Solute Carrier Family 26 Member a2 (Slc26a2) Protein Functions as an Electroneutral SO₄²⁻/OH⁻/Cl⁻ Exchanger Regulated by Extracellular Cl⁻

Background: Slc26a2 is an SO₄²⁻ transporter, mutations in which cause diastrophic dysplasia. How Slc26a2 transports SO₄²⁻ is unknown.

Results: We found that Slc26a2 exchanges SO₄²⁻ for 2OH⁻ or 2Cl⁻ and is regulated by a promiscuous extracellular anion site.

Conclusion: Slc26a2 functions as SO₄²⁻/2OH⁻ or SO₄²⁻/2Cl⁻ exchanger, regulated by extracellular Cl⁻_o.

Significance: The findings should help in understanding aberrant SLC26A2 function in diastrophic dysplasia.

Abstract

Slc26a2 is a ubiquitously expressed SO₄²⁻ transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO₄²⁻ and the ion gradients that mediate SO₄²⁻ uptake are poorly understood. We report here that Slc26a2 functions as an SO₄²⁻/2OH⁻, SO₄²⁻/2Cl⁻, and SO₄²⁻/OH⁻/Cl⁻ exchanger, depending on the Cl⁻ and OH⁻ gradients. At inward Cl⁻ and outward pH gradients (high Cl⁻_o and low pH_o) Slc26a2 functions primarily as an SO₄²⁻_o/2OH⁻_i exchanger. At low Cl⁻_o and high pH_o Slc26a2 functions increasingly as an SO₄²⁻_o/2Cl⁻_i exchanger. The reverse is observed for SO₄²⁻_i/2OH⁻_o and SO₄²⁻_i/2Cl⁻_o exchange. Slc26a2 also exchanges Cl⁻ for I⁻, Br⁻, and NO₃⁻ and Cl⁻_o competes with SO₄²⁻ on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO₄²⁻_i/2OH⁻_o exchange. Slc26a2 can transport oxalate in exchange for OH⁻ and/or Cl⁻ with properties similar to SO₄²⁻ transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence ³⁶⁷GFXXP³⁷¹ between TMD7 and TMD8 close to the conserved Glu⁴¹⁷ in the permeation pathway. Mutation of Glu⁴¹⁷ eliminated transport by Slc26a2, whereas mutation of Phe³⁶⁸ increased the affinity for SO₄²⁻_o 8-fold while reducing the affinity for Cl⁻_o 2 fold, but without affecting regulation by Cl⁻_o. These findings clarify the mechanism of net SO₄²⁻ transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.

Introduction

Protein sulfation, and thus SO₄²⁻ , is essential for cellular and tissue survival. Many proteins undergo post-translational modification by sulfation. Tyrosine sulfation of signaling molecules, like the G protein-coupled receptor chemokine receptors (1), modifies signaling pathways. Protein sulfation contributes to detoxification of endogenous compounds (2). A critical role of protein sulfation is sulfation of proteoglycans (3). Proteoglycans are constituents of the extracellular matrix that mediate the cell response to growth factors (4). Several disorders are caused by mutations in genes that affect proteoglycan synthesis or sulfation. The sulfate groups in proteoglycans are critical in formation of active domains, and the high polyanionic charge density of the proteoglycans is neutralized by SO₄²⁻ (5). Sulfation of secretory proteins, like digestive enzymes and mucins, is essential for their synthesis, processing through the biosynthetic pathway and packaging in secretory granules (6). Hence, understanding SO₄²⁻ homeostasis is essential for understanding cell development and function.

Cells have two sources of SO₄²⁻, a minor source from degradation of cysteine and methionine and active uptake of SO₄²⁻ mediated largely by the SO₄²⁻ transporters Slc26a1 and Slc26a2⁴ (7, 8). Slc26a1 and Slc26a2 belong to the family of the SLC26 transporters, which includes 11 genes with Slc26a10 being a pseudogene (9). Members of the family transport remarkably diverse substrates, including Cl⁻, HCO₃⁻, I⁻, SO₄²⁻, formant, and oxalate, and can function as coupled electroneutral or electrogenic transporters or as ion channels (9, 10). Mutations in several members of the family are associated with human diseases, including autosomal recessively inherited chondrodysplasias (SLC26A2) (11, 12), congenital chloride diarrhea (SLC26A3) (13), Pendred syndrome (SLC26A4) (14), deafness (SLC26A5) (15), and perhaps reduced fertility (SLC26A8) (16). In addition, deletion of Slc26a6 in mice resulted in nephrolithiasis due to aberrant oxalate transport (17) and in aberrant pancreatic and parotid ducts HCO₃⁻ transport (18, 19).

Although Slc26a1 has limited tissue distribution, Slc26a2 is ubiquitously expressed with particularly high levels in developing and mature cartilage as well as in epithelial tissues like pancreas, salivary glands, colon, bronchial glands, tracheal epithelium, and eccrine sweat glands (20, 21). The central role of Slc26a2 in supplying the bulk of cellular SO₄²⁻ is evident from the lethality of deletion of the SLC26A2 gene in humans and mice (20, 22), mainly due to under-sulfation of proteoglycans leading to aberrant development (23). Indeed, measurement of SO₄²⁻ uptake in fibroblast from patients with a severe form of the disease showed reduced or lack of SO₄²⁻ uptake (20, 24). Most mutations causing diastrophic dysplasia are missense mutations that affect either trafficking to the plasma membrane or showed reduced SO₄²⁻ transport (25, 26).

