Structure-function relationships of AE2 regulation by
Ca
-sensitive stimulators NH
and
hypertonicity
Marina N.
Chernova,
Andrew K.
Stewart,
Lianwei
Jiang,
David J.
Friedman,
Yune Z.
Kunes, and
Seth L.
Alper
Molecular Medicine and Renal Units, Beth Israel Deaconess
Medical Center, Department of Medicine, Harvard Medical School,
Boston, Massachusetts 02215
 |
ABSTRACT |
We showed previously that the nonerythroid
anion exchanger AE2 and the erythroid anion exchanger AE1 differ
greatly in their regulation by acute changes in intracellular pH
(pHi) and extracellular pH (pHo). We
have now examined how AE2, but not AE1, is activated by two stimuli
with opposing effects on oocyte pHi: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus,
NH
. We find that both NH2-terminal
cytoplasmic and COOH-terminal transmembrane domains of AE2 are required
for activation by either stimulus. Directed by initial deletion
mutagenesis studies of the NH2-terminal cytoplasmic domain,
an alanine scan of AE2 amino acids 336-347 identified residues
whose individual mutation abolished or severely attenuated sensitivity
to both or only one activating stimulus. Chelation of cytoplasmic
Ca2+ (Ca
) diminished or abolished AE2 stimulation by NH
and by hypertonicity. Calmidazolium
inhibited AE2 activity, but not that of AE1. AE2 was insensitive to
many other modifiers of Ca2+ signaling. Unlike AE2
stimulation by NH
and by hypertonicity, AE2
inhibition by calmidazolium required only AE2's COOH-terminal
transmembrane domain.
band 3; Xenopus oocyte; isotopic flux; site-directed
mutagenesis; chloride-bicarbonate exchange; chloride-bicarbonate
exchanger; intracellular calcium ion
 |
INTRODUCTION |
BICARBONATE IS
TRANSPORTED across plasma membranes of higher vertebrate
cells by the polytopic transmembrane proteins encoded by the SLC4 and
SLC26 gene superfamilies. The SLC4 anion transporter gene superfamily
includes at least three homologous genes encoding Na+-dependent Cl
/HCO
exchangers: AE1 (SLC4A1), AE2 (SLC4A2), and AE3 (SLC4A3). These
polypeptides contribute to regulation of pH/HCO
concentration ([HCO
]), volume, and
[Cl
] in individual cells, in their surrounding
extracellular space, and in compartments separated by epithelial cell
monolayers (1, 2). AE transcripts are expressed in
patterns that display specificity of tissue distribution and
specificity of developmental and homeostatic regulation. Mutations in
the AE1 gene, expressed in erythroid and renal intercalated cells, have
been associated with inherited erythroid dyscrasias such as hereditary
spherocytosis and with autosomal dominant and recessive forms of distal
renal tubular acidosis (2). AE2 and AE3 are expressed more
widely but have not been associated with human disease.
AE polypeptides are characterized by an NH2-terminal
hydrophilic, cytoplasmic domain of ~400-700 amino acids (aa).
This domain is believed, on the basis of studies of red blood cell AE1,
to interact with several proteins of the cytoskeleton and with
metabolic and signaling enzymes. This NH2-terminal
cytoplasmic domain is followed by a polytopic transmembrane domain of
~500 aa that mediates anion exchange and (at least in AE1) binds the
transmembrane carbonic anhydrase CAIV through interaction with an
exofacial loop. Following the transmembrane domain is a short,
hydrophilic COOH-terminal tail of 30 aa or longer that binds the
cytosolic carbonic anhydrase CAII via an acidic motif (38)
and terminates with a candidate type-2 PDZ interaction motif
(14).
The nonerythroid anion exchanger AE2 (SLC4A2) and the
erythroid/renal anion exchanger AE1 (SLC4A1) differ greatly in
their patterns of acute regulation when expressed as heterologous
polypeptides in Xenopus oocytes. Whereas acute decreases in
intracellular (pHi) and extracellular (pHo) pH
each inhibit AE2-mediated Cl
/Cl
and
Cl
/HCO
exchange (23, 31, 32,
43), AE1-mediated Cl
transport is much less
sensitive to pHo and completely insensitive to
pHi over the range tested (31, 32, 43).
Similarly, whereas acute exposure to low concentrations of
NH
(19) and to moderate hypertonicity
(20) activate AE2, AE1 is unaffected by these stimuli.
Curiously, these two AE2 activators produce rapid but opposing changes
in oocyte pHi. Hypertonicity alkalinizes oocytes through cJNK-dependent stimulation of a Cl
-dependent
Na+/H+ exchange activity (18).
Thus activation of AE2 by hypertonicity is consonant with AE2
activation by alkaline pHi, and alkalinization accounts for
at least part of AE2 activation by hypertonicity (20). In
contrast, stimulation of AE2 by NH
occurs in the
presence of a pHi sufficiently acidic so as to inactivate AE2 completely if elicited in any other way (31, 32, 43). The mechanism by which NH
stimulates AE2 in a way
that overrides concomitant inhibition by acidic pHi remains unknown.
Although some of the AE2 structural elements required for AE2 to
exhibit its wild-type sensitivity to inhibition by acidic pHo and to pHi have been defined (31, 32,
43), the structural elements required for stimulation by either
NH
or hypertonicity have not been reported. We
therefore set out to define amino acid residues of AE2 whose presence
is required for AE2 to display its wild-type sensitivity to the acute
stimulators NH
or hypertonicity. We hypothesized that
these two AE2 stimulators with opposing effects on pHi
would require the integrity of different regions of the AE2 polypeptide to produce stimulation.
