©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Band 3-like Proteins and Cl/HCO Exchange in Isolated Cardiomyocytes (*)

(Received for publication, June 9, 1994; and in revised form, October 21, 1994)

Michel Pucéat (§) Irina Korichneva Robert Cassoly (1) Guy Vassort

From the Laboratoire de Physiopathologie Cardiovasculaire, INSERM U-390, Centre Hospitalier Universitaire Arnaud de Villeneuve, 34295 Montpellier, France Institut Jacques Monod, CNRS Unité Mixte de Recherche 9922, Université Paris-7, 75251 Paris, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The identification of the protein that exerts the function of Cl/HCO(3) exchange is still unresolved in cardiac tissue. We have addressed this issue by using a multiple technical approach. Western blotting analysis with an antibody raised against human erythroid whole band 3 protein, the so-called protein that mediates the Cl/HCO(3) exchange in erythrocytes, showed that adult cardiomyocytes expressed two proteins immunologically related to the erythroid band 3. These proteins migrated in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They were specifically found in the membrane but not in the cytosolic or the myofibril fractions of adult cardiomyocytes. Confocal microscopy further indicated that the immunostained proteins were mainly located at the sarcolemma and along T-tubules, typical membrane structures of adult cardiomyocytes. Using an antibody raised against a cardiac amino-terminal domain of rat AE3, we found that the 120-kDa protein is the translation product of the AE3 gene specifically expressed in heart and brain. Using an antiserum raised against a specific domain of mouse erythroid band 3 (AE1), which is not shared by AE3, we showed that the 80-kDa protein is likely to be a truncated translation product of the AE1 gene. Microinjection of the anti-human erythroid whole band 3 antibody into single isolated cardiac cells significantly inhibited the Cl/HCO(3) exchange activity. Furthermore, the anti-AE1 antibody strongly decreased the efficiency of 4,4`-diisothiocyanatostilbene-2,2`-disulfonate to inhibit the ionic exchange. We thus suggest that the 80-kDa or both the 80- and the 120-kDa proteins immunologically related to the erythroid band 3 protein perform the anionic exchange in rat cardiomyocytes.


INTRODUCTION

Intracellular pH (pH) modulates various cellular events including metabolism, ionic conductances, Ca homeostasis, and cell division. In cardiac muscle, both Ca homeostasis and Ca sensitivity of myofilaments, which closely regulate contractility, are affected by changes in pH(1) . pH also modulates gap junction conductances and in turn cardiac conduction(2) . A fine regulation of pH is thus required to maintain cardiac function. To such an end, the cardiac cell possesses membrane ionic transporters sensitive to intracellular pH. Under acid loads, a Na/H antiport is mainly activated to extrude protons from the myocyte(3, 4) . A Na- and HCO(3)-dependent transporter was also recently reported to participate in pHregulation from acidosis(5, 6) . In contrast, a rise in pHactivates a Na-independent Cl/HCO(3) exchanger, which operates as a HCO(3) extruder(7) . Numerous studies have been devoted to the Na/H antiport in many cell types including cardiomyocytes. More specifically, the cDNA encoding the cardiac Na/H antiport was recently cloned, and the protein responsible for the cationic exchange was immunologically identified(8) . Although under physiological conditions the Cl/HCO(3) exchanger is the only ionic transporter able to alleviate alkaline loads in cardiac tissue, much less data are available in regard to the protein responsible for cardiac anionic exchange.

The best characterized Cl/HCO(3) exchanger in tissues so far investigated is the erythrocyte band 3 glycoprotein. The protein is responsible for the extrusion of HCO(3) ions generated by the hydration of intracellular CO(2). The anionic flux allows HCO(3) equilibration between plasma and erythrocyte following CO(2) transport. The glycoprotein represents 30% of erythrocyte membrane proteins and has a molecular mass of 90 kDa(9, 10) . Band 3-like proteins, which share some antigenic properties with erythrocyte band 3, were identified in various nucleated somatic cells but not in cardiac cells(11, 12) . Such proteins were suggested to perform the ionic exchange function in hepatocytes (13) and brain(14, 15) . Numerous studies using molecular biology approaches show that band 3-like proteins belong to a family of broadly distributed proteins in vertebrate cells. They have been classified into three groups, namely the anion exchangers AE, (^1)AE2, and AE3 on the basis of the gene that encodes them(16) . More specifically, the AE1 gene encodes the erythroid band 3 as well as a truncated form of the polypeptide in kidney(17) . AE1 transcripts were also recently found in heart(18) . The AE2 ones are mainly expressed in stomach and intestine, while the AE3 transcripts are the major ones in brain and heart(14, 18) . Cardiac AE3 cDNA giving the most abundant 3.8-kilobase transcript was recently cloned from a rat heart cDNA library. It is identical to brain AE3 cDNA except for one exon, and it predicts a polypeptide of 113 kDa(19) .

