1 Canadian Institutes of Health Research Membrane Protein Research Group, Departments of Physiology and Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; and 2 Laboratorio di Chimca Bioinorganica, Dipartimento di Chemica, Università di Firenze, I-50019 Sesto Fiorentino, Florence, Italy
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ABSTRACT |
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COOH-terminal cytoplasmic tails of chloride/bicarbonate anion exchangers (AE) bind cytosolic carbonic anhydrase II (CAII) to form a bicarbonate transport metabolon, a membrane protein complex that accelerates transmembrane bicarbonate flux. To determine whether interaction with CAII affects the downregulated in adenoma (DRA) chloride/bicarbonate exchanger, anion exchange activity of DRA-transfected HEK-293 cells was monitored by following changes in intracellular pH associated with bicarbonate transport. DRA-mediated bicarbonate transport activity of 18 ± 1 mM H+ equivalents/min was inhibited 53 ± 2% by 100 mM of the CAII inhibitor, acetazolamide, but was unaffected by the membrane-impermeant carbonic anhydrase inhibitor, 1-[5-sulfamoyl-1,3,4-thiadiazol-2-yl-(aminosulfonyl-4-phenyl)]-2,6-dimethyl-4-phenyl-pyridinium perchlorate. Compared with AE1, the COOH-terminal tail of DRA interacted weakly with CAII. Overexpression of a functionally inactive CAII mutant, V143Y, reduced AE1 transport activity by 61 ± 4% without effect on DRA transport activity (105 ± 7% transport activity relative to DRA alone). We conclude that cytosolic CAII is required for full DRA-mediated bicarbonate transport. However, DRA differs from other bicarbonate transport proteins because its transport activity is not stimulated by direct interaction with CAII.
metabolon; chloride/bicarbonate exchanger; downregulated in adenoma
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INTRODUCTION |
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BICARBONATE METABOLISM
is essential in humans, because carbon dioxide is the metabolic
end product of respiratory oxidation and
CO2/HCO/HCO
Several lines of evidence have demonstrated an interaction between cytosolic carbonic anhydrase II (CAII) and the AE1, AE2, and AE3 anion exchanger isoforms. Binding of erythrocyte membranes to CAII increased CAII enzymatic activity (25), which suggests an interaction between these two proteins. CAII can be coimmunoprecipitated with solubilized AE1 and incubation with an extracellular lectin-caused agglutination of AE1 and a similar redistribution of CAII on the cytosolic surface of the erythrocyte membrane (45). A sensitive microtiter binding assay, using truncation and point mutation of the AE1 COOH terminus, led to the identification of the binding site of CAII in AE1 as LDADD (amino acids 886-890) (46) and the basic amino-terminal region of CAII as the binding site for AE1 (44).
The functional consequences of the AE/CAII interaction have been studied (42). Using HEK-293 cells transiently transfected with AE1 cDNA, we determined that inhibition of endogenous CAII activity with acetazolamide resulted in a decrease of AE1 transport activity. Mutation of the AE1, LDADD, and CAII-binding motif caused a loss of CAII binding and a corresponding 90% decrease of AE1 transport activity. Overexpression of the functionally inactive CAII mutant, V143Y (10), displaced wild-type CAII from all three members of the AE family and had a dominant-negative effect on anion transport, inhibiting transport by ~50%. This demonstrated that binding of functional CAII to the COOH terminus of AE1, AE2, and AE3 proteins is required for maximal bicarbonate transport activity. The requirement of a physical interaction between CAII and AE for maximal bicarbonate transport provided the first direct evidence of a functional transport metabolon: a physically associated complex of proteins in a metabolic pathway (38, 39). The metabolon may accelerate the coupled production of bicarbonate and transport by minimization of the diffusional distance between CAII and the bicarbonate transporter. The interaction between CAII and a peptide corresponding to the LDADD motif has also been reported to stimulate CAII activity directly (34). Together, these effects will increase substrate concentration at the transport site, thereby stimulating bicarbonate transport.
