Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain

A. K. Stewart, M. N. Chernova, Y. Z. Kunes, and S. L. Alper

Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Boston 02215; and Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of intracellular pH (pHi) in regulation of AE2 function in Xenopus oocytes remains unclear. We therefore compared AE2-mediated 36Cl- efflux from Xenopus oocytes during imposed variation of extracellular pH (pHo) or variation of pHi at constant pHo. Wild-type AE2-mediated 36Cl- efflux displayed a steep pHo vs. activity curve, with pHo(50) = 6.91 ± 0.04. Sequential NH2-terminal deletion of amino acid residues in two regions, between amino acids 328 and 347 or between amino acids 391 and 510, shifted pHo(50) to more acidic values by nearly 0.6 units. Permeant weak acids were then used to alter oocyte pHi at constant pHo and were shown to be neither substrates nor inhibitors of AE2-mediated Cl- transport. At constant pHo, AE2 was inhibited by intracellular acidification and activated by intracellular alkalinization. Our data define structure-function relationships within the AE2 NH2-terminal cytoplasmic domain, which demonstrates distinct structural requirements for AE2 regulation by intracellular and extracellular protons.

chloride-bicarbonate exchange; weak acids; Xenopus oocytes; isotopic flux; pH-sensitive microelectrodes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ANION EXCHANGER (AE) genes AE1, AE2, and AE3 encode widely expressed plasmalemmal Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger proteins that contribute to the regulation of intracellular pH (pHi), intracellular Cl- concentration, and cell volume in vertebrate cells (1, 2). All AE polypeptides have a COOH-terminal, hydrophobic, polytopic, transmembrane domain of >500 amino acid residues in length, ending in a short COOH-terminal cytoplasmic tail and sharing ~67% sequence identity. This transmembrane domain is preceded by an NH2-terminal hydrophilic, cytoplasmic domain of 400-700 amino acids displaying lower overall sequence homology (1). In the absence of NH2-terminal cytoplasmic domains, the COOH-terminal transmembrane domains suffice to mediate anion exchange (7, 18). The NH2-terminal cytoplasmic domain of erythroid AE1 binds to multiple cytoskeletal proteins and anchors the glycolytic pathway near ATP-consuming and ATP-regulated transporters of the erythroid plasma membrane. In contrast, functions of the cytoplasmic NH2-terminal domains of AE2 and AE3 remain unclear. Binding to cytoskeletal proteins has been postulated by analogy with erythrocyte AE1. However, conflicting data have been presented using different binding assays (14, 19).

Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange mediated by erythrocyte AE1 displays a broad pH vs. activity profile, consistent with its role in facilitating equilibrium of CO2 and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> across the red blood cell membrane to transfer CO2 from metabolizing tissues to the alveolar airspace for excretion (1, 12). In contrast, "nonerythroid" Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange, as measured in tissue culture cells, is sensitively regulated by pHi, consistent with its postulated role in recovery from alkaline loads (22). Similarly, the recombinant, nonerythroid anion exchanger, AE2, is highly sensitive to changes in pHi (10, 13, 26, 29). In contrast, recombinant AE3 has been reported to be insensitive to changes in pH (26).

Regulation of recombinant AE function has been demonstrated in mammalian cells by monitoring Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange (13, 15). However, in these experiments, intracellular acidification produced no measurable inhibition of function. Intracellular alkalinization indeed stimulated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity, but did so only at pHi values 0.2-0.3 units above resting values. These results suggested that nonerythroid AE anion exchangers functioned physiologically at low levels in constitutive mode and underwent alkaline activation only upon exposure to extreme alkali loads. Moreover, the increased intracellular [HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>] (substrate), which accompanied intracellular alkalinization, complicated interpretation of the hypothesized allosteric activation. For these reasons, we have conducted further studies with Xenopus oocytes.

Recombinant AE2-mediated 36Cl- influx and efflux in cRNA-injected Xenopus oocytes (10) were inhibited by acid extracellular pH (pHo) and activated by alkaline pHo under conditions of unclamped, near steady-state pHi (10, 30). A similar pattern of regulation by pH was found for AE2-mediated Cl-/nitrate exchange in mammalian cells in which ionophores were used to equalize pHi and pHo while varying pHo (26). In these systems, AE2 activity at resting pHi was closer to the midpoint of the activity scale such that perturbation of pHi in either direction produced a parallel change in transport activity.

Zhang et al. (30) localized a "pH-sensor" element to the AE2 transmembrane domain. A "pH-modifier" site important in setting the pHo(50) for regulation of transport was also localized to the large region between amino acid 99 and amino acid 510 of the AE2 NH2-terminal cytoplasmic domain (30). In the present work, we first reproduced the previously reported pHo dependence of AE2-mediated 36Cl- influx in groups of oocytes by measurement in single oocytes of AE2-mediated 36Cl- efflux. With this method, we refined the localization of the regions within the NH2-terminal cytoplasmic domain critical to regulation by pHo. We next characterized weak acid permeability of the oocyte and the lack of weak acid transport by oocyte-expressed AE2 to validate a method that allows variation of oocyte pHi at constant pHo. Last, we demonstrated that the regions of the AE2 NH2-terminal cytoplasmic domain required for AE2 inhibition by intracellular protons are not identical to those required for AE2 inhibition by bath acidification. The data suggest that both pHi and pHo regulate AE2 activity but require nonidentical regions of the AE2 NH2-terminal cytoplasmic domain polypeptide to do so.