While Northern blotting studies led to the characterization of the AE mRNA transcripts in whole heart, it is not known whether the respective translation products are present in cardiomyocytes and if so, whether these proteins are anionic exchangers. Therefore, this study was designed to identify band 3-like proteins expressed in cardiac cells and to investigate whether these proteins exert the anionic exchange function. Members of AE family (i.e. AE1, AE2, and AE3) share significant (60%) sequence identity(16) . Between AE1 and AE3, sequence identity reaches 81% in the carboxyl-terminal cytoplasmic domain(20) , a hydrophyllic region expected to be one of the most immunogenic. Accordingly, a polyclonal antibody raised against the purified erythroid band 3 was first used to identify band 3-like proteins in cardiac tissue. Band 3-like proteins of apparent molecular masses of 80 and 120 kDa were found in adult hearts. The two proteins were detected only in the membrane fraction prepared from adult rat isolated cardiac cells, a preparation devoid of contaminating non-muscle cells. Confocal microscopy of immunostained adult rat myocytes showed the location of the proteins at the sarcolemma and T-tubule level. Using an antiserum specific to AE1 and an antibody specific to AE3, the 80- and 120-kDa proteins were tentatively characterized as products of AE1 and AE3 genes, respectively. Furthermore, our results provide evidence that either or both proteins perform the anionic exchange in cardiac cells.


EXPERIMENTAL PROCEDURES

Purification of Human Erythrocyte Band 3 and of Its Cytosolic Domain; Preparation of Antibodies

Unsealed washed erythrocyte ghosts were prepared according to Dodge et al.(21) from freshly collected human blood. Whole band 3 protein was purified taking special care to eliminate the smallest amounts of contaminant glycophorin and phospholipids(22) . Briefly, band 3 was extracted from erythrocyte membrane by 0.5% Triton X-100. The purification of the protein above 95% was achieved by ion-exchange chromatography on a DE52-cellulose column and subsequent elution from a thiol-exchange affinity column (p-carboxymethyl biogel ethylagarose 4B). The protein was eluted by 15 mM beta-mercaptoethanol.

The cytosolic fragment of human band 3 was isolated from the ghosts by the method of Bennett and Stenbuck(23) . Briefly, the ghosts were depleted of most of their extrinsic proteins by incubating them with 10 volumes of 0.17 M acetic acid at 20 °C and subsequent washings with 0.1 M phosphate buffer pH 7. The membranes were collected at 14,000 times g for 20 min and digested with 1 µg of chymotrypsin/ml for 40 min at 0 °C. The proteolysed membranes were discarded after centrifugation, and the supernatant was applied on the top of a DE52-cellulose column. The cytosolic fragment of human band 3 was dialysed against 5 mM phosphate buffer at pH 6.5 and stored in liquid nitrogen. The preparation was checked by SDS-polyacrylamide gel electrophoresis, and the molarity of the cytosolic fragment of human band 3 was measured spectrophotometrically in the dialysis buffer with = 27 mM cm(24) .

Two mg of each of the highly purified antigens were run in 10% SDS-polyacrylamide gel electrophoresis. The 42 and 100 kDa bands corresponding to the cytosolic fragment of human band 3 and whole band 3, respectively, were visualized by Coomassie Blue staining and excised from the gel. The gels were homogeneized in PBS and finally dialysed against PBS containing 10% methanol, 0.1 mM phenylmethylsulfonyl fluoride, 2.5% NaN(3), 0.1% Triton X-100, and 15 g/liter DE52 pre-swollen phosphocellulose. Two New Zealand rabbits were selected for the absence of immunoreaction with their preimmune serum as assessed by Western blotting of human erythroid ghosts. The antigens were emulsified with complete Freund's adjuvant (v/v) for subcutaneously injections. The injections were repeated at 3-week intervals with incomplete Freund's adjuvant. Blood samples were collected 5 and 6 weeks after the first injection. The reactivity and specificity of the sera were monitored by immunoblotting. The antibodies were purified at 4 °C by reversible absorption with their respective antigens coupled to Affi-Gel 15 (Bio-Rad) according to manufacturer's instructions. The pure antibodies were dialyzed versus PBS, 50% glycerol and stored at -20 °C.

