A New Member of the HCO
Transporter Superfamily Is an Apical Anion Exchanger of
-Intercalated Cells in the Kidney*
Hirohiko
Tsuganezawa
,
Kazuo
Kobayashi
,
Masahiro
Iyori
,
Takashi
Araki
,
Amane
Koizumi§,
Shu-Ichi
Watanabe§,
Akimichi
Kaneko§,
Taro
Fukao
,
Toshiaki
Monkawa
,
Tadashi
Yoshida
,
Do Kyong
Kim¶,
Yoshikatsu
Kanai¶,
Hitoshi
Endou¶,
Matsuhiko
Hayashi
**, and
Takao
Saruta
From the
Department of Internal Medicine and the
§ Department of Physiology, Keio University School of
Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 and the
¶ Department of Pharmacology and Toxicology, Kyorin University
School of Medicine, 6-20-2 Shinnkawa, Mitaka City,
Tokyo 181-8611, Japan
Received for publication, May 24, 2000, and in revised form, November 30, 2000
 |
ABSTRACT |
The kidneys play pivotal roles in acid-base
homeostasis, and the acid-secreting (
-type) and
bicarbonate-secreting (
-type) intercalated cells in the collecting
ducts are major sites for the final modulation of urinary acid
secretion. Since the H+-ATPase and anion exchanger
activities in these two types of intercalated cells exhibit opposite
polarities, it has been suggested that the
- and
-intercalated
cells are interchangeable via a cell polarity change.
Immunohistological studies, however, have failed to confirm that the
apical anion exchanger of
-intercalated cells is the band 3 protein
localized to the basolateral membrane of
-intercalated cells. In the
present study, we show the evidence that a novel member of the anion
exchanger and sodium bicarbonate cotransporter superfamily is an apical
anion exchanger of
-intercalated cells. Cloned cDNA from the
-intercalated cells shows about 30% homology with anion exchanger
types 1-3, and functional expression of this protein in COS-7 cells
and Xenopus oocytes showed sodium-independent and
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive anion
exchanger activity. Furthermore, immunohistological studies revealed
that this novel anion exchanger is present on the apical membrane of
-intercalated cells, although some
-intercalated cells were
negative for AE4 staining. We conclude that our newly cloned
transporter is an apical anion exchanger of the
-intercalated cells,
whereas our data do not exclude the possibility that there may be
another form of anion exchanger in these cells.
 |
INTRODUCTION |
It is well known that the kidneys play very important roles in
acid-base balance. Proximal convoluted tubules reabsorb bicarbonate from the primary urine, and the Na+/H+
exchanger and sodium bicarbonate cotransporter
(NBC)1 are the key
transporters for this function. Cortical to medullary collecting ducts
secrete protons and bicarbonate, whereas proton secretion occurs
predominantly under usual conditions. Over the past 2 decades, the
mechanisms of these acid-base transporters have been extensively
studied. In the cortical collecting ducts, there are at least two types
of intercalated cells,
and
, which secrete protons and
bicarbonate, respectively (1). The
-intercalated cells possess
H+-ATPase and an anion exchanger on the apical membrane and
the basolateral membrane, respectively (1, 2, 3). This basolateral anion exchanger in the
-intercalated cells is a truncated form of
band 3 protein (4), anion exchanger (AE) type 1 (5), and its
localization was revealed by immunohistochemical studies (3, 6). On the
other hand,
-intercalated cells possess H+-ATPase and an
anion exchanger on the basolateral and the apical membranes,
respectively. In the early work of Schuster et al. (3), it
was suggested that peanut lectin binds exclusively to
-intercalated
cells. Further immunohistochemical studies (7) revealed that a minority
of the intercalated cells had basolateral band 3 protein labeling with
apical peanut lectin binding, raising the possibility that there might
be a third type of intercalated cell. Functional studies by Emmons and
Kurtz (8) also suggested that about half of the peanut lectin-positive
cells might be a variant of
-intercalated cells, which showed anion
exchanger activities on the both apical and the basolateral membranes.
The apical anion exchanger in the peanut -lectin-positive cells,
however, exhibits several differences in its transporter
characteristics, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic
acid (DIDS) sensitivity (8, 9), as compared with AE1. Furthermore,
since immunohistological studies with a specific antibody directed
against the band 3 protein have not shown positive staining on the
apical membranes of
-intercalated cells (3, 6, 7), it has been suggested that this apical anion exchanger is a transporter distinct from AE1. In contrast to these previous immunocytochemical and functional studies, van Adelsberg et al. (10) reported that purified and immortalized
-intercalated cells express AE1 and have
apical anion exchanger activity, suggesting that the difference in the
characteristics of the apical and basolateral membranes is responsible
for the difference in the immunoreactivity to the anti-AE1 antibody in
immunohistological studies. Fejes-Tóth et al. (11),
however, reported that primary cultures of
-intercalated cells
showed very low levels of AE1 mRNA and protein expression. In the
present study, we examined whether there is indeed a novel anion
exchanger in
-intercalated cells, using the polymerase chain
reaction (PCR)-based cloning method. The obtained cDNA was determined to be a new member of the AE and NBC superfamily, and this
novel transporter is present on the apical membranes of the
-intercalated cells.
 |
EXPERIMENTAL PROCEDURES |
Isolation of
-intercalated Cells--
The
-intercalated
cells were collected by a fluorescence-activated cell sorter, as
previously reported (12). Female New Zealand White rabbits (1.2-1.8
kg) were anesthetized with pentobarbital (35 mg/kg, intravenously).
Both kidneys were immediately removed and perfused through cannulae
inserted into the renal arteries with solution A at 4 °C (which
contained (in mM) NaCl 115, KCl 5.0, MgSO4 1.0, CaCl2 1.8, sodium acetate 10, Na2HPO4 1.6, NaH2PO4 0.4, Na-HEPES 5, H-HEPES 5, and D-glucose 8.3, at pH 7.4)
containing 0.2% collagenase and 0.02% hyaluronidase. The kidney
cortex was separated and cut into small pieces. The pieces were
incubated in the enzyme-containing solution A at 37 °C for 5 min.
After this enzymatic digestion, tubular fragments were obtained by
centrifugation (50 × g) and washed three times with
enzyme-free solution A. The fragments were suspended in 40% Percoll
solution and centrifuged at 16,000 × g for 20 min.
With this centrifugation, the tubular fragments were separated into two
layers, and the distal tubular fragments became concentrated in the
upper layer. This layer was resuspended and washed three times with
solution A. These fragments were further incubated at 37 °C for 30 min under a 100% O2 atmosphere in solution A containing
0.2% collagenase, 0.02% hyaluronidase, and 500 units of DNase. The
cells were then centrifuged at 50 × g for 5 min and
incubated at 4 °C for 30 min in solution A containing fluorescein
isothiocyanate (FITC)-labeled peanut lectin (50 µg/ml). The cell
suspension was passed 2 times through a 26-µm stainless steel mesh.