Our studies of regulated 36Cl
transport by
chimeric AE polypeptides showed that the presence of both the
NH2-terminal cytoplasmic and COOH-terminal transmembrane
domains of AE2 is required for functional activation either by
hypertonicity or by NH
. Systematic, progressive
deletion mutagenesis studies indicated the importance for both modes of
AE2 stimulation of a highly conserved region in the midportion of the
AE2 NH2-terminal cytoplasmic domain. An alanine scan of AE2
aa 336-347 identified residues whose individual mutation abolished
or severely attenuated both types or only one type of stimulation.
Chelation of cytoplasmic Ca2+ (Ca
)
diminished AE2 stimulation by NH
and abrogated
stimulation by hypertonicity. NH
elevated oocyte
cytoplasmic [Ca2+] ([Ca2+]i) in
the presence or nominal absence of bath Ca2+. Calmidazolium
blocked both stimulated and basal AE2 activity. However, many other
modifiers of cell signaling, including other calmodulin inhibitors,
failed to inhibit AE2 or reduce its stimulation by
NH
. In contrast to AE2 stimulation by
NH
and by hypertonicity, AE2 inhibition by
calmidazolium did not require the presence of the AE2
NH2-terminal cytoplasmic domain.
 |
METHODS |
Materials.
Female Xenopus were purchased from NASCO (Madison, WI).
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-acetoxymethyl
ester (BCECF-AM) was from Molecular Probes. Bumetanide was the gift of
P. Feit (Leo Pharmaceutical). 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 2-deoxynucleotide
5'-triphosphates (dNTPs) were purchased from Promega (Madison, WI).
Expand High Fidelity PCR System was obtained from Roche (Indianapolis,
IN). The Sure Escherichia coli strain was from Stratagene
(La Jolla, CA). Oligonucleotides were purchased from Bio-Synthesis
(Lewisville, TX). Anti-calmodulin polyclonal and monoclonal antibodies
were from Sigma.
Solutions.
ND-96 medium consisted of (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and 2.5 sodium
pyruvate with 100 µg/ml gentamicin. All flux media contained 10 µM
bumetanide and lacked pyruvate and gentamicin. Influx media contained
20-38 mM NaCl as Na36Cl (2.8-3.2 µCi/ml). When
indicated, 10 or 20 mM NaCl was replaced with 10 or 20 mM
NH4Cl. Hypertonic media for efflux assays were supplemented
with Na isothionate to a calculated osmolarity of 362 mosM (verified
with a µOsmometer 5004 microosmometer; Precision Systems, Natick,
MA). All media were titrated to a final pH of 7.40 before use.
Mutagenesis, cRNA transcription, and polypeptide expression.
Construction of the mutant AE2 cDNAs was as previously described
(9, 31, 32, 43). The designation AE2
N#
indicates a cDNA encoding an NH2-terminally truncated AE2
in which an introduced Met residue precedes native AE2 residue (#+1).
Thus, in AE2
N248, the introduced initiator Met is
followed by natural AE2 aa residue 249.
Capped cRNA transcripts were synthesized from linearized DNA templates
with the MEGAscript kit (Ambion, Austin, TX), and resuspended in
diethylpyrocarbonate-treated water. RNA integrity was confirmed and
concentration was estimated by formaldehyde-agarose gel
electrophoresis. Handling of Xenopus and preparation of
oocytes were performed as previously described (9, 11,
18-20, 31, 32, 43) as approved by the Institutional Animal
Care and Use Committee of Beth Israel Deaconess Medical Center.
cRNAs or water (50 nl) were injected into defolliculated oocytes and
incubated for 2-5 days at 19°C in ND-96 to allow biosynthesis of
heterologous protein.
36Cl
influx studies.
Groups of 8-12 oocytes previously injected with cRNA or water were
preincubated for 15 min in 150 µl of ND-96 or ND-96 in which 10 mM
NaCl was replaced with 10 mM NH4Cl. Oocytes were then transferred into 150 µl of the same medium containing in addition 10 µM bumetanide and 2.8-3.2 µCi Na36Cl. Two 10-µl
aliquots of influx medium were saved for later determinations of
36Cl
specific activity in each influx group.
36Cl
influx into oocytes was carried out for
15 min and then terminated by three washes in 25 ml of isotonic,
Cl
-free medium. Individual oocytes underwent
scintillation counting of associated 36Cl
.
AE-mediated 36Cl
influx was calculated by
subtracting values for Cl
influx into water-injected
oocytes subjected to identical flux conditions as part of each
experiment. Tests of wild-type (wt) AE2 and/or wt AE1 were included for
comparison in each experiment. cRNA quantities chosen for injection
resulted in rates of AE-mediated 36Cl
influx
that varied across a range of threefold among all tested constructs.
AE-mediated 36Cl
influxes were compared in
the absence and presence of NH4Cl by unpaired two-tailed
t-test (Microsoft Excel).
36Cl
efflux studies.
Efflux studies were carried out as previously described (28,
29). Individual oocytes injected with cRNA or water 2-5
days previously 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 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
timed intervals of 2 or 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 2 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. Stimulation or inhibition of AE2 activity was calculated as percentage of control
[(kpostmodulation/ kpremodulation) × 100, where k is rate constant]. 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 less than threefold from machine background
values. Rates of AE-mediated 36Cl
efflux from
individual oocytes before and after addition of modulator were compared
by paired, two-tailed t-test (Excel or JMP).
Measurements of
[Ca2+]i in Xenopus oocytes.
[Ca2+]i was measured by fluorescence
excitation ratio in fura 2-AM-loaded oocytes or by fluorescence
intensity measurement in dextran-coupled calcium green-injected
oocytes, as previously described (35, 36).
 |
RESULTS |
AE2 stimulation by NH
and by
hypertonicity requires presence of both NH2-terminal
cytoplasmic and COOH-terminal transmembrane domains.
