fig. 9C) compared with −25.4 × 10⁻⁵ pH units*s⁻¹ for WT-PCFT (Fig. 9A). When the ND90 pH was decreased to 4.5, there was a small inward current. Upon addition of MTX, there was a much larger MTX-induced inward current and ∼0.05 unit fall in pH_i. The increase in inward current induced by MTX at pH 4.5 was comparable for the WT-PCFT and H281A mutant. Additionally, at pH 4.5, the dpH_i/dt with addition of MTX was equivalent for H281A (−24.1 × 10⁻⁵ pH units*s⁻¹) and WT-PCFT (−24.5 × 10⁻⁵ pH units*s⁻¹).

FIGURE 9.

Electrophysiological studies of WT-PCFT, H247A-PCFT, and H281A-PCFT expressed in Xenopus oocytes. pH_i changes (upper tracing) and currents (lower tracing) were recorded simultaneously in individual oocytes voltage-clamped at V_h = −90 mV. Oocytes were superfused with ND90 solution (at the indicated pH_o; (5.5 blue shading and 4.5 yellow shading), and then MTX was added (gray shaded column). The general protocol was designed to change one solution component at a time: beginning at pH_o 7.5 (no shading), change pH_o to 5.5 (blue shading), add MTX, remove MTX, change to pH_o of 4.5 (yellow shading), add MTX, remove MTX, return to pH_o 7.5. A, recordings in an oocyte injected with WT-pcft at pH 5.5 and 4.5; 20 μm MTX was utilized, a concentration that saturates WT-pcft. B, a water-injected control oocyte with the identical protocol on the same day. C, the H281A-PCFT-injected oocytes. The MTX concentration used in this protocol was 100 μm to compensate for the high K_t for this mutant. D and E are the H247A-pcft-injected oocytes. In D, oocytes were exposed to pH 5.5 buffer first then to pH 4.5 buffer. In E, the order of exposure was reversed. In both cases the pH was returned to 7.4 between changes in the acid buffers. Recordings are representative of 3–8 separate experiments per experiment type from at least 3 donor Xenopus oocytes. The y axis in C indicates a range of −100 to −500 nA; the other panels indicate a range of 0 to −300 nA. All five experiments have been scaled so that the “5-min” time bar is identical. The text below the time bar indicates the actual experiment number.

The pattern was quite different in oocytes injected with the H247A mutant as indicated in Fig. 9D. There was a small current and drop in pH_i upon exposure of oocytes to the pH 5.5 buffer (initial dpH_i/dt was −65.6 × 10⁻⁵ pH units*s⁻¹). There was little further change when MTX was present in the ND90 (initial dpH_i/dt was −11.6 × 10⁻⁵ pH units*s⁻¹). When the pH was increased to 7.4 the current went to, or slightly above, the baseline and pH_i stabilized (−0.5 × 10⁻⁵ pH units*s⁻¹). When the buffer pH was dropped to 4.5, in the absence of MTX, there was a marked inward current and initiation of acidification (−23.0 × 10⁻⁵ pH units*s⁻¹ in Fig. 9D). Upon exposure to MTX the current was slightly more negative, and the pH_i continued to fall (no change in rate). When the perfusate returned to buffer at pH 4.5, the inward current persisted, and there was a small further decrease in pH_i. The current returned to baseline and pH_i stabilized when the buffer pH was pH 7.4 (+13.5 × 10⁻⁵ pH units*s⁻¹). Fig. 9E illustrates the same pattern when the order of exposure to buffer at pH 5.5 and 4.5 was reversed. Under these conditions the initial exposure to pH 4.5 buffer also acidified the oocyte (−7.9 × 10⁻⁵ pH units*s⁻¹), and addition of MTX at pH 4.5 resulted in further oocyte acidification (−11.6 × 10⁻⁵ pH units*s⁻¹).

Hence, mutation of the His²⁴⁷ residue to Ala resulted in transport of H⁺ mediated by PCFT in the absence of folate substrate. This was also observed in the WT-PCFT and H281A mutant, but to a much lesser extent.

DISCUSSION

This study addressed aspects of the structure-function of PCFT focusing on the role of His residues in transport mediated by this carrier. Of the three fully conserved residues, mutation of only two to Ala, His²⁴⁷ and His²⁸¹, had a substantial impact on transport function. However, the changes that occurred when these sites were mutated were quite different and will be considered separately. Within the context of these studies, electrophysiological measurements encompassed, for the first time, direct assessment of intracellular pH. This demonstrated that (i) current induced by folate transport into Xenopus oocytes is accompanied by cellular acidification as occurs in all proton-coupled transport processes and (ii) PCFT-mediated proton transport can occur in the absence of folate (slippage) particularly when the pH gradient is high.