The final density of the cells was adjusted to 1-1.5 × 107 cells/ml with solution A. The cells were analyzed and
sorted with an Epics cell sorter system (Coulter Electronics, Hialeah, FL) as reported previously (12). Living single cells were distinguished by forward and 90° light scatter, according to the criteria in the
aforementioned report. The purity of the sorted
-intercalated cells
was examined by flow cytometric analysis or counting of FITC-positive
and -negative cells by fluorescence microscopy. These analyses showed
the purity of the cells to be about 95%.
Reverse Transcription-PCR--
Total RNA from
-intercalated
cells was extracted using an RNA extraction kit (Qiagen, GmbH, Hilden,
Germany). One microgram of total RNA was reverse-transcribed with a
random hexamer (Gene Amp RNA PCR Core kit; Applied Biosystems, Foster
City, CA). PCR was performed with a pair of degenerate primers:
5'-AAGGG(C/T)TC(G/C)GG(G/C/T)TTCCA(C/T)CT-3' (forward, F-1) and
5'-GTGAC(G/C/T)CCCATGTA(G/C)AGGAA-3' (reverse, R-1), which were
designed from the consensus sequences of AE1 to AE3
(GenBankTM accession numbers X12609, S45791, and AF031650).
The cycler (DNA Thermal Cycler 480, PerkinElmer Life Sciences) was programmed as follows: 94 °C for 30 s (denaturation), 44 °C
for 30 s (annealing), and 72 °C for 60 s (extension) with
30 cycles. An additional final incubation at 72 °C for 10 min (final
extension) was then performed. A band corresponding to an ~300-bp PCR
product was isolated by gel electrophoresis and subcloned. Two out of 30 clones, which were sequenced with an ABI Dideoxy Terminator Cycle
Sequencing kit (Applied Biosystems), were considered to be a partial
cDNA of the novel anion exchanger, named AE4. Among the 30 sequenced clones, 14 clones were highly homologous to cDNA of human
AE1 (GenBankTM accession number X12609), and 14 clones were
identical to cDNA of rabbit AE2 (GenBankTM accession
number S45791). The partial AE4 cDNA was extended toward the
5'-site end by reverse transcription (RT)-PCR with the AE4-specific
primer, 5'-CTGATGCCCAGGAAGCTGTG-3' (RT-1), for the RT reaction and the
forward primer, 5'-ATCACTTTTGGAGGACTGCTTGG-3' (F-2), which was designed
from the AE consensus sequence, and reverse primer,
5'-GCTCGGCTCTCCCTCCGAAG-3' (AE4-specific, R-2), for PCR. An ~1-kb PCR
product containing the partial AE4 sequence was obtained. The 5'-region
of this 1-kb fragment (5'-GCCTTCTGCAGAGATTACAGCCTG-3', probe-1) was
used for the cDNA library screening. The rabbit kidney uni-ZAP
cDNA library (Stratagene, La Jolla, CA) was purchased and
mass-excised in accordance with the instructions provided, and it was
then screened with the GeneTrapper cDNA-positive Selection System
(Life Technologies, Inc.). Two positive clones (~2.3 and ~2.5 kb)
were obtained, and sequencing analysis showed both clones to contain
the partial AE4 sequence and that the 2.5- and 2.3-kb clones contained
identical sequences.
The 5'-RACE System (Life Technologies, Inc.) was used to obtain
full-length AE4 cDNA with the primer, 5'-AGCACAGCTGAGACACACTG-3' (GSP-31), for the RT reaction. A pair of primers, the forward primer,
5'-RACE Abridged Anchor Primer (included in the kit), and reverse
primer, 5'-TGCAGGGCATCCGAGAAATC-3' (GSP-32), was used for the PCR.
These two primers, GSP-31 and -32, were designed from the 5'-region of
the 2.3-kb AE4 clone. Approximately 1.1-kb PCR products were obtained
and subcloned into the TA cloning vector. Sequencing of several clones
yielded clones of two lengths, one short and one long. The shorter
cDNA was 48 bp shorter than the longer cDNA. The procedure for
the sequencing of full-length AE4 is summarized in Fig. 1.
To clone full-length cDNA of AE4 for functional studies, we
performed RT-PCR again with the primer GSP-31 for the RT reaction, and
forward primer tagged with an EcoRI recognition sequence at the 5'-end, 5'-ATCAGAATTCAGCTGTGCCGCTCCCAGCAT-3', and reverse primer
(GSP-32) for the PCR. Two PCR products of different lengths were
obtained and subcloned. Both clones were ligated at the KpnI site with the 2.3-kb AE4 cDNA, which was obtained by cDNA
library screening, as described above. The sequences of the obtained
cDNA were verified; the longer and shorter clones were named AE4a
and AE4b, respectively. For the functional study, the AE4a cDNA was subcloned into the pcDNA3.1 expression vector at the
EcoRI and HindIII sites (pcDNA AE4a).
Northern Blotting--
Poly(A)+ RNA was extracted by
the acid guanidinium thiocyanate/phenol chloroform method (13) using
oligo(dT)-cellulose from various rabbit tissues. In each lane, 1-5
µg of RNA were electrophoresed. The blot was hybridized for 18 h
in hybridization solution at 63 °C, with a 32P-labeled
probe prepared from the full-length cDNA of the newly cloned anion
exchanger gene. A final high stringency wash was performed at 61 °C
in 0.1× SSC, 0.1% SDS, and the blot was exposed to the Imaging Plate
(Fuji Film, Tokyo, Japan). The blot was re-hybridized with a
glyceraldehyde 3-phosphate probe, which was purchased from Novagen
(Madison, WI).
COS-7 Transfection Procedures--
COS-7 cells were grown and
transfected in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum and maintained in humidified incubators at
37 °C under 5% CO2. The cells were seeded onto
coverslips in 6-cm culture dishes, 18 h prior to transfection with
10 µg of plasmid DNA. pcDNA AE4a, pEGFP N3
(CLONTECH, Palo Alto, CA), or control vector
pcDNA was transiently expressed in COS-7 cells by the LipofectAMINE
method. After 36-42 h of incubation, the transfected COS-7 cells were
used for functional studies, and the efficiency of the GFP transfection
was determined by an epifluorescence microscope (Olympus Optical,
Tokyo, Japan). In each of the transfection studies, 100 cells on the
coverslips with the GFP-transfected COS-7 cells were observed and the
ratio of GFP-positive to total cells was calculated.
Measurement of Intracellular pH of Transfected COS-7
Cells--
36-42 h after the transfection, the transfected COS-7
cells were loaded with 10 µM of an acetoxymethyl ester of
the pH-sensitive fluorescent dye,
2'7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF-AM, Molecular
Probes, Inc., Eugene, OR) for 30 min at 37 °C. To determine the
function of AE4, we studied the effects of Cl
removal on
the intracellular pH (pHi) in AE4- and vector-transfected COS-7
cells. The superfusion solution (solution B), containing (in
mM) NaCl 115, NaHCO3 25, KCl 5, Na2HPO4 1.6, NaH2PO4
0.4, CaCl2 1, MgCl2 1, and
D-glucose 10, at pH 7.4, was gassed with 5%
CO2 and 95% O2 and was changed continuously
with a peristapump at a flow rate of 5 ml/min. The solution was
preheated through a water-jacketed line, and the temperature of the
superfusion chamber was maintained at 37 °C.