    MATERIALS AND METHODS
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Reagents. Na36Cl was purchased from ICN (Irvine, CA). All other chemical reagents were of analytical grade and purchased from Sigma, Calbiochem, or Fluka. Restriction enzymes and T4 DNA ligase were purchased from New England BioLabs (Beverly, MA). Taq DNA polymerase and dNTPs were purchased from Promega (Madison, WI). Oligonucleotides were synthesized on a Milligen Cyclone Plus DNA synthesizer.

Solutions. ND-96 medium consisted of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, and 2.5 sodium pyruvate, pH 7.40. All flux media lacked sodium pyruvate and contained Na36Cl. In ND-96 solutions of different pH, HEPES was used for pH values of 7.0, 8.0, and 8.5. 2-(N-morpholino)ethanesulfonic acid (5 mM) was substituted for pH 5.0 and 6.0, and 5 mM piperazine-N,N'-bis(2-ethanesulfonic acid) was substituted for pH 6.5. In Cl--free solutions, NaCl was replaced isosmotically with 96 mM sodium isethionate and equimolar calcium and magnesium gluconate. When weak acid was added to flux media, it substituted for an equimolar quantity of anion (i.e., Cl-).

Construction of AE2 mutant cDNAs. Murine AE2 encoded in plasmid pDelta X was used as a template for polymerase chain reaction (PCR). The AE2 NH2-terminal truncation mutants Delta N99, Delta N510 (30), and Delta N659 (31) were constructed by a four-primer PCR method as described. AE2 NH2-terminal truncation mutants Delta N310, Delta N328, Delta N347, and Delta N391 were constructed as follows. AE2 PCR products were generated from the common downstream reverse primer yz12 (5'-CAGCCATTAGCACCAGCG-3') encoding AE2 nt 2899-2881 and by one of four AE2 upstream forward primers encoding either nt 1116-1132 (yz31; 5'-TCCCCGCGGCATCATGGCCCCGCATAAGCCCCA-3'), nt 1167-1183 (yz33; 5'-TCCCCGCGGCATCATGGACAAAAACCAGGAGC-3'), nt 1224-1240 (yz34; 5'-TCCCCGCGGCATCATGGATGTGGAAGAGGAGA- 3'), or nt 1356-1372 (zy36; 5'-TCCCCGCGGCATCATGGGGGTGGCCCATCAGGT-3').

Each forward primer contained at its 5' end a SacII restriction site and a Kozak consensus ATG. The resultant PCR products were restricted by SacII (cutting within the upstream primer sequence) and SmaI (cutting at AE2 nt 2860) and subcloned back into the complementary SacII/SmaI acceptor fragment of the AE2-encoding plasmid pDelta X (30). Integrity of all PCR products and ligation junctions was confirmed by DNA sequencing of both strands.

The designation AE2Delta N# indicates a cDNA encoding an NH2-terminally truncated AE2 in which an introduced methionine (Met) residue substitutes for native AE2 residue # and is followed by native residue (#+1). Thus in AE2Delta N310, the introduced initiator Met is followed by natural AE2 amino acid residue 311. In AE2Delta N659, the initiator Met 659, four residues beyond the tryptic cleavage site at Lys-654 to Ala-655 (30), is native to AE2.

cRNA expression in Xenopus oocytes. Mature female Xenopus (NASCO, Madison, WI) were maintained and subjected to partial ovariectomy as described (10). Stage V-VI oocytes were manually defolliculated after incubation of ovarian fragments with 2 mg/ml of collagenase A (Boehringer Mannheim, Germany) for 60 min in ND-96 solution containing 50 ng/ml gentamycin and 2.5 mM sodium pyruvate. Oocytes were injected on the same day with cRNA or 50 nl of H2O and incubated at 19°C.

Capped cRNA was transcribed from linearized cDNA templates with the MEGAscript kit (Ambion, Austin, TX) and resuspended in diethylpyrocarbonate-treated water. RNA integrity was confirmed by formaldehyde gel electrophoresis, and concentration was determined using a spectrophotometer at an absorbance wavelength of 260 nm.

Nominal oocyte surface expression of wild-type and mutant AE2 polypeptide was documented by confocal immunofluorescence microscopy (29) using affinity-purified antibody to mouse AE2 amino acids 1224-1237 (13, 30, 32).

36Cl- efflux measurements. Defolliculated oocytes were injected with cRNA or water (50 nl) and maintained for 2-5 days at 19°C in ND-96. Individual oocytes were injected in Cl--free ND-96 with 50 nl of 130 mM Na36Cl representing 8,000-12,000 counts per minute (cpm). After 5-10 min of recovery from injection, the efflux assay was initiated by transfer of individual oocytes to 6-ml borosilicate glass tubes, each containing 1 ml of efflux solution. At intervals of 3 min, 0.95 ml of this efflux solution was removed for scintillation counting and replaced with an equal volume of fresh efflux solution. After completion of the assay with a final efflux period marked by addition of 200 µM DIDS, each oocyte was lysed in 100 µl of 2% SDS. Samples were counted for 3-5 min. Values for two standard deviations were <6% of the mean.

