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Am J Physiol Cell Physiol 296: C1279C1290, 2009.

First published March 25, 2009; doi:10.1152/ajpcell.00638.2008.

Regulation of intestinal Cl/HCO


3 exchanger SLC26A3 by intracellular pH
Hisayoshi Hayashi,1 Kazuhito Suruga,2 and Yukari Yamashita1
1

Laboratory of Physiology and 2Laboratory of Nutritional Physiology, School of Nutritional Sciences, University of Shizuoka,
Suruga-ku, Shizuoka, Japan

Submitted 15 December 2008; accepted in final form 23 March 2009

Hayashi H, Suruga K, Yamashita Y. Regulation of intestinal


Cl/HCO
3 exchanger SLC26A3 by intracellular pH. Am J Physiol
Cell Physiol 296: C1279 C1290, 2009. First published March 25, 2009;
doi:10.1152/ajpcell.00638.2008.SLC26A3, a Cl/HCO
3 exchanger,
is highly expressed in intestinal epithelial cells, and its mutations
cause congenital chloride diarrhea. This suggests that SLC26A3 plays
a key role in NaCl absorption in the intestine. Electroneutral NaCl
absorption in the intestine is mediated by functional coupling of the
Na/H exchanger and Cl/HCO
3 exchanger. It is proposed that the
coupling of these exchangers may occur as a result of indirect linkage
by changes of intracellular pH (pHi). We therefore investigated
whether SLC26A3 is regulated by pHi. We generated a hemagglutinin
epitope-tagged human SLC26A3 construct and expressed it in Chinese hamster ovary cells. Transport activities were measured with a
fluorescent chloride-sensitive dye dihydro-6-methoxy-N-ethylquinolinium iodide (diH-MEQ). pHi was clamped at a range of values from
6.0 to 7.4. We monitored the transport activity of SLC26A3 by reverse

mode of Cl/HCO
3 and Cl /NO3 exchange. None of these exchange
modes induced membrane potential changes. At constant external pH
7.4, Cl/HCO
3 exchange was steeply inhibited with pHi decrease
between 7.3 and 6.8 as opposed to thermodynamic prediction. In
contrast, however, Cl/NO
3 exchange was essentially insensitive to
pHi within physiological ranges. We also characterized the pHi
dependency of COOH-terminal truncation mutants. Removal of the
entire COOH-terminal resulted in decrease of the transport activity
but did not noticeably affect pHi sensitivity. These results suggest that
Cl/HCO
3 exchange mode of human SLC26A3 is controlled by a
pH-sensitive intracellular modifier site, which is likely in the transmembrane domain. These observations raise the possibility that
SLC26A3 activity may be regulated via Na/H exchanger 3 (NHE3)
through the alteration of pHi under physiological conditions.
electroneutral NaCl absorption; downregulated in adenoma; dihydro6-methoxy-N-ethylquinolinium iodide

play many physiological roles, including regulation of cell volume, fluid secretion, and acid-base balance (3,
10, 32). An efficient absorption of Cl in the intestine is
important to maintain the optimal levels of Cl in the body.
Three chloride absorptive pathways have been proposed (15,
25): 1) a paracellular pathway, which is dependent on potential
difference; 2) an electroneutral pathway involving parallel
functioning of Na/H exchange and Cl/HCO
3 exchange;

and 3) an HCO
3 -dependent Cl absorptive pathway, which is
not coupled to a parallel Na/H exchange. Among these
chloride-absorptive mechanisms, electroneutral NaCl absorption is thought to be a predominant pathway.
NHE3 (SLC9A3) is a major Na/H exchanger contributing
to NaCl absorption, and its regulation was investigated extensively (21, 46). In contrast, the molecular identity of the
CHLORIDE IONS

Address for reprint requests and other correspondence: H. Hayashi, Laboratory


of Physiology, School of Nutritional Sciences, Univ. of Shizuoka, Yada 52-1,
Surugaku, Shizuoka 422-8526, Japan (e-mail: hayashih@smail.u-shizuoka-ken.
ac.jp).
http://www.ajpcell.org

Cl/HCO
3 exchanger involved in NaCl absorption still remains incompletely understood. At least four Cl/HCO
3
(OH) exchangers (SLC4A1, SLC4A2, SLC26A3, and
SLC26A6 ) have been found in intestinal epithelial cells (9, 35,
37, 43). Binder and colleagues (2, 33, 34) have shown that rat
SLC26A3 mediates 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS)-insensitive Cl/OH exchange, and SLC4A1
(AE-1) mediates DIDS-sensitive Cl/HCO
3 exchange in the
rat colon. Genetic analysis studies have shown that mutations
in human SLC26A3 result in congenital chloride-losing diarrhea (CLD), a disease-manifested metabolic alkalosis with
diarrhea containing high chloride concentrations (22). The
phenotype of SLC26A3-deficient mice was similar to CLD in
humans (38). Both human and mouse SLC26A3 have been
shown to function as a Cl/HCO
3 exchanger in the heterologous expression systems (11, 29). These studies suggested that
SLC26A3 serves an important role in intestinal Cl/HCO
3
exchange and Cl absorption. Unfortunately, a specific inhibitor for SLC26A3 is currently not available, although it has
been inhibited by niflumic acid more potently than DIDS in the
heterologus expression systems (11).
The Na/H exchangers and Cl/HCO
3 exchangers are
coupled since they are activated/inactivated under same conditions (1, 13, 17, 31, 42). It is believed that indirect linkage
through changes in intracellular pH (pHi) is involved: changes
in one exchanger could generate a H/HCO
3 gradient locally
that might affect the other exchangers activity (23). This mode
of coupling would have been facilited by close residency to
each other since both NHE3 and SLC26A3 seem to bind to
PDZ domain containing proteins (27, 36, 44). There are two
possible mechanisms for the intracellular H/HCO
3 generated
by one exchanger to affect the other exchangers. First, H/
HCO
3 can act as a substrate by binding to the transport site.
Second, H/HCO
3 might act as a regulation signal by binding
to a modifier site that is independent of H/HCO
3 transport.
NHE3 (and NHE1) is well known to be activated by H
binding to a modifier site that is independent of H transport
site. On the other hand, it has been studied less extensively
whether SLC26A3 has modifier sites that are sensitive to pHi
(or intracellular HCO
3 ) (11).
As a first step toward understanding the details of coupling
between the Na/H exchangers and the Cl/HCO
3 exchangers in the intestine, we conducted experiments to define the pHi
sensitivity of SLC26A3 using a heterologous expression systems in the Chinese hamster ovary (CHO) cells. To measure
the activity of SLC26A3, we used a chloride-sensitive fluorescent dye dihydro-6-methoxy-N-ethylquinolinium iodide (MEQ).
We clamped the pHi at various levels with nigericin in media
containing varying K concentrations. We found that Cl/
HCO
3 exchange mediated by human SLC26A3 was steeply
inhibited by acidic pHi. In addition, we characterized the pHi
dependency of human SLC26A3 mutant in which the entire

0363-6143/09 $8.00 Copyright 2009 the American Physiological Society

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SLC26A3 AND INTRACELLULAR pH

COOH terminal was deleted. Removal of the COOH terminal


decreased transport activity but did not noticeably affect the
pHi sensitivity.
MATERIALS AND METHODS