Fluorescence was measured with an Argus 50 system (Hamamatsu Photonics,
Shizuoka, Japan). Cultured cells loaded with BCECF-AM were excited
alternately at 480 and 430 nm by a computer-controlled shutter for 1/30
of a second, and the fluorescent image at 510 nm was obtained at each
excitation wavelength (a set of fluorescent images). The ratio of the
intensity of fluorescence at 480 nm to that at 430 nm was calculated
for each set of images. The mean of 16 sets of images was taken as one
measurement value and stored and processed in a personal computer. The
16 sets of images were obtained every 10 s. After a 15-min
equilibration period, pHi measurement was started. After
measuring basal pHi for 1-2 min, solution B was quickly
changed to Cl
-free solution, containing (in
mM) sodium gluconate 115, NaHCO3 25, potassium
gluconate 5, Na2HPO4 1.6, NaH2PO4 0.4, calcium gluconate 3.5, magnesium
gluconate 1, and D-glucose 10, at pH 7.4, and gassed with
5% CO2 and 95% O2. After a 4-6-min
superfusion with the Cl
-free solution, the superfusion
solution was changed to solution B. After acquisition of fluorescent
images, the pHi was calibrated by the nigericin method as
reported previously (14). 10-20 cells in each dish were chosen, and
the pHi was calculated by using a calibration curve. In each
independent transfection, patch clamp studies and pHi
measurements on the AE4-transfected cells and mock-transfected cells
were performed on the same day. The rate of pHi change
(dpHi/dt) during the initial 30 s after
Cl
removal from the bath was calculated from
the recording of pHi by a personal computer. The pHi
and dpHi/dt measurements were also performed with
Cl
- and Na+-free solutions. The
Na+-free solution contained (in mM)
tetramethylammonium (TMA)-Cl 115, TMA-HCO3 25, K2HPO4 2, KH2PO4 0.5, CaCl2 1, MgCl2 1, and D-glucose 10. The Na+- and Cl
-free solution contained (in
mM) TMA hydroxide 115, gluconic acid lactone 115, TMA-HCO3 25, K2HPO4 2, KH2PO4 0.5, calcium gluconate 3.5, magnesium
gluconate 1, and D-glucose 10. Both solutions were gassed
with 5% CO2 and 95% O2 and adjusted to pH 7.4 by TMA hydroxide or gluconic acid lactone.
In addition, the pHi and dpHi/dt
measurements were performed with Cl
and
HCO
-free solutions in another series
of experiments with four independent transfections. The HCO
-free solution contained (in
mM) NaCl 115, sodium gluconate 15, Na-HEPES 5, H-HEPES 5, KCl 5, Na2HPO4 1.6, NaH2PO4 0.4, CaCl2 1, MgCl2 1, and D-glucose 10. The
Cl
- and HCO
-free
solution contained (in mM) sodium gluconate 130, Na-HEPES
5, H-HEPES 5, potassium gluconate 5, Na2HPO4
1.6, NaH2PO4 0.4, calcium gluconate 3.5, magnesium gluconate 1, and D-glucose 10. Both solutions
were gassed with 100% O2 and adjusted to pH 7.4 by
Na-HEPES or H-HEPES.
Patch Clamp Experiments on the Transfected COS-7 Cells--
A
coverslip to which cultured cells had adhered was placed in a recording
chamber, and the chamber was mounted on the stage of an inverted
microscope equipped with Nomarski optics (IX-70, Olympus, Japan) and
an × 40 objective lens. The chamber was continuously superfused
with solutions, gravity-fed at a rate of ~1 ml/min at room
temperature (~25 °C). Membrane voltages were recorded by a patch
clamp method in the whole cell configuration. The patch pipette was
made of Pyrex tubing pulled on a micropipette puller (P-87, Sutter
Instrument, Novato, CA). The recording pipette was connected to the
input stage of a patch clamp amplifier (Axopatch 200B, Axon
Instruments, Foster City, CA). An Ag-AgCl wire connected to the bath
via a ceramic bridge served as an indifferent electrode. The pipette
resistance was ~10 M
when filled with pipette solution. The input
capacitance and the series resistance were electrically compensated to
the maximal extent possible. Signals were low-pass filtered (Bessel
filter, cut-off frequency 5 kHz) and sampled at 10 kHz with a DigiData
1200 interface and pCLAMP 8 software (Axon Instruments) or a digital
tape recorder (Sony Precision, Japan). Recorded data were analyzed with
Igor Pro software (WaveMetrics, Lake Oswego, OR).
Three external solutions for the current clamp experiments were used.
The CO2/HCO
-free external solution contained (in mM) NaCl 115, sodium gluconate 15, KCl 4, CaCl2 2, MgCl2 1, Na-HEPES 5, H-HEPES 5, and D-glucose 10 (pH 7.4, gassed with 100%
O2). The
CO2/HCO
external solution
contained (in mM) NaCl 115, NaHCO3 25, KCl 4, CaCl2 2, MgCl2 1, and D-glucose 10 (pH 7.4, gassed with 5% CO2 and 95% O2). The
Na+-free external solution contained (in mM)
TMA-Cl 115, TMA-HCO3 25, KCl 4, CaCl2 2, MgCl2 1, and D-glucose 10 (pH 7.4, gassed with
5% CO2, 95% O2). The pipette solution
contained (in mM) KCl 20, potassium gluconate 110, MgCl2 2, CaCl2 1, EGTA 5, and HEPES 10 (pH
7.3).
cRNA Synthesis and Uptake Experiments Using Xenopus laevis
Oocytes--
cRNA synthesis and uptake measurements were performed as
described previously (15). The capped cRNA was synthesized in
vitro using T7 RNA polymerase from the plasmid DNA linearized with
HindIII. Defolliculated oocytes were injected with 10-50 ng
of the capped AE4 cRNA and incubated in Barth's solution, which
contained (in mM) NaCl 88, KCl 1.0, Ca(NO3)2 0.33, CaCl2 0.44, MgSO4 0.8, NaHCO3 2.4, and HEPES 10 with 10 µg/ml gentamicin at 18 °C. After 2-3 days of incubation, uptake
experiments were performed at room temperature in flux media (which
contained (in mM) NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5) containing 0.1 mM bumetanide
to block the endogenous
Na+-K+-2Cl
cotransporter as
reported previously (16). A group of 4-8 oocytes was placed in
microtiter wells containing 300 µl of the flux medium with 3.52 µCi
of Na36Cl. This quantity of isotope added 14.4 mM to the NaCl concentration of the flux medium. After
incubation, isotopic influx was terminated by rapid transfer through
three 5-ml washes with Cl
-free ND-96 (which contained (in
mM) sodium isethionate 96, potassium gluconate 2, calcium gluconate 1.8, magnesium gluconate 1, HEPES 5, bumetanide 0.1).