We showed previously that 36Cl
influx
mediated by AE2, but not by AE1, can be stimulated by
NH
and by hypertonicity (19,
20). Figure 1 confirms these
findings and extends them to 36Cl
efflux
measurements. Neither the chimera
AE1cyto/AE2memb nor the chimera
AE2cyto/AE1memb retains sensitivity to either
activator. Thus the presence of both the
NH2-terminal cytoplasmic and the COOH-terminal
transmembrane domains of AE2 is required for NH
or
for hypertonicity to stimulate AE2 anion transport activity.

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Fig. 1.
Stimulation of AE2-mediated Cl transport either by
NH or by hypertonicity requires both cytoplasmic and
transmembrane domains of AE2. A: schematic of wild-type AE2
(gray bars), AE1 (open bars), and chimeric constructs. Residue numbers
are black for AE2 and gray for AE1. wt, Wild type; cyto, cytoplasmic;
memb, transmembrane. B: %stimulation by
NH of Cl influx. Means ± SE for
n* influx experiments, each representing 8-10
oocytes, are shown. Also shown is Cl efflux from
n individual oocytes expressing the same cRNA
constructs. Unstimulated (basal) AE-mediated
Cl influx rates for these oocytes ranged between 6 and 17 nmol/h. For n*/n in parentheses, n*
indicates the no. of influx experiments and n indicates the
no. of efflux experiments. C: %stimulation by hypertonicity
of Cl efflux from oocytes expressing the indicated cRNA
constructs. Means ± SE are shown for n oocytes.
P values compare to the basal condition. Mean rate constants
for basal AE-mediated Cl efflux were 0.014 (AE1), 0.017 (AE2), 0.028 (AE1cyto/AE2memb), and 0.010 (AE2cyto/AE1memb).
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|
AE2 NH2-terminal cytoplasmic domain residues required
for stimulation by NH
and by
hypertonicity reside beyond aa 311.
Experiments were performed to define with gradually increasing
precision the residues within the AE2 NH2-terminal
cytoplasmic domain required for sensitivity to each of the two
activators. The first stage of evaluation assessed AE2 constructs in
which increasing portions of the cytoplasmic NH2-terminal
domain were deleted (Fig. 2A).
As shown in Fig 2, B and C, sensitivity to both
stimuli was retained in the mutant AE2 polypeptides
N99,
N248, and
N311. However, stimulation was
absent in the AE2 mutant polypeptides
N410,
N510, and
N659. Thus the presence of AE2 residues between aa 311 and aa 410 is required for wild-type AE2 stimulation by NH
or hypertonicity. AE2
N659 actually exhibited moderate inhibition by the
normally stimulatory (and acidifying) NH
(Fig. 2B), but not by the alkalinizing stimulator, hypertonicity
(Fig. 2C).

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Fig. 2.
Deletion scan of the AE2 NH2-terminal cytoplasmic
domain region localizes a region critical for stimulation of
Cl transport by NH and by
hypertonicity. A: schematic of AE2 NH2-terminal
cytoplasmic domain deletion constructs. B: %stimulation by
NH of Cl influx into oocytes expressing
the indicated cRNAs. Means ± SE are shown for n
experiments, each representing 8-10 oocytes. Unstimulated (basal)
AE-mediated Cl influx rates for these oocytes ranged
between 6 and 20 nmol/h. [%Stimulation by NH of AE2
N659 portrays Cl efflux and can be
directly compared with NH -stimulated Cl
efflux by wt AE2 of 344 ± 101% (n = 4).]
C: %stimulation by hypertonicity of Cl efflux
from oocytes expressing the indicated cRNAs. Means ± SE are shown
for n oocytes. P values compare to the
unstimulated condition. Mean rate constants for basal AE-mediated
Cl efflux were between 0.011 and 0.020.
|
|
AE2 NH2-terminal cytoplasmic domain residues between aa
311 and 347 are required for stimulation by
NH
and by hypertonicity.
Sequential NH2-terminal deletions between AE2 aa 311 and
410 were generated and studied to define more precisely the residues needed for wild-type patterns of AE2 stimulation (Fig.
3A). However, truncation of as
few as 17 additional residues beyond aa 311 abrogated stimulation by
NH
(Fig. 3B) or by hypertonicity (Fig.
3C). Internal deletions within the region between AE2 aa 311 and 347 were constructed and expressed to test whether deletion of
these residues sufficed to abrogate stimulation. Stimulation by both
NH
(Fig. 3B) and hypertonicity (Fig.
3C) was abolished by internal deletion of aa 312-347 or by deletion of either half of that region in the mutants
AE2
(312-328) and
AE2
(329-347).

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Fig. 3.
Deletion scan of the AE2 NH2-terminal cytoplasmic
domain region between residues 311 and 391 further refines localization
of a region critical for stimulation of Cl transport by
NH and by hypertonicity. A: schematic of
AE2 NH2-terminal cytoplasmic domain deletions and internal
deletions. B: %stimulation by NH of
Cl efflux from oocytes expressing the indicated cRNAs.
Means ± SE for n oocytes are shown; P
values compare to the unstimulated condition. Mean rate constants for
unstimulated Cl efflux ranged between 0.0067 and 0.0441. C: %stimulation by hypertonicity of Cl efflux
from oocytes expressing the indicated cRNAs. Means ± SE for
n oocytes are shown; P values compare to the
unstimulated condition. Mean rate constants for unstimulated
Cl efflux ranged between 0.007 and 0.020.
|
|
Integrity of two noncontiguous regions within AE2 aa 312-347
is required for wild-type stimulation by
NH
and by hypertonicity.
Because NH2-terminal or internal deletions might disrupt
protein structure in unanticipated ways, we carried out a systematic hexa-alanine substitution scan within the region of aa 312-347 (Fig. 4A) implicated as
important by the above deletion analysis, as well as in previous
studies of AE2 regulation by pHi and pHo (31, 32). Figure 4, B and C, reveals
similar patterns of alteration in the acute responses of these
A6-substituted mutant AE2 polypeptides to
NH
and to hypertonicity. Alanine substitution of
residues 312-317, 330-335, and 336-341 retained significant stimulation by NH
, although of magnitude
lower than wtAE2. In contrast, alanine substitution of AE2 residues
318-323 or 342-347 completely abolished stimulation by
NH
. Alanine substitution of AE2 residues 324-329
also led to a minimal stimulation by NH
that failed
to reach statistical significance (Fig. 4B).

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Fig. 4.
Hexa-alanine scan of the AE2 region between residues 312 and 348 refines still further localization of regions critical for
stimulation of Cl transport by NH and
by hypertonicity. A: schematic of the hexa-alanine
substitutions in AE2. B: %stimulation by
NH of Cl efflux from oocytes expressing
the indicated cRNAs. Means ± SE for n oocytes are
shown; P values compare to the unstimulated condition. Mean
rate constants for unstimulated AE2-mediated Cl efflux
ranged from 0.014 to 0.046, except for k = 0.0045 for
AE2 (A)6318-323. C: %stimulation by
hypertonicity of Cl efflux from oocytes expressing the
indicated cRNAs. Means ± SE for n oocytes are shown;
P values compare to the unstimulated condition. Mean rate
constants for unstimulated Cl efflux ranged from 0.008 to
0.044, except k = 0.0044 for AE2
(A)6318-323.
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|
Hypertonic stimulation of AE2 was of wild-type magnitude when residues
330-335 or 336-341 were substituted with alanine and only
partially reduced when residues 312-317 were substituted with
alanine. In contrast, AE2 was insensitive to hypertonic stimulation when alanines were substituted for AE2 residues 318-323,
324-329, or 342-347. Thus the integrity of these three short
regions is of particular import to the ability of AE2 to undergo acute
stimulation on exposure to NH
or to hypertonicity.
Individual mutation of AE2 residues within aa 336-347 can
abrogate wild-type stimulation by
NH
and by hypertonicity.
We chose to study the contributions to AE2 regulation of individual
amino acid residues within the critical region of aa 342-347. We
also examined individually the residues 336-341 in view of their
shared importance in AE2 regulation by changing pHi and pHo (28, 29). Figure
5B shows that the AE2 mutant
W336A retains considerable sensitivity to stimulation by
NH
whereas stimulation by hypertonicity is abolished
(Fig. 5C). AE2 R337A remains sensitive to stimulation by
both NH
and hypertonicity, whereas AE2 E338A is
barely responsive to NH
but fully responsive to
hypertonicity. Mutation of the candidate phosphorylation site T339 to
either valine or to glutamate changed stimulation by
NH
minimally and stimulation by hypertonicity not at
all (Fig. 5, B and C, right).
Hypertonic stimulation of AE2 A340G was intact, but stimulation by
NH
was greatly reduced, although not abolished. All
other single alanine substitution mutations in the region of AE2 aa
336-347 abolished or nearly abolished stimulation by
NH
and by hypertonicity, with the sole exception of
AE2 K344A, which retained minimal but significant stimulation by both
regulators. The AE2 double mutant E346A/E347A was unresponsive to
stimulation either by NH
(102 ± 5%,
n = 8) or by hypertonicity (125 ± 12%,
n = 12; not shown). Thus the single substitution
mutation with the greatest discordance of response to
NH
and hypertonicity was W336. AE2 A340G and E338A
also differed in their responses to the two stimuli, although to a
lesser degree.

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Fig. 5.
Alanine scan of the AE2 regulatory region between residues 336 and
347 assesses roles of individual amino acids in the stimulation of
Cl transport by NH and by
hypertonicity. A: schematic of the AE2 region in which the
indicated residues were individually mutated to alanine. B:
%stimulation by NH of Cl efflux from
oocytes expressing the indicated cRNAs. Left: alanine scan.
Right: threonine mutants. Mean rate constants for basal
AE-mediated Cl efflux for each mutant ranged from 0.008 to 0.032. C: %stimulation by hypertonicity of
Cl efflux from oocytes expressing the indicated cRNAs.
Left: alanine scan. Right: threonine mutants.
Means of n oocytes are shown. Mean rate constants for basal
AE-mediated Cl efflux for each mutant ranged from 0.003 to 0.016. *P < 0.05 compared with initial basal rate
in same oocytes; **P < 0.005.
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Individual mutation of AE2 residues within aa 336-347 has
distinct consequences for regulation by
NH
, hypertonicity, pHi,
and pHo.
The WRETARWIKFEE sequence of AE2 aa 336-347 is among the most
highly conserved among the NH2-terminal cytoplasmic domains of the SLC4 gene family (27, 31). Its importance to
regulation of AE2 by pHi and pHo has been
demonstrated previously (32). Figure
6 summarizes the similarities and
differences among the altered responses of these AE2 missense
mutants to four acute regulatory stimuli of AE2. The W336A mutation
disrupted or modified AE2 responses to pHi,
pHo, and hypertonicity but retained substantial sensitivity
to stimulation by NH
. The AE2 missense mutations
E338A, I343A, and F345A exhibited wild-type responses only to changing
pHo, but responses to changing pHi, NH
, or hypertonicity were altered or abolished. In
contrast, AE2 E346A maintained wild-type response to changing
pHi but exhibited shifted sensitivity to pHo
and loss of stimulation by NH
and by hypertonicity. The AE2 mutants R337A, T339V, and T339E maintained wild-type response patterns to all four acute regulatory stimuli. In contrast, the AE2
mutants R341A, W342A, E347A, and (not shown) the double mutant E346A/E347A exhibited mutant phenotypes in response to all four acute
regulatory stimuli.

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Fig. 6.
Effects of mutation of individual residues within the
conserved regulatory region of AE2 [amino acids (aa) 336-347] on
acute regulation by 4 stimuli. Most residues were mutated to Ala. A340
was mutated to Gly. Thr339 was mutated to Glu and to Val, with
indistinguishable results. +, Wild-type (qualitatively unmodified)
phenotype; , regulatory effect abolished or [in the case of
extracellular pH (pHo)] shifted. pHi,
intracellular pH.
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|
Chelation of Ca
in oocytes
attenuates AE2 stimulation by NH
and by hypertonicity.
The mechanism by which NH
activates AE2 despite
acidic pHi remains obscure. Previous studies suggested several factors that were not sufficient to account for the
stimulation. Thus stimulation by NH
was not mimicked by equally depolarizing concentrations of KCl, by concentrations of
chloroquine sufficient to alkalinize the pH of intracellular organelles, or by incubation with or injection of polyamines
(19). The same degree of stimulation is observed whether
NH
is added as NH4Cl, as the
NH
salt of the permeant weak acid acetate, or as the
NH
salt of the impermeant (31) weak acid
formate (n = 4 for each; not shown).
Our previous finding (18) that chelation of
Ca
inhibits hypertonic activation of the endogenous Cl
-dependent Na+/H+ exchange
activity of the oocyte prompted examination of the role of
[Ca2+]i in the acute stimulation of AE2.
Prior injection into oocytes of BAPTA (5 mM) or EGTA (5 mM) attenuated
subsequent AE2 stimulation by exposure to 20 mM NH
(Fig. 7A). Thirty-minute
exposure to 20 mM NH
modestly elevated
[Ca2+]i by 28 ± 2 nM, as measured in
water-injected oocytes loaded with fura 2-AM (n = 3;
mean ± SE) or by 33 ± 2 nM in oocytes injected with 70-kDa
dextran-coupled calcium green (n = 4), and elevated [Ca2+]i by 32 ± 1 nM in AE2-expressing
oocytes (n = 6; fura 2-AM). Injected EGTA inhibited
NH
-induced elevation of
[Ca2+]i by 85% (P < 0.001, n = 8; fura 2-AM), whereas extracellular EGTA alone
reduced neither NH
-stimulated 36Cl
efflux nor elevation of
[Ca2+]i.

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Fig. 7.
Role of cytoplasmic Ca2+
(Ca ) in AE2 stimulation by NH and
by hypertonicity. A: chelation of Ca
attenuates stimulation of AE2 by NH .
*P < 0.005. Mean rate constants for basal
Cl efflux from individual oocytes ranged from 0.017 to
0.029. B. chelation of Ca abolishes or
attenuates stimulation of AE2 by hypertonicity. *P = 0.01; **P < 0.0001. Mean rate constants for basal
Cl efflux from individual oocytes ranged from 0.007 to
0.017. BAPTA and EGTA solutions titrated to pH 7.40 were injected to
final concentrations of 5 mM. Means ± SE for n oocytes
are shown.
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|
Prior injection into oocytes of BAPTA (5 mM) or EGTA (5 mM) also
attenuated or abrogated subsequent AE2 stimulation by exposure to
hypertonic medium (Fig. 7B). Thirty-minute exposure to
hypertonicity (282 mosM) increased oocyte
[Ca2+]i by 80 ± 18 nM
(n = 6; mean ± SE) in water-injected
oocytes and by 70 ± 5 nM (n = 6) in
AE2-expressing oocytes (fura 2 measurements). However, extracellular
EGTA inhibited this hypertonic elevation of
[Ca2+]i by 79% (n = 6), in
contrast to its lack of effect on NH
-induced elevation of [Ca2+]i.
Calmidazolium inhibits both basal and stimulated AE2 activity by
acting on COOH-terminal transmembrane domain of AE2.
The ability of injected Ca2+ chelators to inhibit
NH
stimulation of AE2-mediated Cl
transport led us to test the calmodulin dependence of this stimulation. The calmodulin inhibitor calmidazolium also inhibited AE2-mediated 36Cl
uptake (ID50 ~0.5 µM;
not shown). Interestingly, calmidazolium at slightly higher
concentrations also inhibited basal AE2 function in the absence of
NH
. As shown in Fig. 8,
10 µM calmidazolium inhibited basal activity of wtAE2 to 30% of the
uninhibited value. Higher concentrations of calmidazolium did not
further inhibit AE2. In contrast, wtAE1-mediated
36Cl
efflux in the presence of 10 µM
calmidazolium retained 79 ± 1% of its uninhibited value
(n = 7; not shown). Although 5 mM EGTA added to a
nominally Ca2+-free bath only minimally reduced AE2
inhibition by 10 µM calmidazolium (39 ± 11% of control;
n = 3), that inhibition was greatly attenuated (79 ± 19% of control; n = 3) in oocytes in the same
Ca2+-free bath also injected with EGTA (5 mM final).

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Fig. 8.
