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