The phenotype of chondrodysplasias is highly variable, ranging from mild (27) to lethal before or shortly after birth (11). To better understand the disease and cellular SO₄²⁻ homeostasis, it is necessary to understand transport and regulation of Slc26a2. To date, characterization of transport by Slc26a2 was based on measurement of isotopic fluxes (24, 25, 28) that are the sum of both net and exchange fluxes, with the exchange dominating the fluxes. These studies revealed that Slc26a2 can transport SO₄²⁻, Cl⁻, and oxalate (24, 25, 28), and a recent detailed characterization of the fluxes suggested that Slc26a2 functions as an electroneutral transporter when mediating isotopic fluxes. SO₄²⁻ fluxes appeared to be sensitive to intracellular and extracellular pH (24). An unusual finding was that inhibition of SO₄²⁻ and oxalate isotopic uptake by external Cl⁻ exhibited simple saturation, whereas Slc26a2-mediated exchange of intracellular SO₄²⁻, oxalate, or Cl⁻ for external Cl⁻ was non-saturable (24), suggesting that the measured fluxes, at least isotopic efflux, is mostly exchange rather than net fluxes.

The available information is not sufficient to determine the mode of SO₄²⁻ and other ions transport by Slc26a2 and the cellular ionic gradients that drive net transport. We used Xenopus oocytes expressing Slc26a2 to report that Slc26a2 functions as SO₄²⁻/2OH⁻, SO₄²⁻/2Cl⁻, and SO₄²⁻/OH⁻/Cl⁻ exchanger, depending on the cellular Cl⁻ and OH⁻(H⁺) gradients. Slc26a2 can also mediate Ox²⁻_o/OH⁻_i/Cl⁻_i exchange and transport I⁻, Br⁻, and NO₃⁻. Slc26a2 activity is regulated by an extracellular anion binding site, which is not involved in ion transport. Modeling of the Slc26a2 transmembrane sector identified an extracellular loop, which contains the conserved sequence ³⁶⁷GFXXP³⁷¹ in the vicinity of the gating Glu⁴¹⁷ as a potential part of the permeation pathway. These findings should help in further understanding ion transport by the SLC26 transporters and aberrant SLC26A2 function in diastrophic dysplasia.

EXPERIMENTAL PROCEDURES

Solutions and Reagents

For experiments in oocytes, the standard HEPES-buffered ND96 solution contained (in mm): 96 NaCl, 2 KCl, 1.8 CaCl₂, 1 MgCl₂, and 5 HEPES, pH 7.5. Cl⁻-free solutions were prepared by replacing chloride with gluconate in the presence of calcium cyclamate substituted for CaCl₂. A 100 mm solution of diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) (Invitrogen) dissolved in DMSO was prepared freshly and diluted to a final concentrations of 10 or 50 μm in the relevant solutions. All other chemicals and reagents were purchased from Sigma.

cRNA Preparation

The pCMV-Sport6-Slc26a2 (GenBank^TM/EMBL/DDBJ, accession no. BC028345) was purchased from Open Biosystems and was used as template for cRNA preparation. The plasmid was linearized with NotI and used to transcribe cRNA with an mMESSAGE mMACHINE Sp6 kit (Life Technologies, Applied Biosystems), respectively. Mutation in Slc26a2 were generated by a site-directed mutagenesis kit (Agilent Technologies) and verified by sequencing.

Biotinylation and Western Blot Analysis

To monitor surface expression of Slc26a2 WT, E417A, and E417K, HEK cells transfected with vector alone or Myc-tagged Slc26a2 constructs were incubated with EZ link Sulfo-NHS-LC-Biotin (0.5 mg/ml, Thermo Fisher Scientific) for 30 min at room temperature. Subsequent steps were as previously described (29) with the following modifications: 50 μl of 1:1 slurry of immobilized avidin beads (Thermo Fisher Scientific) was added to 300 μg of protein in 300 μl of cell extract, and the mixture was incubated overnight. To monitor protein expression the PVDF membranes were incubated overnight with anti-Myc antibodies diluted 1:1,000 (Cell Signaling) and for 1 h with HRP-conjugated goat anti-mouse (Invitrogen) diluted 1:2,000. For β-actin detection membranes were incubated for 1 h with monoclonal anti-β-actin peroxidase (Sigma-Aldrich) diluted 1:20,000.

Xenopus laevis Oocyte Preparation

All experiments in this study were conducted under the National Institutes of Health guidelines for research on animals, and experimental protocols were approved by the Institutional Animal Care and Use Committee. Oocytes were isolated by partial ovariectomy of anesthetized female X. laevis (Xenopus Express, Brooksville, FL) and treated by collagenase B (Roche Applied Science), as described previously (30). Stage V–VI oocytes were injected with 10 ng of cRNA using glass micropipettes and a microinjection device (Nanoliter 2000; World Precision Instruments) in a final volume of 27.6 nl. Control oocytes were injected with equal volumes of H₂O. Oocytes were incubated at 18 °C in ND96 supplemented with 2.5 mm pyruvate and antibiotics and were studied 72–144 h after injection.

Voltage, pH, and Cl⁻ Measurement in Oocytes

Voltage recordings were performed at room temperature with two-electrode voltage clamp, exactly as described previously (29, 30). Voltage, pH_i, and Cl⁻_i concentrations were measured as detailed previously (31, 32). In the present study, the Cl⁻-sensitive electrode was also used to record intracellular Br⁻, I⁻, and NO₃⁻ with the resin and the procedure used to measure Cl⁻ (see “Results”).