Materials and solutions. Nigericin, 2,7-bis(2-carboxyethyl)-5(6)carboxyfluorescein (BCECF)-AM, MEQ, Alexa 546-conjugated goat
anti-mouse antibody, Alexa 488-conjugated wheat germ agglutinin
(WGA), Blasticidin, and Zeocin were obtained from Invitrogen
(Carlsbad, CA). Mouse anti-hemagglutinin (HA) antibody was from
Covance (Berkeley, CA). Fluorometric imaging plate reader (FLIPR)
membrane potential kit (red) was from Molecular Devices (Sunnyvale, CA). Tunicamycin was from Sigma (St. Louis, MO).
Isotonic HEPES-buffered medium contained (in mM): 140 KCl, 1
MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with
Tris at 37C). The HCO
3 -buffered solutions contained (in mM) 115
KCl, 25 KHCO3, 1 MgCl2, 1 CaCl2, and 10 glucose, bubbled with 5%
CO2-95%O2. The Cl-free solution was prepared by equimolar substitution with gluconate or nitrate, except that 1 mM CaCl2 was
replaced with 5 mM Ca-gluconate.
Establishment of cell lines harboring wild-type or mutant
SLC26A3. It has been shown that stable transfectant of SLC26A3 is
hard to establish, since SLC26A3 induces growth suppression (8). To
overcome this problem, we established SLC26A3-expressing cells
using inducible gene expression systems (Flp-In T-Rex core kit,
Invitrogen). Full-length coding SLC26A3 transcripts were amplified
from human colon Marathon-Ready cDNA (Clontech, Mountain
View, CA) using the Advantage HF-2 PCR kit (Clontech) with
primers designed from mRNA of human SLC26A3 (NCBI accession
number NM_000111). A PCR was performed with the following
primers: 5-AATGATTGAACCCTTTGGGAATCAGTA-3 and 5CATAGTCAGATGAAGATCCTTCTGAATCAT-3. The PCR conditions were as follows: 94C for 15 s, followed by 30 cycles at 94C
for 10 s, 60C for 30 s, and 68C for 4 min. PCR products were cloned
into the TA vector pGEM T Easy (Promega, Madison, WI) according
to the manufacturers instructions. The full-length of nucleotide sequence of SLC26A3 was confirmed. To facilitate immunological
detection, we constructed a double hemagglutinin (HA)-tagged vector. An HA sequence containing linkers were cloned into HindIII-NotI
sites of pcDNA5/FRT/TO (Invitrogen). The NotI fragment containing
full-length of SLC26A3 was then excised from TA vector and inserted
into the NotI-site of HA double tag containing pcDNA5/FRT/TO,
yielding plasmid pcDNA/FRT/TO/HA-Full-SLC26A3 that was used
to incorporate the full-length SLC26A3 gene into Flp-InCHO cells
(Invitrogen). Flp-InCHO cells were maintained in Dulbeccos modified Eagles medium (DMEM) /F-12 containing 10 g/ml blasticidin
and 100 g/ml Zeocin. Stable cells harboring HA-tagged SLC26A3
protein were generated by cotransfection of pcDNA/FRT/TO/ HAFull-SLC26A3 and the pOG44 plasmid (Invitrogen) into Flp-InCHO
cells by lipofectamine 2000 (Invitrogen) and were selected by limiting
dilution in the presence of hygromycin B (200 g/ml) and blasticidin
(10 g/ml). Expression of HA-tagged SLC26A3 was followed by
immunofluorescence. Protein expression in stable cells was induced
by adding tetracycline or doxycycline to culture media at a final
concentration of 1 g/ml for 24 h at 37C before experiments.
For transient transfection, NotI fragments containing full-length of
SLC26A3 were excised and inserted into mammalian expression
vector D-HA-pRc/CMV (Riken, Japan), which contains a double (HA)
epitope tag, located at the NH2 terminus of the protein of interest. This
resultant plasmid D-HA-pRc/CMV-Full-SLC26A3 was used to generate COOH-terminal deletion mutants of SLC26A3. The COOHterminal deletion mutants were truncated at positions 524, 643, and
705 (constructs 524, 643, and 705, respectively) using KODPlus-Mutagenesis kit (TOYOBO).
Measurement of intracellular chloride. Intracellular Cl concentration ([Cl]i) was determined by the Cl-sensitive fluoroprobe
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dihydro-MEQ (diH-MEQ). DiH-MEQ, the membrane-permeant form


of MEQ, was synthesized from MEQ according to the manufactures
instructions. The synthesized diH-MEQ was dissolved at a concentration of 300 mM in dimethyl sulfoxide and stored at 80C for up
to 1 mo. The cells were seeded onto 25-mm round glass coverslips and
mounted in a custom-made perfusion chamber that allowed continuous superfusion of the cells. The cells were then loaded with diHMEQ by incubation with 300 M diH-MEQ for 10 min at 37C in a
Ca2- and Mg2-supplemented phosphate-buffered saline (PBS).
Microfluorometry was performed as previously described (19) with
modifications. Briefly, the chamber was placed on the stage of an
inverted microscope (TE200-U, Nikon, Tokyo, Japan) equipped with
a microscopic dual-wavelength fluorometer system (CAM-230, Japan
Spectroscopic, Tokyo, Japan), perfused at 6 ml/min with the gasequilibrated solution and maintained at 3537C. The temperature of
the solutions was maintained at 37C using a water-jacketed stainless
tubing inlet. Solutions were delivered by gravity to the chamber
through CO2-less impermeable tubing. Clusters of the cells were
excited at 350 nm for 50 ms every 2 s, and the fluorescence was
measured at 400 nm through a bandpass filter. All these procedures
were controlled by a computer (Macintosh LC), which was equipped
with a data acquisition and analysis system (Lab View 2, National
Instruments, Houston, TX). In situ calibration was performed at the
end of each experiment by measuring the fluorescence intensity F0
obtained when the cells were exposed to the Cl-free solution containing 10 M amphoterin B. At the end of the measurement the cells
were exposed to 140 mM KSCN in the absence of Cl to obtain the
background fluorescence. Changes in F0/F in each experiment were
then converted to changes in [Cl]i using the Stern-Volmer constant
obtained from the pooled data.
Measurement of pHi and membrane potential. Measurement of the
fluorescence of intracellular BCECF or FLIPR red dye was performed
essentially as described for the measurements using diH-MEQ. To
measure cytosolic pH, the cells were loaded with 0.5 M BCECF-AM
for 10 min at 37C in Ca2- and Mg2-supplemented PBS. For
membrane potential measurement, the cells were loaded with FLIPR
red dye for 30 min at 37C according to the manufacturers instructions. Fluorescence intensity was measured at emission wavelength
520 nm and excitation at 440 and 490 nm for BCECF or at excitation
wavelength 530 nm and emission 565 nm for the FLIPR red dye.
Manipulation of pHi. To manipulate pHi, the cells were treated with
K/H-exchanging ionophore nigericin (10 M) and varying extracellular concentrations of K, as previously described (18) with
modifications. Since at equilibrium the ratios of intracellular and
extracellular K and H concentrations are equal ([K]i/[K]o
[H]i/[H]o), the desired pHi can be calculated from the imposed
[K] gradient and pHo, assuming a [K]i of 140 mM. Based on these
considerations, the solutions (in mM) used contained the following:
for pHi 7.4, 140 K; for pHi 7.0, 56 K; for pHi 6.8, 35 K; for pHi
6.4, 14 K; and for pHi 6.0, 5.6 K. N-methyl-D-glucamine was used
to balance the osmolarity.
Immunofluorescence. The cells were plated onto glass coverslips,
rinsed with PBS, and fixed using 4% paraformaldehyde (PFA) in PBS
for 20 min. After fixation, the cells were incubated with 100 mM
glycine in PBS for 20 min. The cells were then incubated with Alexa
488-conjugated WGA (100 g/ml) for 20 min before permeabilization. The cells were next preblocked with 5% skimmed milk and
permeabilized in 0.1% Triton in PBS for 30 min and then incubated
with anti-HA monoclonal antibody (1:1,000 dilution) for 1 h. After
being washed three times to remove unbound antibody, the cells were
incubated with Alexa 546-conjugated donkey anti-mouse antibody
(1:1,000 dilution) for 1 h. After final washes, the samples were
mounted onto glass slides using Dako medium (Dako, Carpinteria,
CA). Images were acquired using a laser-confocal microscope (Zeiss
LSM510).
Immunoblotting. The adherent cells were washed with PBS and
scraped off with a rubber policeman into 1 Laemmli sample buffer,

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SLC26A3 AND INTRACELLULAR pH

and samples were subjected to SDS-PAGE and transferred onto


nitrocellulose filters (Hybond, Amersham Bioscience). Blots were
blocked with 5% skimmed milk in PBS containing 0.1% Triton X-100
and exposed to anti-HA monoclonal antibody (1:5,000 dilution),
followed by horseradish peroxidase-conjugated secondary antibody
(1:5,000 dilution, Pierce, Rockford, IL). Immunoreactive proteins
were visualized by enhanced chemiluminescence (Super Signal,
Pierce) and exposed to Kodak film.
Statistical analysis. Experimental values are given as the means
SE of the indicated number of the determinations. Comparisons
between two groups were made by either unpaired or paired Studentss t-test, as appropriate.
RESULTS

Expression and functional characterization of HA-SLC26A3.


