©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Conserved Glutamate Is Responsible for Ion Selectivity and pH Dependence of the Mammalian Anion Exchangers AE1 and AE2 (*)

(Received for publication, August 1, 1995; and in revised form, September 26, 1995)

Israel Sekler (§) Roger S. Lo(§)(¶) Ron R. Kopito (**)

From the Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The erythrocyte anion exchanger AE1 (band 3) serves as an important model for the study of the mechanism of ion transport. Chemical modification of human erythrocyte AE1 has previously suggested that glutamic acid residue 681 lies within the transport pathway and can cross the permeability barrier. This glutamate is conserved in all anion exchangers sequenced to date. We examined the effect on divalent (sulfate) and monovalent (chloride and bicarbonate) anion transport of mutating the corresponding glutamates in mouse AE1 and the closely related anion exchanger, AE2. Substitution of this conserved glutamate with uncharged or basic amino acids had a negligible effect on the maximal rate of sulfate-sulfate exchange in AE-reconstituted proteoliposomes, but largely abolished the steep pH dependence of sulfate transport observed in wild-type AE1 and AE2. In contrast, exchange of monovalent anions was undetectable in cells expressing these mutants. Replacement of the conserved glutamate with aspartate abolished both monovalent and divalent anion transport. These data suggest that the conserved glutamate residue plays a dual role in determining anion selectivity and in proton coupling to sulfate transport. A model explaining the role of the conserved glutamate in promoting ion selectivity and pH regulation is discussed.


INTRODUCTION

Plasma membrane anion exchangers (AE) (^1)constitute a diverse family of transporters, which, in mammalian cells, contribute to the control of transmembrane anion gradients, cell volume, and acid-base homeostasis. Three members of this family, designated AE1-AE3, have been cloned and demonstrated by functional expression to mediate transmembrane exchange of the physiological substrates, chloride and bicarbonate (reviewed in Kopito(1990) and Alper(1991)). In mammals, AE1-AE3 are expressed in a tissue- and cell-specific manner. The best characterized among these is AE1, which was first identified as the major integral membrane protein of the erythrocyte, known as ``band 3''. AE1-mediated anion exchange is rapid, electrically silent, and tightly coupled (see Passow(1986), Knauf(1986), and Macara and Cantley(1983) for reviews). The latter two features are best explained by a ``ping-pong'' kinetic model in which anion translocation in one direction is temporally distinct from, but dependent upon, anion translocation in the opposing direction. This tight substrate coupling ensures that the flow of one substrate is driven by the electrochemical potential of the other. Thus, anion exchangers can be used by cells to establish Cl gradients at the expense of pH (HCO(3)) (Vaughan-Jones, 1979, 1982) or pH gradients at the expense of Cl (Paradiso et al., 1987).

Anion exchange by AE1 is highly selective, both for anions over cations and among different anions (see Passow(1986), Knauf(1986), and Macara and Cantley(1983) for reviews). AE1 displays strong selectivity for its physiological substrates, transporting chloride 10-250 times more rapidly than other halides. Remarkably, despite their dissimilar size, charge distribution, and atomic structures, chloride and bicarbonate are transported by AE1 at nearly the same rate (Lowe and Lambert, 1982). This contrasts sharply with the transport of the divalent sulfate anion, which, although structurally similar to bicarbonate, is transported by AE1 at a rate nearly 10,000-fold slower than chloride or bicarbonate (Brahm, 1988; Schnell et al., 1977). Protons have opposing effects on the transport by AE1 of monovalent anions such as Cl and HCO(3) and divalent anions like sulfate. These dramatic differences in transport rates and proton dependence between the transport of monovalent and divalent anions have been reconciled by Gunn(1972) who proposed that protonation of a titratable group with pK 5 could convert AE1 from a monovalent to a divalent anion carrier. Milanick and Gunn(1984) further suggested that the transport mechanism requires at least two distinct ion binding sites: one for anions and one for protons. They proposed that proton binding could serve to alter the net charge of the anion binding pocket that crosses the permeability barrier during translocation (Milanick and Gunn, 1984). This model is consistent with the observation of a stoichiometric flux of protons accompanying sulfate during sulfate-Cl countertransport (Jennings, 1976). The AE2 exchanger, which is expressed in a wide variety of cell types (Kopito, 1990), shares key features with AE1, including a preference for Cl and HCO(3) as substrates, sensitivity to stilbene disulfonate inhibitors (Lee et al., 1991), and proton-stimulated sulfate transport (Sekler et al., 1995). These conserved features, therefore, may reflect fundamental aspects of the underlying transport mechanism.

Despite the wealth of kinetic and biophysical data on anion exchanger-mediated transport, and despite the availability of cloned cDNAs for three members of the AE family, the mechanistic details of how anion exchangers maintain a remarkably robust ability to catalyze extremely fast transport of the structurally dissimilar chloride and bicarbonate anions are known. Moreover, little is known about the amino acids specifically involved in ion binding, transport, or pH regulation. Chemical modification studies of anion transport in erythrocytes have provided some support for the involvement of certain amino acids in the anion exchange reaction cycle. These studies (Julien and Zaki, 1988; Wieth et al., 1982) have suggested that at least one unidentified arginine is essential for anion translocation. While modification of lysines by stilbene disulfonate and other lysine-specific reagents inhibits transport activity (Cabantchik and Rothstein, 1974; Jennings and Nicknish, 1985), site-directed mutagenesis of the lysines which constitute the covalent binding site for stilbene disulfonates fails to alter the ion affinity or the rate of ion transport, suggesting that they are not directly involved in the transport process (Garcia and Lodish, 1989; Wood et al., 1992). The participation of other AE1 lysines in anion translocation has not been resolved.