Measurement of Buffer Capacity

To determine OH⁻(H⁺) fluxes by Slc26a2 we determined the buffer capacity of oocytes bathed in HEPES-buffered medium. Because we can measure both Cl⁻_i and pH_i, we determined the buffer capacity directly rather than relying on pH_i changes induced by weak acids. Supplemental Fig. 1A shows that two consecutive injections of the oocytes with 13.8 nl of 100 mm HCl reduced pH_i and increased Cl⁻_i. Similar determination in five experiments and using the pH_i and Cl⁻_i changes of the first injection resulted in a buffer capacity of 17.1 ± 2.2/pH unit, which is similar to that reported by others (33).

Modeling and Prediction of the Slc26a2 Transmembrane Domains Structure

The transmembrane sector of the mouse Slc26a2 model was generated using the Deepview Swiss-PDB viewer by Raw sequence fit of the Slc26a2 sequence (NCBI accession no. NP_031911) onto the putative Slc26a6 model previously generated by us based on structural similarity to the bacterial ClC-ec protein (29). The predicted binding site of DIDS on the Slc26a2 model was performed with the AutoDockVina software (34), according to software tutorial instructions. Briefly, the box grid determining the Slc26a2 region of binding was set using the AutoDockTools software with the following coordinates (center: x = 0.472, y = 1.222, z = 0.472) (size: x = 30, y = 26, z = 24). Exhaustiveness level was set to default. AutoDockTools was further used to select all rotatable bonds of the DIDS molecule. The AutoDockVina software generated nine different models, and herein we present the best model as ranked by the software with a predicted affinity of −8.9 kcal/mol for the binding of Slc26a2 and DIDS. The final model (cartoon and surface representations) was generated using PyMOL (Schrödinger, LLC).

RESULTS AND DISCUSSION

Slc26a2 Functions as an SO₄²⁻/2OH⁻, SO₄²⁻/2Cl⁻, and Possibly SO₄²⁻/OH⁻/Cl⁻ Exchanger

Slc26a2-mediated net fluxes were assayed in Xenopus oocytes by measuring intracellular pH (pH_i) and Cl⁻ (Cl⁻_i) and the membrane potential in the same oocytes. Fig. 1A shows that removal of extracellular Cl⁻ (Cl⁻_o) had no effect on pH_i and the membrane potential with a slow rate of reduction in Cl⁻_i. Exposing Slc26a2-expressing oocytes bathed in Cl⁻-free solution to 0.2 mm SO₄²⁻ resulted in a precipitous reduction in pH_i and Cl⁻_i. Removal of SO₄²⁻_o with the concomitant addition of Cl⁻_o resulted in increased pH_i and Cl⁻_i. Fig. 1C shows almost no change in Cl⁻_i and pH_i in water-injected oocytes under the same conditions.

FIGURE 1.

Slc26a2 functions as an electroneutral SO₄²⁻/OH⁻(H⁺)/Cl⁻ exchanger. pH_i (dark traces) and Cl⁻_i (dark gray traces) and membrane potential (light gray traces) were simultaneously recorded in Slc26a2 (A) or H₂O (C)-injected oocytes. Note that the membrane potential did not change following addition of SO₄²⁻. B, averaged (mean ± S.E. of the indicated number of experiments) transport rates of Cl⁻ and OH⁻(H⁺) in the presence and absence of 0.2 mm SO₄²⁻ were used to determine net transport (rightmost columns). D, pH_i was recorded while clamping membrane potential at −100 , +40, or −30 mV, as indicated.

Reduction in pH_i can be due to H⁺ influx or OH⁻ efflux. Because some of the SO₄²⁻ transport is coupled to Cl⁻, and the Cl⁻ coupling is affected by pH_o (see below), we will refer to the transported ion as OH⁻, although we cannot distinguish between the transport of OH⁻ and H⁺. The average Slc26a2-mediated SO₄²⁻-coupled net Cl⁻ and OH⁻ transports are shown in Fig. 1B and indicate that under the conditions of Fig. 1A ∼40% of SO₄²⁻ is transported in exchange for Cl⁻ and ∼60% in exchange with OH⁻. SO₄²⁻ transport is electroneutral, because it is not associated with a change in membrane potential (Fig. 1), and SO₄²⁻-coupled OH⁻ (Fig. 1D) and Cl⁻ (not shown) fluxes are the same at membrane potentials of +40 and −100 mV. This indicates that the coupling stoichiometry of SO₄²⁻ exchange with Cl⁻ and OH⁻ is likely 1:2 with Slc26a2 functioning as SO₄²⁻/2OH⁻, SO₄²⁻/2Cl⁻ and possible SO₄²⁻/OH⁻/Cl⁻ exchanger.

To further determine the relationship between SO₄²⁻ and Cl⁻ we measured the effect of Cl⁻_o on the apparent affinity for SO₄²⁻_o. Fig. 2A shows an example of the protocol used for these experiments. Oocytes expressing Slc26a2 were exposed to solutions containing the desired Cl⁻_o (0, 5, 20 or 50 mm) and SO₄²⁻ concentration for 5 min to obtain the rate of OH⁻ efflux. Then the oocytes were incubated in Cl⁻-containing solution without SO₄²⁻ to extrude the SO₄²⁻ and recover pH_i before exposure to the subsequent SO₄²⁻ concentration. The plots in Fig. 2B obtained from these experiments were used to calculate the apparent K_m for SO₄²⁻ that were then plotted as a function of Cl⁻_o (Fig. 2C). The linear relationship in Fig. 2C indicates that Cl⁻_o competes with SO₄²⁻_o for interaction with the external substrate site. This competition is different from the non-saturating Cl⁻_o dependence reported for the isotopic exchange of Cl⁻_o with intracellular Cl⁻ (Cl⁻_i) or intracellular oxalate (24). This may reflect the different dependence of Cl⁻_o of the half (exchange) and full turnover cycle (net) of transport by Slc26a2.