Comparison of the amino acid sequences of the COOH-terminal tails of bicarbonate transport proteins shows that, with the exception of DRA (31), all of these proteins contain at least one consensus CAII-binding motif consisting of a hydrophobic residue followed by four amino acids, of which at least two are acidic residues (46). Formation of a transport metabolon with CAII may therefore also occur with other bicarbonate transport proteins. NBCs also physically interact with CAII, and this interaction, as in the case of the AE family, is necessary for their maximal transport activity [B. Alvarez, F. Loiselle, and J. Casey, unpublished observation (NBC1) and F. Loiselle and J. Casey, unpublished observation (NBC3)]. The absence of a potential CAII-binding site in the COOH-terminal region of DRA therefore raises the questions of whether DRA forms a complex with CAII and whether DRA requires the formation of such a complex to maximize the rate of bicarbonate transport. If DRA does not interact with CAII, it differs significantly from the other bicarbonate transporters, particularly in its mode of regulation.
Human DRA, cloned from a colon subtraction library, is expressed in the
normal colon but not in most adenocarcinomas (31). The
protein product of the DRA gene is a membrane glycoprotein predicted to
span the membrane 10-14 times (5). The protein is
related to the sulfate transporters DTSDT (13) and SAT-1 (2) and has been shown to transport sulfate when expressed in Xenopus oocytes (36). Although DRA has
considerably less similarity to the
Cl/HCO
/HCO
Cl/HCO
/HCO
Cystic fibrosis (CF) is an autosomal recessive disease arising from
inactivation or misprocessing of a cAMP-sensitive Cl
channel, known as the CF transmembrane conductance regulator (CFTR)
(29). CF causes defective fluid and electrolyte secretion in secretory epithelia (26, 47), which impairs the
respiratory, pancreatic, hepatobiliary, and genitourinary systems
(29). Two recent papers have shown that the expression of
DRA in both trachea epithelial cells and cultured pancreatic duct cells
increases in the presence of active CFTR. This increase in expression
of DRA is accompanied by an increase in
Cl
/HCO
/HCO
In the present study, we investigated the interaction between DRA and
CAII. Using HEK-293 cells transiently transfected with DRA cDNA, we
determined that inhibition of CAII activity with acetazolamide resulted
in a substantial decrease of DRA transport activity, indicating that
DRA requires the presence of active CAII for maximal bicarbonate
transport. A microtiter plate assay showed that the COOH-terminal tail
of DRA binds CAII with a much lower affinity and capacity than AE1.
Expression of functionally inactive V143Y CAII mutant (10)
displaced wild-type CAII from AE1 and had a dominant-negative effect on
anion transport (42). In contrast, overexpression of V143Y
CAII had no effect on the rate of DRA-mediated bicarbonate transport.
Taken together, these results indicate that although DRA activity
requires the presence of CAII enzymatic activity in the cytosol, DRA
and CAII do not form the physical complex required by AE and NBC.
Therefore, DRA is thus unique among HCO
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MATERIALS AND METHODS |
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Materials. Rabbit anti-glutathione S-transferase (GST) antibody was from Santa Cruz Antibodies (Santa Cruz, CA). Biotinylated anti-rabbit IgG and peroxidase-labeled biotin/avidin, glutathione Sepharose, pGEX-6P-1 expression vector, and 5' pGEX sequencing primer were from Amersham Pharmacia Biotech (Quebec, Canada). Sheep anti-human carbonic anhydrase II antibody was from Serotec (Raleigh, NC). Poly-L-lysine, nigericin, and o-phenylenediamine dihydrochloride were from Sigma Aldrich Canada (Oakville, Canada). Molecular Probes 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) was from Cedarlane Laboratories (Ontario, Canada). Glass coverslips were from Fisher Scientific Products (Nepean, Canada). Dulbecco's modified Eagle's medium (DMEM), and calf serum and fetal bovine serum were from GIBCO-BRL (Burlington, ON, Canada).
