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 |
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 |
THE ANION EXCHANGER
(AE) genes AE1, AE2, and AE3 encode widely expressed plasmalemmal
Cl
/HCO
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
exchange mediated by
erythrocyte AE1 displays a broad pH vs. activity profile, consistent with its role in facilitating equilibrium of CO2 and
HCO
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
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
exchange (13, 15). However, in these experiments,
intracellular acidification produced no measurable inhibition of
function. Intracellular alkalinization indeed stimulated
Cl
/HCO
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
]
(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 |
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 p
X was used as a template for
polymerase chain reaction (PCR). The AE2 NH2-terminal
truncation mutants
N99,
N510
(30), and
N659 (31) were
constructed by a four-primer PCR method as described. AE2
NH2-terminal truncation mutants
N310,
N328,
N347, and
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 p
X (30). Integrity
of all PCR products and ligation junctions was confirmed by DNA
sequencing of both strands.
The designation AE2
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
AE2
N310, the introduced initiator Met is followed by
natural AE2 amino acid residue 311. In AE2
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 M
. 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).
 |
RESULTS |
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.
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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,
AE2 N99, and AE2 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,
AE2 N347, and AE2 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 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.
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|
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 AE2
N99 and AE2
N510. As shown in Fig.
2B, AE2
N99-mediated
36Cl
efflux exhibited a pHo
dependence indistinguishable from that of wild-type AE2, whereas the
pHo(50) value of AE2
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
AE2
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 AE2
N347
and AE2
N391 with wild-type AE2. Whereas the pH vs.
efflux curve for AE2
N391 differed only marginally from
that of wild-type AE2 [
pHo(50) = 0.22 ± 0.07; n = 21, P > 0.05], the curve
for AE2
N347 was significantly shifted to a more acid pH
value [
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
AE2
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.
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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
( ) 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.
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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
) to allow oocyte pHi to reach a new
steady state. Subsequent reintroduction of Cl
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
-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
( ) 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.
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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
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
( ) 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.
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|
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, AE2
N347, or
AE2
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 ( ),
AE2 N347 ( ), and AE2 N391
( ) in the presence of 40 mM butyrate to acidify
pHi and following its removal to alkalinize
pHi. B: pooled experiments with 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 AE2
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 |
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
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
exchange activity were
0.25-0.3 units higher than at rest. This pH-dependent behavior of
recombinant AE2-mediated Cl
/HCO
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
AE2
N391 and the acid-shifted pHo(50) values
of both AE2
N347 and AE2
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
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, AE2
N391 resembled
N347
and
N510 in their lack of activation upon
pHi alkalinization by butyrate removal. In contrast,
whereas AE2
N391 exhibited a near wild-type response to
pHo acidification, both
N347 and
N510 displayed acid-shifted pHo(50) values.
The unresponsiveness of AE2
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-AE2
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
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
(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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