Carboxyl residues have also been implicated in AE-mediated anion transport. Treatment of red blood cells with carbodiimide compounds inhibits monovalent anion transport (Craik and Reithmeier, 1985; Bjerrum et al., 1989). More recently, treatment of red blood cells with carboxyl-specific Woodward's reagent K (WRK), followed by borohydride reduction, inhibited chloride transport by 75-80% while stimulating SO(4)/Cl exchange (Jennings and Al-Rhaiyel, 1988). Significantly, WRK treatment eliminates both the pH dependence of sulfate transport and the proton flux which normally accompanies SO(4)/Cl exchange (Jennings and Al-Rhaiyel, 1988), suggesting that the WRK modified carboxyl may be the same as the titratable group originally proposed by Gunn(1972). The WRK-modified carboxyl residue in human AE1 was recently identified by Edman degradation to be Glu-681, predicted to be located near the cytoplasmic interface of transmembrane helix 8 and is conserved among all anion exchangers known to date (Jennings and Smith, 1992).

The present work examines the functional and structural consequences of mutating the glutamate in mouse AE1 (Glu-699) and AE2 (Glu-1007), which correspond to the WRK-reactive residue (Glu-681) in human erythrocyte AE1. The data support the hypothesis that this glutamate in mouse AE1 and AE2 plays a key role in determining the ion selectivity and kinetics of anion transport by these closely related anion exchangers.


EXPERIMENTAL PROCEDURES

Polymerase Chain Reaction Mutagenesis

Mouse AE1 mutants, AE1E699A, E699Q, and E699D and AE2 mutants AE2E1007K, E1007Q, and E1007D were constructed by megaprimer PCR mutagenesis (Landt et al., 1990; Sarkar and Sommer, 1990). PCR was performed on the double-stranded AE2/pRBG4 expression vector as the template. The reverse mixed primer, 5` GGT GAT CTG TGT CT(G,T) CAT, GAA, GAT, GAG 3`, was the mutagenic primer for AE2E1007K and E1007Q. The reverse primer, 5` GGT GAT CTG TGT ATC CAT GAA GAT GAG 3`, was the mutagenic primer for AE2E1007D. The mutagenic primers and the forward primer, 5` AAC AGC CGG TTC TTC CCT GGC C 3`, (upstream of the mutagenic site) were used to generate a approx 250-bp megaprimer by Vent polymerase. This megaprimer was eluted from an agarose gel and used in conjunction with a reverse primer, 5` CTT TAT TTG TAA CCA TTA TAA GC 3` (downstream of the mutagenic primer on the multiple cloning site of pRBG4), to generate a approx 1-kilobase fragment. This final PCR product harbors two unique restriction sites, one for BalI between the reverse mutagenic primer and the upstream forward primer and another for HindIII in between the megaprimer and the reverse primer on the multiple cloning site of pRBG4. The final PCR product of approx 1 kilobase was eluted from an agarose gel, subjected to a double digest by BalI and HindIII, and the approx970-bp restriction product was then subcloned into the AE2/pRBG4 expression vector. The vector (approx6930 bp) used for ligation was generated by partial restriction digest of the AE2/pRBG4 expression vector (approx7900 bp) using HindIII and BalI, with the latter restriction enzyme having three additional target sites on the AE2/pRBG4 expression vector. All mutants were sequenced throughout their PCR-generated regions (Sanger et al., 1977).

Expression of Anion Exchangers in Cultured Cells

Murine anion exchanger cDNAs were cloned into the expression vector pRBG4 and expressed in human embryonic kidney (HEK) 293 cells (Lee et al., 1991). Cells were grown and transfected in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and maintained in humidified incubators at 37 °C and 5% CO(2). Anion exchangers were expressed by transient transfection of HEK cells as described previously (Lee et al., 1991; Ruetz et al., 1993) except that calcium phosphate-precipitated plasmid was added at 16 µg of plasmid/150-mm tissue culture plate.

ClEfflux Assay in Whole Cells

For a single time course, trypsinized and triturated HEK293 cells from three 150-mm plates were washed in the standard chloride buffer (30 mM NaCl, 16 mM HEPES-KOH, pH 7.4, and 200 mM sucrose). The cells were then resuspended in isotopic standard chloride buffer containing 5 µCi of [Cl] NaCl in the presence of 3 µM valinomycin (to depolarize the membrane potential and thereby increase Cl accumulation and minimize electrogenic Cl currents) and incubated for 20 min at room temperature. The cells were then pelleted and the isotopic supernatant carefully removed. The time course was initiated by resuspending the Cl-loaded cells in degassed, non-isotopic standard reaction buffer and terminated by passing an aliquot through a Dowex column as described in (Sekler et al., 1995) followed by washing with 1.5 ml of ice-cold 300 mM sucrose.