FIGURE 2.

Effect of Cl⁻_o on Slc26a2-mediated SO₄²⁻_o/OH⁻_i exchange. A, Slc26a2-expressing oocytes were alternately perfused with a solution containing 110 mm Cl⁻ and Cl⁻-free solutions containing the indicated SO₄²⁻ concentrations for ∼5 min. The plots in B are the relative average rates of OH⁻ fluxes as a function SO₄²⁻ at Cl⁻_o of 0 (○), 5 (■), 20 (△), and 50 (●) mm. The rates were normalized to the rate at 30 mm SO₄²⁻, which was taken as 100%, and the plots were fitted to the Hill equation. The averages are the mean ± S.E. of 3–5 experiments at each Cl⁻_o concentration. The resulting apparent K_m values for SO₄²⁻ are plotted as a function of Cl⁻_o (C). D, control (H₂O-injected, black trace) and Slc26a2-expressing oocytes (gray trace) bathed in HCO₃⁻-buffered solution were exposed to Cl⁻-free solution at the indicated time to asses Cl⁻/HCO₃⁻ exchange activity. In E the exchange of I⁻, Br⁻, and NO₃⁻ with Cl⁻_i was measured by incubating oocytes in Cl⁻-free solution containing 2 mm of I⁻ (left traces), Br⁻, or NO₃⁻ (right traces). The influx was terminated by removal of the anions from the bath and anion efflux was initiated by addition of Cl⁻ to the bath. The lack or minimal anion efflux in the absence of Cl⁻_o indicates low net and exchange rate of the anions with OH⁻ and the rapid efflux of the anions upon addition of Cl⁻_o indicates fast exchange rate of the anions with Cl⁻. The control in the left panel is representative of oocytes injected with H₂O.

Many of the SLC26 transporters can transport HCO₃⁻ in exchange for Cl⁻ (9). However, Fig. 2D shows that Slc26a2 does not function as a Cl⁻/HCO₃⁻ exchanger. The capacity of Slc26a2 to transport other anions, such as I⁻, Br⁻ and NO₃⁻, in addition to SO₄²⁻, OH⁻ and Cl⁻ was further tested by measuring their intracellular concentration. supplemental Fig. 1B shows that the resin used to detect Cl⁻ can also detect Br⁻ and NO₃⁻ ∼10 times better than Cl⁻ and I⁻ ∼100 times better than Cl⁻ (see also (29)). The left panel of Fig. 2D shows that exposing Slc26a2-expressing oocytes to Cl⁻-free solution containing 2 mm I⁻ resulted in a rapid influx of I⁻. Removal of I⁻ in the absence of Cl⁻_o stopped the influx. To initiate I⁻ efflux it was necessary to add Cl⁻_o, with as little as 1 mm Cl⁻_o resulting is nearly maximal rate of I⁻ efflux. Similar behavior was observed with Br⁻ and NO₃⁻ (Fig. 2D, right panel) and no I⁻ (Fig. 2D, gray trace, left panel), Br⁻ or NO₃⁻ (not shown) fluxes were observed in water-injected oocytes. These findings indicate that the Slc26a2 permeation pathway is not very selective and can accommodate I⁻, Br⁻ and NO₃⁻ to mediate I⁻/Cl⁻, Br⁻/Cl⁻ and NO₃⁻/Cl⁻ exchange.

The Ratio of SO₄²⁻/2OH⁻ and SO₄²⁻/2Cl⁻ Exchange Is Determined by pH_o

Coupling of SO₄²⁻ transport to OH⁻ and Cl⁻ raised the question of how the availability of substrate would affect the coupling. We addressed this question by examining the effect of pH_i and pH_o on SO₄²⁻ transport. Fig. 3A shows example traces of the changes in pH_i (left panel) and of the Cl⁻_i (right panel) as a result of SO₄²⁻ transport at pH_o of 6.5 (black traces) and 8.2 (gray traces). The rates of OH⁻ and Cl⁻ influx and efflux under both conditions are summarized in Fig. 3B. The models in Fig. 3B show the direction of ion fluxes during SO₄²⁻ influx (left) and SO₄²⁻ efflux (right) and the columns show the associated OH⁻(H⁺) and Cl⁻ fluxes at pH_o of 6.5 and 8.2. SO₄²⁻ influx is coupled to Cl⁻ and OH⁻ efflux, while SO₄²⁻ efflux initiated by removal of SO₄²⁻_o and addition of Cl⁻_o is coupled to Cl⁻ and OH⁻ influx. During SO₄²⁻ influx acidic pH_o increases OH⁻ efflux with low Cl⁻ efflux while alkaline pH_o has the opposite effect. On the other hand, during SO₄²⁻ efflux acidic pH_o inhibits OH⁻ efflux and increases Cl⁻ efflux while alkaline pH_o has the opposite effect.

FIGURE 3.

Effect of pH_o on SO₄²⁻_o influx and SO₄²⁻_i efflux. A, example traces for measurement of pH_i and Cl⁻_i in solutions buffered to pH_o of 6.5 (black traces) or 8.2 (gray traces). The models in B show the direction of the fluxes and the average efflux and influx rates of OH⁻(H⁺) (dark columns) and Cl⁻ (gray columns) fluxes are summarized in B. The results are the mean ± S.E. of the number of experiments listed in the columns. In C pH_i and Cl⁻_i were measured at 110 mm Cl⁻_o and first at pH_o of pH 7.5 and then at pH_o of 6.5. The columns on the right show the average rates (mean ± S.E.) of Cl⁻ and OH⁻ influx at pH_i of 6.5 upon removal of SO₄²⁻, and the lower traces are example traces obtained in H₂O-injected oocytes.