Individual oocytes were then quickly transferred to 4-ml scintillation
vials containing 250 µl of 10% SDS. Sodium-independent 36Cl
flux was measured in the
Na+-free flux medium (which contained (in mM)
TMA-Cl 86, TMA hydroxide 20, KCl 2, CaCl2 1.8, MgCl2 1, HEPES 5, bumetanide 0.1, H36Cl 20). In
this experiment, after incubation, the oocytes were washed three times
with 5 ml of Cl
- and Na+-free medium (which
contained (in mM) TMA hydroxide 96, gluconic acid lactone
96, potassium gluconate 2, calcium gluconate 1.8, magnesium gluconate
1, bumetanide 0.1). The 36Cl
flux
was also measured in a flux medium containing 200 µM DIDS (which contained (in mM) NaCl 96, KCl 2, CaCl2
1.8, MgCl2 1, HEPES 5, bumetanide 0.1) with 3.52 µCi of
Na36Cl for each sample. In these experiments, DIDS was
added 20 min before the start of incubation with the flux medium. The
influx values were calculated as nanomoles of chloride per oocyte in the time course experiments. In the experiments on sodium dependence and DIDS sensitivity, the influx values were calculated as nanomoles of
chloride per oocyte per min.
Preparation of Specific Antibody to AE4--
Rat polyclonal
antibody was raised against a synthetic peptide corresponding to the 15 C-terminal amino acids of AE4 (residues LMYQPKAPEINISVN). A cysteine
residue was attached to the N terminus of the peptide to introduce an
SH residue for coupling. The 16-amino acid CLMYQPKAPEINISVN was
synthesized by the 9-fluorenylmethyloxycarbonyl method. A synthetic
peptide conjugated with keyhole limpet hemocyanin by the
sulfo-maleimidobenzoyl-N-hydroxysuccinimide ester method was
used to immunize two Wistar rats with Freund's complete adjuvant (first injection) or incomplete adjuvant (from the second injection). After five injections, a sufficient increase of the antibody titer was
confirmed by enzyme-linked immunosorbent assay, and serum was
collected. Specific antibodies were prepared from the antiserum by
affinity column chromatography using the antigen peptide coupled to
2-fluoro-1-methylpyridinium-toluene-4-sulfonate-activated Cellulofine (Seikagaku Kogyo, Tokyo, Japan).
Immunoblotting--
Membrane fractions were prepared from the
kidneys of female New Zealand White rabbits. The kidneys were perfused
with phosphate-buffered saline (PBS) via the renal artery. After
washing as much blood as possible out of the kidney, the cortex, outer
medulla, and inner medulla were dissected. Crude tissue homogenates
were prepared by homogenization in ice-cold buffer (which contained (in
mM) sucrose 320, Tris-HCl 10, EDTA 2, PMSF 0.1, pepstatin A
1 µg/ml, leupeptin 1 µg/ml) with a Teflon-glass homogenizer. Crude
homogenates were centrifuged at 1,000 × g for 20 min
to remove nuclei and tissue debris, and a supernatant was obtained. The
supernatant was further centrifuged at 105,000 × g for
20 min, and the pellet was suspended in the same buffer (membrane
fraction). Total protein concentrations were determined by the Lowry method.
Fifty micrograms of the membrane fraction from each part of the kidney
were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
followed by immunoblotting. SDS-PAGE was performed as described by
Laemmli (17) on 5% gels. Proteins were transferred onto a Hybond ECL
nitrocellulose filter (Amersham Pharmacia Biotech) and probed with
affinity-purified anti-AE4 antibody. Bound antibodies were visualized
by an ECL Western blotting system (Amersham Pharmacia Biotech) with
peroxidase-conjugated goat anti-rat IgG (Cappel, ICN Pharmaceuticals
Inc., Aurora, OH) as the second antibody. To determine antigen
specificity, the antibody solutions (0.5 µg/ml of IgG) were
preabsorbed with the antigen peptide (50 µg/ml).
Immunohistochemical Studies--
Kidneys from female New Zealand
White rabbits were fixed by perfusion of a
periodate/lysine/paraformaldehyde mixture via the renal artery. The
kidney pieces were embedded in OCT compound in dry ice and isopentane,
and thin sections (~6 µm) were prepared with a cryotome. The kidney
sections were stained with affinity-purified polyclonal rat antibody
raised against the C terminus of rabbit AE4 and post-stained with
peroxidase-conjugated goat anti-rat IgG (Cappel) as follows. Endogenous
peroxidase was blocked by incubation in 0.3% hydrogen peroxide in
methanol. The sections were washed in PBS and incubated overnight at
4 °C with affinity-purified polyclonal rat antibody against rabbit
AE4 (IgG concentration 4 µg/ml). The tissue sections were then washed
in PBS (4 times) and incubated for 2 h in peroxidase-conjugated
goat anti-rat IgG diluted 1:200 in PBS. The sections were washed in PBS
(4 times), and peroxidase was detected by incubation in solution
containing 0.1% 3,3'-diaminobenzidine and 0.01% hydrogen peroxide.
After color development, the samples were washed in PBS. For light
microscopy, the sections were counterstained with methyl green,
dehydrated, and coverslipped. For immunofluorescence observations,
rhodamine-labeled goat anti-rat IgG (Cappel) was used as the second
antibody. After incubation with the first antibody, the tissue sections
were stained in PBS containing rhodamine-labeled antibody and
FITC-labeled peanut lectin (10 µg/ml) for 2 h. The sections were
washed in PBS (4 times) and viewed by confocal laser scanning
microscopy (LM510, Carl Zeiss Co. Ltd., Jena, Germany). For FITC, the
excitation and emission wavelengths were 488 and 505 nm, respectively.
For rhodamine, the excitation and emission wavelengths were 568 and 585 nm, respectively. To determine antigen specificity, the antibody solutions were preabsorbed with the antigen peptide (100 µg/ml).
Chemicals and Statistics--
The results are expressed as
means ± S.E. in the text and figures. Statistical analysis was
performed by one-way analysis of variance and Fisher's test.
p < 0.05 was considered to denote statistical
significance. Unless stated otherwise, all the chemicals employed were
purchased from Sigma or Wako Junyaku Co. (Osaka, Tokyo, Japan).
 |
RESULTS |
As described under "Experimental Procedures" and Fig.
1, the full-length cDNA of AE4 was
cloned and sequenced. Among the 30 clones sequenced, only 2 clones
contained a novel cDNA sequence of AE. Since the sequences of the
degenerate primers contained sequences completely matching those of AE1
and AE2 and were 80-90% homologous with the cloned AE4, the
relatively low frequency of the AE4 clone among the 30 clones does not
necessarily mean that AE4 mRNA expression is low in the sorted
-intercalated cells. The nucleotide sequences obtained were 3164 and
3116 bp in length and contained open reading frames of 2865 and 2817 bp, respectively. The shorter form (AE4b) of the cDNA lacked 48 bp
of the longer form (AE4a) and was considered to have been derived from
alternative splicing. The proteins predicted for AE4a and AE4b were
composed of 955 and 939 amino acids, respectively, and AE4a showed 48 and 34% sequence identities with human NBC1 (GenBankTM
accession number AF007216) and rabbit AE2, respectively (Fig. 2). The homologies of AE4a with human
NBC2 (AB012130), human NBC3 (AF047033), rat NBCn1 (AF069511), and
Drosophila Na+-driven anion exchanger (AF047468)
were 41, 38, 37, and 40%, respectively.