Preparation of Crude Microsomes

Microsomes were prepared as described previously (Sekler et al., 1995). Unless otherwise stated all steps were performed at 0-4 °C. Medium was removed from 5 to 10 150-mm culture dishes, and the cells were harvested using a plastic cell scraper, resuspended in 3 ml of wash buffer (140 mM NaCl, 10 mM Tris-HCl, pH 7.5) for each plate, pooled, and centrifuged at 1500 rpm (551 times g) for 10 min in a H-6000A Sorvall rotor. The resulting pellet was resuspended in 20-40 ml of wash buffer, and cells were centrifuged at 1000 rpm (291 times g) for 10 min. The cellular pellet was resuspended in 10-20 ml of swelling buffer (10 mM Tris-HCl, pH 7.5, supplemented with 1 mM phenylmethylsulfonyl fluoride) and incubated on ice for 20 min. The suspension was then subjected to centrifugation at 4000 rpm (4660 times g). The resulting pellet was resuspended in 10-20 ml of swelling buffer, homogenized with 50 strokes using a 5-ml glass Dounce homogenizer, and sonicated twice for 1 s using a G112SPIT cylinder-shaped bath sonicator (Laboratory Supplies Co.) that was operated at 80 times 10^3 cycles/s at 300 watts. Sucrose solution was then added from a 70%, w/v, stock to achieve a final concentration of 10%, w/v. The nuclear fraction was removed by centrifugation at 1200 rpm (400 times g) for 5 min, and the supernatant was spun at 3700 rpm (4100 times g) for 5 min. The resulting supernatant was made to 50 mM KCl and 10 mM HEPES-KOH, pH 7.4. This mixture was spun at 35,000 rpm (140 times 10^3 times g) in a Ti 45 Beckman rotor for 50 min, and the final microsomal pellet was resuspended in buffer containing 7%, w/v, sucrose, 25 mM KCl, 1 mM MgCl(2), 15 mM HEPES-KOH, pH 7.5. Crude microsomes derived from 10 150-mm plates were resuspended in 0.5-1.2 ml at a protein concentration of about 1 mg/ml, homogenized using a 2-ml tight Teflon glass homogenizer, and immediately frozen and kept in liquid nitrogen for up to 60 days.

Reconstitution of the Anion Exchanger

Reconstitution into proteolipisomes was performed as described previously (Sekler et al., 1995). Unless otherwise stated, all steps were performed at 0-4 °C. Crude microsomes (40-80 µg of protein in 40-80 µl) were mixed with a 50-fold excess (w/w) of sonicated soybean asolectin (Sigma) in the standard sulfate buffer (20 mM Na(2)SO(4), 10 mM MES-KOH, pH 6.0, and 2 mM MgSO(4)) with 70 µCi of [S]Na(2)SO(4) (1050-1600 Ci/mmol (DuPont NEN) in a final volume of 0.5-1 ml in the presence or absence of 300 µM DIDS. The reconstitution mixture was subjected to one cycle of rapid freezing in liquid nitrogen and thawing at room temperature, followed by 5 times 1 s of sonication cycles using a G112SPIT cylinder-shaped bath sonicator (Laboratory Supplies Co.) that was operated at 80 times 10^3 cycles/s. To remove tracer not trapped inside the proteoliposomes and to exchange to the desired extravesicular buffer, the sample was spun for 5 min through a prechilled 10-ml Sephadex G-50 fine (Pharmacia) column at 1200 rpm (400 times g) in a H-6000A Sorvall rotor that was semi-dried by centrifugation for 3 min at 900 rpm (200 times g) in the same rotor. Prior to the semi-dry processing the resin was preswollen by boiling 2 h in water then equilibrated with at least 2 column volumes of desired buffer without the isotopic tracer.

Sulfate Transport Assays

Sulfate transport assays were performed as described previously (Sekler et al., 1995). [S]SO(4) transport was initiated by transferring the proteoliposomes to a 30 °C water bath and terminated by passing 50-100-µl aliquots through Dowex-1 (Bio-Rad) anion exchange columns that were converted into a formate form and packed into 1.5-ml Pasteur pipettes as described (Gasko et al., 1976). Loading of the sample was followed by washing with 1.5 ml of ice-cold isomolar sucrose solution. Recoveries of the anion exchanger after the Sephadex spin and Dowex columns were determined by immunoblot to be between 85 and 95%. Time 0 of each reaction was determined by passing ice-cold proteoliposomes on the Dowex column. Different preparations showed sulfate efflux specific activity that ranged from 150 to 450 times 10^3 cpm of [S]SO(4)/mg of protein times min at 20 mM SO(4), pH 6, for AE1 and AE2. These values correspond to 4-12 nmol of SO(4)/mg protein times min. For sulfate influx experiments, proteoliposomes were formed in the standard sulfate buffer as described above with nonradioactive sulfate. Transport was initiated by adding 70-100 µCi of [S]SO(4) to 1 ml of proteoliposomes and passing the proteoliposomes through a 3-ml Dowex column. The transport data represent an average of at least three independent experiments each performed in triplicate.

pH Dependence of Sulfate Efflux

Proteoliposomes were formed as described above and passed through a semi-dry Sephadex spin-down column, equilibrated with standard sulfate buffer in which 10 mM MES was replaced by 10 mM MES and 10 mM HEPES brought to the desired pH with KOH. To avoid formation of a DeltapH across the proteoliposomes, 10 µM nigericin (Sigma) and 5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (Sigma) were added prior to passage of the proteoliposomes over the semi-dry spin column, and sulfate flux experiments were performed as described above. The dissipation of the pH gradient was verified by spectrofluorimetric analysis of intravesicular pH in proteoliposomes reconstituted in the presence of the acid form of 1 µM BCECF (Molecular Probes).