Fig. 3C further illustrates the reciprocal effect of pH_o on OH⁻ and Cl⁻ fluxes. Exposing Slc26a2-expressing oocytes to a solution buffered to pH 7.5 and containing 110 mm Cl⁻ and 2 mm SO₄²⁻ resulted in a reduction in pH_i at a rate of ∼0.18 ± 0.03 mm/min (n = 8), with no change in Cl⁻_i. Removal of SO₄²⁻ resulted in recovery of pH_i. H₂O injected oocytes showed no response to SO₄^2−. Hence, at high Cl⁻_o and pH_o of 7.4 all the Slc26a2-mediated SO₄²⁻_iflux is mediated by SO₄²⁻/2OH⁻ exchange (or SO₄²⁻/2H⁺ cotransport). When the same oocytes were exposed to the same solution containing 110 Cl⁻ and 2 mm SO₄²⁻, but now buffered to pH of 6.5, SO₄²⁻ uptake resulted in a large reduction in pH_i with no change in Cl⁻_i, while SO₄²⁻ efflux initiated by removal of SO₄²⁻_o resulted in a small increase in pH_i and a large Cl⁻ influx (Fig. 3C). Thus, at low pH_o SO₄²⁻ uptake is predominantly mediated by SO₄²⁻/2OH⁻(2H⁺) exchange, while SO₄²⁻ efflux is dominated by SO₄²⁻_i/Cl⁻_o exchange.

The sulfate transported species can be SO₄²⁻ or HSO₃⁻. Although we did not examine this in great detail, the results in Figs. 1–3 favor SO₄^2−. Thus, if the transported species is HSO₃⁻ then acidic pH_o should markedly enhance Sulfate influx. Fig. 3B indicates that is not the case. Second, SO₄²⁻ efflux after removal of SO₄²⁻_o should be independent of pH_o since pH_o should have no effect of the transported SO₄²⁻ species. Again, this is not the case. Third, changes on pH_o have the same effect on SO₄²⁻ and Ox²⁻ transport (see below), suggesting that the transport rate follows the pH gradient rather than substrate species.

Coupling of SO₄²⁻ transport to both Cl⁻ and OH⁻ may function to ensure SO₄²⁻ uptake under acidic and alkaline conditions. Slc26a2 is expressed in the luminal membrane of polarized cells (31, 35) that can be exposed to acidic and alkaline pH. In the stomach and synovial fluid pH is acidic (36, 37) and SO₄²⁻_o/2OH⁻_i exchange mediates most SO₄²⁻ uptake. On the other hand, in secretory glands, like the pancreas (38) and salivary glands (39), luminal pH is alkaline, which inhibits SO₄²⁻_o/OH⁻_i exchange (Fig. 3B) and most SO₄²⁻ uptake is by SO₄²⁻_o/2Cl⁻_i exchange.

Regulation of Slc26a2 by Cl⁻_o

While measuring net SO₄²⁻ efflux we noticed that removal of SO₄²⁻_i in the continuous absence of Cl⁻_o never resulted in SO₄²⁻ efflux, as would be expected from SO₄²⁻_i/2OH⁻_o exchange. This is illustrated in the period bordered by the dashed box of Fig. 4A. However, addition of as little as 1 mm Cl⁻_o triggered a robust SO₄²⁻_i/2OH⁻_o exchange and a small Cl⁻ influx (Fig. 4A, period marked by gray box). The dependence of the SO₄²⁻_i/2OH⁻_o exchange rate of Cl⁻_o followed simple saturation curve with apparent K_m of 3.7 ± 0.9 mm (Fig. 4B). Activation of the exchange was not specific for Cl⁻. Fig. 4C shows that 1 mm external Cl⁻, Br⁻, I⁻, NO₃⁻ and SCN⁻ similarly activated SO₄²⁻_i/2OH⁻_o exchange. Only 1 mm F⁻ did not activate the exchange (Fig. 4C), but actually inhibited the exchange initiated by the other anions (not shown).

FIGURE 4.

Activation of Slc26a2-mediated SO₄²⁻_i/OH⁻_o exchange by Cl⁻_o. A, example traces of Cl⁻_i (black) and pH_i (gray) in oocytes expressing Slc26a2. SO₄²⁻_iflux was terminated by removal of SO₄²⁻_o (period bordered by dashed line). SO₄²⁻ efflux did not start until the addition of 1 mm Cl⁻_o (period bordered by gray square), which triggered robust SO₄²⁻_i/OH⁻_o exchange with minimal SO₄²⁻_i/Cl⁻_o exchange. B, the relative rate of SO₄²⁻_i/OH⁻_o exchange is plotted as a function of the activating Cl⁻_o. The rates at each Cl⁻_o were normalized to the rate measured at 110 mm Cl⁻_o, which was taken as 100%. The plot is the average of 3–5 experiments and fitted to the Hill equation. C, the protocol in A was used to measure activation of SO₄²⁻_i/OH⁻_o exchange by 1 mm of the indicated anions (period marked by gray square). The results are mean ± S.E. of the number of experiments indicated in the columns.