Molecular biology. An expression construct for the human DRA protein was received as a generous gift from Dr. Manoocher Soleimani (12), and V143Y CAII mutant was from Dr. Carol Fierke (10). Expression constructs for CA and AE have been described previously (6, 42). Plasmid DNA for transfections was prepared by using Qiagen columns (Qiagen, Mississauga, Canada).
Tissue culture. Proteins were expressed by transient transfection of HEK-293 cells (11) by using the calcium phosphate method (30). Cells were grown at 37°C in an air/CO2 (19:1) environment in DMEM supplemented with 5% (vol/vol) fetal bovine serum and 5% (vol/vol) calf serum.
Anion exchange activity assay.
Anion exchange activity was monitored by using a fluorescence assay
described previously (41). Briefly, HEK-293 cells grown on
poly-L-lysine-coated coverslips were transiently
transfected as described earlier. Two days posttransfection, coverslips
were rinsed in serum-free DMEM and incubated in 4 ml of serum-free medium containing 2 µM BCECF-AM (37°C, 15 min). Coverslips were then mounted in a fluorescence cuvette and perfused alternately with
Ringer's buffer (5 mM glucose, 5 mM potassium gluconate, 1 mM calcium
gluconate, 1 mM MgSO4, 2.5 mM
NaH2PO4, 25 mM NaHCO3, and 10 mM
HEPES, pH 7.4) containing either 140 mM NaCl or 140 mM sodium gluconate
bubbled with air/CO2 (19:1). Fluorescence was
monitored by using a Photon Technologies International RCR fluorimeter
at excitation wavelengths 440 and 502.5 nm and emission wavelength
528.7 nm. After calibration using the high potassium/nigericin technique (43) at three pH values between 6.5 and 7.5, fluorescence ratios were converted to intracellular pH
(pHi). Rates of change of pHi were determined
by linear regression (Kaleidagraph software) of the initial
HCOtotal ×
pHi (28),
where, as determined previously,
total = 57.5 mM
(41). In all cases, the transport activity of
sham-transfected cells was subtracted from the total rate to ensure
that these rates consist only of AE transport activity.
GST-fusion protein construction and purification. A GST-fusion protein consisting of the cDNA for GST fused to the 33 amino acid COOH-terminal tail of human AE1 was a generous gift from Dr. Reinhart Reithmeier (45). The GST-fusion protein, consisting of the COOH-terminal 42 amino acids of DRA, was constructed by using PCR to amplify the appropriate cDNA sequence. With the use of human DRA cDNA as template, the forward and reverse primers CGCGGATCCAAGAAAGATTACAGTACTTCAAAGTTTAATCC and CGCGGATCCGAATTTTGTTTCAACTGGCACCTCATATACCCACT, respectively, introduced a BamHI restriction site at both ends of the amplified product. The PCR product was digested with BamHI and cloned into the pGEX-6P-1 expression vector, digested in the same way. The construct GST-DRAct was verified by sequencing with a Beckman Instruments CEQ2000 DNA sequencer, and plasmid DNA was purified by using Qiagen columns. The GST-DRAct construct was transformed into Escherichia coli strain BL21-codon plus (Stratagene), and a single colony was used to inoculate 50 ml of Luria-Bertani (LB) medium. After overnight growth at 37°C with shaking, this culture was used to innoculate 1.2 l LB medium (5 ml/200 ml). The culture was grown at 37°C with shaking until the A600 was 0.6-1.0. Isopropylthiogalactoside (1 mM final) was added, and growth was allowed to continue for 2-6 h. The culture was then centrifuged at 10,000 g for 10 min, and bacterial pellets were resuspended in 4°C PBS (150 mM NaCl and 5 mM Na2HPO4, pH 7.5) containing protease inhibitors (Complete mini-protease inhibitor cocktail; Roche Applied Science, Quebec, Canada). Resuspended cells were disrupted by sonication [4 × 1 min, power level 9.5 with model W185 probe sonifier (heat systems; Ultrasonics, Plainview, NY)], and Triton X-100 was added to a final concentration of 1% (vol/vol) with slow stirring for 30 min. After centrifugation at 15,000 g for 10 min, the supernatant was added to 1.3 ml of glutathione Sepharose 4B (50% slurry equilibrated with PBS) and incubated at room temperature with gentle agitation for 1-2 h. The sample was centrifuged at 500 g for 5 min, and the pellet was washed three times with PBS. The fusion protein was eluted by using 10 mM reduced glutathione in 50 mM Tris · HCl, pH 8.0.