We first studied the expression of HA-SLC26A3 by immnoblotting. As shown on Fig. 1A, two immunoreactive bands were
detected in the HA-SLC26A3-expressing CHO cells using
anti-HA antibody: a lower band (85 kDa) and upper band
(120 kDa). The upper band migrated as a wide band, likely
attributable to glycosylation. This was confirmed by treatment
of the cells with an N-glycosylation inhibitor tunicamycin.
Treatment with tunicamycin resulted in the virtual disappearance of the two bands and the appearance of a single 78-kDa

Fig. 1. Expression of SLC26A3 in Flp-In Chinese hamster ovary (CHO) cells.


A: detection of HA-SLC26A3 by immunoblotting. The cells were cultured in
the absence (Doxy) or presence (Doxy) of doxycycline for 24 h. Where
noted, the cells were treated with 5 g/ml tunicamycin. The whole cell lysates
were solubilized in Laemmli sample buffer and used for SDS-PAGE and
immunoblotting using anti-hemagglutin (HA) antibodies. Immunoreactive
bands were visualized by enhanced chemiluminescence. BD: subcellular
localization of HA-tagged SLC26A3. Flp-In CHO cells harboring HA-tagged
SLC26A3 were cultured with or without (insets) 1 g/ml doxycycline for 24 h.
The cells were then fixed with paraformaldehyde (PFA) and stained with Alexa
488-labeled wheat germ agglutinin (WGA) (green) before permeabilization
(B). After permeabilization, the cells were immunostained using anti-HA
antibody followed by Alexa 546-labeled (red) anti-mouse secondary antibody
to visualize SLC26A3 (C). Confocal slices acquired near the middle of the
cells are shown. The letters with apostrophe are the corresponding x versus z
reconstructions. The images are representative of at least 4 similar experiments. Bar in D, 5 m.
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band, in agreement with a previous study (14). This indicates


that similar to the wild-type parental protein, HA-SLC26A3 is
modified posttranslationally by N-linked glycosylation (7).
This also verifies that the expressed protein undergoes normal
maturation in CHO cells. We next determined the subcellular
distribution of HA-SLC26A3 with confocal microscopy (Fig.
1, BD). To confirm the cell surface expression, the plasma
membrane was stained with WGA before permeabilization of
the cells. As shown in Fig. 1D, the epitope-tagged protein was
colocalized with WGA, suggesting that it is expressed on the
cell surface. Transverse reconstructions confirmed the surface
staining (Fig. 1, BD). Some fraction of HA-SLC26A3 appeared to be intracellular, possibly reflecting the immature
protein that can be detected by SDS-PAGE as a lower molecular weight band (85 kDa).
We next verified whether the protein tagged with HAepitope retained its functional properties by fluorometry in
diH-MEQ-loaded cells. Figure 2A shows a typical calibration
of diH-MEQ by exposing the cells to the amphotericin B and
by varying the perfusate Cl concentration. To calibrate
[Cl]i, we used amphoterin B to vary [Cl]i. Since these
antibiotics form Cl-permeable pores, it is a convenient
method to use for altering [Cl]i in the cells (4, 16). Figure 2B
is a Stern-Volmer plot of the pooled data. The Stern-Volmer
constant was 82.3 M1. We first assessed the effect of expression of SLC26A3 on resting pHi and [Cl]i (Table 1). Expression of HA-SLC26A3 did not significantly change the resting
level of [Cl]i compared with the wild-type CHO cells (P
0.19). These values are within the range of 29 47 mM, which
was measured by other methods in the CHO cells (6, 40),
demonstrating the validity of our measurements. Upon expression of HA-SLC26A3, basal resting pHi was slightly but not
significantly decreased (P 0.25) (Table 1). Antiport activity
was assessed by the removal of extracellular Cl from the
perfusate. Since a small DIDS-insensitive Cl/HCO
3 exchange activity was occasionally seen before induction of the
exchanger (data not shown), probably due to contamination of
doxycycline or leakiness of the inducible expression systems,
we used the wild-type CHO cells as a control for functional
experiments. In wild-type CHO cells, upon removal of extracellular Cl from the perfusate only a small reduction of [Cl]i
was observed (1.6 1.7 mM/min, n 6, Fig. 2C). As shown
in Fig. 2D, HA-SLC26A3-expressing cells responded to removal of Cl with a vigorous reduction of Cl (61.7 11.7
mM/min, final [Cl]i 19.8 1.4 mM, n 10) . The initial
rate of [Cl]i decrease was almost completely inhibited by 50
M niflumic acid (by 89%, n 3, Fig. 2, D and F). In contrast,
H2-DIDS did not significantly inhibit the rate of [Cl]i decrease induced by Cl removal, the rate being 41.8 11.6 and
40.4 6.7 mM/min in the cells incubated with or without 250
M H2-DIDS, respectively (Fig. 2, E and F). HA-SLC26A3expressing cells responded to removal of Cl with a large
intracellular alkalinization (pH 0.69 0.05, n 5, Fig.
2G). Furthermore, pHi increase induced by removal of extracellular Cl was virtually not observed in nominally HCO
3free HEPES-buffered solution (0.11 0.03, n 3, Fig. 2H).
Jointly, these observations indicated that HA-tagged SLC26A3
retained Cl/HCO
3 exchanger in agreements with previous
studies (11).

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Fig. 2. Functional properties of SLC26A3 in Flp-In CHO cells. A: representative trace of dihydro-6-methoxy-N-ethylquinolinium iodide (diH-MEQ) calibration
performed by exposing the cells to the Cl-permeable ionophore amphotericin B and by varying the perfusate Cl concentration. At the end of the measurement, the
cells were perfused with 150 mM KSCN containing solution to obtain background fluorescence. B: Stern-Volmer plot of calibration data calculated from six experiments
like that in A. Data are means SE. Where error bars are not apparent, they are within the size of the symbol. Fo and F are the values of the fluorescence intensity in
the absence and presence of Cl, respectively. D and E: the intracellular Cl changes were determined fluorometrically with diH-MEQ after induction of SLC26A3.

Measurements were done in the presence of CO2/HCO


3 . The cells were stained with diH-MEQ, perfused with 120 mM Na -containing, HCO3 -buffered solution.
Where indicated by the bars, Cl was removed from the perfusate (by replacing with gluconate). To determine the effect of niflumic acid on SLC26A3 activity, 50 M
niflumic acid was added to the perfusate where indicated. Arrows indicate initial rate of intracellular Cl concentration ([Cl]i) decrease. Similar measurements were
done in wild-type CHO cells (C). E: representative trace showing the effect of H2-DIDS on SLC26A3 activity. Traces are representative of at least three experiments.
F: summary of effects of inhibitors on SLC26A3 activity. *P 0.05 compared with control. Representative traces showing the effect of removal of extracellular Cl
on intracellular pH (pHi) in the CO2/HCO
3 -buffered solution (G) and HEPES-buffered solution (H) in the SLC26A3-expressing cells.

