(Received for publication, August 4, 1995; and in revised form, November 14, 1995)
From the
We have compared regulation by pH of AE1 (band 3)- and
AE2-mediated Cl
uptake into Xenopus oocytes.
Cl
influx was assayed at
varying extracellular pH (pH
) values between 9.0
and 5.0 under conditions in which corresponding intracellular pH
(pH
) values were at or near steady-state. Wild
type (WT) AE1 displayed a broad convex pH versus activity
curve, with peak activity at pH
7.0 and 63% of
maximal activity at pH
5.0. In contrast, WT AE2
displayed a steep pH versus activity curve, with peak activity
at pH
9.0 and full suppression at pH
5.0. The structural basis of these differing pH
sensitivities was examined by expression of cRNAs encoding chimeric and
truncated proteins. Mutant polypeptides were expressed in oocytes and
detected at the cell surface. The AE2
/AE1
polypeptide displayed a broad pH versus activity curve
similar to that of WT AE1. In contrast, the
AE1
/AE2
polypeptide displayed a steep pH versus activity curve, which was shifted toward acid pH values
from that of WT AE2 by 0.69 ± 0.04 pH
units. Moreover, whereas the pH versus activity
curves of AE2
99 and WT AE2 were indistinguishable, AE2
510
exhibited a pH versus activity curve acid-shifted from that of
WT AE2 by 0.66 ± 0.13 pH
units
(indistinguishable from that of AE1
/AE2
).
The data suggest that a pH sensor resides within the transmembrane
region of AE2. The affinity for protons of this pH sensor is influenced
by a modifier site located between residues 99 and 510 of the
N-terminal cytoplasmic domain of AE2. Acidification of oocytes with
acetate suggested that pH
accounted for some but
not all of the measured pH dependence of AE2.
The band 3-related AE anion exchanger gene family comprises at
least three genes, each of which encodes polypeptides that mediate
anion exchange when expressed in heterologous expression systems
(reviewed in (1) ). The prototype red cell band 3 (AE1) serves
both as a chloride/HCO exchange mechanism and as a major
membrane anchor for the spectrin/ankyrin/actin
cytoskeleton(2) . The polypeptide products of the AE2 and AE3
genes are postulated to serve similar roles(1) . Their
polytopic transmembrane domains of
530 C-terminal amino acids
suffice to mediate anion exchange(3, 4, 5) .
Their N-terminal cytoplasmic domains of
700 amino acids in length
may, by analogy with red cell AE1, bind to cytoskeleton proteins of
nonerythroid cells(6) . Chloride/HCO
exchange
contributes widely to maintenance of cellular pH, to secondary active
chloride loading, and to transepithelial movement of chloride and
bicarbonate (1) .
Whereas chloride/monovalent anion exchange
by red cell AE1 displays a broad pH versus activity
profile(7) , which serves to maintain relatively constant
activity across the pH range of the capillary from its beginning to the
end of its course through CO- and acid-generating tissues,
chloride/HCO
exchange and
Cl
influxes measured in tissue culture cells display a steep pH
dependence(8, 9, 10) . Tissue culture cells,
as well as most nonerythroid cell types, generally express AE2 and/or
AE3 ion exchangers rather than
AE1(1, 11, 12, 13, 14, 15, 16, 17, 18) .
The steep pH dependence of the nonerythroid anion exchangers has led to
the proposal that they contribute not only to cellular defense against
alkaline loads, but also in some tissues to the maintenance of resting
pH
(1, 19) as well as to
maintenance of resting
[Cl]
(20) . However, when the pH
dependence of AE2-mediated chloride/HCO
exchange was tested
in transiently transfected mammalian cells(21, 22) ,
in infected insect cells(23) , or in intact gastric
glands(24) , activation by alkaline pH
(when noted) required alkalinization beyond 0.25 pH units.
These findings suggested that AE2 might be inadequately sensitive to
intracellular alkalinization to play a physiological role in the
regulation of resting pH
. In contrast, AE2
expressed in Xenopus oocytes was activated by exposure to
alkaline media and inhibited by exposure to acidic
media(25, 26) . This pH dependence of AE2-mediated
chloride/base exchange contrasted with AE1-mediated chloride transport
which, in preliminary experiments, showed considerably less sensitivity
to pH(46) .