Experimental data were plotted as ln(%cpm remaining) vs. time. 36Cl- efflux rate constants were measured from linear fits to data from the last three time points for each condition. Within each experiment, water-injected and AE2 cRNA-injected oocytes from the same frog were subjected to parallel measurements. All single values for 36Cl- efflux from AE2 cRNA-injected oocytes in Cl- medium exceeded 150 cpm. Efflux cpm values for water-injected oocytes or for AE2 cRNA-injected oocytes in the presence of DIDS differed approximately threefold from machine background values (i.e., <100 cpm). For each experimental day, activity of AE2 truncation constructs was compared with wild-type AE2 activity at pH 7.4. Each AE2 mutant was tested in oocytes from at least three frogs, using multiple cRNA preparations.

Rate constants measured at each pHo value for wild-type AE2 and the AE2 truncation mutants in each individual experiment were fit (SigmaPlot) to the following first-order logistic sigmoidal equation: v = (Vmax × 10-K)/(10-K + 10-x), where v = AE2-mediated Cl- efflux rate constant, Vmax = the maximum AE2-mediated Cl- efflux rate constant, x = pHo at time of measurement of rate constant, and K = pHo(50), the pHo at which v is half-maximal. Data for each mutant were normalized to the fit parameter Vmax calculated for each individual oocyte (100%), and the normalized data were fit to the same equation. Differences in mean pHo(50) values for individual mutants were subjected to analysis of variance (30) and comparison for all pairs using Tukey-Kramer analysis (JMP for Macintosh).

To vary pHi at constant pHo, oocytes were preincubated for 30-60 min before the start of the experiment in weak acid-containing solution in the presence or absence of Cl-. 36Cl- efflux was then initiated and continued after oocyte transfer into efflux medium lacking weak acid. pHi sensitivity of AE2-mediated 36Cl- efflux was expressed for individual oocytes as fold stimulation of AE2 activity following weak acid removal [(rateweak acid removal/rateweak acid presence) · 100].

Measurement of oocyte pHi. Oocyte pHi was measured using pH microelectrodes as described previously (23). pH electrodes were pulled on a vertical electrode puller (model 730; Kopf Instruments) from borosilicate glass tubing (M1B150F-6; World Precision Instruments) prewashed with nitric acid and ethanol. pH electrodes were silanized at 200°C for 5 min using 50 µl bis(dimethylamino)dimethyl silane (cat. no. 14755; Fluka Chemical, Milwaukee, WI), and the tips were coated with hardened Sylgard (Dow Corning) to reduce electrical noise. The tips of the electrode were backfilled with hydrogen ionophore I cocktail B (Fluka 95923) and filled with pH 7.0 buffer containing (in mM) 40 KH2PO4, 23 NaOH, and 150 NaCl. pH electrodes inserted into an Ag-AgCl half-cell electrode holder and calibrated at pH 6.0, 7.0, and 8.0 exhibited slopes of 55-63 mV/pH unit. A single point adjustment in pH 7.4 solution was made to calibrate the pH electrode before oocyte insertion.

Voltage electrodes pulled as earlier and filled with 3 M KCl had resistances <5 MOmega . Voltage electrodes and pH electrodes were connected to a high-impedance electrometer (FD-223; World Precision Instruments). Online recordings of voltage from both electrodes were displayed via a PC running DUO-18 software (World Precision Instruments). Voltage due to pHi was obtained by subtraction of the Vm signal (oocyte membrane potential) from the pH electrode signal. All experiments were performed at room temperature (21°-23°C).


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NH2-terminal truncation mutants of AE2 retain basal anion efflux function in Xenopus oocytes. Figure 1A shows the NH2-terminal truncation mutants of AE2 expressed and studied in Xenopus oocytes. All mutants were functional, as measured by 36Cl- efflux (Fig. 1B) and by 36Cl- influx (not shown), consistent with previous influx measurements (30). Because both 36Cl- influx and efflux mediated by AE2 exhibit trans-anion dependence and DIDS (10, 30), the activity preserved in the NH2-terminal truncation mutants represented Cl-/anion exchange. With the experimental conditions of Fig. 1 and those to follow, the bulk of AE2 activity represented Cl-/Cl- exchange.


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Fig. 1.   36Cl- efflux activity of wild-type (WT) and mutant anion exchanger type 2 (AE2) polypeptides expressed in Xenopus oocytes. A: schematic diagram of wild-type AE2 and AE2 NH2-terminal truncation mutants studied. B: efflux rate constants of wild-type and mutant AE polypeptides expressed in Xenopus oocytes. Mutant cRNA quantities for injection (8-20 ng) were selected to yield efflux rate constants comparable to that of wild-type AE2 (8 ng). Numbers in parentheses indicate number of oocytes.

To facilitate functional comparison of wild-type AE2 with AE2 NH2-terminal truncation mutants, oocytes were injected with cRNA quantities over an approximate fourfold range chosen to yield equivalent rates of 36Cl- efflux activity at pHo 7.4. As a result, transport rates at pHo 7.4 differed among wild-type and mutant polypeptides by no more than 2.5-fold in individual experiments. AE2-mediated Cl- efflux rate constants were routinely 0.04-0.06 min-1 at pHo 8.5 for all constructs. pHo-dependence experiments were included in subsequent analyses only when rate constants at pHo 8.5 were >0.02 min-1 to allow reliable measurement of rates at lower pHo values. This exclusion criterion resulted in elimination from the analysis of <15% of experiments. Wild-type and mutant AE2 polypeptides were detected by confocal immunofluorescence microscopy at the oocyte surface (not shown).