Analysis of chimeras and of NH2-terminal cytoplasmic
domain sequential deletion constructs defines the AE2 transmembrane
domain as critical for inhibition by calmidazolium. Means ± SE
for n oocytes are shown; P values compare each
group with the uninhibited condition. Mean rate constants for
uninhibited Cl efflux ranged from 0.064 to 0.009.
|
|
The AE2 structural requirements for inhibition by calmidazolium were
compared with those of stimulation by NH
and by
hypertonicity. Figure 8 shows that although the
AE1cyto/AE2memb chimera was inhibited by
calmidazolium, the converse chimera
AE2cyto/AE1memb was insensitive to inhibition
by the drug. These data suggested both the importance of the AE2
membrane domain for calmidazolium action and the lack of importance of
the NH2-terminal cytoplasmic domain of AE2. Analysis of
mutants progressively truncated across the NH2-terminal
cytoplasmic domain supported this hypothesis. Removal of the
NH2-terminal 99 aa was without any effect on calmidazolium inhibition. Even with removal of the NH2-terminal 311, 410, 510, or 659 aa (nearly all of the NH2-terminal cytoplasmic
domain), most of the inhibitory effect of calmidazolium was maintained. Thus the AE2 transmembrane domain was both necessary and sufficient to
exhibit sensitivity to inhibition by calmidazolium. This differed from
the requirement of both cytoplasmic and membrane domains of AE2 for
stimulation by NH
or hypertonicity.
Because calmodulin sometimes acts via CaM-kinases or calcineurin
phosphatases, the AE2 mutants T339V and T339E were also tested for
calmidazolium responsiveness. As shown in Fig. 8, AE2 T339V exhibited
wild-type sensitivity to calmidazolium. AE2 T339E showed calmidazolium
sensitivity equivalent to that shown by the NH2-terminal truncation mutants
N311, -410, -510, and -659.
Effect of agents that modulate
[Ca
]i
signaling on acute stimulation of AE2.
Although calmidazolium potently inhibited both basal and
NH
-stimulated AE2-mediated Cl
transport, the calmodulin inhibitor W13 was inactive as an inhibitor of
basal or NH
-stimulated AE2 activity when exposed in
the bath solution (92 µM; n = 3) or injected to an
estimated final intracellular concentration of 200 µM
(n = 9). However, injection of CaMKII inhibitor peptide
(40) to final intracellular concentrations of 1 (n = 5) or 50 (n = 5) µM in each case
modestly inhibited AE2 stimulation by NH
by 30%
(P < 0.05 for the pooled data; n = 10). Nonetheless, stable interaction of calmodulin with AE2
was not detected by several approaches (not shown). Calmodulin-agarose
did not bind AE2 in 1% Triton X-100 lysates of transiently transfected
and muristerone-induced 293-EcR cells or in lysates of light membranes
from pig gastric mucosa (45), under conditions in which
the matrix binds KCNN1 (SK1) K+ channel polypeptide
(30). Moreover, anti-calmodulin antibody (Sigma) failed to
coimmunoprecipitate AE2 from these same lysates.
Several modifiers of organellar Ca2+ stores were without
significant effect on basal or NH
-stimulated AE2-mediated 36Cl
efflux (not shown). Among
antagonists of inositol 1,3,4-trisphosphate receptor-mediated
Ca2+ release tested were heparin sulfate [injected to
final 5 mg/ml, indistinguishable in the presence (n = 22) or absence (n = 21) of bath Ca2+ from
the inactive analog heparan sulfate]; pyridoxal
phosphate-6-azo(benzene-2,4-disulfonic acid) (injected to final 50 µM, n = 5); 2 aminophenylborate [bath 250 µM
(n = 5) or injected to final 100 µM
(n = 4)]; xestospongin C (injected to final 10 µM;
n = 5); and MDL-12,330A (injected to 100 µM;
n = 5). Agents that elevate oocyte
[Ca2+]i without altering basal or
NH
-stimulated AE2-mediated
36Cl
efflux included thapsigargin (bath 10 µM; n = 5) and A-23187 (bath 30 µM;
n = 5). These data suggest that, although elevation of
[Ca2+]i is required for maximal
NH
stimulation of AE2, it is not sufficient itself to
mediate AE2 activation.
The c-JNK inhibitor SP-600125 blocks hypertonic activation of the
endogenous oocyte Cl
-dependent
Na+/H+ exchanger (18). In
contrast, SP-600125 (final injected concentration 25 µM) did not
inhibit basal function (n = 13) or hypertonic
stimulation (n = 14) of AE2 whether injected acutely or
2 h before the efflux assay. At the nonspecific (final injected)
concentration of 50 µM SP-600125, hypertonic stimulation was reduced
by 50% (n = 3), whereas NH
stimulation of AE2 was unaffected (n = 11). Neither the
p42/44 MAPK inhibitor PD-98059 (10 µM injected; n = 5) nor the p38 MAPK inhibitor SB-220025 (2 µM injected;
n = 4) reduced hypertonic stimulation of AE2. The
general protein kinase inhibitor staurosporine (1 µM in bath;
n = 6) modestly inhibited
NH
-stimulated AE2-mediated 36Cl
efflux by 24 ± 6%
(P < 0.01).
Additional modifiers of intracellular signaling that were without
effect on basal or NH4+-stimulated AE2-mediated
36Cl
efflux (data not shown) included the
phospholipase C inhibitor U-73122 (injected to final 12 µM;
n = 5) and calcineurin (protein phosphatase 2B,
injected 0.025 U, nominal 7.5 µM; n = 5). Among additional bath-applied drugs without effect on basal AE2-mediated 36Cl
influx were the
phosphatidylinositol-3-kinase inhibitor wortmannin (10 µM;
n = 6), the calcineurin inhibitor cyclosporin A (1 µM; n = 6), the protein serine phosphatase inhibitor
okadaic acid (100 nM; n = 7), the protein kinase C
inhibitor chelerythrine (10 µM; n = 8), the tyrosine
kinase inhibitors genistein (10 µM; n = 8),
calphostin (1 µM; n = 8), tyrphostin AG126
(n = 8), and tyrphostin S1 (n = 11),
and the lipid mediators sphingosine (100 µM, n = 4 in
bath, n = 4 injected) and its antagonist,
dihydrosphingosine (100 µM; n = 4 in bath,
n = 4 injected).
 |
DISCUSSION |
We have defined structural elements of AE2 required for wild-type
stimulation by NH
and by hypertonicity. Each
stimulation requires both NH2-terminal cytoplasmic and
transmembrane domains of AE2. The regions of the
NH2-terminal cytoplasmic domain required for each
stimulation are the same. However, within the highly conserved AE2 aa
336-347, stimulation by NH
and by hypertonicity
each exhibit slightly different structural requirements. Chelation of
intracellular Ca
inhibits both stimuli.