The findings in Fig. 4 suggest that SO₄²⁻ transport by Slc26a2 is regulated by interaction of an anion with a regulatory site. The regulatory site is not selective for Cl⁻, but because Cl⁻ is the major extracellular anion, Slc26a2 is likely regulated by Cl⁻_o interaction with the regulatory site. The Cl⁻_o regulatory site is likely different from the transport site since increased Cl⁻_o should increase SO₄²⁻_i/2Cl⁻_o exchange while reducing SO₄²⁻_i/2OH⁻_o exchange. However, the opposite is observed. Activation of Slc26a2-mediated SO₄²⁻_i/2OH⁻_o by Cl⁻_o may be by stabilization of an active Slc26a2 conformation. However, the exact mechanism remains to be elucidated. The physiological significance of regulation of Slc26a2 activity by Cl⁻_o is not known at present. The Cl⁻ content in the GI tract is high in the range of 100–150 mm and is determined largely by acid secretion (40). On the other hand, urine Cl⁻ can be below 4 mm when prerenal azotemia occurres with metabolic alkalosis (41) and regulation of Slc26a2 by Cl⁻_o can become significant. In addition, the luminal membrane-localized Slc26a2 is exposed to variable Cl⁻ concentrations, as low Cl⁻ in ducts that absorb the Cl⁻, such as the pancreatic (38) and salivary (32) ducts, the intestine (42) and the vas deferens (43). One possibility is that SO₄²⁻ absorption take place only when there is some luminal Cl⁻. Perhaps in Cl⁻ absorbing epithelia completion of Cl⁻ absorption may be used to signal termination of the SO₄²⁻ absorptive activity. Additionally, regulation by Cl⁻_o may be used to stop reverse Slc26a2 transport to prevent SO₄²⁻ loss by SO₄²⁻ efflux across the luminal membrane due to SO₄²⁻_i/2OH⁻_o when luminal Cl⁻ becomes very low due to Cl⁻ absorption.

A Potential Slc26a2 Permeation Pathway

In a previous study we developed a model of the Slc26a6 transmembrane sector to search for motifs that determine the function of the electrogenic Slc26 transporters as coupled and uncoupled transporters (29). The modeling identified a glutamate (Glu⁻) conserved in all Slc26 transporters that has the same orientation as Glu⁻ E148 in the Cl⁻ permeation pathway of the ClC transporters (44–47). Interestingly, a recent study utilized the predicted Slc26a6 model and the crystal structure of the Slc26a5 STAS domain to assemble a detailed putative structure of Slc26 transporters (48). This structure showed a surprising similarity to the low resolution structure of a bacterial Slc26 homologue obtained using SANS (small angle neutron scattering) method in terms of symmetry and size. Notably, this study suggested that Slc26 functions as a dimer, as was previously suggested based on the predicted similarity of Slc26a6 to the bacterial ClC-ec dimeric crystal structure. Therefore, assuming a similar overall architecture for Slc26 transporters, we thread the Slc26a2 transmembrane sector on the Slc26a6 model to determine the localization of the conserved Glu⁻ Glu⁴¹⁷ (Fig. 5). Another purpose of the modeling was to identify additional determinants of the Slc26a2 ion permeation pathway and perhaps the extracellular Cl⁻ regulatory site. The motif GSGIP was identified as a potential anion (Cl⁻) binding site that is conserved in the ClC transporters (44, 49). Mutations of residues within this motif altered ionic selectivity and coupling in the yeast and mammalian ClCs (50, 51). We searched for a similar motif in the Slc26 transporters. Although identical motif is not present in the Slc26 transporters, supplemental Fig. 2 shows the presence of the well conserved sequence GFXXP. The structural model in Fig. 5 shows the predicted localization of the Slc26a2 conserved Glu⁻ Glu⁴¹⁷ and phenylalanine Phe³⁶⁸ and of a potential DIDS binding site.

FIGURE 5.

In silico model of the putative structure of the Slc26a2 TMDs. The model was derived by threading the TMD sector of Slc26a2 on the TMDs of Slc26a6 reported before (29) (see “Experimental Procedures”). A, shows the predicted position of the 12 transmembrane helices of Slc26a2. B, shows the space filling of the GFMPG sequence. Highlighted in C are the positions of the extracellular loop located between TMDs 7 and 8 (light blue), the conserved phenylalanine Phe³⁶⁸ (blue), the conserved Glu⁻ Glu⁴¹⁷ (green), and the putative position of DIDS binding site (red). D, illustrates a cross-section (∼20 Å) through the surface representation of the putative Slc26a2 revealing a potential binding cavity. Interestingly, Glu⁻ 417 (green) and the EC loop (orange) are constituents of the binding pocket (outlined in cyan) in which the inhibitor DIDS (red) is also bound.

To test the prediction in Fig. 5 we first determined the sensitivity of Slc26a2 to DIDS. Fig. 6A shows that 50 μm DIDS completely inhibited SO₄²⁻-driven OH⁻(H⁺) efflux and most of the SO₄²⁻-driven Cl⁻ efflux. The residual DIDS-insensitive Cl⁻ efflux is likely not mediated by Slc26a2 but by a DIDS-insensitive transporter native to the oocytes. Fig. 6B shows that DIDS inhibited SO₄²⁻ efflux when added after SO₄²⁻ uptake. Also in this case DIDS completely inhibited OH⁻(H⁺) influx, but with a residual Cl⁻ influx. Similar results were obtained with 10 and 50 μm DIDS, indicating that the DIDS sensitivity of Slc26a2 is in the same range of that reported for Slc26a6 (52). Fig. 6C summarizes the rates of OH⁻ and Cl⁻ fluxes in the absence and presence of SO₄²⁻ and DIDS, indicating that at pH_o of 7.5 and the absence of Cl⁻_o ∼60% of SO₄²⁻ uptake is coupled to OH⁻ efflux and 40% to Cl⁻ efflux. Fig. 6D test another prediction of the model in Fig. 5 by neutralizing (Slc26a2(E417A)) or reversing (Slc26a2(E417K)) the charge of the conserved Glu⁴¹⁷. Both mutations eliminated SO₄²⁻ (Fig. 6D) and I⁻ (not shown) transport activity. Inhibition of transport was not due to altered trafficking of the mutants to the plasma membrane (Fig. 6E).