Preparation of an Anti-rat AE3 Antibody

An antibody was raised against a synthetic peptide corresponding to the predicted (19) amino-terminal domain of rat cardiac AE3 (sequence PGDTEDRGPGRN), which is not shared by brain AE3. The peptide was coupled to keyhole limpet hemocyanin and to BSA. Two New-Zealand rabbits were selected for the absence of immunoreactivity of their preimmune sera as assessed by Western blotting of cardiac membranes. The rabbits were immunized with keyhole limpet hemocyanin-peptide emulsified with complete Freund's adjuvant following three subcutaneous injections every 3 weeks. Blood was collected 1 week after the last injection and every 10 days. The antibody was affinity purified by reversible absorption on the BSA-peptide coupled to CNBr-activated Sepharose. The pure antibody was dialyzed versus PBS, 50% glycerol and stored at -20 °C.

Preparation of Rat Ghosts, Whole Organ Homogenates, and Isolated Cardiac Cells

Rat ghosts were prepared from freshly collected blood according to Fairbanks et al.(9) .

Hearts and brains from two adult rats were quickly removed from anesthetized animals, and the hearts were perfused for 5 min in a Ca-containing solution to wash out blood. The organs were cut into small pieces, extensively washed, and homogenized using a glass homogenizer in a Tris-HCl buffer, pH 7.5, containing 50 mM Tris, 2 mM EDTA, 10 mM EGTA, 5 mM dithiothreitol, 250 mM sucrose, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µM E64. The homogenates were centrifugated for 15 min at 15,000 times g to discard the myofilaments. The supernatants were saved and added with 4 times Laemmli electrophoresis buffer.

Isolated rat ventricular cells were prepared as described previously (25) with the following minor modifications. Briefly, the hearts from 250-g Wistar male rats were quickly removed and perfused in a nominal Ca-free medium for 5 min and then with 1.2 mg/ml of collagenase added with 30 µM CaCl(2). After 1 h, the heart was removed and cut into several pieces. The tissue was then gently dissociated through a wide-bore tip pipette. The cells were filtered on 250-µm nylon mesh and incubated for 15 min at 37 °C. Meanwhile, Ca concentration of the incubation medium was increased step by step up to 1 mM. The preparation provided at least 6 times 10^6 rod-shaped cells. Cells were kept at 37 °C in Hepes-buffered medium adjusted to pH 7.4 and containing 117 mM NaCl, 5.7 mM KCl, 4.4 mM NaHCO(3), 1.5 mM KH(2)PO(4), 1.7 mM MgCl(2), 1 mM CaCl(2), 21 mM Hepes, 11 mM glucose, 10 mM creatine, 20 mM taurine, and 0.5% BSA for at least 8 h.

Preparation of Whole Cell Lysate, Cytosolic and Membrane Fractions from Isolated Cells

After spinning down the adult rat cardiac cells, the pellet was resuspended in a 50 mM glycerophosphate buffer adjusted to pH 7.4 and added with 1 mM EGTA, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µM E64. The lysate was incubated at 4 °C for 15 min and centrifugated at 15,000 times g for 15 min. The supernatant was added with 4 times Laemmli electrophoresis buffer. In some experiments designed to fully prevent proteolysis, the cells were quickly pelleted and boiled in Laemmli electrophoresis buffer.

For subcellular fractionation, the cells were homogenized in a buffer containing 50 mM glycerophosphate, 250 mM sucrose, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 100 µM E64 using a glass homogenizer. The homogenate was centrifugated at 15,000 times g for 30 min. The supernatant was saved as the cytosolic fraction. The pellet was resuspended in the same buffer added with 1% Triton X-100. The sample was kept on ice for 30 min and was spun down at 15,000 times g for 45 min. The supernatant was saved as the crude membrane fraction. The myofibril pellet was rinsed with the Triton-containing buffer and resuspended in Laemmli buffer. Both cytosolic and membrane fractions were added with Laemmli electrophoresis buffer prior to Western blotting.

