|
|
|
|||||||
|
|||||||||
(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 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 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 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
The
AE2 The AE2 truncation mutants
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. Individual oocytes underwent scintillation counting of associated AE-mediated
Cl
Figure 5:
Chloride influx versus pH curves
for WT and chimeric AE polypeptides. Oocytes expressing the indicated
polypeptides were subjected to Cl
Figure 6:
Cl
influx versus pH curves for WT and truncated AE2 polypeptides.
Oocytes expressing the indicated polypeptides were subjected to
Cl
Figure 4:
Time course of pH
Mean values measured for WT
AE1 and for AE2
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
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 [
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, The amount of AE2
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
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
Table 2summarizes the
near-steady-state pH
WT AE1 and WT AE2 differed significantly in their regulation by pH (Fig. 5). WT AE1 (closed triangles) displayed a broad
pH
As shown in Fig. 5,
AE2 pH dependence of chloride transport
activity of the converse chimera,
AE1 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
Figure 7:
Chloride influx versus extracellular acetate concentration for WT AE2, at constant
extracellular [Cl
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 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 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 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 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 Thus,
pH When AE2 transport activity was plotted as a function of
pH These responses of the AE
constructs to combined variation of pH The postulated pH-sensing function could
have one or more of the following three topographical dispositions. The
pH sensor might detect pH 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
Volume 271,
Number 10,
Issue of March 8, 1996 pp. 5741-5749
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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) .
- 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) .
/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.
Materials
Female Xenopus were purchased
from NASCO (Madison, WI) and maintained as described
previously(25) .
2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester
(BCECF-AM) (
)was from Molecular Probes. Bumetanide was the
gift of P. Feit (Leo Pharmaceutical). NaCl 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). Sure
Escherichia coli strain was from Stratagene (La Jolla, CA). Oligonucleotides were
synthesized on a Milligen Cyclone Plus DNA synthesizer.
Solutions
ND-96 medium consisted of (in
mM) 96 NaCl, 2 KCl, 1.8 CaCl, 1 MgCl,
5 HEPES, and 2.5 sodium pyruvate. All flux media lacked pyruvate and
contained 38 mM NaCl as NaCl. Some flux media
substituted equimolar MES or bis-tris propane for HEPES. Flux media
were titrated to the indicated pH values before use.
PCR Products and cDNA Construction
Murine AE1
encoded in plasmid pBL (26) and murine AE2 encoded in plasmid
pX (11, 25, 26) were used as templates
for polymerase chain reaction (PCR) and for RNA transcription. Chimeric
cDNAs were constructed by a four-primer PCR method employing two rounds
of PCR amplification(33) . The AE1/AE2
chimera was constructed as follows (Table 1). AE1 served as
the first-round template for primers yz5 and yz2. AE2 served as the
first-round template for primers yz1 and yz8. The two first-round
amplification products were coannealed to serve as second-round
template for the bracketing primers yz5 and yz8. The second-round
chimeric amplification product joined AE1 nucleotide 1395 to AE2
nucleotide 2292 in a fragment of 841 nucleotides in length. This PCR
product was completely digested by NcoI and partially digested
by StuI, and the central fragment of 543 base pairs was
gel-purified. To reconstruct the full-length chimera, this fragment
underwent three-way ligation with the 3.8-kb NcoI/HindIII fragment of pBL (AE1) and the 1.6-kb StuI/HindIII fragment of p
X (AE2). The final
AE1/AE2
cDNA encoded mouse AE1 cDNA from
its translational initiation codon to nucleotide 1395 (corresponding to
amino acids 1-422) fused to mouse AE2 cDNA from nucleotide 2292
to its 3` end (corresponding to amino acids 704-1237, the entire
transmembrane (TM)-spanning region beginning at TM1).
/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).
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 pX. The
reconstructed AE299 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 pX.
The reconstructed AE2510 encoded an engineered ATG followed by
amino acids 511-1237.DNA Sequence Analysis
Sequencing reactions were
carried out using the DyePrimer cycle sequencing kit (Applied
Biosystems), and the reaction products were sequenced on the Applied
Biosystems model 371 automated sequenator. PCR-amplified fragments and
their ligation junctions in all reconstructed cDNAs were completely
sequenced on both strands to verify absence of introduced mutations.In Vitro Transcription and Translation
cDNAs
encoding WT AE1 and AE2/AE1
were
linearized with HindIII. All other cDNAs were linearized with ClaI. Capped cRNA transcripts were synthesized from these
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 by A
. In vitro translation of cRNAs in the
absence and presence of dog pancreas microsomes was with rabbit
reticulocyte lysate (Promega).