FIGURE 6.

Inhibition of Slc26a2 by DIDS and by mutations of Glu⁴¹⁷. Example traces depicting inhibition of SO₄²⁻_iflux (A) and SO₄²⁻ efflux (B) by 50 μm DIDS. Note the complete inhibition of the coupled OH⁻ but not of Cl⁻ fluxes. C, the average rates (mean ± S.E. of the indicated number of experiments) of OH⁻ and Cl⁻ fluxes. In D shown are examples of oocytes expressing either wild-type Slc26a2, Slc26a2(E417A), or Slc26a2(E417K) that were used to measure the SO₄²⁻-associated OH⁻ and Cl⁻ fluxes. E, shows the surface expression of Slc26a2 and mutants with actin used as a control for the biotinylation.

The sequence GFXXP is predicted to be in the extracellular loop between transmembrane domains (TMDs) 7 and 8, with Phe³⁶⁸ predicted to be in the entrance of the permeation pathway (Fig. 5). The mutations G367A and P371A had no effect on SO₄²⁻ transport or its coupling to Cl⁻ and OH⁻ (not shown). However the F368A mutation had multiple effects. Fig. 7A shows that Slc26a2(F368A) is ∼50% less active than wild-type Slc26a2 in exchanging SO₄²⁻ for OH⁻ (left traces) and Cl⁻ (right traces). Most notably, the F368A mutation increased the apparent affinity of Slc26a2 for SO₄²⁻ by ∼8-fold to reduce the apparent K_m for SO₄²⁻_o from 79 ± 7 to 9.7 ± 0.7 μm. Unexpectedly from competition between SO₄²⁻_o and Cl⁻_o (Fig. 2), the F368A mutation increased the apparent K_m for inhibition of SO₄²⁻ uptake by Cl⁻_o from 26 to 50 mm (Fig. 7C). Hence, Phe³⁶⁸ appears to control the access of SO₄²⁻ and Cl⁻ to the permeation pathway. Interestingly, Fig. 7D shows that the F368A mutation had no effect of the apparent affinity for the Cl⁻_o regulatory site that activates SO₄²⁻_i/OH⁻_o exchange. This finding provides the strongest evidence that inhibition of SO₄²⁻ uptake by Cl⁻_o (Figs. 4B and 7C) and activation of SO₄²⁻_i/OH⁻_o exchange by Cl⁻_o probably involves interaction of Cl⁻_o with two separate sites.

FIGURE 7.

Phe³⁶⁸ in Slc26a2 permeation pathway. A, example traces for pH_i and Cl⁻_i measurement in oocytes expressing either wild-type (gray traces) or Slc26a2(F368A) (black traces). This protocol was used to monitor Slc26a2-mediated the SO₄²⁻ dependence of SO₄²⁻_o/OH⁻_i exchange (B) and inhibition of the exchange by Cl⁻_o (C). SO₄²⁻_i/OH⁻_o exchange was used to monitor activation of the reverse exchange by Cl⁻_o (D). All plots (B–D) were fitted to the Hill equation, and K_m values are given as mean ± S.E.

The findings in Fig. 7, A–C provide additional evidence for the importance of the GSGIP or the GFXXP motifs in the function of the Cl⁻ transporters, in addition to the two additional GXXXP motifs that participate in Cl⁻ transport in the bacterial ClCs (44). The bacterial ClC-ec1 crystal structure shows that the permeation pathway has three Cl⁻ interacting sites (44–46, 49). Ser¹⁰⁷ and Gly¹⁰⁸ in the GSGIP motif coordinate the Cl⁻ ion in the internal substrate site, and the side chain of Ser¹⁰⁷ participates in binding of the middle Cl⁻ (45, 49). In Slc26a2 Phe³⁶⁸ appears to control the affinity for the substrate (SO₄²⁻ ), suggesting that Phe³⁶⁸ may participate in the access of SO₄²⁻ to the permeation pathway or in shaping the external SO₄²⁻ binding site. The increased apparent affinity for SO₄²⁻ and reduced apparent affinity for Cl⁻ by the F368A mutation suggests that Phe³⁶⁸ may hinder access of SO₄²⁻ and facilitate access of Cl⁻ to the permeation pathway or reduces the time SO₄²⁻ spends in the external binding site on its way across the plasma membrane. Perhaps this is necessary to allow SO₄²⁻_i/Cl⁻_o exchange at high SO₄²⁻_i when SO₄²⁻ efflux is required. Irrespective of the exact role of Phe³⁶⁸, the present findings further support the notion of similarities between the ClC and SLC26 transporters permeation pathways and that the opening of the permeation pathway is situated in the region of TMDs 7 and 8.