Heterologous expression of AE1 in mammalian cells has to date not allowed study of AE1 transport function at the cell surface(27) . As with AE1, AE1/AE2 chimeric polypeptides also did not traffic to the cell surface. Study of microsomes from transfected cells (27) and of proteoliposomes prepared by reconstitution of these microsomes in the presence of exogenous phospholipid (28) has allowed comparison of sulfate fluxes mediated by heterologous AE1 and AE2. However, the sulfate/anion exchange measured in these studies is activated by acid pH, consistent with the proton/sulfate cotransport earlier described in red cells(29) . The alkaline activation (or acid suppression) of AE-mediated chloride/base exchange in these vesicle systems has not been studied with the requisite time resolution. In contrast, AE1 is expressed at the surface of the Xenopus oocyte (30, 31) at least as efficiently as AE2(25) . Thus, the oocyte lends itself to the comparative study of the regulation of heterologous AE gene products in an intact cell system.
We have compared the differences in pH dependence of chloride/base
exchange mediated by AE1 and by AE2 in Xenopus oocytes. The
transmembrane domains of AE1 and AE2 are 65% identical in amino acid
sequence. In contrast, the overlapping regions of N-terminal
cytoplasmic domain are only 33% identical, and the 250 N-terminal amino
acids of AE2 correspond to none in AE1. Inspection of the sequences
suggests the possibility that the greater differences in the N-terminal
cytoplasmic domains of AE1 and AE2 might explain the associated
differences in pH sensitivity of chloride transport. However, analysis
of the Na/H
exchanger, NHE1, provided
evidence for a regulatory pH sensor in the transmembrane domain of the
polypeptide(32) . In addition, these authors localized a
modulatory function to the cytoplasmic domain of NHE1. In order to
define and localize structures in the AE2 polypeptide that mediate
regulation of transport activity by pH, we expressed chimeric and
truncated AE anion exchangers in Xenopus oocytes and assayed
Cl
transport across an extracellular pH range from
5.0 to 9.0. Our data suggest a model in which the transmembrane domain
of AE2 contains a pH sensor whose affinity for protons is modulated by
a structure residing between amino acids 99 and 510 of the AE2
N-terminal cytoplasmic domain.
The
AE2/AE1
chimera was constructed as
follows (Table 1). The AE2 plasmid p
X served as first-round
PCR template for primers yz7 and yz4, and the AE1 plasmid pBL served as
first-round template for primers yz3 and yz9. The two first-round
amplification products were coannealed to serve as second-round
template for the bracketing primers yz7 and yz9. The second-round
chimeric amplification product joined AE2 nucleotide 2291 to AE1
nucleotide 1396 in a fragment of 1.7 kb in length. This PCR product was
digested by NaeI and SphI, and the central fragment
of 1.4 kb was gel-purified. To reconstruct the full-length chimera, a
plasmid containing a tandem head-to-tail insertion of AE2 and AE1
underwent partial digestion with NaeI and complete digestion
with SphI to yield a 6.3-kb NaeI/SphI
fragment, which was also gel-purified. The 1.4-kb PCR fragment and the
6.3-kb cDNA fragment were ligated to construct
AE2
/AE1
. Sequence analysis of the
amplified plasmid demonstrated a missense mutation in the AE1 portion
of the PCR product. This mutant cDNA was partially digested with BsmI and completely digested with HindIII. The
resultant 5-kb fragment was ligated with the 2.5-kb BsmI/HindIII fragment from the AE1 plasmid, pBL. The
final AE2
/AE1
cDNA encoded AE2
nucleotides 105-2291 (corresponding to amino acids 1-703) fused
to the AE1 sequence from nucleotide 1396 to its 3` end (corresponding
to amino acids 423-929, the entire transmembrane-spanning region
beginning at TM1).
The AE2 truncation mutants 99 and
510
were constructed as follows (Table 1). The AE2 PCR product
generated from primers yz13 and sz8 was digested with SacII
and AvrII and ligated to the large SacII/AvrII fragment of the AE2 plasmid p
X. The
reconstructed AE2
99 cDNA encoded an engineered ATG followed by
amino acids 100-1237. The AE2 PCR product generated from primers yz11
and yz12 was digested with SacII and SmaI and ligated
to the large SacII/SmaI digestion product of p
X.