In some experiments designed to further investigate the presence of band 3-like proteins in the cytoskeleton, the Triton X-100 insoluble extract was washed in imidazole buffer containing 30 mM imidazole, 108 mM potassium acetate, 4.2 mM magnesium acetate, 30 mM sodium acetate, 0.3 mM dithiothreitol, 10 mM EGTA adjusted to pH 7.1 and incubated in high ionic strength buffer (0.6 M KCl) added with 1% Triton X-100 for 30 min at 4 °C. The extract was spun at 12,000 times g for 15 min. The pellet was discarded, and the supernatant added with 4 times Laemmli buffer prior to Western blotting.

Electrophoresis and Western Blot Analysis

Protein content was estimated according to Bradford(26) . Proteins were separated on 6, 7.5, or 8.5% SDS-polyacrylamide gel electrophoresis according to Laemmli(27) . Immediately after electrophoresis, proteins were electrophoretically transferred to a nitrocellulose membrane (70 V, 1.5 h) according to Towbin et al.(28) . Nonspecific binding sites of nitrocellulose sheets were then blocked for 1 h at 37 °C with 5% nonfat milk in PBS, 0.1 M, pH 7.4, 0.1% Tween 20. After washing with PBS, 0.1% Tween 20, the sheets were incubated overnight at 4 °C with the primary antibody or antiserum (rabbit anti-human erythrocyte band 3, 1/500; anti-mouse AE1 1/200, anti-rat cardiac AE3 1/1000). After washing, the nitrocellulose membranes were probed with the secondary antibody coupled to peroxydase (anti-rabbit IgG 1/2000) or to biotin (anti rabbit IgG 1/20,000) for 1 h at 37 °C. In the latter case, the sheets were washed and finally incubated with 10 µg/ml extravidin-peroxydase for 1 h at 20 °C. Proteins were revealed using nitro blue tetrazolium as substrate for peroxydase or the ECL detection system used according to the manufacturer's instructions. The ECL films were exposed for 3-30 min or for 10-30 s when the extravidin-biotin system was used.

Immunostaining of Cells and Analysis in Confocal Microscopy

For immunostaining, the cells were attached to laminin-coated coverslips. They were quickly rinsed with PBS and fixed for 15 min at room temperature with 3.7% paraformaldehyde in PBS. The cells were then incubated in 0.1 M glycine in PBS for 15 min at room temperature, washed with PBS, and permeabilized with 0.2% Triton X-100 (w/v) for 10 min at room temperature. After washing in PBS containing 1% BSA and blocking nonspecific sites by incubating in PBS added with 1% BSA, 0.1% Tween 20 and 5 mM MgCl(2) for 20 min at 37 °C, the cells were probed overnight at 4 °C with the primary antibody (anti-human erythroid whole band 3 1/50 or anti-myosin light chain 1/200 diluted in blocking buffer). After washing the cells with PBS containing 1% BSA, they were incubated for 30 min at 37 °C in the presence of Texas red- or fluorescein-conjugated affinity purified goat anti-rabbit IgG used at a dilution of 1/100 and 1/200, respectively, followed by extensive washing. For double immunostaining, sheep anti-mouse fluorescein-conjugated secondary antibody was used to label myosin. The coverslips were mounted on glass slides in citifluor.

The immunostained cells were observed in confocal laser scanning microscopy using a Bio-Rad MRC-600 confocal imaging system (Bio-Rad) mounted on an Optiphot II Nikon microscope. Images were collected using an oil immersion 60times objective (numerical aperture, 1.4, plan Apochromat). An argon ion laser (514-nm line) and an helium-neon laser (543-nm line) were used for fluorescein- and Texas-red excitation, respectively. Confocal system was set to a resolution of 0.5-µm field depth. Kalman filtering was used to integrate the signal collected over eight frames to decrease the noise with a scan time of 4 s/frame and a rastor size of 512 lines. Photographs were made using a camera mounted on a VM1710 Lucius and Baer film recorder.

Measurement of Intracellular pH

Intracellular pH was measured in single isolated cells attached to laminin-coated coverslips and loaded with the pH-sensitive fluorescent indicator SNARF1/AM as described previously(29) . For such experiments, the cells were bathed in a control solution containing 120 mM NaCl, 5.7 mM KHCO(3), 1.2 mM NaH(2)PO(4), 1.7 mM MgSO(4), 1 mM NaHCO(3), 1 mM CaCO(3), and 20 mM Hepes adjusted to pH 7.4 at 32 °C. In the Cl-free solution used to reverse the Cl/HCO(3) exchanger, sodium gluconate replaced NaCl. All of the experiments were performed in the presence of 1 µM ethylisopropyl amiloride to prevent the Na/H antiport activity. pH(i) was calibrated in situ using the nigericin method(29) .