Effect of Cl/HCO
3 exchange activity on membrane potential. Shcheynikov et al. (39) found earlier that mouse SLC26A3
is an electrogenic transporter with a 2Cl/1HCO
3 exchange
stoichiometory in the oocytes. In contrast, human SLC26A3
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has not been shown to be electrogenic in the oocytes and


HEK293 cells (11, 26). We therefore assessed the electrogenicity of SLC26A3 using a membrane potential-sensitive dye.
Since fluorescence response is larger at depolarized membrane

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Table 1. Effect of expression of SLC26A3 on resting


intracellular pH and Cl concentration

Wild-type
SLC26A3-expressing

Cli, mM

pHi

43.12.7 (6)
38.22.1 (10)*

7.002.7 (4)
6.890.09 (5)*

Each value represents the mean SE. The number in parentheses indicates
the number of experiments. Cli, intracellular Cl concentration; pHi,
intracellular pH. *Not significant when compared with wild-type.

potentials with this dye, we performed experiments in media


that were containing 100 mM K. As illustrated in Fig. 3B (left
of the trace), the cells expressing SLC26A3 depolarized membrane potential upon removal of extracellular Cl in the
HCO
3 -buffered solutions. However, the magnitude of this depolarization did not differ significantly from that in the wild-type
CHO cells (Fig. 3, A and C). These membrane potential
changes could be due to endogenous Cl conductive pathway(s) in the wild-type CHO cells. The FLIPR membrane
potential assay kit contains not only an oxonol-type voltagesensitive dye but also a fluorescent quencher (see review, 30).
It is conceivable that Cl/HCO
3 exchange activity may be
inhibited by the quencher. To rule out a potential artifact, we
measured the rate of Cl/HCO
3 exchange with BCECF after
the cells were loaded with FLIPR kit. The magnitude of
alkalinization induced by the Cl-free solution was virtually
not changed after FLIPR dye loading (pHi 0.7, n 2)

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(not shown). These observations suggest that Cl/HCO


3
exchange mode of SLC26A3 is electroneutral. Furthermore,
the rates of Cl/HCO
3 exchange activity, as measured by
diH-MEQ, were not affected by changing the perfusate K
concentration (Fig. 3D).
Cl/HCO
3 exchange mode of SLC26A3 is sensitive to pHi.
Having established that SLC26A3 expressed in the CHO cells
is fully functional, we next wanted to investigate the sensitivity
of the exchanger to pHi. Therefore, we monitored the rate of
[Cl]i decrease, reflecting chloride exchange for HCO
3 , at
varying pHi in the CO2/HCO
3 -buffered solution. We first
established the conditions for clamping pHi. All measurements
were performed in media that were Na free, to minimize the
contribution of Na-dependent pHi regulators. By adding an
electroneutral K/H-exchanging ionophore nigericin at sufficiently high concentrations and by setting the transmembrane
K gradient and extracellular pH to predetermined levels, pHi
can be clamped to any desired value. As shown in Fig. 4, upon
changing the extracellular K concentration from 145 to 35
mM, pHi was changed from 7.2 to 6.8. This result suggested
that nigericin is present in high enough concentrations for pHi
clamping in the CO2/HCO
3 -buffered solution. However, it
took 5 min to equilibrate pH by setting the transmembrane
K gradient alone in the CO2/HCO
3 -buffered solution. Therefore, we clamped pHi by using HEPES- or 4-morpholinoethanesulfonic acid (MES)-buffered-solutions before each
measurement (Fig. 5A). Under this condition, pHi reached

Fig. 3. The changes of membrane potential induced by removal of extracellular Cl. Representative traces showing membrane potential changes induced by
extracellular Cl removal in wild-type CHO cells (A) and in cells expressing SLC26A3 (B). Membrane potential was monitored fluorometrically with FLIPR
red dye. Where indicated, the concentration of Cl in the perfusate was altered. Cl-free solution was prepared by equimolar substitution with gluconate or
nitrate, in the CO2/HCO
3 -buffered solution (left part of the trace) and HEPES-buffered solution (middle of the trace), respectively. Finally, to calibrate the
membrane potential, the cells were perfused with varying K concentrations (right part of the trace). To facilitate comparison between experiments, the data were
normalized to the fluorescence intensity where the cells were perfused with 150 mM K. The traces are representatives of 3 experiments for each experiment.
C: summary of membrane potential changes induced by extracellular Cl removal in the CO2/HCO
3 -buffered solution and HEPES-buffered solution. D: effect
of extracellular K concentrations on Cl/HCO
3 exchange activity as measured by diH-MEQ.
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SLC26A3 AND INTRACELLULAR pH

Fig. 4. Effect of varying extracellular K concentrations on pHi in the


CO2/HCO
3 -buffered solution. The efficiency of the clamping procedures was
verified by measuring pHi. The cells were treated with 10 M nigericin, and
the concentration of K in the perfusate was altered in the CO2/HCO
3buffered solution. The trace is representative of three experiments in the
SLC26A3-expressing cells.

equilibrium within 2 min. Switching the perfusate from a


pHi-clamping solution to the experimental one containing CO2/
HCO
3 caused only a small shift of pHi (Fig. 5A). When the
Cl/HCO
3 exchange was initiated by removal of extracellular
Cl, pHi was increased and reached its nadir, suggesting that
the Cl/HCO
3 exchange activity is greater than the nigericin
exchanging activity. Recovery of pHi was observed upon
readdition of extracellular Cl returned to the original level
(Fig. 5B, open squares), indicating that pHi can be clamped
stably at the desired pHi level. As summarized in Fig. 5B, pHi
was clamped nearly at the desired levels at acid pHi in both the
wild-type (open squares) and the SLC26A3-expressing CHO
cells (closed circles). We next monitored the Cl/HCO
3 exchange activity by using Cl-sensitive dye under the same
experimental condition (Fig. 5C). Three measurements were
performed consecutively on the same cells. The mean value of
the initial rate of [Cl]i decreases from the first and third
measurements was taken as the control value, which were
clamped at pHi 7.4 and compared with the rate in the acidified
cells. When the cells were clamped at clamping pH of 6.4, the
resting [Cl]i was slightly but significantly increased compare
with the levels at clamping pH of 7.4 (31 1 and 35 1 mM,
n 7, P 0.02). In addition, the initial rate of [Cl]i decrease
upon removal of extracellular Cl was inhibited by 50% (Fig.
5C). The open squares in Fig. 8A summarizes the results of
experiments performed with clamping pHi at various levels, the
initial rate being steeply inhibited with pHi decreased between 6.8
and 7.3. It should be noted here that under the present experimental condition acid-loaded cells would be expected to show a
greater rate of Cl/HCO
3 exchange activity due to the increased
inward-directed HCO
3 gradient. Nevertheless, we found that the
Cl/HCO
3 exchange activity was decreased in the acid-loaded
cells.
Cl/NO
3 exchange mode of SLC26A3. In the preceding
experiments, we could not clamp the pHi alone, because
intracellular HCO
3 was also changed inevitably under the
conditions used. To overcome this problem, we took advantage
of the ability of SLC26A3 to carry NO
3 . Therefore, we next
monitored the changes of [Cl]i as chloride exchanges for
nitrate at varying pHi in nominally HCO
3 -free HEPES-buffered solution. We first verified that NO
3 was transported via
HA-SLC26A3 in diH-MEQ-loaded cells. Fluorescence of
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MEQ is quenched in the presence of Cl but not NO