Antibodies
Affinity-purified polyclonal antibody
to the 12 carboxyl-terminal amino acids of murine AE2 has been
described(16, 17) . Murine monoclonal antibody to the
12 carboxyl-terminal amino acids, 1224-1237, of murine AE2 ()was purified by fast protein liquid chromatography
(Pharmacia Biotech Inc.) with protein A-agarose affinity
chromatography. Polyclonal antiserum to the 12 carboxyl-terminal amino
acids of murine AE1 was the gift of Dr. A.-M. Garcia.Immunoprecipitation of Total and Surface AE Proteins from
Xenopus Oocytes
Groups of 10-12 oocytes, injected either
with 20 ng of cRNA or with water, were incubated for 48 h at 19 °C
in 200 µl of ND-96, pH 7.4, containing 1 mCi/ml (30
µM) of S-methionine. Metabolically labeled
oocytes were washed in modified ND-96, pH 8.0, in which 48 mM NaCl was replaced with 100 mM sucrose, and then incubated
for 3 h at room temperature in the same medium in the presence or
absence of 5 mg/ml papain. Subsequent washing, homogenization in Triton
X-100, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis
fluorography were as described previously(31) .
Defolliculated oocytes were injected with cRNA or
water, and incubated in ND-96 for 2-4 days at 19 °C. Groups
of 8-12 injected oocytes were preincubated for 15 min in 1 ml of
ND-96 at the indicated pH and then transferred into 150 µl of the
same medium containing in addition 10 µM bumetanide and
2.8-3.2 µCi of NaCl
Influx
Measurements
Cl. Total NaCl concentration
was constant at 96 mM (38 mM of which was from
Na
Cl). Two 10-µl aliquots of mixed influx medium were
removed for later determinations of
Cl-specific activity
in each influx group.
Cl
influx into
oocytes was carried out for 15 or 60 min in the absence or presence,
respectively, of 100 µM amiloride and then terminated by
three washes in 25 ml of isotope-free ND-96. Each construct was tested
under both influx conditions, with similar results.
Cl
influx was then performed as above.
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.
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 AE2510 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.
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.
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
AE2510 (squares, n = 5), data
from each independent experiment were fitted to a logistic sigmoid
equation, normalized to fitted saturation activities, and then
averaged.
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.
/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.Measurement of Oocyte Intracellular pH
(pH
Oocyte pH
) was measured by BCECF
fluorescence ratio imaging as described
previously(25, 26) . Oocytes were loaded with
2-5 µM BCECF-AM for 1 h and mounted in a customized
superfusion chamber mounted on an inverted microscope
stage(25) . Oocytes were irradiated, and fluorescence ratios
were acquired and recorded to hard disk with an Image 1 digital ratio
imaging system (Universal Imaging, West Chester, PA). The plane of
focus of 20 µm (
10 objective, N.A. 0.4) was set under
the vegetal pole surface (25) for the measurements presented or
at different focal planes (26) with similar results. In
situ calibration of the BCECF fluorescence ratio (35, 36) was performed as described(25) .
cDNA Constructs of AE Mutations
In order to
localize those protein domains within AE2 and AE1 that are necessary
for expression of their different sensitivities to pH, domain-swap
chimeras and truncated AE2 cDNAs were generated as described under
``Experimental Procedures'' and in Table 1. The
constructs are presented in schematic form in Fig. 1. The
AE1/AE2
cDNA encoded a chimeric
polypeptide consisting of mouse AE1 cytoplasmic amino acids 1-422
fused to mouse AE2 TM domain amino acids 704-1237. The
AE2
AE1
cDNA encoded a chimeric
polypeptide consisting of mouse AE2 cytoplasmic amino acids 1-703
fused to mouse AE1 TM domain amino acids 423-929. AE2
99 cDNA
encoded an engineered ATG followed by amino acids 100-1237. AE2510
encoded an engineered ATG followed by amino acids 511-1237.