Calmidazolium inhibits AE2 profoundly but inhibits AE1 only minimally.
AE2 inhibition by calmidazolium does not require the presence of the
AE2 NH2-terminal cytoplasmic domain but does require the
AE2 transmembrane domain.
Role of NH
in physiological
function of AE2.
NH
is present in several tissues that express AE2,
although often absent from in vitro studies of cellular ion transport.
AE2 in the basolateral membranes of surface enterocytes
(3) is exposed in the interstitium of the portal
circulation to 1-3 mM (total [NH
]), reflecting
much higher concentrations in the gut lumen. These concentrations
approximate the ED50 for stimulation of AE2 by NH
(19). AE2 in the basolateral
membranes of parietal cells is likely exposed to similar
[NH
], because it resides on the other side of the
tight junction from gastric luminal [NH
] of up to
20 mM when the lumen is colonized with urease-positive
Helicobacter pylori (19).
NH
can reach high concentrations in the renal
medullary interstitium, which provides
NH3/NH
for secretion and "trapping"
in the acidic collecting duct lumen. NH
stimulates
HCO
absorption by isolated perfused rabbit cortical
collecting duct, due in part to activation of basolateral
Cl
/HCO
exchange
(41). Luminal NH
in control or
acidotic rat cortical tubule segments sampled by micropuncture lies
near or below the ED50 for AE2 stimulation by
NH
(23a). Basolateral NH
stimulates
Cl
secretion in isolated, perfused rat inner medullary
collecting duct (39), consistent with stimulation of
basolateral AE2 (4). AE2 is also abundant in the
medullary thick ascending limb of rat (4, 16), where it
can impact on uptake of residual tubular HCO
. Renal
medullary [NH
] likely remains at levels that are
maximal with respect to AE2 stimulation, rendering AE2 insensitive to
inhibition by acidic pHi (23a). Both in gut and in kidney,
AE2 may rely on NH
to maintain function during
fluctuations in pHo and/or pHi. The apical
membranes of these gastrointestinal and renal epithelia potentially
exposed to very high luminal [NH
] tend to share
with the Xenopus oocyte plasma membrane its high ratio of
NH
to NH3 permeabilities (7, 8, 19,
25, 29). This similarity supports the relevance to mammalian
epithelial physiology of AE2 stimulation by NH
in
Xenopus oocytes. NH
modulation of AE2
function may be relevant not only at the plasma membrane but also in
the Golgi apparatus (4, 18a), where AE2 may play a role in the
regulation of organellar pH or [Cl
].
Regulation of anion exchange by NH
is not restricted
to SLC4 anion exchangers. The SLC26A3 (DRA) anion exchanger of the
ileocolonic apical membrane is also stimulated acutely by
NH
and inhibited by acidic pHi (Chernova
et al., unpublished observations), whereas the SLC26A4 (pendrin) anion
exchanger of the type B intercalated cell apical membrane appears
insensitive both to NH
and acidic pHi
(A. K. Stewart and S. L. Alper, unpublished observations). NH
regulates anion exchange chronically as well as
acutely. Thus prolonged hyperammonemia increased intracellular [Cl
] in hippocampus by mechanisms sensitive to
inhibition by stilbene disulfonates and by acetazolamide. This change
coincided with upregulation of AE3 mRNA and protein. Both the changes
in intracellular [Cl
] and in AE3 abundance were blocked
by inhibition of protein kinase C and stimulated by phorbol ester
(21). Interestingly, dietary NH
loading
exacerbates polycystic kidney disease in the Han:SPRD rat and in the
CD1:pcy/pcy mouse (34), although the mechanisms remain unknown.
Structural regions of AE2 required for responsiveness to
NH
and hypertonicity.
Although NH
and hypertonicity change oocyte
pHi in opposing directions, the same regions of AE2 are
required for AE2 to exhibit sensitivity to activation in their presence. Effectiveness of both stimuli requires the presence of the
AE2 NH2-terminal cytoplasmic and COOH-terminal
transmembrane domains. This fact is emphasized by the unequivocal
inhibition of AE2
N659 by NH
and by a
trend toward inhibition of the chimera
AE1cyto/AE2memb. This inhibition may represent
unmasking of AE2 inhibition by acidic pHi in the absence of
residues crucial for AE2 activation by NH
. Moreover,
a wide series of truncation and alanine substitution mutations within
the NH2-terminal cytoplasmic domain preserve, reduce, or
abolish AE2 responses to NH
and to hypertonicity in
strict parallel.
Only AE2 aa 336-347 (32) reveals a single difference
in structural dependence of these two stimuli: the AE2 mutant W336A lacks responsiveness to hypertonicity while maintaining sensitivity to
activation by NH
(Figs. 5 and 6). In addition, the
alanine scan of this region uncovers one difference in the structural
dependence of sensitivity to hypertonicity and to pHi,
evident in the AE2 mutant E346A, which retains sensitivity to
inhibition by acidic pHi but loses responsiveness to
hypertonicity. This difference suggests that intracellular
alkalinization may not suffice to explain activation of AE2 by
hypertonicity, despite its requirement for oocyte
Na+/H+ exchange activity (18).