Distinct regions of the AE2 NH2-terminal cytoplasmic domain contribute to the acute regulation of AE2 by varying pHo. Figure 2A shows representative 36Cl- efflux traces from four AE2-expressing oocytes and from one water-injected oocyte as a function of sequentially increasing pHo. Wild-type AE2-mediated 36Cl- efflux was minimal at low pHo and increased at higher pHo values. The pHo value at which 36Cl- efflux was half-maximal [pHo(50)] was 6.91 ± 0.04 (n = 37 oocytes; Fig. 2, B-D). Similar efflux results were obtained when the order of pHo variation was reversed (data not shown). Thus the pHo(50) values measured for AE2-mediated 36Cl- efflux matched that previously measured for AE2-mediated 36Cl- influx (30). In both that study and the current study, changing pHo was accompanied by smaller changes in oocyte pHi (Fig. 2C). The measured regulation of AE2 thus reflected changes in both pHo and pHi.


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Fig. 2.   Extracellular pH (pHo) regulation of 36Cl- efflux from oocytes expressing wild-type and mutant AE2 polypeptides. A: 36Cl- efflux from individual water-injected oocytes () and wild-type AE2-expressing oocytes (triangles and circles). 36Cl- was injected 5 min before time 0, and pHo was changed as indicated. DIDS (D, 200 µM) was added during the final efflux period at pHo 8.5. The same experimental protocol generated the normalized data presented in B-D. B: normalized Cl- efflux plotted as a function of pHo for oocytes expressing wild-type AE2, AE2Delta N99, and AE2Delta N510. Lines are fit as described in MATERIALS AND METHODS. C: normalized Cl- efflux plotted as a function of pHo for oocytes expressing wild-type AE2, AE2Delta N347, and AE2Delta N391. Near steady-state oocyte intracellular pH (pHi) values at each pHo value are as measured in Ref. 30. Values in B and C are means ± SE. D: pH(50) values exhibited by wild-type AE2 and the indicated Delta N mutants, calculated from curve fits of pHo vs. activity plots for (n) individual oocytes (means ± SE). *P < 0.05 compared with wild-type AE2.

Zhang et al. (30) showed that a region between AE2 amino acids 99 and 510 in the NH2-terminal cytoplasmic domain was critical in determination of the apparent pHo(50) value for regulation of AE2-mediated 36Cl- influx. We therefore tested the pHo dependence of wild-type AE2-mediated 36Cl- efflux with that of AE2Delta N99 and AE2Delta N510. As shown in Fig. 2B, AE2Delta N99-mediated 36Cl- efflux exhibited a pHo dependence indistinguishable from that of wild-type AE2, whereas the pHo(50) value of AE2Delta N510 was shifted to a more acidic pH value by 0.52 ± 0.10 units (n = 20, P < 0.002). This shift, measured as 36Cl- efflux from individual oocytes exposed to all pHo values, was similar to the pHo(50) shift toward acidic values of 0.69 ± 0.13 exhibited by AE2Delta N510-mediated 36Cl- influx into oocyte groups, each exposed to a single pHo value (30). These data validate the AE2-mediated 36Cl- efflux method for structure-function studies of AE2-mediated Cl- transport in individual Xenopus oocytes and confirm the importance of pH regulation to AE2 of the central region of the AE2 NH2-terminal cytoplasmic domain.

We extended analysis of the AE2 NH2-terminal cytoplasmic domain's role in AE2 regulation by pHo by examining additional truncated polypeptides. Figure 2C compares pH vs. 36Cl- efflux curves for AE2Delta N347 and AE2Delta N391 with wild-type AE2. Whereas the pH vs. efflux curve for AE2Delta N391 differed only marginally from that of wild-type AE2 [Delta pHo(50) = 0.22 ± 0.07; n = 21, P > 0.05], the curve for AE2Delta N347 was significantly shifted to a more acid pH value [Delta pHo(50) = 0.52 ± 0.10; n = 26, P < 0.002]. Figure 2D summarizes the pHo(50) values obtained as shown in Fig. 2, B and C, for all tested NH2-terminal truncation mutants. The data reveal three important regions of the NH2-terminal cytoplasmic domain. Removal of the first region, between amino acid residues 328 and 347, produced an acid pH-shifted pHo(50) value. Incremental removal of the second region, residues 348-391, largely reversed pHo(50) toward the wild-type value. Incremental removal of the third region, between residues 391 and 510, restored the acidic pHo(50) value for AE2-mediated 36Cl- transport. This acidic pHo(50) value was not further modified in AE2Delta N659.

Weak acids as regulators of oocyte pHi. The above experiments, as well as those of Zhang et al. (30), were performed under conditions where the experimentally imposed change of 1 unit of pHo was accompanied by ~0.13 units of change in oocyte pHi. Thus despite the demonstrated importance of regions of the NH2-terminal cytoplasmic domain in regulation of AE2-mediated Cl- transport by pH, the relative contributions of pHo and pHi to this regulation could not be determined. Zhang et al. (30) also used sodium acetate exposure to inhibit wild-type AE2 activity under conditions of acidified oocyte pHi at constant pHo. We therefore examined in greater detail the effects of weak acids on oocyte pHi and used the weak acids to measure regulation of wild-type and mutant AE2-mediated 36Cl- efflux by pHi at constant pHo.