3 (5). As
shown in Fig. 6A, when perfusate Cl was totally replaced

with NO
3 in wild-type CHO cells, a small [Cl ]i decrease was
observed and this was totally abolished by H2-DIDS (4.4 0.7
vs. 0.4 0.9 mM/min in the absence and presence of 125 M
H2-DIDS, respectively, n 3), suggesting that this Cl/NO
3
exchange is via endogenous H2-DIDS-sensitive anion exchange. In contrast, in SLC26A3-expressing cells, replacement
of extracellular Cl with NO
3 led to a rapid and reversible
decrease of [Cl]i (39.8 6.3 mM/min, final [Cl]i 24.3
1.8 mM, n 9). This [Cl]i decrease was attenuated by
nifulumic acid (Fig. 6, C and D) but insensitive to H2-DIDS
(Fig. 6, B and D). These results suggested that SLC26A3 can

Fig. 5. Effect of intracellular acidification on Cl/HCO


3 exchange activity.
A: representative trace of pHi in the SLC26A3-expressing cells. pHi was
clamped at the desired values by using pHi-clamping solutions that contained
the K/H exchanging ionophore nigericin (Nig, 10 M) in HEPES- or
MES-buffered solution. A pHi-clamping solution was applied intermittently.
Switching the perfusate from the pHi-clamping solution to the experimental

one containing CO2/HCO


3 (pH 7.4) and varying [K ] caused only small
shift of pHi. In the experimental solution, the removal of extracellular Cl still

caused an increase in pHi, reflecting HCO


exit.
3 uptake in exchange for Cl
However, these pHi changes cannot be used for quantitative estimation of
Cl/HCO
3 exchange activity since they are distorted by Nig. B: summary of
the relationship between expected pHi, which is calculated from imposed [K]

and [H ] gradients (see MATERIALS AND METHODS), and observed pHi in


wild-type CHO cells (open squares, n 35) and in SLC26A3-expressing
cells (before and after Cl/HCO
3 exchange, closed and open circles respectively, n 38) when perfused with experimental solutions. Where error bars
are absent, they are smaller than the symbol used. C: representative trace of
[Cl]i to determine the effect of intracellular acidification on Cl/HCO
3
exchange activity in the SLC26A3-expressing cells. Arrows indicate initial rate

of [Cl ]i decrease.

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Fig. 6. Cl/NO3 exchange activity in HEPES-buffered solution. Representative traces showing the effect of extracellular Cl removal on [Cl]i (A) and pHi
(E) in wild-type CHO cells. The cells were perfused with 130 mM Na containing HEPES-buffered solution, followed by perfusing with Cl-free solution (Cl
was replaced with nitrate). Representative traces of effects of extracellular Cl removal on [Cl]i (B) and pHi (F) in SLC26A3-expressing cells. Where indicated
by the bar, 125 M H2-DIDS was added. Each trace is representative of at least 3 experiments. C and A: representative trace showing the effect of niflumic acid

on Cl/NO
3 exchange activity. Traces are representative of three experiments. D: summary of effects of inhibitors on Cl /NO3 exchange activity in the
SLC26A3-expressing cells. *P 0.05 compared with control.

mediate Cl/NO
3 exchange. It was reported that mouse
SLC26A3 can also mediate Cl/OH exchange (24). Therefore to evaluate a possible Cl/OH exchange, we measured
pHi using pH-sensitive dye BCECF using the same conditions
as for the [Cl]i measurements (Fig. 6, E and F). When the
perfusate Cl was totally replaced with NO
3 , a small decrease
of pHi was observed in the wild-type CHO cells (0.22 0.02,
n 3), this magnitude of acidification was not significantly
different from that in the SLC26A3-expressing cells (0.23
0.06, n 3, P 0.91), suggesting that Cl/OH exchange
mode is not operating. Although we did not study further this
NO
3 -induced acidification mechanism, it might be due to
proton nitrate cotransporter (12). It was reported previously
that mouse SLC26A3 operates as uncoupled anion transport
when NO
3 was used as substrates (39). We therefore measured
membrane potential when Cl was replaced with NO
3 in the
HEPES-buffered solution. As illustrated in Fig. 3B, the cells
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expressing SLC26A3 were hyperpolarized when Cl was removed from the perfusate (0.15 0.03 arbitrary unit, n 3).
However, this magnitude of hyperpolarization did not differ
significantly from that in the wild-type CHO cells (0.11 0.01
arbitrary unit, n 3, P 0.26, Fig. 3, A and C). These results
excluded that Cl exit in the SLC26A3-expressing cells occurred via conductive pathway(s). These membrane potential
changes could be due to the entry of NO
3 via endogenous
conductive pathway(s) in the wild-type CHO cells. Furthermore, varying transmembrane K ratios did not affect the rate
of intracellular Cl decrease (data not shown). Together, these
results suggest that SLC26A3 can transport NO
3 in exchange
for Cl exit via an electroneutral process.
We next examined pHi dependency of SLC26A3 in the

Cl/NO
3 exchange mode in nominal HCO3 -free HEPESbuffered solution. In this buffer solution, pHi was clamped at a
desired level in SLC26A3-expressing cells (Fig. 7A). As shown

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SLC26A3 AND INTRACELLULAR pH

immunobloting as two distinct bands (53, 90 kDa) (Fig. 9J).


This suggests that the upper band was fully glycosylated. To
confirm this, the cells were treated with tunicamycin, resulting
in the disappearance of the two bands and their converge to a
single 49-kDa band. In contrast, as shown in Figs. 9, E and H,
643 and 709 were diffusely distributed throughout the
cytoplasm and did not localize to the cell surface membrane.
Immunobloting revealed that these mutants migrate as only one
band, observed at the predicted molecular size of 71 and 81
kDa, respectively. This suggests that proper trafficking of these
mutants to the plasma membrane is prevented.
We next established cells harboring truncated mutants of
SLC26A3 using inducible gene expression systems. Using
these cells we measured the Cl/HCO
3 exchange activity by
diH-MEQ. As shown in Fig. 9K, the removal of the entire
COOH-terminal domain resulted in a decrease in the Cl/
HCO
3 exchange activity in the 524 mutant (12 3 mM/min,
n 4) compared with wild-type SLC26A3. However, the pHi
sensitivity was still observed when the pHi was clamped at
clamping pH of 6.4 (3 1 mM/min, n 4 Fig. 8A, closed
circle). In contrast, we could not detect any Cl/HCO
3 exFig. 7. Effect of intracellular acidification on Cl/NO
3 exchange activity. A
representative trace showing pHi changes induced by removal of extracellular
Cl ([Cl]o). Effect of acidification on pHi changes induced by removal of
[Cl]o in the SLC26A3-expressing cells (A). Cl-free solution was prepared
by equimolar substitution of Cl to nitrate. Where indicated, the concentration
of K and Cl, and pH in the perfusate was altered. A representative trace of
the effect of intracellular acidification on Cl/NO
3 exchange activity (B). pHi
was clamped at clamping pH of 6.4 as in Fig. 6A in separate cells. Arrows
indicate initial rate of [Cl]i decrease.

in Fig. 7B, the rate of [Cl]i decrease was not inhibited when
the cells were acidified at clamping pH of 6.4. The open
squares in Fig. 8B summarizes the results of experiments at
various clamping pH. The rate of Cl/NO
3 exchange activity
was not significantly inhibited at pHi values between 7.4 and
6.4. When the cells were challenged by a clamping pH of 6
(observed pHi, 6.2), the initial rate was inhibited only by 25%
(P 0.01, n 5). In contrast to Cl/HCO
3 exchange activity,
there was no apparent inhibition by acidic pHi for Cl/NO
3
exchange activity. This could be due to the absence of extracellular/intracellular CO2/HCO
3 -buffered condition. However, the rates of Cl exit when CO2/HCO
3 -buffered solution
containing 25 mM Cl was switched to the one containing no
Cl and 25 mM NO
3 did not differ between control and
acid-loaded cells (P 0.35, n 3, see Fig. 8B, closed square).
COOH terminus of SLC26A3 does not contribute to pH
regulation. It has been shown that removal of the entire COOH
terminal cytoplasmic domain of SLC26A3 abolished the sensitivity of pHi in the oocytes (11). It is thought that histidine
residue is a candidate for a pH sensor, since their imidazole
side chain have a pKa value of near 7. Comparison of amino
acid sequences of the COOH terminal of SLC26A3 reveals that
three histidine residues (H647, H714, and H719) are highly
conserved among several species. To get insights into the pHi
sensor of SLC26A3, we made various COOH terminally truncated mutants. Immunofluorescence confocal microscopy was
used to define the subcellular distribution of the truncated
mutants. As shown in Figs. 9, B and B, 524: the mutant
lacking the entire COOH-terminal cytoplasmic domain appeared to be as punctate spots at the plasma membrane and
intracellular compartments. The 524 mutant was detected by
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Fig. 8. Summary of the effects of pHi on Cl/HCO


3 and Cl /NO3 exchange.