The reconstructed AE2
510 encoded an engineered ATG followed by
amino acids 511-1237.
Some oocytes
were acidified by acetate exposure. The incubation medium for these
experiments was a modified ND-96 in which 38 mM NaCl was
substituted with 38 mM sodium isethionate. Oocytes were
preincubated for 30 min in this modified medium or in solutions in
which sodium acetate was substituted for equimolar sodium isethionate. Cl
influx was then performed as above.
Individual oocytes underwent scintillation counting of associated Cl
. AE-mediated
Cl
influx was calculated by subtracting
values for Cl
influx into water-injected oocytes
subjected to identical flux conditions as part of each experiment.
Every experiment that tested chimeric constructs compared the chimeras
with both WT AE1 and WT AE2 in the same experiment. Every experiment
that tested AE2 truncation constructs compared the mutants with WT AE2
in the same experiment. Each construct was tested in oocytes from at
least four frogs, using multiple RNA preparations.
AE-mediated
Cl influx values determined in flux media of pH
5.0-9.0 were plotted as a function of pH
. Mean
values measured for WT AE2, AE1
/AE2
,
AE2
99, and AE2
510 in each individual experiment were fit to
the following first-order logistic sigmoid equation using Ultrafit 2.1
(Elsevier): v = (V
10
)/(10
+
10
), where v = measured AE-mediated
Cl
influx, V
= the
maximum value for AE-mediated Cl
influx, x = pH
of the experiment, and K =
pH
, the pH
at which v is
half-maximal. Results of individual experiments were pooled (see Fig. 5and Fig. 6) by plotting data normalized to the fit
parameter V
calculated for each individual
experiment. Differences in mean pH
values of individual
constructs (derived from curves of activity versus pH
) were subjected to statistical analysis by analysis
of variance (34) and by two-tailed t tests (Microsoft
Excel, version 5.0 for Macintosh). Results did not differ when influx
assays were stratified according to performance of 15- or 60-min
uptakes (n = 7 and n = 6, respectively,
for WT AE2). Results also did not differ when influx assays were
performed at 4 or 5 pH
values per experiment (n = 4 and n = 9, respectively for WT AE2).
Therefore, pooled results were analyzed.
Figure 5:
Chloride influx versus pH curves
for WT and chimeric AE polypeptides. Oocytes expressing the indicated
polypeptides were subjected to Cl influx assays at
varying pH
values between 5.0 and 9.0. The
corresponding steady-state pH
values on the lower x axis were measured in four experiments such as that of Fig. 4. For WT AE2 (open triangles, n =
13 independent experiments) and for AE1
/AE2
(open circles, n = 13), data from each
independent experiment were fitted to a logistic sigmoid equation,
normalized to fitted saturation activities, and then averaged. For WT
AE1 (filled triangles, n = 6) and
AE2
/AE1
(filled circles, n = 4), data from each independent experiment were normalized
to the highest Cl
influx activities and then
averaged.
Figure 6:
Cl
influx versus pH curves for WT and truncated AE2 polypeptides.
Oocytes expressing the indicated polypeptides were subjected to
Cl influx assays at varying pH
values between 5.0 and 9.0. The corresponding steady-state
pH
values on the lower x axis were
measured in identically treated oocytes. For WT AE2 (triangles, n = 6 independent experiments),
AE2
99 (circles, n = 5), and
AE2
510 (squares, n = 5), data
from each independent experiment were fitted to a logistic sigmoid
equation, normalized to fitted saturation activities, and then
averaged.
Figure 4:
Time course of pH change in WT AE2-expressing oocytes exposed to changes in
pH
. Oocytes were loaded with BCECF-AM, mounted on
the stage of an inverted microscope, and video-imaged by ratio
fluorometry. Superfusate was changed from pH 7.4 to the indicated
pH
at the time noted in the lower bars.
The upper bars mark the addition of bumetanide and amiloride
to the superfusate to replicate isotopic influx assay
conditions.
Mean values measured for WT
AE1 and for AE2/AE1
were also pooled by
normalization to V
for each individual
experiment. The transport values for these two polypeptides were not
well fit by linear or logistic sigmoid functions and were plotted by
hand.
Figure 1: Schematic diagram of the WT and mutant AE polypeptides studied.