A range of weak acids (formate, acetate, propionate, butyrate, and benzoate) was screened for the ability to acidify Xenopus laevis oocytes. Oocyte pHi was recorded using pH microelectrodes at a constant external pH of 7.4 for all experiments. Resting pHi of oocytes was 7.27 ± 0.02 (n = 27). Figure 3A shows a representative pHi trace recorded from a single oocyte in which addition to the superfusate of 40 mM butyrate, but not 40 mM formate (inset), elicited a reversible decrease in oocyte pHi of 0.50 ± 0.03 pH units (n = 8). Subsequent removal of weak acid from the superfusate was followed by reversal of oocyte acidification to resting values or with a small pHi overshoot. The time constant (t0.5) for butyrate-induced acidification was 403 ± 55 s (n = 8), and upon removal of weak acid, pHi recovered with a time constant of 315 ± 15 s (n = 6).


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Fig. 3.   Weak acid transport in Xenopus oocytes. A: recording of pHi in a Xenopus laevis oocyte by pH-sensitive microelectrode. Substitution of 40 mM NaCl by sodium butyrate at pHo 7.4 reversibly acidified pHi from resting value of 7.20 to a new steady state of 6.67, whereas sodium formate did not change oocyte pHi (inset). B: mean decrease in oocyte pHi 20-25 min following substitution of 40 mM NaCl with the indicated weak acid sodium salts in the superfusate (means ± SE). Butyrate produced the greatest degree of acidification. Numbers in parentheses indicate number of oocytes. C: comparison of [14C]formate efflux (n = 4) with that of [14C]butyrate (n = 6) from oocytes injected 2 days previously with water.

Figure 3B summarizes peak oocyte acidification achieved by 20-25 min of exposure to 40 mM of the indicated weak acid salts. Sodium butyrate produced the greatest acidification, as recently described (16). Efflux rates of 14C-weak acid (Fig. 3C) demonstrated that the failure of formate to acidify oocytes is paralleled by a much slower efflux of [14C]formate than of [14C]butyrate from water-injected oocytes.1 The rates of [14C]butyrate efflux from water-injected and AE2-expressing oocytes into Cl--containing medium were indistinguishable, showing that butyrate is not transported at measurable rates by AE2. Therefore, we employed weak acids to test the pHi dependence of AE2 activity.

Changing pHi at constant pHo with weak acids regulates AE2 function. Oocytes previously injected with water or AE2 cRNA were preincubated with 40 mM butyrate, pH 7.4, for 30-60 min before measurement of 36Cl- efflux (Fig. 4A). Butyrate inhibited AE2-mediated 36Cl efflux by 80.9 ± 1.9% (n = 35, P < 0.005). Removal of butyrate enhanced AE2-mediated 36Cl- efflux after a brief lag period but had no effect on water-injected oocytes. Figure 4B summarizes the stimulation of AE2-mediated 36Cl- efflux elicited by removal of the indicated weak acid salts. All weak acids tested except formate substantially suppressed AE2-mediated 36Cl- efflux. Conversely, removal of all weak acids tested except formate stimulated AE2-mediated 36Cl- efflux.


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Fig. 4.   Weak acid removal stimulates AE2-mediated 36Cl- efflux. A: representative 36Cl- efflux traces from water-injected (open circle ) and AE2-expressing () oocytes in the presence of butyrate and during its subsequent removal, followed by addition of the inhibitor DIDS (means + SE, n = 5). B: fold stimulation of wild-type AE2-mediated 36Cl- efflux following removal of the indicated weak acids (40 mM). Only formate had no effect. Acetate, butryate, propionate, and benzoate each inhibited 36Cl- efflux, and their removal stimulated 36Cl- efflux (means ± SE). Numbers in parentheses indicate number of oocytes.

These data support the hypothesis that changes in pHi at constant pHo regulate AE2 function and predict that graded variation of extracellular weak acid concentration should produce graded variation in AE2 function. Figure 5A shows a representative time course of 36Cl- efflux from an AE2-expressing oocyte preincubated for 2 h with 40 mM butyrate and exposed to progressive reduction in butyrate concentrations. Each change of butryate concentration was made in the absence of bath chloride (Cl<UP><SUB>o</SUB><SUP>−</SUP></UP>) to allow oocyte pHi to reach a new steady state. Subsequent reintroduction of Cl<UP><SUB>o</SUB><SUP>−</SUP></UP> reactivated AE2-mediated 36Cl- efflux in each condition. Oocyte pHi in a representative oocyte subjected to the same sequence of graded reductions in butyrate concentration (Fig. 5B) reached new steady-state pHi values sufficiently rapid to explain each graded increase in AE2-mediated 36Cl- efflux. Figure 5C summarizes data from similar experiments in five oocytes. The lower x-axis shows the pHi values measured at each corresponding extracellular butyrate concentration. The results were similar when the Cl<UP><SUB>o</SUB><SUP>−</SUP></UP>-free incubation periods were omitted (not shown). These data provide further support for the hypothesis that variations in pHi at constant pHo can regulate AE2 function in individual oocytes.


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Fig. 5.   Regulation of AE2-mediated 36Cl- efflux in an individual oocyte by varying pHi at constant pHo. A: representative time course of 36Cl- efflux from a water-injected (open circle ) and from an AE2-expressing () oocyte in the presence of sequentially decreasing bath concentrations of sodium butyrate, each in the absence and presence of bath Cl-. Efflux was terminated by addition of DIDS. B: changes in oocyte pHi measured by pH-sensitive microelectrode during sequential exposure of oocyte to sodium butyrate at the indicated concentrations. C: rate constant of AE2-mediated 36Cl- efflux (n = 5) during sequential decreases in bath butyrate concentration as in B. Bottom x-axis indicates measured pHi at each butyrate concentration.