Biosynthesis and Surface Expression of WT and Mutant AE
Polypeptides
cRNAs transcribed from the above described cDNAs
were used as templates for in vitro translation reactions in
reticulocyte lysate. Polypeptides of predicted size were translated
from each cRNA. Each polypeptide was inserted into microsomal membranes
and underwent an increase in M
and bandwidth
suggestive of core N-glycosylation (not shown). Thus, the
mutant cRNAs encoded polypeptides of the predicted length that folded
in such a way as to permit core glycosylation.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.
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.
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, AE2510, and AE1/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.
Functional Analysis of Expressed Mutant cDNAs in Xenopus
Oocytes
Measurement of Cl
influx
in oocytes expressing AE2
/AE1
polypeptide
provided a functional demonstration of surface expression of the
mutant. Fig. 3shows that injection of increasing mass
quantities of AE2
/AE1
cRNA produced
increasing AE-mediated
Cl
influx
activity. However, the nmol of chloride uptake/ng of injected cRNA for
this chimera, as also for AE2
510, was very low compared with those
measured for WT AE2 and WT AE1. In contrast, AE299 and
AE1/AE2
chimera were both considerably
more active per ng of injected cRNA than either WT cRNA. Operationally
defined specific activities of chloride transport among the cRNAs
spanned a range of 150-fold, from 17.8 nmol/oocyte
hng (for
AE1/AE2
) to 0.12
nmol/oocyte
hng (for AE2/AE1
and for AE2
510). These differences were replicated using
cRNA from multiple transcription reactions and from multiple cDNA
templates. The rank order of specific activities of the AE polypeptides
tested correlated with the rank order of amounts of AE polypeptide
detected at the surface by proteolytic cleavage (Fig. 2).
influx into H
O-injected
oocytes, 0.6 ± 0.1
nmol/oocyteh.
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.
Determination of Flux Assay Conditions in Which Oocyte
pH
When oocytes are placed into
media of varied pH Is Near Steady State, pH changes in response.
When pH is shifted from neutral to acidic values, oocyte
pH acidifies to a minimum and then recovers toward resting
pH(25) . 40% of this recovery is due to
endogenous sodium/hydrogen exchange activity (26, 36, 38) of the oocyte. We exploited
these data to devise flux assay conditions in which AE-mediated
chloride transport could be measured at near-steady-state oocyte
pH
values. pH of individual oocytes expressing
WT AE2 was monitored via BCECF fluorescence ratio imaging during the
transition from ND-96, pH 7.4, to preincubation medium of
varying pH for 15 min and then into flux medium of the same
pH containing in addition 100 µM amiloride and
10 µM bumetanide. As shown in Fig. 4, oocytes
exposed to varying pH under these conditions showed
considerable pH change within 15 min, achieved
near-steady-state pH values within 30 min, and
displayed minimal recovery over the ensuing 40-80 min ( Fig. 4and data not shown). At acid pH
this steady
state reflected the balance between ongoing proton entry and the
previously described (38) amiloride-resistant acid extrusion
system(s) of the oocyte. () 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) ).
Regulation by pH of WT AE-mediated
The pH dependence of
chloride transport mediated by WT AE1 and by WT AE2 was compared under
the assay conditions described in Fig. 4. Transport activities
were measured at pHCl
Uptake
values from 5.0 to 9.0.
Near-steady-state pH values measured in different oocytes
in the same conditions ranged from 7.13 to 7.60 (Fig. 5).
pH determined by glass pH electrode and by BCECF
fluorometry did not change during oocyte incubations of 15 and 60 min
(not shown). Nonetheless, since the pH range chosen
exceeded the useful pH range of HEPES buffer, identical experiments
were also performed in media buffered by 5 mM MES and by 5
mM bis-tris propane. The results obtained did not differ from
those measured in the presence of HEPES (not shown). In addition, all
AE constructs were subjected to pHversus activity
assays in which 60-min Cl
influx in the
presence of 100 µM amiloride was compared to 15-min
Cl
influx in the absence of amiloride.
pH dependence of transport in the two assay conditions did not differ.
versus 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).