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Fig. 1.
Cloning strategy for AE4 cDNA.
(1), RT-PCR of total RNA from -intercalated cells was
performed with the degenerate primers, F-1 and R-1, and an ~300-bp
clone was obtained. (2), RT was performed with an
AE4-specific primer (RT-1). A specific reverse primer was
designed from the sequence of the ~300-bp clone (R-2).
With a forward primer designed from the consensus sequence of the AE
family (F-2), PCR was again performed, and an ~1-kb clone
was obtained. (3), a probe (probe-1) was designed
from the sequence of the 1-kb clone, and a cDNA library was
screened. An ~2.3-kb clone was obtained and sequenced.
(4), to obtain the 5'-end of AE4, RT was performed with
AE4-specific primer (GSP-31), and 5'-RACE was performed with
a specific reverse primer (GSP-32) designed from the
sequence of the 2.3-kb clone and the Abridged Anchor Primer
(AAP). An ~1.1-kb clone was obtained and ligated with the
2.3-kb clone at the KpnI site.
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Fig. 2.
The deduced amino acid sequence of AE4a and
its homology to AE1 to AE3. In the multiple sequence alignment, AE
sequences (GenBankTM accession numbers X12609, S45791, and
AF031650) identical to AE4a are highlighted by the shaded
areas. The deduced amino acid sequence of AE4b lacks 16 residues
in the N-terminal region (underlined). The amino acid
sequence for the antibody is double underlined. The
predicted membrane-spanning domains are indicated by the dotted
line. The GenBankTM accession numbers for AE4a and
AE4b are AB038263 and AB038264, respectively.
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As shown in Fig. 3a, the
hydropathy profile of AE4 is very similar to that of the other anion
exchangers. The newly cloned AE4 has an extensive hydrophilic
N-terminal region with about 400 amino acids and a C-terminal region of
about 500 amino acids, which is rich in hydrophobic residues. Twelve
membrane-spanning domains were predicted from the primary amino acid
sequence using a Kyte-Doolittle algorithm. The topology showed a marked
similarity to that of the AE and NBC superfamily (18-20). In the
region homologous to the AE-consensus DIDS-binding motif (21),
KLXK (where X is I, V or Y), AE4 has the sequence
KMLN (amino acids 517-520), and there are a few protein kinase A and
protein kinase C sites in the cytoplasmic domain (Fig.
3b).

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Fig. 3.
a, hydropathy plot of AE4. Twelve
membrane-spanning domains were predicted from the primary amino acid
sequence using a Kyte-Doolittle algorithm (window size, 18 amino
acids). b, membrane model of the AE4 protein. The putative
membrane-spanning domains are indicated by numbers. The
DIDS-binding motifs are indicated by diamonds. Three
consensus N-linked glycosylation (N-glyc) sites
are present in AE4a, although only two are predicted to be
extracellular (amino acids 548 and 572). Of the three consensus protein
kinase A (PKA) sites, two are predicted to lie in the
intracellular region (Ser-275 and Ser-370; circles). Of the
six consensus sites for protein kinase C (PKC), five are
predicted to be intracellular (Ser-169, Ser-270, Thr-302, Thr-354, and
Thr-622; square).
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Northern blots of rabbit tissue poly(A)+ RNA hybridized
with an AE4a cDNA probe detected a major transcript of ~3.2 kb
with three additional minor bands in the kidney cortex (Fig.
4a) at the lengths of ~3.8,
~3.4, and 2.8 kb. These minor bands might be products of alternative
splicing, although we identified only one shorter transcript by
cDNA sequencing, which lacked 16 residues in the N-terminal region.
We detected no expression of the ~3.2-kb AE4a mRNA from the
kidney medulla, spleen, liver, skeletal muscle, lung, brain, heart,
stomach, and small intestine, whereas a very faint band at ~3.2 kb
was observed in the case of the large intestine. In the inner medulla,
small intestine, and spleen, however, faint bands at ~3.8 kb, also
seen in the kidney cortex, were revealed. In addition, in the heart and
skeletal muscle, faint bands were observed at ~4 kb. It is possible
that these ~4-, ~3.8-, ~3.4-, and 2.8-kb lengths bands may
reflect cross-hybridization of the AE4a probe with isoforms of the AE
and NBC superfamily. It is also possible that they are novel isoforms
of AE4.

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Fig. 4.
Northern blot analysis of AE4 expression in
rabbit tissues. Lanes 1-12, 1, kidney
cortex; 2, kidney outer medulla; 3, kidney inner
medulla; 4, stomach; 5, small intestine;
6, large intestine; 7, pancreas; 8,
liver; 9, spleen; 10, heart; 11,
skeletal muscle; 12, brain. Each lane was loaded with 1-5
µg of poly(A)+ RNA. The AE4 probe was used in
a, and the probe for glyceraldehyde 3-phosphate in
b.
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Expression of the AE4a cDNA in COS-7 cells caused ~60% of the
cells on the coverslips to undergo an increase in pHi in
response to Cl
removal from the bath, whereas ~40% of
the cells showed a small increase in pHi (Fig.
5b). Cell histograms, which
are classified based on the dpHi/dt value, are shown
in Fig. 6. All of the cells with mock
transfection showed dpHi/dt below 3.0 × 10
3 pH units/s, whereas about 60% of the
cells with AE4a transfection showed dpHi/dt higher
than 4.0 × 10
3 pH units/s. In each
transfection, the frequency of the cells with
dpHi/dt values higher than 4.0 × 10
3 pHi units/s was calculated. The
frequency of cells with higher dpHi/dt was 63.2 ± 6.3%, whereas GFP transfection indicated transfection efficiency to
be 68.8 ± 7.2%. Since the transfection efficiencies determined
by functional and GFP studies were in good agreement, it is assumed
that the high dpHi/dt values observed in a portion
of the transfected cells reflect the function of transfected AE4. Rapid
alkalinization after bath Cl
removal was also seen in the
Na+-free medium (Fig. 5d), indicating that cells
showing transient expression of AE4a possess sodium-independent anion
exchanger activity on their membranes. Mock-transfected cells, on the
other hand, showed very little alkalinization in response to
Cl
removal from the bath in either
Na+-containing or Na+-free solution (Fig. 5,
a and c). In Fig. 5, the basal pHi of
mock-transfected cells and cells with lower dpHi/dt was higher than that of cells with higher dpHi/dt.
The basal pHi in the mock-transfected and AE4-transfected cells
are summarized in Table I. Theses values
did not differ significantly among three groups of cells in either
Na+-free or Na+-containing solutions.

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Fig. 5.
Representative pHi changes in
response to bath Cl removal in the presence or absence of
Na+. a, the representative pHi
changes in the mock-transfected cells. Each plot denotes the mean
pHi values of three cells on one coverslip. b, the
representative pHi changes in the AE4-transfected cells. In
this experiment, about 60% of the pcDNA AE4a-transfected cells on
the coverslip showed rapid alkalinization, and 40% showed a slight
degree of alkalinization after the removal of Cl from the
bath. The representative pHi changes in the cells with rapid
alkalinization (closed circles) and a slight degree of
alkalinization (open circles) on the same coverslip were
shown. Each plot denotes the mean values of the three cells of each
type on one coverslip. c, the representative pHi
changes in the mock-transfected cells after bath Cl
removal in the absence of Na+. Each plot denotes the mean
pHi values of the three cells on one coverslip. d,
the representative pHi changes in the AE4-transfected cells
after bath Cl removal in the absence of Na+.