Weak acid anions are neither AE2 transport substrates nor AE2 inhibitors. The above-demonstrated inhibition of AE2-mediated 36Cl- efflux function by weak acids might be explained if the weak acid anions served as transported substrates of AE2 (weak agonist inhibitors) or as pure inhibitors of AE2. Figure 6, A and B, suggests that butyrate anion is not a substrate at the exofacial transport site of AE2, since in the absence of Cl-, 40 mM butyrate did not stimulate 36Cl- efflux. After butyrate removal, subsequent restoration of Cl<UP><SUB>o</SUB><SUP>−</SUP></UP> increased 36Cl- efflux activity to control rates (see Fig. 1B). In addition, 0.5 mM [14C]butyrate influx into AE2-expressing oocytes was no greater than into water-injected oocytes (Fig. 6D); neither is butyrate a substrate at the cytoplasmic transport site of AE2, as shown by the AE2 independence and DIDS insensitivity of [14C]butyrate efflux (Fig. 3C).


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Fig. 6.   Weak acid anions are neither transport substrates nor inhibitors of AE2 in Xenopus oocytes. A: 36Cl- efflux from a water-injected (open circle ) and an AE2-expressing () oocyte in the presence and absence of bath butyrate, as a function of the presence and absence of bath Cl-. B: summary of 5 experiments (means ± SE) similar to that in A. AE2 does not mediate measurable exchange of intracellular Cl- for extracellular butyrate. C: although 2 h of exposure to 40 mM acetate reduced AE2-mediated 36Cl- efflux, after 24 h of acetate exposure, AE2 activity returned to baseline values. This reflected recovery of oocyte pHi (not shown) and shows that acetate itself did not inhibit AE2-mediated 36Cl- transport. Similar results were noted with 40 mM butyrate (not shown). D: butyrate is not a substrate for AE2. [14C]butyrate influx was indistinguishable in water-injected and in AE2-expressing oocytes, whether without or with 24-h prior exposure (pre-inc) to 40 mM butyrate. Results were similar for [14C]butyrate efflux (not shown). Numbers in parentheses indicate number of oocytes.

The failure of extracellular butyrate alone to support 36Cl- efflux may have been secondary to intracellular acidification. Therefore, oocytes were incubated in the presence of weak acid (pHo 7.4) for 24 h to allow pHi recovery to near-initial values (resting pHi 7.01 ± 0.01, n = 5). Figure 6C shows that AE2-mediated 36Cl- efflux was inhibited after 2 h of exposure, but not after 24 h of exposure, to 40 mM acetate. Similar results were obtained with butyrate. These data show that weak acid anions are not themselves "substrate site inhibitors" of AE2-mediated 36Cl- efflux at resting pH. [14C]butyrate influx into AE2-expressing oocytes previously exposed for 24 h to 40 mM butyrate was not greater than in water-injected oocytes and not different from oocytes freshly exposed to only 0.5 mM butyrate (Fig. 6D). These data show that weak acid anions are neither direct inhibitors of AE2 nor transported substrates of AE2 in Xenopus oocytes.

AE2 is activated by hypertonic shrinkage of Xenopus oocytes by a process requiring endogenous Na+/H+ exchanger (NHE) activity (9). Because exposure of oocytes to isotonic 40 mM butyrate may increase total intracellular solute, and removal of extracellular butyrate may promote solute loss from oocytes that parallel pHi increase, we assessed a possible contribution of shrinkage-activated NHE activity to activation of AE2 by butyrate removal. Basal AE2-mediated 36Cl- efflux was unaffected by bath Na+ removal (n = 4, not shown). Figure 7 shows that AE2 activation by butyrate removal was similarly uninhibited by the absence of bath Na+ or by the addition of 1 mM amiloride, conditions that completely inhibit hypertonic activation of endogenous oocyte NHE (6). The data suggest that weak acid removal activates AE2 by increasing pHi (see Fig. 5) without involvement of endogenous NHE activity.


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Fig. 7.   Stimulation of AE2 by butyrate removal does not involve shrinkage-induced endogenous Na+/H+ exchange activity. Neither removal of bath Na+ nor addition of amiloride to Na+-free bath inhibited AE2 stimulation by butyrate removal. Numbers in parentheses indicate number of oocytes.

Regulation of AE2 NH2-terminal truncation mutants by weak acid removal. Figure 2B showed the importance of regions' COOH-terminal to amino acid residue 328 in the regulation of AE2 activity by pHo. The weak acid removal method was used to compare the structural elements required for AE2 regulation by pHo with the requirements for AE2 regulation by pHi at constant pHo. Figure 8A shows 36Cl- efflux traces from representative oocytes expressing either wild-type AE2, AE2Delta N347, or AE2Delta N391. Whereas wild-type AE2-mediated 36Cl- efflux (closed circles) was reduced by the butyrate-induced fall of pHi and subsequently stimulated by the butyrate removal-induced rise in pHi, the AE2 truncation mutants were insensitive to butyrate-induced changes in pHi.