The X-ray structure of the AE1 region homologous to AE2 aa 336-347
(
2-loop-
3) predicts a similar
-sheet-loop-
-sheet structure in AE2 (32, 42). We postulate that the various acute
regulatory stimuli of NH
, hypertonicity,
pHi, and pHo alter the structure of this region
in AE2 and/or the structure of one or more complementary interaction
surfaces. These interaction surfaces may reside within AE2 or in one or
more distinct polypeptides.
Mechanism of NH
stimulation of
AE2.
NH
is a first messenger for the slime mold
Dictyostelium. In yeast, extracellular NH
currently is thought to signal nitrogen balance only via transmembrane transport and metabolism (17). Although similar
NH
signaling in mammalian cells has not yet been
described, the Mep/Amt gene family of
alkylammonium/NH
transport proteins may provide new
approaches to investigation of this possibility. Extracellular
NH
enters the oocyte via nonspecific cation
channel(s) (7, 8, 25) that can be activated by reactive
oxygen species (13). A possible link between
NH
entry into the cell and NH
stimulation of AE2 is suggested by the interaction (direct or indirect)
between AE1 and the RhAg polypeptide in the erythrocyte
(6), likely mediated in part by protein 4.2 (24). Analogous interaction might exist in the type A
intercalated cell basolateral membrane between AE1 and the putative
NH
transporter RhBG (37) and between AE2
and a related transporter.
Although chelation of Ca
inhibits AE2 stimulation by
NH
and by hypertonicity, additional data show that
elevation of [Ca2+]i does not suffice to
explain stimulation of AE2. These include the lack of AE2 stimulation
by thapsigargin or by A-23187, the slow, modest increase in
[Ca2+]i observed during exposure to
NH
, the modest (although significant) inhibitory
effects of CaMKII inhibitory peptide on NH
stimulation of AE2, and the lack of effect of many pharmacological
modifiers of Ca2+ signaling. Although pharmacological
inhibition of cJNK, p38, and p42/44 did not support a role for MAPKs in
the action of NH
or hypertonicity on AE2, a modest
inhibitory effect of staurosporine suggested possible additional
involvement of a kinase. Direct evidence suggesting regulatory
phosphorylation of recombinant bAE3 has been reported (5),
and suggestive evidence favors phosphoregulation of native AE2
(28).
Mechanism of AE2 inhibition by calmidazolium.
The requirement of [Ca2+]i elevation for
stimulation of AE2 by NH
and by hypertonicity led to
the test of calmidzolium as an AE2 modulator. Although at lower
concentrations calmidazolium indeed inhibited stimulation by
NH
, at modestly higher concentrations inhibition of
basal activity was also evident. The AE2 structure-function
relationship of the inhibition of basal activity suggested a mechanism
of action distinct from that of the stimulators. Although both
cytoplasmic and transmembrane domains of AE2 are required for wild-type
patterns of stimulation, only the transmembrane domain appears to be
required for inhibition by calmidazolium.
Despite the ability of Ca
chelation to attenuate
inhibition of basal AE2 function by calmidazolium, several observations
raise the question of an inhibitory mechanism unrelated to calmodulin.
These include calmidazolium's moderate potency and lack of inhibition
by other calmodulin inhibitors. Calmidazolium is an imidazole, and it
has a host of pharmacological activities in addition to inhibition of
calmodulin. These include inhibition of P-450 cytochromes
(44), the P2X7 receptor (12), the maitotoxin
receptor (15), and constitutive nitric oxide synthase
(33). Although Ca
was elevated in MDCK
cells by 2-5 µM (22), 25 µM calmidazolium did not
suffice to activate Ca
-dependent Cl
current in oocytes (26). It also remains possible that
calmidazolium directly inhibits AE2-mediated Cl
transport, with either an internal or external site of action.
In conclusion, at least three conditions differentially regulate
AE2-mediated anion exchange via mechanisms influenced by [Ca2+]i. NH
and
hypertonicity both stimulate AE2 despite their opposing effects on
oocyte pHi. Each stimulus requires the presence of both the
AE2 NH2-terminal cytoplasmic domain and the transmembrane
domain. Within the AE2 NH2-terminal cytoplasmic domain,
each stimulus requires the integrity of select (but not identical)
amino acid residues in the critical region of aa 336-347. Each
stimulus is inhibited by chelation of Ca
. Investigation of the role played by Ca
in AE2
regulation led to the discovery that calmidazolium inhibits AE2
function through a process requiring only the AE2 transmembrane domain.
This inhibition is itself attenuated by chelation of
Ca
. Additional experiments will be required to
elucidate the mechanisms of these AE2 regulators and to define their
relationship to the physiological functions of AE2-mediated anion exchange.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Institutes of Health (NIH)
Grants DK-43495 and DK-34854 (the Harvard Digestive Diseases Center) to
S. L. Alper and NIH fellowships T32-HL-07516 and F32-DK-09136 to
Y. Z. Kunes. A. K. Stewart was supported by an International Prize Traveling Fellowship of the Wellcome Trust.
 |
FOOTNOTES |
Present addresses: A. K. Stewart, University Laboratory of
Physiology, Parks Rd., Oxford OX1 3PT, UK; Y. Z. Kunes, Millenium Pharmaceuticals, 640 Memorial Dr., Cambridge, MA 02139
Address for reprint requests and other correspondence:
S. L. Alper, Molecular Medicine and Renal Units, RW763 East
Campus, Beth Israel Deaconess Medical Center, 330 Brookline
Ave., Boston, MA 02215 (E-mail:
salper{at}bidmc.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.
First published January 15, 2003;10.1152/ajpcell.00522.2002
Received 11 November 2002; accepted in final form 9 January 2003.
 |
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