In this experiment, the pattern of pHi changes was divided in
two types, as shown in c. About 40% of the cells showed
slow and slight alkalinization, whereas about 60% showed rapid
alkalinization after Cl removal from the bath in the
absence of Na+. The representative pHi changes in
the cells with rapid alkalinization (closed circles) and a
slight degree of alkalinization (open circles) on the same
coverslip were shown. Each plot denotes the mean values of three cells
of each type on one coverslip.
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Fig. 6.
Cell histograms based on the rates of
pHi changes after bath Cl removal.
Open bar indicates the mock-transfected cells. Shaded
bar indicates the AE4-transfected cells. As shown in the figure,
dpHi/dt in the mock-transfected cells was less than
3.0 × 10 3 pH units/s. On the other
hand, about 60% of the cells with AE4 transfection had
dpHi/dt values higher than 4.0 × 10 3 pH units/s.
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Table I
Basal intracellular pH in the AE4-transfected and mock-transfected
cells
AE4 transfected cells were classified by the rate of intracellular pH
change (dpHi/dt) after bath Cl removal.
High dpHi/dt is defined as a value 4.0 × 10 3 pH units/s and low dpHi/dt as a value
lower than <3.0 pH units/s.
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In a different series of experiments, the dpHi/dt
values were measured with or without bath
CO2/HCO
. In the absence
of CO2/HCO
,
dpHi/dt values were still significantly higher in
AE4a-transfected cells (2.2 ± 0.32 × 10
3 pH units, n = 98 cells of
all examined cells on the coverslips) than in mock-transfected cells
(0.29 ± 0.31 × 10
3 pH units,
n = 54 cells, p < 0.05), although, in
AE4a-transfected cells, the dpHi/dt values in the
presence of CO2/HCO
(12.0 ± 0.68 × 10
3 pH unit,
n = 56 cells with high dpHi/dt,
p < 0.001 compared with values in the absence of
CO2/HCO
) were much higher
than in its absence. Furthermore, since COS-7 cells possess small
endogenous anion exchanger activity as shown in Figs. 5 and 6, the
responses between AE4 expressed and nonexpressed cells in the absence
of CO2/bicarbonate were not obviously separated from each other among
AE4-transfected cells. These responses were markedly different from the
experiments with CO2/HCO
(Fig. 6). These data, however, strongly suggest that AE4 mediates Cl
/HCO
exchange,
whereas it is also suggested that AE4 mediates Cl
/base
exchange in the absence of HCO
.
To investigate whether this protein transports Cl
, AE4a
was expressed in Xenopus oocytes, and the
36Cl
uptake was measured. As shown in Fig.
7a,
36Cl
uptake was linear up to 15 min of
incubation. Therefore, the oocytes were incubated for 15 min in
subsequent studies. As shown in Fig. 7b, the oocytes
injected with the cRNA of AE4a showed significantly higher
36Cl
uptake than that in the control oocytes,
and this increase in 36Cl
uptake by cRNA
injection of AE4a was not significantly changed by 200 µM
DIDS. Furthermore, in the oocytes injected with the cRNA of AE4a,
36Cl
uptake in Na+-free medium
was similar to that in control flux medium. These 36Cl
uptake data reveal that this anion
exchanger is sodium-independent and DIDS-insensitive.

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Fig. 7.
36Cl uptake by the
AE4 cRNA-injected Xenopus oocytes. A, in
the oocytes injected with AE4 cRNA, 36Cl
uptake was linear up to 15 min. Open circles, AE4
cRNA-injected oocytes; closed circles, control oocytes. The
data from four independent experiments were summarized, and each plot
denotes the mean value of 20-30 cells. b, the
36Cl uptake in sodium-free medium and the
medium containing 200 µM DIDS. The
36Cl uptake values were expressed as
nanomoles of Cl /min/oocyte.
36Cl uptake values do not differ
significantly among the three groups in either AE4 cRNA-injected cells
or control cells. Shaded column indicates the mean
36Cl uptake values of AE4 cRNA-injected
oocytes in the control, Na+-free, and 200 µM
DIDS solutions. Open column indicates the mean
36Cl uptake values of control oocytes in the
control, Na+-free, and 200 µM DIDS solutions.
n = 14-18 in each group. The values from three
independent experiments were pooled. Vertical bars indicate
standard errors. *, p < 0.05, compared with the
corresponding control oocytes in the control, Na+-free, and
200 µM DIDS solutions.
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Since AE4 showed significant homology to NBC, we investigated the
effect of Na+ removal from the bath on membrane voltage in
the presence of HCO
by the patch
clamp method (Figs. 8, a and
b). The basal membrane voltage of the mock-transfected cell
was approximately
20 mV (
23 ± 1.6 mV, n = 6 cells) and was not significantly different from the membrane voltage of
the AE4-transfected cells (
25 ± 1.3 mV, n = 32 cells). The membrane voltage of mock-transfected cells was not
significantly changed by bath Na+ removal (Fig.
8a), and an AE4-transfected cell showed a similar response
(Fig. 8b). Examining 32 AE4-transfected and 6 mock-transfected cells from four independent transfections, no cells
showed significant membrane voltage changes in response to bath
Na+ removal. In the patch clamp study, the cell viability
was verified by the response to high K+-external solution,
and all cells were shown to retain K+ sensitivity. In these
four transfection studies, since the efficiency of GFP transfection and
the frequency of cells with high dpHi/dt were 68.8 and 63.2%, respectively, it is extremely unlikely that all of the
cells examined by patch clamp expressed no AE4 protein. These patch
clamp studies confirmed that the AE4a-transfected COS-7 cells do not
possess electrogenic NBC activity.

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Fig. 8.
Representative recording of the membrane
voltage by the whole cell patch clamp technique. a, a
sample recording of a mock-transfected cell. The cell did not show any
significant changes in membrane voltage by the removal of bath
Na+, whereas it was depolarized by high K+
solution. The other five cells also showed similar behavior.
b, a sample recording of an AE4-transfected cell. Similar to
the mock-transfected cells, this cell did not show any voltage changes
by the removal of bath Na+, whereas it was depolarized by
high K+ solution. Thirty two cells in four independent
transfections were examined, and no cell showed significant changes in
membrane voltage with the removal of bath Na+. In these
four independent transfections, the pHi changes after bath
Cl removal were also examined, and 63.2% of the cells
had anion exchanger activity, as shown in Fig. 5.
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We generated a polyclonal antibody specific for AE4 to examine the
localization of this protein in the kidney. The specificity of the
antibody was examined by immunoblot analysis with membrane fractions
prepared from rabbit kidneys (Fig. 9).