Polypeptide
biosynthesis was next analyzed in Xenopus oocytes (Fig. 2). Oocytes injected with WT AE2 cRNA produced
polypeptides of 135 (core-glycosylated) and 160 kDa
(complex-glycosylated) as shown previously(25) , while WT AE1
cRNA produced the typical broad band at 100 kDa, which contains
within it both core- and complex-glycosylated
species(30, 31) . All constructs that contained the
AE2 TM domain exhibited the expected two polypeptide bands, whereas all
those containing the AE1 TM domain displayed the expected single band.
The truncated AE2
99 cRNA produced polypeptides of 125 and 150
kDa, and
510 cRNA produced polypeptides of
80 and 115 kDa.
Chimeric AE1
/AE2
cRNA produced
polypeptides of 110 and
135 kDa. Chimeric
AE2
/AE1
cRNA produced a single
polypeptide of
130 kDa, which accumulated to the lowest level
among the AE mutants tested.
Figure 2:
Immunoprecipitation of full-length and
surface-proteolized AE polypeptides. Oocytes previously injected with
the indicated cRNA constructs or with water (top) were
metabolically labeled with [S]methionine and
then incubated with 5 mg/ml papain for 3 h at room temperature, washed
free of the protease, and lysed in Triton X-100 with protease
inhibitors. Clarified lysate was subjected to immunoprecipitation with
anti-AE2 C-terminal amino acids 1224-1237 (left panel)
or with anti-AE1 C-terminal amino acids 918-929 (right
panel). The C-terminal papain fragment of the AE2 transmembrane
domain is labeled P35. Size standards are marked between
panels.
The presence of these heterologous AE
polypeptides at the oocyte surface was assessed by incubation of
oocytes in the absence or presence of 5 mg/ml papain for 3 h at room
temperature. Papain digestion of surface-exposed WT AE2 in Xenopus oocytes produced a carboxyl-terminal AE2 fragment of 32 kDa (Fig. 2). Accumulation of the 32-kDa fragment was maximal at 3
h. The same fragment was produced by papain digestion of porcine
basolateral gastric microsomes, in which the amino acid
sequence of the C-terminal fragment defined a 35-kDa fragment cleaved
within the nonglycosylated ectoplasmic loop linking transmembrane spans
7 and 8. This papain treatment protocol did not increase
Cl
influx in water-injected oocytes (not
shown). Thus, papain digestion provided biochemical evidence for the
presence at the oocyte surface of polypeptides encoding WT AE2,
AE2
99, AE2
510, and AE1
/AE2
.
The amount of AE2/AE1
at the oocyte
surface was below the threshold of detection by polyclonal antibody to
the carboxyl-terminal 12 amino acids of murine AE1. The corresponding
carboxyl-terminal
35-kDa fragment of human AE1 produced by papain
digestion of intact human red cell ghosts is barely detectable as a
broad, very faint smear by Coomassie Blue stain(37) . Although
this fragment of WT human AE1 has been immunoprecipitated from
metabolically labeled Xenopus oocytes with a particular
monoclonal antibody(30) , the comparable fragment from mouse
AE1 has not yet been detected in Xenopus oocyte lysates.
Nonetheless, expression of both WT mouse AE1 and
AE2
/AE1
lead to increased Cl
uptake, as shown below.
Figure 3:
Specific transport activity of WT and
mutant AE transporter cRNAs expressed in Xenopus oocytes. 72 h
after injection with the indicated amounts of cRNA, chloride influx was
measured over 60 min in ND-96, pH 7.4, as described under
``Experimental Procedures.'' The data shown reflect
subtraction of Cl influx into H
O-injected
oocytes, 0.6 ± 0.1
nmol/oocyte
h.
The
considerable range of AE-mediated chloride uptake/ng of injected cRNA
among WT and mutant AE polypeptides suggested that prior titration of
functional activity would be important for functional comparison of
multiple cRNAs in different lots of oocytes. Dose-response curves such
as those of Fig. 3allowed selection of quantities of each cRNA
for injection, which reliably yielded absolute transport rates
differing by no more than 3-fold among the various cRNAs in individual
experiments. In the experiments summarized in Fig. 5and Fig. 6, maximal AE-mediated Cl uptakes at
alkaline pH
were routinely 5-7 nmol/oocyte and never
below 2 nmol/oocyte. The Cl
uptakes measured for all
AE polypeptide constructs in pH sensitivity experiments represented
initial rates.