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Fig. 8.   Effect of AE2 sequential NH2-terminal deletions on stimulation by butyrate removal. A: 36Cl- efflux at constant pHo from single oocytes expressing wild-type AE2 (), AE2Delta N347 (triangle ), and AE2Delta N391 (open circle ) in the presence of 40 mM butyrate to acidify pHi and following its removal to alkalinize pHi. B: pooled experiments with Delta N mutants of AE2, performed as in A, and expressed as %stimulation of 36Cl- efflux following butyrate removal (means ± SE). Numbers in parentheses indicate number of oocytes.

Similar experiments performed with AE2 NH2-terminal truncation mutants are summarized in Fig. 8B. Removal of the NH2-terminal 99 or 310 amino acids moderately reduced the degree of AE2 activation by butyrate removal. However, removal of 328 amino acids nearly abolished AE2 activation by butyrate removal. Incremental removal of additional NH2-terminal sequence, including that generating AE2Delta N391, failed to restore the ability of butyrate removal to activate AE2. These data suggest that AE2 residues between amino acids 310 and 328 are required for normal inhibition of AE2-mediated Cl- transport by intracellular acidification. A possible role for residues' NH2-terminal to amino acid 310 is also suggested. Moreover, AE2 NH2-terminal truncation mutants differ in their functional responses to variation of bath pHo and to variation of pHi by introduction and removal of weak acids.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present work has more precisely defined the structural loci of pH sensitivity of AE2-mediated monovalent anion exchange in Xenopus oocytes. We have confirmed and extended results on regulation of AE2 by varying pHo in conditions of unclamped, near steady-state pHi. We then standardized the use of weak acids to vary oocyte pHi at constant pHo in the context of electroneutral anion-exchange experiments. We used this system to establish that changes in pHi alone within the physiological range suffice to regulate AE2 function. Last, we found that restricted regions of the AE2 NH2-terminal cytoplasmic domain, whose presence is required for inhibition of transport activity by acidification of bath pHo, are not identical to those required for inhibition of transport activity when pHi is acidified at constant pHo. These results extend the utility of the Xenopus oocyte as a system in which to study the structure-function relationships of anion exchangers. In addition, they suggest a model of greater complexity than previously envisioned to describe AE2 regulation by protons.

Host systems and assays used to study regulation of AE2 by protons. Transport activity of recombinant AE2 has been detected in transiently transfected cells from monkey (17), human (15, 26), and hamster (13) and in infected insect cells (8) as well as in Xenopus oocytes (10, 30). AE2 is much more sensitively regulated by pH than is AE1 (10, 26, 30). pHi sensitivity at constant pHo has been demonstrated for AE2-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange by pH ratio fluorimetry in transiently transfected mammalian cells in culture. However, AE2 was not inhibited by pHi values more acidic than resting values, and the alkaline pHi values required to increase AE2-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange activity were 0.25-0.3 units higher than at rest. This pH-dependent behavior of recombinant AE2-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in transiently transfected mammalian cells suggests that, under these serum-free conditions, AE2 is constitutively active at low levels at resting and acidic pHi and activated only during extreme alkaline load.2 AE2-mediated Cl--nitrate exchange monitored in transiently transfected HEK-293 cells by 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) fluorescence exhibited a larger degree of pH dependence of AE2 under conditions of nigericin/high-K+ pH clamp in which pHo = pHi (26). However, the selectivity of Cl--sensitive fluorophores has precluded measuring exchange of Cl- with halides or bicarbonate.

pH dependence of AE2 function in Xenopus oocytes has been studied by measurement of unidirectional 36Cl- flux in exchange for nonbicarbonate anions.3 The previously established pH dependence of AE2-mediated 36Cl- influx has been extended in the present work to pH dependence of AE2-mediated 36Cl- efflux. Measurement of 36Cl- efflux offers the advantage of allowing each oocyte to serve as its own control for multiple subsequent experimental conditions. The studies of Zhang et al. (30) and Fig. 2 of the present work tested pH dependence of AE2 function by varying pHo over 3-4 units, while oocyte pHi varied in response to reach near steady-state values extending over a range of ~0.5-0.6 units. Under these experimental conditions, acidification reduced AE2 activity, and alkalinization increased AE2 activity. However, even this improved approach still could not resolve the regulatory effects of pHo from those of pHi.

To investigate pHi regulation of AE2 function at constant pHo, we have shown that the weak acid method of acidifying oocytes is suitable for analysis of wild-type and mutant AE2-mediated 36Cl- efflux experiments. Whereas weak acids have been used in oocytes to study pH sensitivity of ion channels (4, 16, 18, 28), and, to a limited degree, cotransporters (3, 23), their application to the study of electroneutral anion exchangers has been limited (30). We have found that although formate is not permeant in X. laevis oocytes, acetate, propionate, butyrate, and benzoate are both permeant and useful for acidification of oocyte pHi at constant pHo (Fig. 3). Butyrate and acetate acidified oocytes without serving either as transport substrates or as direct inhibitors of AE2 (Fig. 6). Moreover, the effect of weak acid removal on AE2 was independent of endogenous (shrinkage-activated) NHE activity. Thus regulation of AE2-mediated 36Cl- efflux by weak acid addition and removal can be interpreted as reflecting AE2 regulation by pHi (Figs. 4-6).