A: initial rates of [Cl ]i decrease were measured in Cl /HCO3 exchange mode


in SLC26A3-expressing cells at various pHi as in Fig. 5. Closed circles,
measurements with the 524 mutant. B: initial rates of [Cl]i decrease were
measured in Cl/NO
3 exchange mode in SLC26A3-expressing cells at various
pHi as in Fig. 7. Closed squares in B indicate measurements in Cl/NO
3
exchange mode in the presence of 5% CO2 and 25 mM HCO
3 . Experiments

were done similarly as shown in Fig. 5, but the Cl concentration was reduced
to 25 mM (the rest was replaced by gluconate), and the initial rates of [Cl]i
decrease after 25 mM Cl was replaced by 25 mM NO
3 was measured. For
all experiments, the mean values of initial rate of [Cl]i decrease from the first
and the third measurements at clamping pH at 7.4 were taken as the control
value and compared with the initial rate under acidified condition. Data are
means SE from 3 to 7 experiments. Where error bars are absent, these are
smaller than the symbol used. *P 0.05 compared with at clamping pH of 7.4.

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change activity in the 643 and 709 mutants, in agreement


with their inability to localize to the surface as shown by the
immunofluorescence images described above.
DISCUSSION

The mechanism of coupling between the Na/H exchangers and the Cl/HCO
3 exchangers in the intestine remains
only partially understood. It is thought that change of exchanger activities by pHi (intracellular [H]) through affecting
modifier site is one possible mechanism. We therefore developed a method to change pHi in the presence of CO2/HCO
3.
We conducted experiments to define the pHi sensitivity of
human SLC26A3 using heterologous expression in CHO cells.
Our results suggest that Cl/HCO
3 exchange mode of
SLC26A3 is controlled by a pH-sensitive intracellular modifier
site. Inhibition of Cl/HCO
3 exchange activity by intracellular
acidification does not result from general toxicity of acidic pHi
because this inhibition was reversible. In addition, we could
observe robust Cl/HCO
3 exchange activity after acidification
(Fig. 5). We assessed the effect of acidification on Cl/HCO
3
exchange activity in Cl outward and HCO
3 inward mode. In
this mode, acid-loaded cells would be expected to show a
greater rate of Cl efflux as a result of the larger inwardly

directed HCO
exit/
3 gradient. Therefore, inhibition of Cl

HCO3 entry by acid pHi indicates that SLC26A3 possesses a


H modifier site. Membrane potential changes could regulate
SLC26A3 exchange activity, since SLC26A3 was shown to be
a Cl/HCO
3 exchanger with a 2:1 stoichiometry (39) and
varying transmembrane [K] ratios altered pHi. In contrast, our
results do not support the electrogenic nature of SLC26A3.
First, although large [Cl]i changes (20 mM) occurred following removal and readdition of extracellular Cl in
SLC26A3 expressing cells, there was no discernable membrane potential change compared with that in the wild-type
CHO cells (Fig. 3). Second, extracellular K concentrations

did not affect Cl/HCO


3 or Cl /NO3 exchange activity.
Our immunofluorescence data show that SLC26A3 was
present both on the surface membrane and intracellular compartments (Fig. 1C). This implies that SLC26A3 activity may
be regulated by altering the number of available molecules at
the plasma membrane. However, this explanation was rendered
unlikely by the observation that Cl/NO
3 exchange activity
was not changed in acid-loaded cells (Fig. 7), suggesting that
the number of transporters on the surface is unchanged.
Other investigators have demonstrated that removal of the
entire COOH-terminal cytoplasmic domain of SLC26A3 abolished its sensitivity to pHi in oocytes (11), suggesting that a pH

Fig. 9. Comparison of the intracellular distribution of COOH terminally


truncated mutants. CHO cells were transiently transfected with cDNA encoding various truncated mutants. Cells were then fixed with paraformaldehyde
(PFA) and stained with wheat germ agglutinin (WGA) (A, D, G) and antihemagglutinin (HA) antibodies (B, E, H), as in Fig. 1. Confocal slices were
acquired near the middle of the cells. The letters with apostrophe show the
corresponding x versus z reconstructions. The images are representative of at
least 3 similar experiments of each type. Bar in I, 10 m. J: detection of
truncated mutants by immunoblotting. CHO cells were transiently transfected,
and whole cell lysates were solubilized in Laemmli sample buffer and used for
SDS-PAGE and immunoblotting using anti-HA antibodies. The images are
representative of 3 similar experiments. K: representative trace showing effect
of intracellular acidification on Cl/HCO
3 exchange activity in the 524
mutant. Traces are representative of four experiments.
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SLC26A3 AND INTRACELLULAR pH

sensor might reside within the cytoplasmic domain. However,


our results are in contrast to those findings, suggesting that pH
sensor of SLC26A3 resides in the transmembrane domain. This
discrepancy may be explained by the use of different expression system and different exchange mode of SLC26A3.
Interestingly, intracellular Cl decrease under acid pHi was
stopped approximately halfway to two-thirds of the way toward the values in more neutral condition, although pHi was
already increased to 7.4 during Cl/HCO
3 exchange (compare the middle part of the traces with the left and right parts
in Fig. 5, A and C). These results imply that the release from
the inhibition of Cl/HCO
3 exchange by pHi is slow. These
slow responses to pHi changes for activity of SLC26A3 are
reminiscent of that of pH-dependent activation for NHE3 (20).
Our results failed to demonstrate pHi sensitivity of
SLC26A3 activity when anion exchange activity was monitored by Cl/NO
3 exchange mode (Figs. 7 and 8). However,
we could observe the modest inhibition when the cells were
clamped at pHi 6.2 (Fig. 8). It is conceivable, therefore, that the
pHi-sensitivity curve is shifted to the acidic side when anion
exchange activity was monitored by Cl/NO
3 exchange mode.
We measured Cl/NO
3 exchange activity where pHi was
clamped at 5. Both extracellular and intracellular pH were
simultaneously changed. However, this condition was toxic,
and Cl/NO
3 exchange activity was totally diminished after
acidification (data not shown). Another possibility is that the
cells are exposed to large gradients of transported ions (extracellular NO
3 144 mM) in these experiments, and this may
mask pHi sensitivity of SLC26A3. To exclude this, we reduced
extracellular NO
3 concentration to 25 mM. Even under this
condition, Cl/NO
3 exchange activity was not inhibited when
pHi was clamped at 6.4 (data not shown). Since CHO wild-type
cells have endogenous Cl/NO
3 exchange activity (Fig. 6A),
we analyzed pHi dependency of this anion exchange. The
endogenous Cl/NO
3 exchange activity was completely inhibited at pHi 6.4 (data not shown). Jointly, we conclude that
the Cl/NO
3 exchange mode of SLC26A3 was essentially
insensitive to physiological pHi ranges. It is noteworthy that
the activity of AE3 was stimulated at alkaline pHi as measured
in Cl/HCO
3 exchange mode (28) but not as measured in
Cl/NO
3 exchange mode (41).
Our data indicated that the pHi dependence of SLC26A3 is
steepest in the range of pH between 6.8 and 7.3, which is well
within the physiological pHi range in the cells. Actually, this
value is in good agreement with the values for the resting pHi
in the native enterocytes (19). What are the physiological
significances of this pHi dependency of SLC26A3? One of the
functions of SLC26A3 would be to stabilize pHi of epithelial
cells. The increase in pHi would accelerate SLC26A3 to
extrude HCO
3 by affecting the modifier site, a property suggested in the present study. In addition, the increase in intracellular HCO
3 would energetically stimulate the activity of the
exchanger. On the other hand, when pHi decreases, SLC26A3
would possibly mediate HCO
3 uptake that would in turn
decrease pHi. Another and probably most important function of
SLC26A3 is to mediate Cl absorption in the intestinal epithelia. Intestinal Cl absorption is, in most cases, coupled to
Na absorption that is mediated by the apical Na/H exchanger. The Na/H exchanger and SLC26A3 transports H
or HCO
3 , respectively, are both derived from hydration of
CO2. Importantly, NHE3, which is suggested to be a major
AJP-Cell Physiol VOL

