Properties of the Arg376 residue of the proton-coupled folate transporter (PCFT-SLC46A1) and a glutamine mutant causing hereditary folate malabsorption

Abstract

The proton-coupled folate transporter (PCFT-SLC46A1) is required for intestinal folate absorption and is mutated in the autosomal recessive disorder, hereditary folate malabsorption (HFM). This report characterizes properties and requirements of the R376 residue in PCFT function, including a R376Q mutant associated with HFM. Gln, Cys, and Ala substitutions resulted in markedly impaired transport of 5-formyltetrahydrofolate (5-FTHF) and 5-methyltetrahydrofolate (5-MTHF) due to an increase in K(m) and decrease in V(max) in HeLa R1-11 transfectants lacking endogenous folate transport function. In contrast, although the influx K(m) for pemetrexed was increased, transport was fully preserved at saturating concentrations and enhanced for the like-charged R376K- and R376H-PCFT. Pemetrexed and 5-FTHF influx mediated by R376Q-PCFT was markedly decreased at pH 5.5 compared with wild-type PCFT. However, while pemetrexed transport was substantially preserved at low pH (4.5-5.0), 5-FTHF transport remained very low. Electrophysiological studies in Xenopus oocytes demonstrated that 1) the R376Q mutant, like wild-type PCFT, transports protons in the absence of folate substrate, and in this respect has channel-like properties; and 2) the influx K(m) mediated by R376Q-PCFT is increased for 5-MTHF, 5-FTHF, and pemetrexed. The data suggest that mutation of the R376 residue to Gln impairs proton binding which, in turn, modulates the folate-binding pocket and depresses the rate of conformational alteration of the carrier, a change that appears to be, in part, substrate dependent.

Fig. 1.

Proton-coupled folate transporter (PCFT) protein expression and trafficking to the cell membrane. Top: Western blot of crude membrane preparations of wild-type (WT) PCFT and a variety of mutants with substitutions at the R376 residue, along with actin-loading controls. Middle: Western blot analysis performed after biotinylation of PCFT accessible at the cell surface followed by streptavidin pull-down. The data in both top and middle panels are representative of five Western blots. There was some variability in R376Q PCFT levels in the crude membrane preparations and biotinylation at the cell surface among the experiments. In several cases, expression was comparable to that of wild-type PCFT. Bottom: Western blot following dilution of the wild-type PCFT pull-down sample to more accurately quantify, by comparison, levels of the mutant R376Q PCFT. Ten microliters of the biotinylated wild-type PCFT protein fraction (shown in middle panel) was added to 10, 30, and 70 μl (1:2, 1:4, and 1:8 dilution, respectively) of 2× SDS-loading buffer, and an equal volume of the samples was analyzed by Western blot analysis.

Fig. 2.

Influx of tritiated folate compounds mediated by PCFT mutants with substitutions at the R376 residue. HeLa cells were transfected with a variety of constructs, and influx was assessed over 1 min at 37°C, pH 5.5, and a substrate concentration of 0.5 μM. A: [³H]folic acid. B: [³H]5-methyltetrahydrofolate ([³H]5-MTHF). C: [³H]5-formyltetrahydrofolate ([³H]5-FTHF). D: [³H]methotrexate (MTX). M and WT, mock- and wild-type-transfected cells, respectively. The data are means ± SE from 3 independent experiments.

Fig. 3.

[³H]pemetrexed influx mediated by PCFT mutants in HeLa R1–11 cells. Left: influx of 0.5 μM pemetrexed. Right: influx compared at pemetrexed concentrations of 0.5, 2.5, and 5 μM. Conditions were the same as in Fig. 2. The data are means ± SE from 3 independent experiments.

Fig. 4.

Influx kinetics mediated by wild-type PCFT and the R376Q mutant as assessed with [³H]pemetrexed in HeLa R1–11 cell transfectants. Inset: [³H]pemetrexed influx kinetics measured over a low concentration range (0.05–6.0 μM) in cells transfected with wild-type PCFT. Influx was assessed over 1 min at 37°C and pH 5.5. The data are representative of 3 independent experiments.

Fig. 5.

Determination of 5-FTHF and 5-MTHF influx K_i values. Influx of [³H]pemetrexed was assessed at concentrations of 0.4 μM or 2.0 μM in HeLa R1–11 cells transfected with either wild-type or R376Q PCFT, respectively. Concentrations of 5-FTHF and 5-MTHF were chosen that produced 40–60% inhibition of [³H]pemetrexed influx. K_i values were determined on the basis of the measured pemetrexed influx K_m and the known concentrations of substrate and inhibitors according to the Michaelis-Menten equation. The data are means ± SE from 3 independent experiments.

Fig. 6.

Pemetrexed and 5-FTHF induced currents and proton-slippage. Wild-type PCFT (A) and R376Q-PCFT (B) were expressed in Xenopus oocytes clamped to −90 mV, and the elicited currents were recorded. The solution pH was initially 7.5, then switched to pH 5.5 (gray shading) to assess for folate substrate-independent currents. In this low-pH solution, oocytes were then exposed to 50 μM 5-FTHF or 50 μM pemetrexed (PMX), white bars, for 2–3 min following which the sequence was reversed. The data are representative of 4–5 experiments from at least 3 separate donor Xenopus.

Fig. 7.

Transport kinetics assessed electrophysiologically in Xenopus oocytes. A–C: current (I) as a function of concentration for pemetrexed (PMX; A), 5-FTHF (B), and 5-MTHF (C) measured with a holding potential of 90 mV. The final points are the calculated maximum current (I_max; V_max). Wild-type PCFT data are indicated by round symbols; data for the R376Q-PCFT mutant are indicated by the square symbols. The ordinate scales are expanded in the insets to B and C to clarify the initial rates for the wild-type PCFT. D: comparison of the influx K_m values. E: influx I_max values among the PCFT folate substrates. The data are representative of 4–5 experiments from at least 3 separate donor Xenopus. Wild-type PCFT is indicated by open bars; the R376Q PCFT mutant is indicated by the filled bars.

Fig. 8.

The pH dependence of transport mediated by wild-type and R376Q PCFT. Left: pH profile for [³H]5-FTHF. Right: pH profile for [³H]pemetrexed. The concentration of both substrates was 0.5 μM. Uptake was assessed over 1 min at 37°C. The data are means ± SE from 3 independent experiments.