Structure-function relationships of AE2 regulation by combined changes in pHo/pHi and by pHi at constant pHo. Zhang et al. (30) demonstrated that the AE2 pHo(50) value derived from the plot of normalized 36Cl- influx vs. pHo depended on the presence of amino acid residues in the NH2-terminal cytoplasmic domain between amino acids 99 and 510. We have confirmed this conclusion with 36Cl- efflux experiments in which each oocyte served as its own control. In addition, we narrowed down this large region to a short stretch between amino acids 328 and 347 and a larger region between amino acids 391 and 510 (Fig. 2).

The difference between the near wild-type pHo(50) value of AE2Delta N391 and the acid-shifted pHo(50) values of both AE2Delta N347 and AE2Delta N510 is reminiscent of studies of recombinant human NHE1 in which at least two separate regions of the long COOH-terminal cytoplasmic domain collaborate to interact with an undefined region within the transmembrane domain to mediate regulation of NHE1 function by pHi (11). Similarly, regulation by pH of several K+ channels involves interactions between distinct sites in NH2-terminal and COOH-terminal cytoplasmic tails (21, 24, 25). The current results suggest that amino acid residues between amino acids 391 and 510 suffice to confer near wild-type proton affinity to the AE2 transmembrane domain. Residues between amino acids 347 and 391 serve in Delta N347 to decrease this proton sensitivity. However, in wild-type AE2 and in less extreme NH2-terminal truncation mutants, this influence is countered by the presence of residues between amino acids 328 and 347, consistent either with their direct interaction or with a conformational change in this or another part of AE2.

Importantly, functional evaluation of the same AE2 NH2-terminal deletion mutants studied during butyrate exposure and removal (pHi variation during constant pHo, Fig. 8) yielded results that differed from those observed during variation of pHo (Fig. 2). In particular, residues between amino acids 310 and 328 that were not required for a wild-type AE2 pattern inhibition of acidic pHo were required for AE2 stimulation upon butyrate removal (pHi alkalinization from an acidic value). Two other differences were evident. First, removal of the NH2-terminal 99 amino acids and 310 amino acids produced graded decreases in AE2 activation by butyrate removal but had no effect on responsiveness to pHo change. Second, AE2Delta N391 resembled Delta N347 and Delta N510 in their lack of activation upon pHi alkalinization by butyrate removal. In contrast, whereas AE2Delta N391 exhibited a near wild-type response to pHo acidification, both Delta N347 and Delta N510 displayed acid-shifted pHo(50) values.

The unresponsiveness of AE2Delta N328 and progressively more truncated AE2 NH2-terminal mutants to intracellular alkalinization reflects a lack of inhibition by butyrate-induced intracellular acidification. We propose that loss of this inhibition by pHi reflects an acid shift in the pHi vs. activity curve larger than detectable in the present experiments. The data together suggest a complex interaction of extracellular and intracellular protons in regulating AE2 activity through multiple regions in the midportion of the AE2 NH2-terminal cytoplasmic domain. We postulate that one or more of these regions interact(s) with the AE2 transmembrane domain, directly or through associated polypeptides, to mediate AE2 regulation by both pHi and pHo. We further suggest that changes in pHo are detected by as yet unidentified residue(s) in the transmembrane domain and are reflected in altered pKa of the transmembrane pHi sensor by pH "modifier/sensor" elements in the NH2-terminal cytoplasmic domain. Figure 9 summarizes these conclusions in schematic form. pHo variation detected by transmembrane domain pH sensor residue(s) could (if residing at the permeability barrier of the AE2 anion translocation pathway) directly modify that translocation pathway. Alternatively, the anion translocation pathway could be indirectly modified from a distance via transmitted conformational change. Future experiments may exploit this system to assess the influence of differing values of invariant pHo on AE2 regulation by pHi.


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Fig. 9.   Schematic of AE2. Influence of the indicated regions of the NH2-terminal cytoplasmic domain upon pH regulation of anion transport mediated in this model by residues in the transmembrane domain. Bottom diagram shows consequences to the apparent pKa of anion transport of truncation of residues NH2-terminal to the indicated amino acids. +, Truncation exhibits a wild-type pKa for 36Cl- efflux; -, truncation exhibits an acid-shifted pKa. Shaded boxes (top) are regions NH2-terminal to which truncation elicits different responses to the 2 acidification protocols.


    ACKNOWLEDGEMENTS

We thank Drs. Alexander Zolotarev and Boris Shmukler for the plasmid pXT7-AE2Delta N659.


    FOOTNOTES

A. K. Stewart was supported by an International Prize Travelling Fellowship of Wellcome Trust. S. L. Alper was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43495 and DK-34854 and the Harvard Digestive Diseases Center.

Present address of Y. Z. Kunes: Millennium Pharmaceuticals, 640 Memorial Dr., Cambridge, MA 02139.

1  In contrast (data not shown), oocytes expressing pendrin mediated extracellular Cl--dependent [14C]formate efflux at very high rates.

2  AE2-mediated Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchange in CHOP cells is activated by addition of serum (13); anion exchange in mesangial cells (5) and in cardiomyocytes (20, 27) is activated by angiotensin II and by ATP.

3  AE2 in Xenopus oocytes does mediate 36Cl- efflux in exchange for extracellular HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (unpublished data), but pH dependence of this mode of AE2 function remains to be studied.

Address for reprint requests and other correspondence: S. L. Alper, RW763 East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215 (E-mail: salper{at}caregroup.harvard.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 15 February 2001; accepted in final form 11 May 2001.


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DISCUSSION
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