Immunoblotting showed three bands in the cortex and two bands each in
the outer medulla and the inner medulla. Among the three bands in the
cortex, those at ~96 and ~90 kDa were also seen in the outer
medulla and the inner medulla, whereas the band at ~110 kDa was seen
only in the cortex. The band at ~110 kDa was abolished by
preabsorption of the antibody with the synthetic peptides used for
immunization. The ~96- and ~90-kDa bands were considered to be
nonspecific, since these bands were not affected by the preabsorption
of the antibody with the antigen peptides. As shown in Fig.
10, a and b, the
immunohistological study revealed AE4 immunoreactivity only in certain
types of cells in the kidney cortex. Moreover, AE4 immunostaining was
detected only on apical cell membranes (Fig. 10b). This
staining was completely abolished by preabsorption of the antibody with
an excess of the synthetic peptide used for immunization (Fig.
11). In this Fig. 11, the serial
sections of the same kidney were used. Since we used ~6-µm sections
of the kidney, it was difficult to determine precisely which cells were preserved in the two consecutive sections. The same collecting duct seen in the left panel of Fig. 11, however, was
preserved in the right panel, and the AE4 labeling in this
tubule was not seen by preabsorption of the antibody with an excess of
the synthetic peptide used for immunization. The immunohistochemical
studies with preabsorbed antibody were performed three times, and all three experiments gave the same results.

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Fig. 9.
Representative Western blot analysis.
Western blot analysis was performed on the membrane fractions prepared
from rabbit kidney cortex (C), outer medulla
(OM), and inner medulla (IM). Left
panel, the affinity-purified anti-AE4 antibody was used as the
first antibody. There were three bands in the cortex and two bands each
in the outer medulla and the inner medulla. Among the three bands in
the cortex, the bands at ~96 and ~90 kDa were also seen in the
outer medulla and the inner medulla, whereas the band at ~110 kDa was
seen only in the cortex. Right panel, the affinity-purified
anti-AE4 antibody was preincubated with the synthetic peptides used for
immunization (50 µg/ml). The band at ~110 kDa in the cortex was
abolished by this preincubation, whereas the ~96- and ~90-kDa bands
were not affected by preabsorption of the antibody with antigen
peptides, indicating that these bands are nonspecific.
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Fig. 10.
Immunohistological study with
affinity-purified antibody directed against the C terminus of AE4.
a, AE4-positive-cells are seen only in the cortex in a
punctate pattern. b, at higher magnification, the labeling
is mainly localized to the apical membrane
(arrowheads).
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Fig. 11.
When affinity-purified anti-AE4 antibody was
preabsorbed with an excess of the synthetic peptide used for
immunization (100 µg/ml), apical staining by
the antibody (arrowheads, left panel) was completely
abolished (right panel, a serial section of the
left panel).
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Double staining with FITC-labeled peanut lectin and rhodamine-labeled
anti-rat IgG was performed to characterize the AE4-positive cells. In
the rabbit kidney, peanut lectin is known to bind exclusively to the
apical membrane of
-intercalated cells (1, 3). As illustrated in
Fig. 12, double staining studies
clearly showed that all of the AE4-positive cells (red) were also
peanut lectin-positive (green), indicating that AE4 is only expressed
in
-intercalated cells. There were also a few cells that were peanut
lectin-positive and AE4-negative. These cells may represent variants of
the intercalated cells (7). Rhodamine labeling is shown to be localized
exclusively on the apical membrane by confocal microscopic observation,
whereas the subapical region is also weakly stained in some cells. The immunostaining by anti-AE4 antibody, however, was not detected in the
basolateral membrane of any cells. On the other hand, the double
staining studies with preabsorbed antibody were also performed three
times, and all three experiments gave no significant staining in the
tubules except red blood cells (data not shown). In these experiments,
red blood cells showed rhodamine fluorescence even without the first
antibody, indicating that this staining is nonspecific binding of the
second antibody to red blood cells.

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Fig. 12.
Confocal laser microscopy showed all
AE4-positive cells to also be positive for peanut lectin
(arrowheads). Two cells in this photomicrograph
(arrows) are positive for peanut lectin (green)
but negative for AE4 (red).
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DISCUSSION |
In the present study, we identified the apical anion exchanger in
-intercalated cells of the kidney as a new member of the bicarbonate
transporter superfamily. The newly cloned transporter showed homology
to members of the anion exchanger family and the NBC family, and the
homology to the NBC family was higher than that to the anion exchanger
family. The expressed protein in COS-7 cells and Xenopus
oocytes, however, showed anion exchanger activity. Immunohistological
study confirmed this protein to be localized to the apical membranes of
-intercalated cells.
We have cloned a novel anion exchanger from the mRNA of sorted
peanut lectin-positive cells, assuming that peanut lectin is one of the
best markers of the
-intercalated cells. The nomenclature for the
subtypes of the intercalated cells is somewhat confusing, although it
is common for the acid-secreting type to be called
-type and for the
bicarbonate-secreting type to be called
-type (1). Previous
functional studies suggested that
-intercalated cells possess a
basolateral anion exchanger and apical H+-ATPase for acid
secretion, whereas
-intercalated cells possess an apical anion
exchanger and basolateral H+-ATPase for bicarbonate
secretion. The immunohistochemical study by Schuster et al.
(3) revealed the basolateral membrane of a certain type of intercalated
cells to be labeled by anti-AE1 antibody. Deducing from the functional
studies, it was suggested that
-type intercalated cells possess AE1
on their basolateral membranes. On the other hand, the intercalated
cells without AE1 labeling showed apical peanut lectin binding,
suggesting that peanut lectin is a marker for
-intercalated cells.
Consistent with this immunohistochemical study,
electrophysiological analysis of the intercalated cells (22, 23)
indicated that there were two types of the intercalated cells, the
cells with apical anion exchanger and the cells with basolateral anion
exchanger, and that both types of the cells had basolateral
Cl
conductance. On the other hand, with the measurement
of the intracellular pH, Emmons and Kurtz (8) showed the existence of a
third type of intercalated cells,
-type cells, which possess anion
exchanger activity on the both apical and the basolateral membranes. In their microperfusion study, the response of pHi to
Cl
removal from the bath or luminal perfusate was
determined in all of the peanut lectin-positive cells. In the rabbit
outer cortical collecting ducts, 4% of the intercalated cells were
considered to be
-type, whereas 96% were peanut lectin-positive.
Among the intercalated cells, 57% showed peanut lectin labeling and
anion exchanger activities on both the apical and the basolateral
membranes, and this type of the cell was defined as
-type. Thirty
nine percent of the intercalated cells showed anion exchanger activity
only on the apical membrane as well as apical peanut lectin binding, and this type of the cell was defined as
-type. The anion exchangers on basolateral and apical membranes in
- and
-type cells,
however, shared common characteristics, i.e. they were both
sodium-independent and DIDS-insensitive. Moreover, the functional
studies by Weiner et al. (24) revealed all peanut
lectin-positive cells with apical anion exchanger activity to also have
anion exchanger activity on their basolateral membranes, whereas
basolateral anion exchanger was also insensitive to DIDS and was
Na+-independent. The reason for the discrepancy between
these two in vitro microperfusion studies is not clear,
although the authors raised the possibility that a sex difference of
the rabbits, male in Emmons' study and female in Weiner's study,
might have resulted in a difference in the proportion of intercalated
cells with anion exchanger activity on both the apical and the
basolateral membranes. Both functional studies, however, demonstrated
that all peanut lectin-positive cells had apical anion exchanger
activity, which is required for bicarbonate secretion. It is likely
that peanut lectin-positive cells including
-type cells work as
bicarbonate secreting, i.e.