Binding assay. The ability of CAII to bind the COOH-terminal tail of DRA was investigated by using a sensitive microtiter assay, described previously (42, 46). Briefly, 200 ng of purified human CAII (Sigma Aldrich) were chemically coupled to wells of a 96-well plate by using 1-cyclohexyl-3-(2-morpholino-ethyl)carbodimide metho-p-toluenesulfanate (Sigma Aldrich). Wells were washed with PBS and blocked with 2% (wt/vol) BSA in PBS. GST fusion proteins of the COOH-terminal tail of AE1 and DRA were purified as described above. After washing with antibody buffer [100 mM NaCl, 5 mM EDTA, 0.25% gelatin (wt/vol), 0.05% Triton X-100 (wt/vol), and 50 mM Tris, pH 7.5], plates were incubated with various concentrations (0-200 nM) of purified GST fusion proteins or GST alone. After being washed, bound fusion proteins were detected by sequential incubation with rabbit anti-GST antibody (Santa Cruz Antibodies), biotinylated anti-rabbit IgG (Amersham), and peroxidase-labeled biotin/avidin (Amersham). Plates were then incubated with the peroxidase substrate o-phenylenediamine dihydrochloride (Sigma), and product formation was detected at 450 nm in a Labsystems Mutiskan MCC microplate reader. Binding data were fitted by using Kaleidagraph software (Synergy Software).
Statistical analysis. Values are expressed as means ± SE. Statistical significance was determined by using a Student's paired t-test with P < 0.05 considered significant.
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RESULTS |
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HCO
|
Effects of CA inhibition on DRA
Cl/HCO
and
NO
/NO
-sensitive fluorescent dye, SPQ (17).
SDPPP and PTSP are membrane-impermeant, broad-spectrum CA inhibitors
that inhibit CAIV with Ki of 0.4 nM and 8 nM, respectively
(33). Cl
/NO
/NO
/HCO
|
Binding of CAII to DRA.
The amino acid sequences of the cytoplasmic COOH-terminal regions of
HCO
|
Effect of carbonic anhydrase on DRA
Cl/HCO
|
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DISCUSSION |
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In this study, we have examined the physical and functional
relationships between DRA and CAII. The lack of a CAII-binding site
motif in the COOH-terminal tail of DRA thus far makes DRA a unique
bicarbonate transport protein. To investigate whether CAII activity had
any impact on the transport capability of DRA, we monitored
DRA-mediated bicarbonate transport activity before and after incubation
with 100 µM acetazolamide, a membrane-permeant CA inhibitor (7,
8). Our data showed that inhibition of CAII by acetazolamide
impaired DRA transport activity, which indicates that CAII activity has
a substantial effect on DRA Cl/HCO
/NO
/HCO
/HCO
Binding assays showed that the ability of the DRA COOH-terminal tail to bind CAII is much less than the bicarbonate transporter, AE1. Because the binding assay showed some limited interaction between CAII and DRA, it is possible that interaction with CAII activates DRA. However, overexpression of the V143Y CAII mutant had no dominant-negative effect on DRA transport activity. This indicates that DRA bicarbonate transport activity is not activated by CAII binding at any site on DRA. We conclude that DRA may bind to CAII to some limited degree but that any physical interaction between the two proteins has no impact on the bicarbonate transport activity of DRA.