His²⁴⁷ is located in the central region of the large cytoplasmic loop between the sixth and seventh TMDs. This predicted loop consists of 31 residues but only one other residue, Arg²⁶⁴ (at the interface of the seventh TMD) is fully conserved across species. In general, this region is poorly conserved among the families of solute transporters; as was the case for the other eukaryotic folate carrier, the reduced folate carrier, rfc (40). However, conserved His residues in cytoplasmic loops are found in other proton-coupled transporters. His-260 in the proton-coupled peptide transporter (PEPT1; Slc15a1) and His-278 in PEPT2 (Slc15a2) (14) are located in the large central cytoplasmic loop between TMDs 6 and 7, but their functions are not known. Similar to the H247Q-PCFT mutant, the H260Q PEPT1 mutant was functional, but the impact of other mutations at this residue on transport kinetics was not studied (14). Indeed, had His²⁴⁷ of PCFT been initially mutated to Gln or Glu, amino acids that retain substantial transport activity, the unique properties of this residue might not have been pursued. The monocarboxylic acid transporter (MCT1 and Slc16a1) has a large cytoplasmic loop between TMDs 6 and 7 that contains a His residue. However, in this case, Arg¹⁴³ in the second cytoplasmic loop was proposed to be involved in substrate selection by forming an ion pair with Glu³⁶⁹ in the fifth cytoplasmic loop (41). His¹³⁴ in the mouse proton-coupled amino acid transporter (PAT1; Slc36a1), located in the first intracellular loop, is identical in all species but its functional role has not been reported (42).

Two major changes occurred with substitutions at His²⁴⁷. First, there was a marked increase in the affinity of PCFT for its substrates. Second, there was a dissociation between transport of folates and H⁺ such that the H247A mutant transported H⁺ (↓pH_i after shift to low pH bath solution) in the absence of the folates (Fig. 9, D and E). Although WT-PCFT has different affinities for folic acid, 5-formyl-THF, and 5-methylTHF, the K_i values for these substrates were all markedly reduced and comparable in the H247A mutant. This increase in affinity occurred irrespective of the charge, size, or polarity of the substituted residue. Consistent with this was the marked fall (∼8.4-fold) in the folic acid influx K_t of the H247A mutant. Although the influx V_max mediated by the H247A mutant was decreased, accounting for the absolute fall in influx at a folic acid concentration of 0.5 μm, influx was much better preserved for the H247Q and H247E mutants. Hence, the enhanced affinity of the His²⁴⁷ mutants was independent of changes in maximum velocity. The role that His²⁴⁷ might play in carrier function was explored using a structural model.

No experimentally solved three-dimensional structure is available for a eukaryotic member of the superfamily of facilitative solute carriers. However, the structures of the E. coli GlpT antiporter (29) and lactose symporter (LacY) (43) have been solved and are quite similar despite sequence identities as low as ∼12%. Extrapolating from these bacterial structures, several functionally diverse eukaryotic facilitative transporters have been modeled (44). Using a comparative protein structural model as well as electrostatic calculations, we found that the most suitable bacterial structural template was GlpT sharing 14% sequence identity to PCFT. This is consistent with the computational analysis reported by another group (45). This model suggests a structure in which an inner bundle of transmembrane helices provides a water-filled translocation pathway, highly electropositive at either end, through which folate substrates and accompanying proton(s) pass. Electrostatic calculations suggested that the region around His²⁴⁷, in particular, is highly electropositive and located at the cytoplasmic “opening” to the translocation pathway. The model predicted His²⁴⁷ to be sufficiently close to Ser¹⁷², in the second cytoplasmic loop and also fully conserved, to form a hydrogen bond (Fig. 7). Based on this prediction, Ser¹⁷² was mutated to Ala, which resulted in a similar high affinity phenotype as H247A. Although the correspondence between the H247A and S172A phenotype provides some support for the homology model's prediction, further evidence will be required to validate this interaction along with the predicted locations of the TMDs, or the residues that might appear to interact across trans-membrane segments.

These observations can be interpreted within the context of the “alternate access model” (46–49) that has been applied as a paradigm for eukaryotic solute transporters in general (50). In this model, intra- or extracellular intramolecular interactions stabilize specific carrier conformations during the transport cycle serving as “gates” that prevent or facilitate substrate access to the binding cavity within the translocation pathway by occluding or exposing the binding sites, respectively. Transitions between consecutive conformational states occur during a translocation cycle that alternate the accessibility of the translocation pathway. The ordered binding of the substrate and the co-transported ion to the outward facing carrier triggers a conformational change resulting in the transposition to an inward facing carrier and an ordered release of substrates into the cytoplasm.