Properties of Slc26a2-mediated Oxalate Transport

Slc26a2 was reported to transport Oxalate (Ox²⁻) by mediate Ox²⁻/SO₄²⁻ exchange (8, 24–26, 28) and that Ox²⁻/SO₄²⁻ exchange is 10 times slower than SO₄²⁻/Cl⁻ exchange (24). However, the properties and mode of Ox²⁻ transport and the capacity of net Ox²⁻ transport by Slc26a2 are not known. We set to estimate net Ox²⁻ transport by measuring Ox²⁻-mediated OH⁻ and Cl⁻ fluxes. Fig. 8A shows that Slc26a2 mediates net Ox²⁻_o/OH⁻_i and Ox²⁻_o/Cl⁻_i exchange in oocytes bathed in Cl⁻-free solution containing 1 mm Ox²⁻, pH 7.5. Removal of Ox²⁻ was not followed by Ox²⁻ efflux until the addition of 1 mm Cl⁻_o to activate the efflux. Importantly, addition of 1 mm Cl⁻_o resulted in minimal Ox²⁻_i/Cl⁻_o exchange but near maximal Ox²⁻_i/OH⁻_o exchange. Increasing Cl⁻_o to 110 mm caused a small additional increase in Ox²⁻_i/OH⁻_o exchange and modest Ox²⁻_i/Cl⁻_o exchange. As expected, Fig. 8B shows that reducing pH_o to 6.5 increased the rate of Ox²⁻_o/OH⁻_i exchange and increasing pH_o to 8.5 inhibited the Ox²⁻_o/OH⁻_i exchange. Fig. 8C shows the opposite effect of pH_o on the Ox²⁻_i/OH⁻_o exchange. Finally, the F368A mutation increased the apparent affinity for Ox²⁻ and reduced the apparent K_m for Ox²⁻ from 90 ± 12 to 50 ± 8 μm. Although this was not as prominent as the increased apparent affinity for SO₄²⁻ (Fig. 7), it was in the same direction. The results in Fig. 8, A–D indicate that the properties of Ox²⁻ transport closely resemble those of SO₄²⁻ transport, although at the same conditions the Ox²⁻ transport rate was ∼50% slower than the SO₄²⁻ transport rate.

FIGURE 8.

Properties of Slc26a2-mediated oxalate (Ox²⁻ ) transport. A, example traces of the Slc26a2-mediated OH⁻ (black trace) and Cl⁻ (gray trace) efflux in response to addition of 1 mm Ox²⁻_o to Cl⁻-free solution at pH 7.5. Removal of Ox²⁻_o terminated the influx (dashed box) and addition of 1 mm Cl⁻_o was required to initiate Ox²⁻_i/OH⁻_o exchange (gray box) with minimal Ox²⁻_i/Cl⁻_o exchange. B and C, effect of pH_o of Ox²⁻ fluxes was as in A, except that the Ox²⁻ efflux was initiated by the addition of 110 mm Cl⁻_o and pH_o was set at 6.5 (dark columns), 7.5 (gray columns), or 8.2 (empty columns). The results are the mean ± S.E. of the number of experiments indicated in the columns. D, the dependence of Ox²⁻_o/OH⁻_i exchange on Ox²⁻_o concentrations was measured with wild-type Slc26a2 (○) and Slc26a2(F368A) (■). The apparent K_m values are given as the mean ± S.E. of 4–5 experiments.

In summary, the present study reports the mechanism of SO₄²⁻ and Ox²⁻ transport by Slc26a2. Both anions are transported in exchange for Cl⁻ and OH⁻ or by cotransport with H⁺. Based on the rate of the coupled OH⁻(H⁺) fluxes in the absence of Cl⁻_o and at substrate concentration of 1 mm, net SO₄²⁻ transport by Slc26a2 is about twice faster than net Ox²⁻ transport. Under normal conditions plasma oxalate is in the micromolar range and even in patients with primary hyperoxaluria plasma oxalate is around 40 μm (53). Moreover, although Slc26a2 is expressed at high level in the luminal membrane of colonic crypts (21), SO₄²⁻ in the colon can be in the millimolar range both in human (54) and animals (55) that will favor SO₄²⁻ uptake by Slc26a2. Indeed, the colon is a major site of SO₄²⁻ absorption (54, 56) that is likely mediated by Slc26a2. Similarly, although Slc26a2 is expressed in the proximal tubule luminal membrane (31), SO₄²⁻ concentration in the proximal tubule is in the millimolar range, and although the role of Slc26a2 in the kidney is not know, if any it is likely to function mainly as an SO₄²⁻ transporter (8). The only possible scenario where Slc26a2 can affect Ox²⁻ homeostasis is by mediating Ox²⁻ secretion in exchange for external SO₄²⁻ when external Cl⁻ is low and pH is high. Even then, this process will be inhibited by the high cytoplasmic Cl⁻ typical of epithelia and by intracellular SO₄²⁻. Thus Slc26a2 is not likely to play a major role in oxalate metabolism in the colon or the kidney.

The permeation pathway includes the conserved SLC26 transporter Glu⁻ and may lay between TMD7 and TMD8, where a phenylalanine conserved in the loop predicted to connect the TMDs may control SO₄²⁻ and Cl⁻ access to the permeation pathway. As yet, mutations of these residues, or even in the vicinity of these residues, have not been found in patients with diastrophic dysplasia (57). This is most likely because Slc26a2 is an essential gene and the mutations that markedly affect Slc26a2 activity may not be compatible with life. Indeed, analysis of several disease causing Slc26a2 mutations showed retention of some SO₄²⁻ transport capacity by the mutants and a good correlation between loss of SO₄²⁻ transport and disease severity (25, 26). The coupling of SO₄²⁻ transport to both OH⁻ and Cl⁻ likely serves to ensure transport at both acidic pH when most SO₄²⁻ uptake is mediated by SO₄²⁻/2OH⁻ exchange and alkaline pH when most SO₄²⁻ uptake is mediated by SO₄²⁻/2Cl⁻ exchange. Slc26a2 is also regulated by an extracellular anion binding site different from the transport site, the physiological function of which remains to be determined, although it may control SO₄²⁻ uptake when Cl⁻_o is very low.