The bicarbonate transport metabolon is emerging as an important mechanism to accelerate and regulate bicarbonate transport activity. A transport metabolon is a physical complex of an enzyme and a transporter, which maximizes and may regulate substrate flux through the enzyme and across the membrane. The first example of a bicarbonate transport metabolon was provided by anion exchangers of the AE family, which form a functional and physical complex with cytosolic CAII (18, 25, 45) and with the extracellular enzyme, CAIV (40). NBC1 and NBC3 also form a metabolon with CAII [B. Alvarez, F. Loiselle, and J. Casey, unpublished observation (NBC1) and F. Loiselle and J. Casey, unpublished observation (NBC3)]. Comparison of the COOH-terminal tails of other bicarbonate transport proteins indicates that, with the exception of DRA, they all contain at least one potential CAII-binding site. The analysis performed in this report indicates that, consistent with the absence of a CAII-binding motif, binding of CAII or CAIV had no effect on DRA transport activity.
This is the first reported examination of the effect of carbonic
anhydrase inhibitors on DRA transport activity. Because carbonic anhydrases and anion exchangers share HCO/NO
When DRA was first cloned from mouse (23), the
Cl/HCO
, which was attributed to a high
specificity at the intracellular substrate site for
HCO
The data presented here show that DRA is unique among bicarbonate
transporters because it does not interact with either CAII or CAIV. One
reason for this may be that DRA does not function solely as a
Cl/HCO
/HCO
,
suggesting that the colonic Cl
/HCO
/NO
/HCO
The lack of interaction between DRA and carbonic anhydrases also suggests that DRA is regulated differently from other bicarbonate transporters. Direct interaction between carbonic anhydrase and a bicarbonate transporter accelerates the bicarbonate transport rate (42). Modulation of the interaction therefore presents a rapid way to alter the rate of bicarbonate transport, but DRA cannot use this mode of regulation. In the physiological context, cells may contain a membrane protein that binds CAII and localizes the enzyme near the DRA transport site. If such a protein exists, HEK-293 cells do not express it because dominant-negative V143Y CAII had no effect on DRA activity.
DRA is expressed on the apical membrane of pancreatic epithelia
(12). In response to the release of secretin, the pancreas produces a HCO/HCO
With the exception of DRA, the COOH-terminal tail of every bicarbonate transport protein contains a potential CAII-binding motif. Formation of a complex with CAII potentiates bicarbonate transport activity, and modulation of the interaction provides a cell with a potential regulatory mechanism to control bicarbonate flux (42). DRA is the only bicarbonate transport protein identified to date in which the CAII-binding site is absent. In this study, we investigated the physical and functional interaction between DRA and CAII. On the basis of our results, we conclude that although there is a need for the presence of CAII enzymatic activity in the cytosol, the weak physical interaction between the two proteins does not affect the functional activity of DRA. For full transport activity, DRA may require an intermediary protein to bind CAII and bring the enzyme close to DRA in the plasma membrane. This makes DRA unique among the bicarbonate transport protein superfamily examined to date.
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ACKNOWLEDGEMENTS |
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We thank Dr. George Schwartz for CAIV cDNA.
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FOOTNOTES |
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This work was funded by an operating grant from the Heart and Stroke Foundation (HSF) of Canada. D. Sterling holds studentship trainee awards from the HSF and Alberta Heritage Foundation for Medical Research (AHFMR). N. Brown was supported by an AHFMR summer studentship. J. Casey is a Senior Scholar of AHFMR.
Address for reprint requests and other correspondence: J. R. Casey, Dept. of Physiology, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2H7 (E-mail: joe.casey{at}ualberta.ca).
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.
July 24, 2002;10.1152/ajpcell.00115.2002
Received 12 March 2002; accepted in final form 17 July 2002.
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