Surface Labeling of AE2

Cell surface biotinylation was performed essentially as described (Loo and Clarke, 1995). Briefly, HEK cells transfected with the various plasmids and grown on 100-mm dishes were incubated for 10 min with 10 mM NaIO(4) on ice. The cells were then washed twice in phosphate-buffered saline (150 mM NaCl, 10 mM HEPES, pH 7.4), incubated for 30 min on ice with 2 mM biocytin hydrazine (Pierce), and washed three times in phosphate-buffered saline. The treated cells were then lysed and immunoprecipitated as described previously (Ward and Kopito, 1994) with anti-AE antibody (Thomas et al., 1989). Immunocomplexes were separated on SDS-polyacrylamide gel electrophoresis, transferred to nitrocellullose, and probed with streptavidin peroxidase. Signals were detected using chemiluminescence (Amersham Corp.).

Intracellular pH Determination

Transiently transfected 293 HEK cells were grown for 48-72 h on 7 times 12-mm glass coverslips coated with 100 µg/ml poly-L-lysine. The cells were loaded with 5 mM of acetoxymethyl ester form of BCECF (Molecular Probes) for 15 min at 37 °C and mounted in a flow cuvette. The coverslip was continuously perfused with standard chloride or chloride-free (gluconate substituted) Ringer's solution as described previously (Kopito et al., 1989). Intracellular pH was recorded by monitoring the fluorescence emission at 530 nm from alternate excitation at 499 and 439 nm using a SPEX-DM3000 spectrofluorometer. Intracellular pH was calibrated by the nigericin high K method as described previously (Thomas et al., 1979).

Miscellaneous Determinations

SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) immunoblot processing was performed as described (Ruetz et al., 1993). Protein concentration was determined with bicinchoninic acid reagent (Sigma) in the presence of SDS using bovine serum albumin as a standard.


RESULTS

Mutation of the WRK Glutamate Alters the Kinetics and pH Dependence of Divalent Anion Transport

The initial rate of [S]sulfate efflux from AE-reconstituted, sulfate-loaded proteoliposomes was used to assess the divalent anion exchange capacity of anion exchanger mutants (Fig. 1). Proteoliposomes were reconstituted with crude microsomal membranes from HEK cells expressing the designated wild-type or mutant anion exchanger. We have previously shown that SO(4) efflux by wild-type AE1 and AE2 proteoliposomes is trans-anion-dependent, activated by protons and is sensitive to inhibition by disulfonic stilbenes (Sekler et al., 1995). In the present study the initial rates of sulfate efflux mediated at pH 6 by wild-type AE1 (Fig. 1A) and wild-type AE2 (Fig. 1B) were 194 ± 27 and 272 ± 35 cpm times 10^5 times mg protein times min, similar to values reported previously (Sekler et al., 1995). This assay was used to examine the effect on sulfate transport of mutations in the Woodward's reagent K-reactive glutamate (Glu-699) in AE1 and the corresponding residue (Glu-1007) in AE2. Sulfate efflux in all cases was inhibited by the disulfonic stilbene, DIDS. Mutation of Glu-699 and Glu-1007 to glutamine (AE1 and AE2), lysine (AE2), or alanine (AE1) had no significant effect on the initial rate of sulfate transport. Remarkably, the most conservative substitution, glutamate aspartate, virtually abolished sulfate transport, reducing it to levels indistinguishable from the basal (DIDS-inhibited) values.


Figure 1: Expression level and sulfate transport by wild-type and mutant anion exchangers. Crude microsomes prepared from cells transfected with wild-type and mutant AE1 (A) or AE2 (B) were analyzed by immunoblotting (top) or for sulfate-sulfate exchange (bottom). Amino acid at position 699 (AE1) or 1007 (AE2) is indicated across the bottom; Glu is wild-type. Efflux of vesicular [S]SO(4) into SO(4) containing medium was determined at pH 6 in the presence (shaded bars) or absence (open bars) of the anion exchange inhibitor DIDS as described under ``Experimental Procedures.'' Values indicated represent the mean ± S.E. of triplicate measurements.