-intercalated, cells.
Consistent with the functional studies of peanut lectin-positive cells
(8, 9), our newly cloned anion exchanger, AE4, was sodium-independent
and DIDS-insensitive, and this result strongly indicates that AE4 is an
apical anion exchanger of
-intercalated cells. The results of the
present immunohistological study with anti-AE4 antibody and peanut
lectin (Fig. 12) also support this possibility.
The antibody to AE4, however, failed to label some of the peanut
lectin-positive cells. These peanut lectin-positive and AE4-negative cells may represent a variant of
-intercalated cells (7). The
results from the further immunohistochemical studies with anti-H+-ATPase and anti-AE1 antibodies and peanut lectin by
Schuster et al. (7) were somewhat different from their
previous work (3). In cortical collecting ducts, double staining with
anti-H+-ATPase and anti-AE1 antibodies showed that ~40%
of the intercalated cells had diffuse or apical H+-ATPase
staining with basolateral AE1 labeling, indicating that these cells are
-type intercalated. In the rabbit kidney, the H+-ATPase
staining was diffuse in the remaining of the intercalated cells but not
exclusively present on the basolateral membrane, and this staining
pattern in the rabbit kidney was different from that in the rat kidney
(2). The results of double staining with peanut lectin and
anti-H+-ATPase antibody yielded results different from
those of their previous report (3). Seventy five percent of the
intercalated cells showed diffuse H+-ATPase staining and
apical peanut lectin labeling, and these are considered to be
-type
intercalated cells. Seventeen percent of peanut lectin-positive cells
showed apical H+-ATPase staining or no labeling by
anti-H+-ATPase antibody. Furthermore,
fluorescence-activated cell sorting showed 6-18% of the peanut
lectin-positive cells to also be positive for AE1. The functions of
these variants of peanut lectin-positive cells were not clarified in
their study, although the authors raised the possibility that there may
be hybrids of
- and
-intercalated cells. In these studies and the
immunohistological study by Madsen et al. (6), however, it
is consistent that apical membrane of the intercalated cells has never
been labeled by anti AE1 antibody.
Considering these functional (8, 9, 24) and immunohistological studies
(3, 7), AE4 is considered to be an apical anion exchanger in
-intercalated cells, which are defined as cells with anion exchanger
activity on the apical membrane, whereas we cannot deny a possibility
that AE4 may be also the apical anion exchanger of
-intercalated
cells in the study by Emmons et al. (8) or of the hybrid
cells reported by Schuster et al. (7). Furthermore, from our
present immunohistological study, it is highly likely that there is
another form of apical anion exchanger in
-intercalated cells. On
the other hand, it is unlikely that AE4 is a basolateral anion
exchanger of
- and
-intercalated cells, since our
immunohistological data did not show any basolateral labeling by
anti-AE4 antibody. These possibilities and molecular characteristics of
the basolateral anion exchanger in
-intercalated cells remain to be
determined in future studies.
The amino acid sequence of AE4 showed 30-48% homology with the known
members of the sodium bicarbonate cotransporter family. Among these
transporters, AE4 showed the highest homology to human NBC1, which
induces electrogenic bicarbonate efflux in a
sodium-dependent manner (19). In contrast to the homology
with NBC, when AE4 was expressed in COS-7 cells and Xenopus
oocytes, AE4 revealed sodium-independent and DIDS-insensitive anion
exchanger activity, as described under "Results." The experiments
with and without CO2/bicarbonate in the superfusion
solution indicated that high dpHi/dt values in
AE4a-transfected cells with CO2/bicarbonate were caused by
Cl
/bicarbonate exchanger activity of AE4. On the other
hand, dpHi/dt values were small but significantly
higher in AE4a-transfected cells than those in mock-transfected cells
without bath CO2/bicarbonate, suggesting that AE4 mediates
Cl
/base exchange. This transporter activity of AE4,
Cl
/base exchanger activity in the absence of
CO2/bicarbonate, is consistent with the previous report in
the apical anion exchanger of the
-intercalated cells (25), and
Cl
/base exchanger activity has been also reported in the
other types of anion exchangers in various cells (26-28). Furthermore,
to confirm that AE4 is not an electrogenic sodium bicarbonate
cotransporter, we performed patch clamp experiments. With sodium
removal from the bath in the presence of bicarbonate, the transfected
cells did not show changes in membrane voltage, suggesting that AE4 does not possess electrogenic sodium-dependent transporter
activity, which is a characteristic feature of NBC1 (19). Regarding
NBCn1 (29), NBC3 (20), and nDAE1 (30), the sodium-independent feature
of AE4 transporter activity distinguishes it from these transporters.
These results indicate that AE4 is a transporter distinct from the
previously known members of the NBC family. Since the transporter
function of this novel protein is a sodium-independent anion exchanger,
we named the newly cloned transporter as AE4, despite its homology
being highest to NBC1.
Based on its localization, AE4 probably mediates
HCO
efflux from
-intercalated
cells into the luminal fluid. In regard to the cell polarity change
theory (31, 32), since the immunohistological study with anti-AE4
antibody showed no labeling of the basolateral membranes in the
collecting ducts, a total cell polarity change of intercalated cells is
unlikely. Instead, it is possible that the two types of intercalated
cells interchange by switching their expressions of the
AE1 and AE4 genes and by sorting of
H+-ATPase. The cloning of AE4 should shed light upon the
molecular mechanisms underlying the cell polarity change.
In conclusion, we have succeeded in the molecular cloning of an apical
anion exchanger from
-intercalated cells of the kidney collecting
ducts, and this finding should provide insight into both the molecular
and the cell biological mechanisms that regulate acid-base balance in
the kidney.
 |
ACKNOWLEDGEMENT |
We thank Dr. Shingo Kato for technical support.
 |
FOOTNOTES |
*
This work was supported in part by grants from the Ministry
of Education, Science, and Culture of Japan, and a National
grant-in-aid for the Establishment of High-Tech Research Center in a
Private University.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB038263 and AB038264.
**
To whom correspondence should be addressed. Tel.: 81-3-5363-3796;
Fax: 81-3-3359-2745; E-mail: matuhiko@mc.med.keio.ac.jp.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M004513200
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ABBREVIATIONS |
The abbreviations used are:
NBC, sodium
bicarbonate cotransporter;
AE, anion exchanger;
RT, reverse
transcription;
PCR, polymerase chain reaction;
FITC, fluorescein
isothiocyanate;
PBS, phosphate-buffered saline;
BCECF-AM, 2'7'-bis(carboxyethyl)-5,6-carboxyfluorescein;
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid;
RACE, rapid
amplification of cDNA ends;
TMA, tetramethylammonium;
pHi, intracellular pH;
dpHi/dt, rate of intracellular pH
change;
bp, base pair;
kb, kilobase pair;
GFP, green fluorescent
protein.
 |
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