Regulation by pH of Chimeric AE-mediated
This major difference
between pHCl
Uptake
versus activity curves of WT AE2 and WT
AE1 allowed us to test the hypothesis that a single region of AE2
distinct in amino acid sequence from AE1 is responsible for the
difference. Therefore, the pair of domain-swap chimeras pictured in Fig. 1was examined for pH sensitivity of Cl
uptake into oocytes. The hypothesis
that the N-terminal cytoplasmic domain of AE2 was necessary and
sufficient for the increased pH sensitivity of AE2-mediated
Cl
uptake predicted in its simplest form
that the AE2
/AE1
chimera would resemble
AE2 in its regulation by pH. The converse hypothesis that the
C-terminal transmembrane domain of AE2 was necessary and sufficient for
the increased pH sensitivity of AE2-mediated
Cl
uptake predicted in its simplest form
that the AE1
/AE2
chimera would resemble
AE2 in its regulation by pH. The experimental data partially supported
this second hypothesis. The C-terminal transmembrane domain of AE2
proved necessary, but not sufficient, to reproduce the pH versus activity phenotype of WT 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.
/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 pH50 value was 6.34 ±
0.06 (n = 13). The mean of pH (the
difference between pH
50 for WT AE2 and pH50 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
.
Regulation by pH of
The hypothesis of modifier function predicts in
simplest form that sufficient truncation of the AE2 cytoplasmic domain
should produce a pH versus activity phenotype that resembles
that of the AE1Cl
Uptake Mediated by N-terminally Truncated AE2
Polypeptides
/AE2
chimera. In order to
localize this proposed modifier function within the N-terminal
cytoplasmic domain, two cytoplasmic domain truncation mutants of AE2
were examined. The pH dependence of AE2
99 and
AE2510 were assayed under the same experimental
conditions as had been used to study the chimeric polypeptides. As
shown in Fig. 6, the pH versus activity curves for
these cytoplasmic domain truncation mutants were of similar shape to
that of WT AE2, with complete inhibition of transport activity at
pH 5.0. Whereas the pH dependence curve for AE299 was
indistinguishable from that of WT AE2, the curve for
AE2510 was shifted to a more acidic pH value
(pH between wt AE2 and AE2
510
= 0.66 ± 0.13 (n = 5); p <
0.005). This result was unchanged when calculated as the difference
between mean pH values for WT AE2 and
AE2
510. The pH dependence curve of
AE2510 did not differ detectably from that of
AE1/AE2
. Thus the region between AE2
amino acids 100 and 510 contained within it a modifier site required
for enhancing the proton sensitivity of the pH sensor.
Role of pH
Since the experiments presented in Fig. 5and Fig. 6were performed under conditions under which both pH in the Regulation of AE2 Activity
by pH and pH were varied, the relative contributions of
pH and pH to the regulation of AE2 by pH were
not addressed. However, the localization of a modifier site to the
cytoplasmic N-terminal hydrophilic domain of AE2 suggested an important
role for pH. Earlier experiments that demonstrated
regulation by pH of AE2-mediated chloride/HCO exchange in mammalian cells were performed in the presence of
CO/bicarbonate(21, 25) . In these
experiments activation of AE2 by alkaline pH never reached
plateau values, probably due to parallel allosteric activation and
increasing concentrations of substrate bicarbonate. Therefore, we
sought to examine AE2-mediated chloride/base exchange activity in
oocytes under conditions that would alter oocyte pH at
constant 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).
] and ionic strength (n = 4). The corresponding steady-state pH
values on the lower x axis were measured in
identically treated oocytes (n =
4).
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).
/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 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./AE2
,
AE2
99, and AE2510. 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).
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. (
) 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. , 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. 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. 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.
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.
), extracellular pH;
pH
, intracellular pH;
AE2
/AE1
, cytoplasmic domain of AE2
attached to the transmembrane domain of AE1;
AE1
/AE2
, cytoplasmic domain of AE1
attached to the transmembrane domain of AE2; TM, transmembrane;
bis-tris propane,
1,3-bis[tris(hydroxymethyl)methylamino]propane hemichloride
salt; kb, kilobase(s); PCR, polymerase chain reaction; MES,
4-morpholineethanesulfonic acid.