apica1 Na/H exchanger, was demonstrated to be activated


by intracellular H via a modifier site, with the half-maximal
value of 0.11 0.13 M (pH 6.89 6.96) for H. These
properties are likely to promote coupling between apica1
NHE3 and SLC26A3 since the increase in the apical SLC26A3
activity (Cl uptake) would cause pHi decrease, which then
increases apical NHE3 activity (Na uptake). By the same
mechanism, the decrease of SLC26A3 activity would induce a
reduction of NHE3 activity. The present finding that SLC26A3
activity is regulated by pHi through modifier sites would
suggest the existence of a similar but reciprocal coupling
mechanism. The increase or decrease of apical NHE3 activity
would increase or decrease pHi, which then activates or inhibits SLC26A3 and promotes the coupled Cl and Na absorption. The existence of a coupling mechanism is also suggested
by the well-known finding that increases in intracellular cAMP
in the intestinal epithelium inhibit both Na and Cl absorption simultaneously (1, 31). However, SLC26A3 does not
possess consensus site for PKA. In accordance with previous
findings (11) treatment of the cells with 8-bromo-cAMP did
not affect the Cl/HCO
3 exchange activity of SLC26A3 in the
present expression system (unpublished observation). It is well
known that NHE3 activity is acutely inhibited by activation of
cAMP-dependent protein kinase (45). Therefore, upon an increase in intracellular cAMP in the enterocytes, intracellular
acidification resulting from inhibition of NHE3 could inhibit
the Cl/HCO
3 exchange activity via the pHi-sensitive modifier sites. Whereas regulation through pHi could serve as a
potent coupling mechanism, our observations do not rule out
the possibility that an additional cofactor (e.g., sodium/hydrogen exchanger regulatory factor) is required in cAMP-mediated inhibition of SLC26A3 activity.
We have examined the subcellular distribution of various
SLC26A3 mutant proteins. Our findings show that although the
524-truncated mutant, which lacks the entire COOH-terminal
part, was expressed at the plasma membrane, its level at the
plasma membrane was lower than that of the wild-type
SLC26A3. In addition, 643 and 709 mutants showed the
lack of maturation and proper trafficking to the plasma membrane (Fig. 9). These results suggest that the COOH-terminal
cytoplasmic domain is needed for proper expression at the
plasma membrane. This interpretation is in accordance with
previous findings showing that disruption of the COOH-terminal STAS (sulfate transporters and anti-sigma-factor) domain
affects steps that are involved in the folding and/or trafficking
pathway (14).
In summary, we have described the pHi dependency of
human SLC26A3 activity, which was regulated within physiological pH ranges. Our findings contribute to the understanding of functional coupling between the Na/H exchangers
and the Cl/HCO
3 exchangers in the intestine. These observations raise the possibility that, under physiological circumstances, SLC26A3 activity may be regulated by NHE3 via the
changes in pHi.
ACKNOWLEDGMENTS
The authors thank Drs. Y. Suzuki (University of Shizuoka) and K. Szaszi
(St. Michaels Hospital, Toronto, Canada) for helpful comments and discussions.
Present address of K. Suruga: Division of Nutritional Science, University of
Nagasaki, Siebold 1-1-1 Manabino, Nagayo-cho Nishisonogi-gun, Nagasakiken, Japan.

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SLC26A3 AND INTRACELLULAR pH


GRANTS
This study was supported in part by grants from the Salt Science Research
Foundation, No. 0340, and the Houansha Foundation (to H. Hayashi).
REFERENCES
1. Argenzio RA, Armstrong M. ANP inhibits NaCl absorption and elicits
Cl secretion in porcine colon: evidence for cGMP and Ca mediation. Am J
Physiol Regul Integr Comp Physiol 265: R57R65, 1993.
2. Barmeyer C, Ye JH, Sidani S, Geibel J, Binder HJ, Rajendran VM.
Characteristics of rat downregulated in adenoma (rDRA) expressed in
HEK 293 cells. Pflugers Arch 454: 441 450, 2007.
3. Barrett KE, Keely SJ. Chloride secretion by the intestinal epithelium:
molecular basis and regulatory aspects. Annu Rev Physiol 62: 535572,
2000.
4. Best L. Glucose-induced electrical activity in rat pancreatic beta-cells:
dependence on intracellular chloride concentration. J Physiol 568: 137
144, 2005.
5. Biwersi J, Verkman AS. Cell-permeable fluorescent indicator for cytosolic chloride. Biochemistry 30: 7879 7883, 1991.
6. Breuer WV, Mack E, Rothstein A. Enhancement of conductive anion
permeability in cultured cells by cetiedil. Biochim Biophys Acta 897:
127134, 1987.
7. Byeon MK, Westerman MA, Maroulakou IG, Henderson KW, Suster
S, Zhang XK, Papas TS, Vesely J, Willingham MC, Green JE,
Schweinfest CW. The down-regulated in adenoma (DRA) gene encodes
an intestine-specific membrane glycoprotein. Oncogene 12: 387396,
1996.
8. Chapman JM, Knoepp SM, Byeon MK, Henderson KW, Schweinfest
CW. The colon anion transporter, down-regulated in adenoma, induces
growth suppression that is abrogated by E1A. Cancer Res 62: 50835088,
2002.
9. Charney AN, Egnor RW, Henner D, Rashid H, Cassai N, Sidhu GS.
Acid-base effects on intestinal Cl- absorption and vesicular trafficking.
Am J Physiol Cell Physiol 286: C1062C1070, 2004.
10. Charney AN, Feldman GM. Systemic acid-base disorders and intestinal
electrolyte transport. Am J Physiol Gastrointest Liver Physiol 247: G1
G12, 1984.
11. Chernova MN, Jiang L, Shmukler BE, Schweinfest CW, Blanco P,
Freedman SD, Stewart AK, Alper SL. Acute regulation of the SLC26A3
congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes. J Physiol 549: 319, 2003.
12. Chow CW, Kapus A, Romanek R, Grinstein S. NO3induced pH
changes in mammalian cells. Evidence for an NO3H cotransporter.
J Gen Physiol 110: 185200, 1997.
13. Donowitz M, Wicks J, Madara JL, Sharp GW. Studies on role of
calmodulin in Ca2 regulation of rabbit ileal Na and Cl transport. Am J
Physiol Gastrointest Liver Physiol 248: G726 G740, 1985.
14. Dorwart MR, Shcheynikov N, Baker JM, Forman-Kay JD, Muallem
S, Thomas PJ. Congenital chloride-losing diarrhea causing mutations in
the STAS domain result in misfolding and mistrafficking of SLC26A3.
J Biol Chem 283: 8711 8722, 2008.
15. Dudeja PK, Ramaswamy K. Intestinal anion absorption. In: Physiology
of the Gastrointestinal Tract (4th ed). Burlington: Academic, 2006, p.
18811915.
16. Ermishkin LN, Kasumov KM, Potseluyev VM. Properties of amphotericin B channels in a lipid bilayer. Biochim Biophys Acta 470: 357367,
1977.
17. Freedman J, Rasmussen H, Dobbins JW. Somatostatin stimulates coupled sodium chloride influx across the brush border of the rabbit ileum.
Biochem Biophys Res Commun 97: 243247, 1980.
18. Hayashi H, Aharonovitz O, Alexander RT, Touret N, Furuya W,
Orlowski J, Grinstein S. Na/H exchange and pH regulation in the
control of neutrophil chemokinesis and chemotaxis. Am J Physiol Cell
Physiol 294: C526 C534, 2008.
19. Hayashi H, Suzuki Y. Regulation of intracellular pH during H-coupled
oligopeptide absorption in enterocytes from guinea-pig ileum. J Physiol
511: 573586, 1998.
20. Hayashi H, Szaszi K, Coady-Osberg N, Orlowski J, Kinsella JL,
Grinstein S. A slow pH-dependent conformational transition underlies a
novel mode of activation of the epithelial Na/H exchanger-3 isoform.
J Biol Chem 277: 11090 11096, 2002.
21. Hayashi H, Szaszi K, Grinstein S. Multiple modes of regulation of
Na/H exchangers. Ann NY Acad Sci 976: 248 258, 2002.
AJP-Cell Physiol VOL