To exclude the possibility that the observed differences were attributable to variation in the expression levels of the wild-type and mutant proteins, the AE content of the proteoliposomes used in this experiment was assessed by immunoblotting using an antibody that recognizes the C-terminal domain of AE1 and is cross-reactive with AE2 (Thomas et al., 1989) (Fig. 1). The data indicate that all anion exchangers and mutants within each class (AE1 and AE2) were expressed at similar levels. The doublet of AE2 bands at 160 and 140 kDa correspond to mature (bearing complex oligosaccharides) and immature (high-mannose oligosaccharides) AE2 (Ruetz et al., 1993), suggesting that wild-type and mutant AE2 proteins were similarly distributed in pre- and post-Golgi compartments. Together, these data suggest that the capacity to transport divalent anions by AE1 is not explained simply by the charge or protonation state of the side chain at position 699, as suggested previously by chemical modification studies. Moreover, these data suggest that the structural requirements at this site are largely conserved between the related anion exchangers AE1 and AE2.

Sulfate transport by AE1 has been reported previously to be steeply pH-dependent, suggesting the participation of protonation of a specific acidic residue in the transport process (Schnell et al., 1977). Because we have previously observed a similar pH dependence of sulfate transport by AE2 (Sekler et al., 1995), we examined the effects of the above-described mutants on the pH dependence of sulfate transport (Fig. 2). Initial rates of sulfate transport were determined under pH-clamp conditions between pH 5.5 and 7.5, such that intravesicular and extravesicular pH were matched. As observed previously, the pH dependence of wild-type AE1 and AE2 was characteristically steep. At pH 7.5, sulfate transport rates by wild-type AE1 and AE2 at pH 7.5 were reduced by 75-80% of the rate at pH 5.5. By contrast, the pH dependence of AE1 (Glu-699) and AE2 (Glu-1007) mutants was significantly diminished. The initial rates of sulfate efflux of AE1E699Q and AE1E699A mutants were only 20% lower at pH 7.5 than at pH 5.5. This pH insensitivity of sulfate transport was even more dramatic for the AE21007Q and AE21007K mutants; we could detect no significant difference in sulfate transport rate between pH 5.5 and 7.5. We conclude that this conserved glutamate plays a key role in determining the pH dependence of sulfate exchange mediated by AE1 and AE2.


Figure 2: pH dependence of sulfate/sulfate exchange mediated by wild-type and mutant anion exchangers. Tracer [S]SO(4) efflux from proteoliposomes reconstituted with AE1 (left panel) or AE2 (right panel). Amino acids at AE1 position 699 were glutamate (wild type, squares); glutamine (circles), or alanine (diamonds). Amino acids at AE2 position 1007 were glutamate (wild type) (squares), glutamine (circles), or lysine (diamonds). Intravesicular and extravesicular pH were clamped with nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to the values indicated on the abscissa, and sulfate transport rates were determined as described in the legend to Fig. 1and under ``Experimental Procedures.'' Values indicated represent the means of triplicate measurements. The specific activity of sulfate efflux for AE1 and AE2 at pH 6 were 213 ± 27 and 264 ± 35 cpm of [S]SO(4)/mg of protein times min, respectively.



A Critical Role for AE2 Glu-1007 in Monovalent Anion Transport

Two whole cell assays were used to determine the effects of mutation of the conserved glutamate residue on the transport of monovalent anions. The small internal volume of proteoliposomes and the rapid kinetics of chloride as compared with sulfate transport precluded our ability to use the reconstituted anion exchangers for investigation of monovalent anion exchange kinetics. Since mouse AE1 is retained in intracellular compartments (Ruetz et al., 1993), these assays could be performed only for AE2, which is efficiently delivered to and stable at the plasma membrane (Ruetz et al., 1993). To confirm the surface expression of AE2 and to determine whether the AE2 Glu-1007 mutants were also present at the cell surface, transfected HEK cells were surface labeled with a membrane-impermeant biotinylation reagent. Following labeling, anion exchangers were immunoprecipitated, fractionated on SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Surface-accessible anion exchangers were then detected by streptavidin-peroxidase staining (Fig. 3). The data show that wild-type AE2 and all of the glutamate mutants were labeled to a similar extent. By contrast, no labeling was observed for AE1, which is known to be retained in an intracellular compartment (data not shown) (Ruetz et al., 1993). Similarly, the reagent failed to react with the 140-kDa band corresponding to the ``immature'' form of AE2 that is restricted to the endoplasmic reticulum (Ruetz et al., 1993), indicating that the label was specific for proteins expressed at the cell surface. These data demonstrate that mutation of Glu-1007 does not influence either the expression level or the fraction of anion exchanger expressed at the cell surface of HEK cells.


Figure 3: Expression of AE2 at the cell surface is unaffected by mutations at position Glu-1007. HEK cells expressing wild-type (Glu) AE2 or AE2 substituted at position 1007 with the indicated residues were surface labeled by biocytin hydrazine, lysed, and immunoprecipitated with AE antibody. Immunocomplexes containing labeled AE2 were separated on SDS-polyacrylamide gel electrophoresis, transferred to nitrocellullose, and detected using streptavidin peroxidase as described under ``Experimental Procedures.''



Expression of wild-type AE2 resulted in a marked acceleration of the initial rate of Cl efflux from HEK cells compared with vector transfected control cells (Fig. 4A). Moreover, the transport was completely dependent on the presence of trans-Cl, was inhibited by the impermeable anion gluconate, and was completely inhibited by the anion exchange inhibitor, DIDS. Increasing external Cl from 30 to 50 mM stimulated a significant increase in the rate of Cl efflux from 1.54 ± 0.22 to 2.2 ± 0.23 times 10 cpm Cl times mg protein times min. These experiments show that AE2-mediated Cl efflux from HEK cells is inhibited by disulfonic stilbenes and is stimulated by the presence of a trans-anion, suggesting that AE2 in these cells is functionally expressed at the cell surface.


Figure 4: AE2 glutamate 1007 is essential for Cl/Cl exchange. A, characterization of the Cl efflux assay. HEK cells expressing wild-type AE2 were loaded with Cl and the rate of isotope efflux was determined in the presence of media containing 20 mM (squares) or 50 mM chloride (triangles). Control efflux determinations on AE2-transfected (circles) or mock-transfected cells (diamonds) were performed in the presence of 30 mM Cl alone (diamonds), 30 mM gluconate (open circles), or 30 mM Cl containing 300 µM DIDS (closed circles). The specific activities for the [Cl] efflux measurement for wild-type AE2 in 30 and 50 mM Cl, respectively, were 1.54 times 10 ± 0.22 and 2.2 times 10 ± 0.23 cpm [Cl]/mg of protein times min. B, effect of substitution of Glu-1007 (squares) with aspartate (triangles), lysine (circles), or glutamine (diamonds) on tracer [Cl] efflux into media containing 20 mM Cl. The specific activity for the chloride efflux measurement for wild-type AE2 was 1.4 times 10 ± 0.017 cpm of [Cl]/mg of protein times min.



To determine the effect of Glu-1007 substitution on Cl transport mediated by surface-expressed AE2, we measured the rate of Cl efflux from cells expressing wild-type AE2 or Glu-1007 mutants thereof (Fig. 4B). The data indicate that substitution of Glu-1007 with Gln, Lys, or Asp decreased the capacity of AE2 to stimulate Cl efflux above background levels. These data suggest that, in contrast to transport of the divalent anion sulfate, chloride transport by AE2 is extremely sensitive to the nature of amino acid side chain at position 1007.

The physiological substrates of the AE class of anion exchangers are chloride and bicarbonate, which bind to AE1 with similar affinities and are transported at similar rates. Because of the marked structural and electrostatic differences between Cl, a halide, and HCO(3), a planar oxyanion, it was important to investigate the effect of Glu-1007 mutations on Cl/HCO(3) exchange, using an assay for intracellular pH which exploits the pH-sensitive intracellular fluorescent indicator BCECF. Using this assay, cells expressing wild-type AE2 responded to the application of a large outward Cl gradient by a rapid (50 s) increase in intracellular pH of 0.6 pH unit (Fig. 5). We have demonstrated previously that this pH increase is the result of the AE-mediated influx of bicarbonate (Lee et al., 1991). This pH change is similar in magnitude to previously observed values (Lindsey et al., 1990) and was reversed upon restoring extracellular [Cl]. There was no significant response in cells transfected with vector plasmid. None of the cells expressing mutant AE2, substituted with either Asp, Lys, or Gln at position 1007, showed detectable HCO(3) transport stimulated by either inward or outward directed Cl gradients. Moreover, we were unable to detect significant HCO(3) transport driven by outward or inward gradients of nitrate, a rapidly transported substrate for wild-type AE1 and AE2 (data not shown). These findings confirm that Glu-1007 contributes a critical role to the transport of monovalent halides or oxyanion substrates in either direction across the membrane. Because the level of expression of AE2 and AE2 mutants at the cell surface of these cells was similar (Fig. 3), we conclude that the specific amino acid side chain at position 1007 of AE2 contributes critically to the transport in either direction of structurally unrelated monovalent anion substrates.


Figure 5: AE2 glutamate 1007 is essential for Cl/HCO(3) exchange. Intracellular pH was monitored in HEK cells expressing the indicated AE2 constructs (or vector) and loaded with the intracellular pH indicator dye BCECF. Cells were equilibrated in normal Ringer's solution buffered with HCO(3)/CO(2), pH 7.4. At the time indicated the normal Ringer's solution was replaced with isoosmotic solution lacking Cl (replaced with gluconate). Intracellular pH and time scales are indicated.




DISCUSSION

The above data show that a conserved glutamate at position 699 of mouse AE1 and position 1007 of mouse AE2 plays a key role in determining the ion selectivity and kinetics of anion transport by these closely related anion exchangers. Substitution of this residue with amino acids containing basic or neutral side chains effectively eliminated the capacity for exchange of the physiological monovalent anions chloride and bicarbonate. By contrast, although the same mutations had no measurable effect on the rate of transport of the divalent anion sulfate at acidic pH, these mutations sharply attenuated the steep pH dependence of sulfate transport. The different consequences of these mutations for monovalent and divalent anion transport suggest a critical role for this glutamate in distinct steps of the chloride and sulfate transport cycles and provide some insight into the basic mechanism by which these exchangers bind and translocate anions.

Previous studies using the carboxyl-specific WRK followed by reductive cleavage permitted ``chemical mutagenesis'' of human erythrocyte AE1 in which one or more glutamic acid carboxyls on the protein were converted to the corresponding alcohol, 5-hydroxynorvaline (Jennings and Anderson, 1987). This effect was later shown to be due to modification of a single glutamate, Glu-681, corresponding to position 699 in mouse AE1 (Jennings and Smith, 1992). WRK/NaBH(4) modification of AE1 inhibited Cl/Cl exchange while stimulating SO(4)/SO(4) exchange (Jennings and Al-Rhaiyel, 1988). Moreover, this modification, which is structurally similar to permanent protonation of the carboxyl, abolished the pH dependence of sulfate transport and eliminated the proton flux normally associated with divalent anion movement (Jennings and Al-Rhaiyel, 1988; Jennings and Smith, 1992). These findings led Jennings to propose that Glu-681 is the titratable site on AE1 the protonation state of which appears to switch AE1 between monovalent and divalent anion selectivity (Jennings and Smith, 1992). However elegant, these data left unresolved several key issues. First, the presence of 20% of unmodified AE1 significantly complicated the interpretation of the chemical modification data. Is the residual Cl/Cl exchange following WRK modification entirely explained by the presence of unmodified protein, or is there residual Cl/Cl exchange following modification? Second, the borohydride reduction reaction is difficult to control; it is apparently not possible to ensure cleavage of all of the arylsulfonate adduct without risking reduction of other groups on the protein (Jennings and Smith, 1992). Hence it is difficult to attribute all the properties of WRK-BH4 modified AE1 to the effects exclusively on Glu-681. Third, although the conversion of E681 to an alcohol would indeed be expected to render the residue uncharged, such chemical modification does not permit the substitution of other amino acids at that position. Consequently, it is not possible to determine whether the WRK effect is to ``protonate'' Glu-681 or to introduce detrimental steric consequences by conversion to 5-hydroxynorvaline. These concerns have all been addressed in the present work using site-directed mutagenesis, which ensures that all copies of the expressed mutant protein will have the mutant phenotype and that any amino acid substitution may be made at will. Moreover, in this paper we have extended the analysis to include the non-erythroid anion exchanger AE2, in which the residue corresponding to human AE1 Glu-681 is highly conserved by Glu-1007. This issue is of particular interest, since it has been suggested that AE2 transport proceeds by a fundamentally different, non-ping-pong mechanism from AE1 (Restrepo et al., 1991). Our data suggest a fundamental mechanistic conservation not only among anion exchangers of different species (i.e. human and mouse) but also of different members (AE1 and AE2). Indeed, functionally reconstituted mouse AE3 displays a strikingly similar pH dependence of sulfate transport to that of AE1 and AE2, (^2)suggesting that the conserved glutamate at position 998 is also the major titratable acidic group in AE3.

Role of the WRK-Glutamate in Anion Binding

A goal of these investigations is to begin to understand the mechanism underlying AE anion selectivity. Anion exchangers maintain the ability to rapidly transport structurally dissimilar anions such as the halide, Cl, and the oxyanions bicarbonate and nitrate. It has been proposed previously that protonation of the key AE1 carboxyl (presumably the WRK glutamate, Glu-681) alters the net charge in the binding pocket, neutralizing the extra charge on the bound sulfate and hence facilitating carrier reorientation (Milanick and Gunn, 1984). Our data suggest that net charge alone cannot account for the role of this glutamate residue, as the rate of sulfate transport was similar in AE1 or AE2 mutants substituted with either the neutral glutamine or the positively charged lysine. Indeed, the only substitution that did not support sulfate transport was the conservative replacement with aspartate, in which the net charge is preserved, suggesting that steric as well as electrostatic interactions at this position contribute to sulfate transport.

This steric constraint suggests a major role for glutamate 699/1007 in the precise coordination of anions in the binding pocket. It is possible that the side chain of glutamate 699/1007 participates in accepting hydrogen bonds from residues that either donate or accept hydrogen bonds to or from chloride. It is also possible that the side chain of glutamate 699/1007 salt bridges with a guanidino group of an arginine which would in turn bind the negative charge of chloride. These indirect roles could allow glutamate 699/1007 to influence the proper geometry of the peptide backbone(s) and or side chain(s) that binds chloride. Networks of hydrogen bonds, primarily main chain peptide NH and CO groups in addition to hydroxyl side chains, are capable of dissipating the charge(s) on a substrate. For instance, the ligand-bound crystal structures of sulfate- and phosphate-binding proteins, which are involved in active transport in Salmonella typhimurium, revealed that single OH groups from side chains of residues such as serines and threonines, together with peptide backbone NH hydrogen donor groups and CO hydrogen acceptor groups form multiple hydrogen bonds which give rise to an extensive and stable network (Pflugrath and Quiocho, 1988; Luecke and Quiocho, 1990; He and Quiocho, 1991). Such an array of interactions can potentially moderate opposite charge attractions and facilitate ion movement. That aspartate cannot replace glutamate to support chloride transport may be explained by the fact that aspartate and glutamate have markedly different effects on the conformation and chemical reactivity of neighboring peptide backbone(s) (Creighton, 1992) and emphasizes that simple net charge considerations cannot account solely for the observed tight Cl binding.