)))
We thank Drs. Ann Rittenhouse and Jeffrey Matthews for
comments on the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. K. Stewart, C. E. Kurschat, R. D. Vaughan-Jones, and S. L. Alper Putative Re-entrant Loop 1 of AE2 Transmembrane Domain Has a Major Role in Acute Regulation of Anion Exchange by pH J. Biol. Chem., March 6, 2009; 284(10): 6126 - 6139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Stewart, C. E. Kurschat, R. D. Vaughan-Jones, B. E. Shmukler, and S. L. Alper Acute regulation of mouse AE2 anion exchanger requires isoform-specific amino acid residues from most of the transmembrane domain J. Physiol., October 1, 2007; 584(1): 59 - 73. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. K. Stewart, C. E. Kurschat, D. Burns, N. Banger, R. D. Vaughan-Jones, and S. L. Alper Transmembrane domain histidines contribute to regulation of AE2-mediated anion exchange by pH Am J Physiol Cell Physiol, February 1, 2007; 292(2): C909 - C918. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Pushkin and I. Kurtz SLC4 base (HCO3-, CO32-) transporters: classification, function, structure, genetic diseases, and knockout models Am J Physiol Renal Physiol, March 1, 2006; 290(3): F580 - F599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Kurschat, B. E. Shmukler, L. Jiang, S. Wilhelm, E. H. Kim, M. N. Chernova, R. K. H. Kinne, A. K. Stewart, and S. L. Alper Alkaline-shifted pHo Sensitivity of AE2c1-mediated Anion Exchange Reveals Novel Regulatory Determinants in the AE2 N-terminal Cytoplasmic Domain J. Biol. Chem., January 27, 2006; 281(4): 1885 - 1896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Chernova, L. Jiang, D. J. Friedman, R. B. Darman, H. Lohi, J. Kere, D. H. Vandorpe, and S. L. Alper Functional Comparison of Mouse slc26a6 Anion Exchanger with Human SLC26A6 Polypeptide Variants: DIFFERENCES IN ANION SELECTIVITY, REGULATION, AND ELECTROGENICITY J. Biol. Chem., March 4, 2005; 280(9): 8564 - 8580. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Stewart, N. Kerr, M. N. Chernova, S. L. Alper, and R. D. Vaughan-Jones Acute pH-dependent Regulation of AE2-mediated Anion Exchange Involves Discrete Local Surfaces of the NH2-terminal Cytoplasmic Domain J. Biol. Chem., December 10, 2004; 279(50): 52664 - 52676. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. K. Dahl, L. Jiang, M. N. Chernova, A. K. Stuart-Tilley, B. E. Shmukler, and S. L. Alper Deficient HCO3- Transport in an AE1 Mutant with Normal Cl- Transport Can be Rescued by Carbonic Anhydrase II Presented on an Adjacent AE1 Protomer J. Biol. Chem., November 7, 2003; 278(45): 44949 - 44958. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N Chernova, L. Jiang, B. E Shmukler, C. W Schweinfest, P. Blanco, S. D Freedman, A. K Stewart, and S. L Alper Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes J. Physiol., May 15, 2003; 549(1): 3 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Chernova, A. K. Stewart, L. Jiang, D. J. Friedman, Y. Z. Kunes, and S. L. Alper Structure-function relationships of AE2 regulation by Ca2+i-sensitive stimulators NH+4 and hypertonicity Am J Physiol Cell Physiol, May 1, 2003; 284(5): C1235 - C1246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L Brett, T. Kelly, C. Sheldon, and J. Church Regulation of Cl--HCO3- exchangers by cAMP-dependent protein kinase in adult rat hippocampal CA1 neurons J. Physiol., December 15, 2002; 545(3): 837 - 853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.K. Stewart, M.N. Chernova, B.E. Shmukler, S. Wilhelm, and S.L. Alper Regulation of AE2-mediated Cl- Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH2-terminal Cytoplasmic Domain J. Gen. Physiol., October 29, 2002; 120(5): 707 - 722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Goss, L. Jiang, D. H. Vandorpe, D. Kieller, M. N. Chernova, M. Robertson, and S. L. Alper Role of JNK in hypertonic activation of Cl--dependent Na+/H+ exchange in Xenopus oocytes Am J Physiol Cell Physiol, December 1, 2001; 281(6): C1978 - C1990. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Markovich Physiological Roles and Regulation of Mammalian Sulfate Transporters Physiol Rev, October 1, 2001; 81(4): 1499 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Stewart, M. N. Chernova, Y. Z. Kunes, and S. L. Alper Regulation of AE2 anion exchanger by intracellular pH: critical regions of the NH2-terminal cytoplasmic domain Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1344 - C1354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. KARUMANCHI, L. JIANG, B. KNEBELMANN, A. K. STUART-TILLEY, S. L. ALPER, and V. P. SUKHATME VHL tumor suppressor regulates Cl-/HCO3- exchange and Na+/H+ exchange activities in renal carcinoma cells Physiol Genomics, April 2, 2001; 5(3): 119 - 128. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tosco, M. N. Orsenigo, G. Gastaldi, and A. Faelli An endogenous monocarboxylate transport in Xenopus laevis oocytes Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2000; 278(5): R1190 - R1195. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. de la Horra, N. Hernando, G. Lambert, I. Forster, J. Biber, and H. Murer Molecular Determinants of pH Sensitivity of the Type IIa Na/Pi Cotransporter J. Biol. Chem., February 25, 2000; 275(9): 6284 - 6287. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. J. Jensen, A. K. Stuart-Tilley, L. L. Peters, S. E. Lux, S. L. Alper, and S. Breton Immunolocalization of AE2 Anion Exchanger in Rat and Mouse Epididymis Biol Reprod, October 1, 1999; 61(4): 973 - 980. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. L. Alper, H. Rossmann, S. Wilhelm, A. K. Stuart-Tilley, B. E. Shmukler, and U. Seidler Expression of AE2 anion exchanger in mouse intestine Am J Physiol Gastrointest Liver Physiol, August 1, 1999; 277(2): G321 - G332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Leem, D. Lagadic-Gossmann, and R. D Vaughan-Jones Characterization of intracellular pH regulation in the guinea-pig ventricular myocyte J. Physiol., May 15, 1999; 517(1): 159 - 180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. M. Schmitt, D. Biemesderfer, M. F. Romero, E. L. Boulpaep, and W. F. Boron Immunolocalization of the electrogenic Na+-HCO-3 cotransporter in mammalian and amphibian kidney Am J Physiol Renal Physiol, January 1, 1999; 276(1): F27 - F38. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Eladari, A. Blanchard, F. Leviel, M. Paillard, A. K. Stuart-Tilley, S. L. Alper, and R.-A. Podevin Functional and molecular characterization of luminal and basolateral Cl-/HCO-3 exchangers of rat thick limbs Am J Physiol Renal Physiol, September 1, 1998; 275(3): F334 - F342. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. M. Maglova, W. E. Crowe, A. A. Altamirano, and J. M. Russell Human cytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in human fibroblasts Am J Physiol Cell Physiol, August 1, 1998; 275(2): C515 - C526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. Milton and I. D. Weiner Regulation of B-type intercalated cell apical anion exchange activity by CO2/HCO-3 Am J Physiol Renal Physiol, June 1, 1998; 274(6): F1086 - F1094. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Jarolim, C. Shayakul, D. Prabakaran, L. Jiang, A. Stuart-Tilley, H. L. Rubin, S. Simova, J. Zavadil, J. T. Herrin, J. Brouillette, et al. Autosomal Dominant Distal Renal Tubular Acidosis Is Associated in Three Families with Heterozygosity for the R589H Mutation in the AE1 (Band 3) Cl-/HCO3- Exchanger J. Biol. Chem., March 13, 1998; 273(11): 6380 - 6388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. C. Brosius III, R. L. Pisoni, X. Cao, G. Deshmukh, D. Yannoukakos, Alan. K. Stuart-Tilley, C. Haller, and S. L. Alper AE anion exchanger mRNA and protein expression in vascular smooth muscle cells, aorta, and renal microvessels Am J Physiol Renal Physiol, December 1, 1997; 273(6): F1039 - F1047. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Alper, A. K. Stuart-Tilley, D. Biemesderfer, B. E. Shmukler, and D. Brown Immunolocalization of AE2 anion exchanger in rat kidney Am J Physiol Renal Physiol, October 1, 1997; 273(4): F601 - F614. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. R.M. Lima, J. A.R. Gontijo, J. B. Lopes de Faria, F. F. Costa, and S. T.O. Saad Band 3 Campinas: A Novel Splicing Mutation in the Band 3 Gene (AE1 ) Associated With Hereditary Spherocytosis, Hyperactivity of Na+/Li+ Countertransport and an Abnormal Renal Bicarbonate Handling Blood, October 1, 1997; 90(7): 2810 - 2818. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |