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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
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE ANION EXCHANGER
(AE) genes AE1, AE2, and AE3 encode widely expressed plasmalemmal
Cl/HCO
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/HCO
Regulation of recombinant AE function has been demonstrated in
mammalian cells by monitoring Cl/HCO/HCO
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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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 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').
X (30). Integrity
of all PCR products and ligation junctions was confirmed by DNA
sequencing of both strands.
The designation AE2N# 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
AE2N310, the introduced initiator Met is followed by
natural AE2 amino acid residue 311. In AE2N659, 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.
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 × 10K)/(10K + 10x), 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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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 min1
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 min1 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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influx. We therefore tested the pHo dependence of
wild-type AE2-mediated 36Cl efflux with that
of AE2N99 and AE2N510. As shown in Fig.
2B, AE2N99-mediated
36Cl efflux exhibited a pHo
dependence indistinguishable from that of wild-type AE2, whereas the
pHo(50) value of AE2N510 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
AE2N510-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 AE2N347
and AE2N391 with wild-type AE2. Whereas the pH vs.
efflux curve for AE2N391 differed only marginally from
that of wild-type AE2 [pHo(50) = 0.22 ± 0.07; n = 21, P > 0.05], the curve
for AE2N347 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
AE2N659.
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.
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-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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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
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
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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 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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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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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, AE2N347, or
AE2N391. 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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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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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/HCO/HCO-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.
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).
N391 and the acid-shifted pHo(50) values
of both AE2N347 and AE2N510 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, AE2N391 resembled N347
and N510 in their lack of activation upon
pHi alkalinization by butyrate removal. In contrast,
whereas AE2N391 exhibited a near wild-type response to
pHo acidification, both N347 and
N510 displayed acid-shifted pHo(50) values.
The unresponsiveness of AE2N328 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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ACKNOWLEDGEMENTS |
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We thank Drs. Alexander Zolotarev and Boris Shmukler for the
plasmid pXT7-AE2N659.
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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
3
AE2 in Xenopus oocytes does mediate
36Cl efflux in exchange for extracellular
HCO
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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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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) and wild-type AE2-expressing oocytes
(triangles and circles). 36Cl
) 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
), and AE2
, truncation exhibits an
acid-shifted pKa. Shaded boxes (top)
are regions NH2-terminal to which truncation elicits
different responses to the 2 acidification protocols.