A Model for the Distinct Roles of a Conserved Glutamate in Chloride and Sulfate Transport by the Anion Exchangers

The preceding discussion argues that the key contribution of the WRK glutamate to AE selectivity is via an indirect rather than a direct interaction with substrate anions. Selectivity of AE1 is best reflected by the maximum transport rate for a given substrate, rather than the apparent binding affinities of the substrates. For example, the physiological substrates chloride and bicarbonate are transported most rapidly and at comparable rates, despite their dissimilar structures. Other halides are transported between 10 and 250 times more slowly, while sulfate and phosphate are transported nearly 10,000 times more slowly. By contrast, the measured binding affinities for these different anions vary by less than one order of magnitude. Size alone cannot account for the observed differences in maximum transport rates, as the distribution in maximal transport rates is not well correlated with molecular geometry. These observations led Krupka to propose that stabilization of the transition state in carrier reorientation is the physical basis for anion selectivity (Krupka, 1989). In this context, our data suggest that the WRK glutamate contributes critically to this stabilization.

The differential effect of Glu-699/1007 substitutions on monovalent and divalent anion transport suggests two mutually exclusive pathways for carrier reorientation, which are outlined in Fig. 6. Preferred substrates, e.g. Cl and HCO(3) (Fig. 6A), form an initial loose complex with the carrier (step 1), which is followed by a conformational change (step 2), leading to a tightly bound (or occluded) form. According to this model, the specificity of ion transport depends on the ``tightness'' of the tightly bound state and is, therefore, correlated with the transport rates rather than with the apparent affinity. We propose that entry into this tightly bound state requires the participation of the unprotonated WRK glutamate, perhaps by forming an essential salt bridge or by contributing to the precise coordination of the substrate anion in the transition state. There is elegant evidence from the crystal structure of the S. typhimurium periplasmic sulfate-binding protein that salt bridges span an opening in the deep cleft wherein a single dehydrated sulfate is completely engulfed. Removal of these salt bridges by site-directed mutagenesis reveals that they function to stabilize the closed liganded form and modulate the kinetics of cleft opening (Jacobson et al., 1991, 1992). Protonation, or substitution by mutagenesis, of the WRK Glu in anion exchangers would interfere with its ability to form salt bridges and hence reduce the probability of forming the tightly bound occluded state (step 2).


Figure 6: A model for anion exchanger-mediated anion transport. Details are given in the text.



On the other hand, substrates like sulfate fail to achieve the tightly bound state necessary for rapid reorientation (Fig. 6B). Binding of sulfate to the protonated form of the carrier is permissive for a slow conformational reorientation in which both the substrate and the proton are released to the trans side of the membrane (steps 2a and 2b). Three distinct mechanisms can be envisioned to account for the effect of protonation at the conserved glutamate in enhancing sulfate transport. First, protonation could enhance the loose binding of sulfate (steps 1 and 5), as suggested by the data of Jennings(1989). However, Milanick and Gunn(1984) observed only a relatively modest effect of pH on the K(m) for sulfate, while over the same pH range, the rate of sulfate transport varied by as much as 100-fold (Milanick and Gunn, 1984; Sekler et al., 1995). Therefore, an effect of protonation of Glu-699/1007 on sulfate affinity cannot be ruled out. Second, protonation could allow a tighter occlusion of sulfate for enhanced stabilization of the transition state (step 2a). However, substituting the conserved glutamate with alanine, asparagine, and even lysine had no major effect on the initial rate of sulfate transport, which reflects the tightness of sulfate occlusion. Residues involved, either directly or indirectly, in sulfate binding are predicted to be very sterically sensitive. We propose that protonation could facilitate the conformational changes associated with sulfate translocation by reducing the energy barrier to movement (step 2b).

In summary, our data support a model in which exchange coupling and substrate specificity are linked to the tightness of substrate binding in the transition state. The data suggest further that two types of conformational reorientations, distinguished by their sensitivity to protonation or mutation at Glu-699/1007, can promote anion exchange. The first is a rapid pathway (represented in Fig. 6A by steps 3a and 3b), which requires precise coordination of monovalent anion binding to stabilize the transition state. This pathway is extremely sensitive to the side chain and even the protonation state at position Glu-699/1007. In the second pathway, divalent anion binding is followed by slow (10^3- to 10^4-fold slower) reorientation only if protonation of the glutamate side chain or mutagenesis reduces the energy barrier to movement.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Research Grant GM38543 and American Cancer Society Grant BE-164 (to R. R. K.). This material is based upon work supported by National Science Foundation Grant MCB-8957340 (to R. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The first two authors contributed equally to this work.

Recipient of grants from the Stanford University Undergraduate Research Opportunities Program.

**
This work was done in part during the tenure of an Established Investigatorship of the American Heart Association. To whom correspondence should be addressed. Tel.: 415-723-7581; Fax: 415-723-8475.

(^1)
The abbreviations used are: AE, anion exchanger; WRK, Woodward's reagent K; PCR, polymerase chain reaction; HEK, human embryonic kidney 293 cells; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonate; BCECF, 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; MES, 4-morpholineethanesulfonic acid; bp, base pair(s).

(^2)
R. S. Lo and R. R. Kopito, unpublished observations.


ACKNOWLEDGEMENTS

We thank Joe Casey for helpful discussion and critical reading of the manuscript.


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