Table 2summarizes the
near-steady-state pH values attained in these conditions by
oocytes exposed to extracellular media of the indicated pH
values. Steady-state pH
in the presence of amiloride
changed 0.12 units/unit pH
, similar to the value of 0.13
for maximal
pH
previously determined in the absence of
amiloride(25) . This buffering of oocyte pH
contrasts with the minimal buffering of human erythrocyte
pH
( (39) and (40) ; Table 2), where
the pH-dependence of AE1 measured previously reflected simultaneous and
nearly equivalent variation of pH
and pH
(summarized in (7) ).
WT AE1 and WT AE2 differed significantly in their regulation by pH (Fig. 5). WT AE1 (closed triangles) displayed a broad
pHversus transport activity curve (n = 6). Activity was maximal at pH
7.0, decreased
to 62.7 ± 6.9% of maximal value at pH
5.0 (p < 0.005), and decreased to 82.7 ± 5.3% of maximal value
at pH
9.0 (p = 0.16). In contrast, WT AE2
activity (open triangles) displayed a steep dependence on
pH
. Activity was maximal at pH
9.0 and
decreased as pH
decreased, with a pH
at which
activity was half-maximal (pH
) of 7.03 ± 0.08 (n = 13). AE2 activity was completely suppressed at
pH
5.0, approximately corresponding to pH
7.13
(see Table 2). The data relating pH
and AE2-mediated
Cl
influx were well fit by a first order
logistic sigmoid equation (Ultrafit goodness of fit index = 0.93
± 0.03, n = 13).
As shown in Fig. 5,
AE2/AE1
(closed circles)
displayed peak activity at pH
8.0 and 9.0 and retained 61.9
± 4.7% maximal activity at pH
5.0. (n = 4). This pH dependence of AE2
/AE1
resembled that of WT AE1 to a much greater degree than that of WT
AE2. Therefore, the AE2 cytoplasmic domain was not sufficient to
produce an AE2 pH-regulatory phenotype in this assay. In addition, the
presence of the AE1 transmembrane domain sufficed to preserve most of
the AE1 pH-regulatory phenotype.
pH dependence of chloride transport
activity of the converse chimera,
AE1/AE2
, was also examined (open
circles, Fig. 5). AE1
/AE2
almost precisely resembled WT AE2 in its pH
versus activity curve, except for a shift to acid pH
values. AE1
/AE2
was also maximal at
pH
8.0, but the pH
50 value was 6.34 ±
0.06 (n = 13). The mean of
pH
(the
difference between pH
50 for WT AE2 and pH
50 for
AE1
/AE2
) values measured in each of 13
individual experiments was 0.69 ± 0.04 units (p <
0.001). This value and its statistical significance were not different,
whether calculated only from the nine experiments in which transport
was assayed at all five pH
values or (as presented) from
these nine plus four additional experiments in which transport was
assayed at only four pH
values. Results were also
indistinguishable whether calculated as the mean of
pH
values calculated from individual experiments or as the
pH
calculated as the difference between mean
pH
values for WT AE2 and for
AE1
/AE2
.
This result suggested that the AE2 carboxyl-terminal transmembrane domain contained a pH sensor sufficient for expression of most of the WT AE2 pH-regulatory phenotype. However, substitution of the AE1 cytoplasmic domain for that of AE2 in the presence of the AE2 transmembrane domain led to a decreased sensitivity to inhibition of transport activity by protons. Thus, the AE2 amino-terminal cytoplasmic domain exerted a modifier function that was necessary for complete replication of the WT AE2 pH-regulatory phenotype.
Substitution of increasing amounts of
extracellular anion with equimolar acetate (Fig. 7, upper
horizontal axis) produced graded increases in acidification of
oocytes, as measured with fluorescence ratio imaging of BCECF (Fig. 7, lower horizontal axis). Increasing acetate
concentrations were also accompanied by dose-dependent inhibition of
AE2-mediated chloride uptake. At pH 7.4, half-maximal
inhibition of AE2 by acetate substitution occurred at 13.5 mM acetate (n = 3), corresponding to an approximate
reduction in pH
of 0.16 units (n = 4). In
contrast, half-maximal inhibition of AE2 by extracellular acidification
(normalized to activity at pH
7.4 in order to compare the
data of Fig. 5with those of Fig. 7) occurred at pH
6.83 (n = 13), corresponding to a pH
reduction of only 0.06 units (n = 4).
Figure 7:
Chloride influx versus extracellular acetate concentration for WT AE2, at constant
extracellular [Cl] and ionic strength (n = 4). The corresponding steady-state pH
values on the lower x axis were measured in
identically treated oocytes (n =
4).
Injection of sodium acetate, pH 7.4, to an estimated final
intracellular concentration of 13 mM neither acidified the
oocytes nor inhibited AE2-mediated uptake of Cl
(not shown). Thus, the inhibitory
effect of extracellular acetate substitution was not likely due to
simple inhibition of AE2 by competition at an anion substrate site but
rather reflected an effect of intracellular acidification. Considered
together, these data support the conclusion that pH
contributed significantly, but not entirely, to the regulation of
AE2 by pH under conditions in which both pH
and pH
were changed (as presented in Fig. 5and Fig. 6).
The present work has used analysis of recombinant chimeric
and truncated polypeptides to initiate determination of the structural
loci of pH sensitivity of AE2-mediated monovalent anion exchange.
Functional studies of recombinant AE polypeptides have been reported in
several heterologous expression systems. Although WT AE2 has been
functionally expressed in mammalian cells(21, 25) ,
heterologous WT AE1 and a version of the
AE1/AE2
chimera did not reach the cell
surface. In contrast, functional plasmalemmal expression not only of
AE2 (25, 26) but also of heterologous WT and truncated
AE1 (30, 31, 41) has been reported in Xenopus oocytes. The present study establishes that Xenopus oocytes can also express functional chimeric AE
polypeptides at the cell surface and so has set the stage for
structure-function studies designed to delineate the amino acid
sequences responsible for AE isoform-specific transport properties. We
have begun this process with the study of the structural domains
responsible for the different pH sensitivities of AE1 and AE2.
In
contrast to the experiments that first demonstrated pH sensitivity of
heterologous AE2 in Xenopus oocytes(25) , the current
flux assay was carried out at near-steady-state pH. In the
experiments of Fig. 5and Fig. 6, oocytes were
preincubated for 15 min at the desired pH
prior to
initiation of the
Cl
influx assay at the
same pH
. Oocytes displayed minimal recovery from alkaline
pH
during the time course of the experiment in these
nominally CO
-free conditions. The presence of amiloride
during 60-min influx assays to block the endogenous oocyte
Na
/H
exchanger (26, 36, 38) , or the performance of 15-min
influx assays, achieved near-steady-state pH
during influx
periods. As pH
was changed between 9.0 and 5.0, the
corresponding steady-state oocyte pH
values ranged from
7.60 to 7.13, respectively. Some oocytes had not achieved maximal
pH
by the end of the 15-min preincubation period (Fig. 4); for these oocytes the influx period included time at
pre-steady-state pH
values. Thus, the observed pH-dependent
changes in AE-mediated transport activity represented minimal estimates
compared with ideal pH
clamp conditions.
The experiments
demonstrated the requirement for the transmembrane domain of AE2 for
display of the characteristic steep pH versus activity curve
in WT AE2, AE1/AE2
,
AE2
99, and AE2
510. In contrast, the
presence in any polypeptide of the AE1 transmembrane domain produced a
broad pH dependence of chloride uptake. Moreover, this pH sensitivity
associated with the presence of the AE1 transmembrane domain was only
minimally changed by substitution of the AE2 N-terminal cytoplasmic
domain for its AE1 counterpart. This apparent lack of cytoplasmic
domain specificity differed from the pH sensitivity of the AE2
transmembrane domain, which displayed modulation of affinity for proton
equivalents by the contiguous N-terminal cytoplasmic domain of AE2, but
not by that of AE1 (Fig. 5). Thus, attachment of the AE1
cytoplasmic domain onto the AE2 transmembrane domain led to an acid
shift of 0.7 units in the pH
value of the pH versus activity curve. This shift was reproduced by removal of the first
510 amino acids from the AE2 cytoplasmic domain. However, restricting
the truncation to only 99 amino acids led to reproduction of the WT AE2
pH dependence phenotype (Fig. 6).