C1289

22. Hoglund P, Haila S, Socha J, Tomaszewski L, Saarialho-Kere U,


Karjalainen-Lindsberg ML, Airola K, Holmberg C, de la Chapelle A,
Kere J. Mutations of the Downregulated in adenoma (DRA) gene cause
congenital chloride diarrhoea. Nat Genet 14: 316 319, 1996.
23. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins JW.
Sodium and chloride transport across rabbit ileal brush border. II: Evidence for Cl-HCO3 exchange and mechanism of coupling. Am J Physiol
Gastrointest Liver Physiol 249: G236 G245, 1985.
24. Ko SB, Shcheynikov N, Choi JY, Luo X, Ishibashi K, Thomas PJ, Kim
JY, Kim KH, Lee MG, Naruse S, Muallem S. A molecular mechanism
for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis.
EMBO J 21: 56625672, 2002.
25. Kunzelmann K, Mall M. Electrolyte transport in the mammalian colon:
mechanisms and implications for disease. Physiol Rev 82: 245289, 2002.
26. Lamprecht G, Baisch S, Schoenleber E, Gregor M. Transport properties
of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells. Pflugers Arch 449: 479 490, 2005.
27. Lamprecht G, Heil A, Baisch S, Lin-Wu E, Yun CC, Kalbacher H,
Gregor M, Seidler U. The down regulated in adenoma (dra) gene product
binds to the second PDZ domain of the NHE3 kinase A regulatory protein
(E3KARP), potentially linking intestinal Cl-/HCO
3 exchange to Na/H
exchange. Biochemistry 41: 12336 12342, 2002.
28. Lee BS, Gunn RB, Kopito RR. Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line. J Biol
Chem 266: 11448 11454, 1991.
29. Melvin JE, Park K, Richardson L, Schultheis PJ, Shull GE. Mouse
down-regulated in adenoma (DRA) is an intestinal Cl(-)/HCO(3)(-) exchanger and is up-regulated in colon of mice lacking the NHE3 Na()/
H() exchanger. J Biol Chem 274: 2285522861, 1999.
30. Molokanova E, Savchenko A. Bright future of optical assays for ion
channel drug discovery. Drug Discov Today 13: 14 22, 2008.
31. Nellans HN, Frizzell RA, Schultz SG. Coupled sodium-chloride influx
across the brush border of rabbit ileum. Am J Physiol 225: 467 475, 1973.
32. ONeill WC. Physiological significance of volume-regulatory transporters. Am J Physiol Cell Physiol 276: C995C1011, 1999.
33. Rajendran VM, Binder HJ. Cl-HCO3 and Cl-OH exchanges mediate Cl
uptake in apical membrane vesicles of rat distal colon. Am J Physiol
Gastrointest Liver Physiol 264: G874 G879, 1993.
34. Rajendran VM, Binder HJ. Distribution and regulation of apical Cl/
anion exchanges in surface and crypt cells of rat distal colon. Am J Physiol
Gastrointest Liver Physiol 276: G132G137, 1999.
35. Rajendran VM, Black J, Ardito TA, Sangan P, Alper SL, Schweinfest
C, Kashgarian M, Binder HJ. Regulation of DRA and AE1 in rat colon
by dietary Na depletion. Am J Physiol Gastrointest Liver Physiol 279:
G931G942, 2000.
36. Rossmann H, Jacob P, Baisch S, Hassoun R, Meier J, Natour D,
Yahya K, Yun C, Biber J, Lackner KJ, Fiehn W, Gregor M, Seidler
U, Lamprecht G. The CFTR associated protein CAP70 interacts with the
apical Cl-/HCO
3 exchanger DRA in rabbit small intestinal mucosa.
Biochemistry 44: 4477 4487, 2005.
37. Schweinfest CW, Henderson KW, Suster S, Kondoh N, Papas TS.
Identification of a colon mucosa gene that is down-regulated in colon
adenomas and adenocarcinomas. Proc Natl Acad Sci USA 90: 4166 4170,
1993.
38. Schweinfest CW, Spyropoulos DD, Henderson KW, Kim JH, Chapman JM, Barone S, Worrell RT, Wang Z, Soleimani M. slc26a3
(dra)-deficient mice display chloride-losing diarrhea, enhanced colonic
proliferation, and distinct up-regulation of ion transporters in the colon.
J Biol Chem 281: 3796237971, 2006.
39. Shcheynikov N, Wang Y, Park M, Ko SB, Dorwart M, Naruse S,
Thomas PJ, Muallem S. Coupling modes and stoichiometry of Cl-/
HCO
3 exchange by slc26a3 and slc26a6. J Gen Physiol 127: 511524,
2006.
40. Sonawane ND, Thiagarajah JR, Verkman AS. Chloride concentration
in endosomes measured using a ratioable fluorescent Cl- indicator: evidence for chloride accumulation during acidification. J Biol Chem 277:
5506 5513, 2002.
41. Sterling D, Casey JR. Transport activity of AE3 chloride/bicarbonate
anion-exchange proteins and their regulation by intracellular pH. Biochem
J 344: 221229, 1999.
42. Sundaram U. Mechanism of intestinal absorption. Effect of clonidine on
rabbit ileal villus and crypt cells. J Clin Invest 95: 21872194, 1995.

296 JUNE 2009

www.ajpcell.org

C1290

SLC26A3 AND INTRACELLULAR pH

43. Wang Z, Petrovic S, Mann E, Soleimani M. Identification of an apical


Cl-/HCO
3 exchanger in the small intestine. Am J Physiol Gastrointest
Liver Physiol 282: G573G579, 2002.
44. Yun CH, Lamprecht G, Forster DV, Sidor A. NHE3 kinase A regulatory
protein E3KARP binds the epithelial brush border Na/H exchanger NHE3
and the cytoskeletal protein ezrin. J Biol Chem 273: 2585625863, 1998.

AJP-Cell Physiol VOL

45. Yun CH, Oh S, Zizak M, Steplock D, Tsao S, Tse CM, Weinman EJ,
Donowitz M. cAMP-mediated inhibition of the epithelial brush border
Na/H exchanger, NHE3, requires an associated regulatory protein.
Proc Natl Acad Sci USA 94: 3010 3015, 1997.
46. Zachos NC, Tse M, Donowitz M. Molecular physiology of intestinal
Na/H exchange. Annu Rev Physiol 67: 411 443, 2005.

296 JUNE 2009

www.ajpcell.org