Within the context of this model the data suggest that when the His²⁴⁷-Ser¹⁷² interaction is disrupted and its “tethering and filtering” role is lost, the carrier shifts to a conformation that increases the general unfettered accessibility of all folate substrates to a high affinity binding pocket accounting for the loss of selectivity and increase in affinity. This may be accompanied by a reduced rate at which the carrier oscillates, which depends upon the specific amino acid substituted and may account for the differences in transport function observed with the various His²⁴⁷ mutants. Such an alteration in conformational equilibrium was observed for G-protein-coupled receptors when substitution of Ala²⁹³ in the third cytoplasmic loop with 19 other amino acids produced a similar change in phenotype (51). Likewise, a change in conformational equilibrium was proposed to account for functional changes that occurred with a Tyr³³⁵ mutation in the intracellular loop between the sixth and seventh TMDs in the human dopamine transporter (52).

The disruption of the Ser¹⁷²–His²⁴⁷ bond, and the associated conformational change, might also explain the dissociation of folate and H⁺ transport seen with the H247A mutant by facilitating H⁺ entry into, and movement through, the translocation pathway without the requirement for co-transport with a folate molecule. Hence, this conformation may enhance a uniporter or channel-like property of the carrier with respect to H⁺. There are a variety of electrogenic transporters, along with many integral membrane proteins, that have channel-like properties (53). Indeed, it has been proposed that “channel-like fluxes are integral to the mechanism of molecules identified essentially as transporters” (54). For example, neurotransmitter transporters such as EAAT4 (Slc1a6), DAT1 (Slc6a3), and SERT (Slc6a4) have neurotransmitter-gated anion channel currents (55–59). EAAT4 has also been reported to show an arachidonate- and transmitter-stimulated H⁺ conductance (60). OPT3 is a “peptide” transporter (related to PepT1; Slc15a1), which is uncoupled from peptide transport (61). NBCn1 (Slc4a7) has an Na⁺“channel” mode (62); D555E-NBCe1 (Slc4a4) has a Cl⁻ conductance mode (63). Slc26a9 functions dually as an electrogenic nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel (64). DMT1 (Slc11a2; NRAMP2) mediates proton fluxes in the absence of Fe²⁺ substrate (65). The G185R mutation in DMT1 in the Belgrade mouse augments a Ca²⁺ channel (66). On the other hand DMT1 transports Fe²⁺ in the absence of a proton gradient (27). This was observed also for folate transport mediated by PCFT (1, 4).

In contrast to His²⁴⁷, His²⁸¹ is predicted to be located within the seventh TMD near the extracellular end of the translocation pathway. Mutation of this residue to Ala produced a substantially different transport phenotype: a marked increase, rather than a decrease in the influx K_t. This is consistent with a fall in the affinity of PCFT for its folate substrates with retention of a specificity profile similar to that of WT-PCFT. Several possibilities exist as a basis for the alteration in function of the H281A mutant. Although the substrate binding order for PCFT is not defined, in all other proton-coupled transporters, the proton(s) binds to the carrier first (27, 41, 67–69). In transporters operating at the acidic microclimate of the human intestine like PAT1 (SLC36A1) (69), DMT1 (SLC11A2) (27), EAAC1 (SLC1A1) (67), MCT1 (SLC16A1) (41), and PEPT1 (SLC15A1) (68), the affinity of the carrier for its substrate(s) is increased when protons are bound first. Hence, the influx kinetics for PCFT are consistent with proton(s) binding to the carrier first causing an allosteric change in the binding pocket that results in an increased affinity for folate substrates. For the WT-PCFT, maximum affinity for its folate substrates was achieved at pH 5.5. A further reduction in pH produces only a small further increase in activity and a minimal change in K_t, suggesting saturation of one or more H⁺-binding sites. For the H281A mutant, the magnitude of the increase in folic acid transport activity as the pH was decreased from 7.5 to 5.5 was far less. However with a further decrease in pH, there was a substantial further increase in folic acid transport. However, at pH 4.5, the folic acid influx K_t for the H281A mutant was still ∼3.5-fold higher than that of the wild-type influx K_t. The decreased pH required to enhance folate binding may be due to the increase in the H⁺ concentration required to achieve a conformation/accessibility of the binding pocket in the H281A mutant carrier that is similar to WT-PCFT at a higher pH. This could occur if, for instance, there was protonation of a negatively charged residue at another site in the H281A mutant that favorably affects the binding pocket. It is clear that His²⁸¹ is not absolutely required for H⁺-coupling, because folate-induced current in Xenopus oocytes in the absence of this residue was associated with cellular acidification. Rather, protonation of the His²⁸¹ residue may result in increased binding of folate substrates to PCFT as occurs with several proton-coupled transporters: DMT1 (12), PepT1 (68), and the pH-dependent anion transporter mouse band 3 protein (70, 71).