Definitive assignment of
the AE2 transmembrane domain pH sensor to the endofacial or exofacial
aspect of the plasmalemma is not yet possible. The combined variation
of pH and pH
did not allow unambiguous
assignment of regulatory function to intra- or extracellular protons.
Inhibition of AE2-mediated chloride uptake by acetate-induced
intracellular acidification at constant pH
(Fig. 7)
indeed suggested a role for pH
in this regulation. However,
AE2 inhibition by acetate was accompanied by a reduction in pH
of 0.16 units, whereas AE2 inhibition by extracellular
acidification was accompanied by a 0.06-unit reduction of
pH
. In addition, recent experiments have indicated that
variation of pH
under conditions of minimal pH
change also can regulate AE2 activity. (
)
Thus,
pH very likely could not account completely for the AE2
inhibition produced by lowering pH
in Fig. 5and Fig. 6. These data together allow the proposal that AE2 can be
regulated both by pH
and by pH
. Coincident
variation of pH
and pH
is a common
pathophysiological derangement in the setting of ischemia and hypoxia
and thus might cooperatively regulate AE2 activity in vivo.
When AE2 transport activity was plotted as a function of
pH, pH dependence fit simple logistic sigmoid kinetics. 80%
of the range of measured AE2 activity was traversed across the 100-fold
range of extracellular proton concentration between pH
6.0
and 8.0 (Fig. 5, upper x axis). pH
could
not be easily measured in the same oocytes in which influx measurements
were performed, but pH
was measured in separate oocytes
otherwise treated identically. When AE2 transport activity was plotted
against the pH
values measured in identically treated
oocytes (Fig. 5, lower x axis), 80% of the range of
measured AE2 activity was traversed across a
2-fold range of
intracellular proton concentration, between 28 and 51 nM ( Table 2and Fig. 5). This degree of cooperativity with
respect to pH
allows the suggestion of multiple
protonatable sensor sites in AE2.
These responses of the AE
constructs to combined variation of pH and pH
suggested a model for pH regulation of AE2 reminiscent of that
proposed for NHE1(32, 42) . The model proposes that a
pH sensor resides within the transmembrane domain. This sensor must be
of sufficiently high proton affinity to account for a large part of the
difference between the pH sensitivities of WT AE2 and WT AE1. In
addition, the AE2 N-terminal cytoplasmic segment harbors a modifier
domain whose removal reduces the proton affinity of the transmembrane
domain pH sensor. The reported deletion experiments locate this
modifier domain between mouse AE2 cytoplasmic amino acids 100 and 510 (Fig. 6). Whether the modulator directly recognizes the sensor
or interacts with it indirectly or via additional polypeptide(s)
remains to be determined.
The postulated pH-sensing function could
have one or more of the following three topographical dispositions. The
pH sensor might detect pH from the endofacial surface of
AE2. Location of the sensor at the AE2 cytoplasmic face is suggested
(but not required) by the cytoplasmic location of the modifier domain.
Such a sensor might reside within cytoplasmic loops connecting
transmembrane spans, within a water-accessible vestibule structure in
the plane of the lipid bilayer, or in the C-terminal cytoplasmic tail.
The same or a different pH sensor could detect pH
at
exofacial residues, including a postulated vestibule structure such as
that which might accommodate the sulfonate moieties of the stilbene
inhibitors of transport(43) . Alternatively, the sensor(s)
might be located in a position that allows proton sensing of both
intracellular and extracellular solutions, thus providing for dual
sensing of pH
and pH
. Glu
(44) and Lys
(45) of human AE1 may
be exposed to both sides of the red cell lipid bilayer. Such residues
very likely contribute to the anion binding and translocation pathway
through AE1.
In summary, AE2 and AE1 function in Xenopus oocytes differ in their regulation by pH. The steeper pH
dependence of AE2 function is mediated by its C-terminal transmembrane
domain. Modulation of that pH dependence requires a region of the AE2
N-terminal cytoplasmic domain (the modifier site) between residues 100
and 510. Future experiments will address the individual and cooperative
roles of pH and pH
in regulation of AE2
activity, define more precisely those residues that comprise the pH
sensor(s) and modifier site of AE2, and examine the mechanisms by which
these two functional domains interact with one another and with other
regulatory sites within the AE2 polypeptide.