Microinjection of Cardiac Cells

To prevent Ca overloading and contracture during microinjection, the cells attached to laminin-coated coverslips were loaded with BAPTA by incubating them for 20 min in 25 µM BAPTA/AM. BAPTA slightly decreases resting intracellular Ca and buffers any further variation in cytosolic Ca concentration(30) . Then, the cardiomyocytes were kept at 37 °C in Hepes-buffered medium containing 5 mM 2,3-butanedionemonoxime. 2,3-Butanedionemonoxime inhibits the generation of actomyosin cross-bridges and thus further prevents cell contracture. In preliminary experiments, we checked that BAPTA loading of noninjected cells did not affect Cl/HCO(3) activity. 2,3-Butanedionemonoxime was washed out 30 min before the pH(i) measurement experiments and appeared not to affect the Cl/HCO(3) exchanger activity as assessed by the lack of significant difference in the ionic exchange activity between untreated and 2,3-butanedionemonoxime-treated cells.

Isolated cells were transferred to the stage of an inverted microscope (Diaphot, Nikon) and were bathed in control medium containing only 0.5 mM Ca. The anti-human erythroid band 3 antibody as well as rabbit affinity-purified IgG were diluted to a final concentration of 90 µg/ml in the injection buffer containing 150 mM KCl, 0.1 mM EDTA, 0.025 mM EGTA, and 1 mM Pipes adjusted to pH 7.2 with KOH. The micropipettes were pulled on a vertical puller, and the tips (diameter about 1 µm) filled by soaking them into a small drop of either the anti-human whole erythroid band 3 antibody or the IgG solutions allowing a short column of solution to enter the pipette by capillarity. Cells were injected using a Picopritzer II microinjection system (Biologic, France). The antibody or control rabbit affinity-purified IgG was expelled from the pipette by increasing the pressure into the pipette up to 2.5 atm for 100 ms. Transient swelling of the injection area indicated that the cell had been injected. Injected cells were then allowed to incubate for at least 1 h at 37 °C before the experiments.

Statistical Analysis

Data are presented as means ± S.E. Statistical significances were estimated by Student's t test. Results are considered significant at p < 0.01.

Materials

Collagenase (CLS4) was purchased from Worthington (Freehold, NJ). Ethylisopropyl amiloride was from RBI (Natick, MA). E64 was from Boehringer Mannheim. SNARF1/AM, BAPTA/AM, and Texas red-conjugated goat anti-rabbit IgG antibody were from Molecular Probes (Eugene, OR). ECL detection system reagents were from Amersham Corp. Rabbit affinity-purified IgG and all other chemicals were purchased from Sigma.


RESULTS AND DISCUSSION

Western blotting analysis with an anti-human whole erythroid band 3 antibody was used to investigate the presence of band 3-like proteins in total homogenates prepared from adult hearts. The purified human band 3 recognized by the antibody appeared as a wide band around 100 kDa characteristic of the glycoprotein. The antibody also reacted with rat band 3 in rat ghosts (Fig. 1A). Fig. 1B further shows that in adult cardiac homogenate, the antibody strongly reacted with two proteins that migrated with apparent molecular masses of 80 and 120 kDa. Both these immunoreactive bands were no more demonstrable when the anti-human erythroid band 3 antibody was used in the presence of 10 µg/ml of purified human band 3 (Fig. 1B). The antibody also reacted with the 80 and 120 kDa proteins in a whole cell lysate prepared from adult rat isolated cardiac cells in EGTA and protease inhibitors containing buffer (Fig. 1B) or in isolated cells quickly denaturated in boiling Laemmli electrophoresis buffer (not shown). Altogether, these findings suggest that the 80 kDa band is not a proteolytic product of the 120-kDa protein.


Figure 1: Immunological identification and subcellular localization of band 3-like proteins in heart. A, Western blot of proteins of rat ghosts (lane1) and of purified human band 3 (lane2) using the the anti-human erythroid band 3 antibody. 50 and 3 µg of protein were loaded in lanes1 and 2, respectively. The blot was probed with peroxydase-conjugated anti-rabbit IgG, and the proteins were revealed by ECL detection. B, Western blot of proteins of adult rat cardiac homogenate (lane1), whole ventricular cell lysate (lane2), and of brain homogenate (lane3) probed with the antibody raised against the human whole erythroid band 3-protein; lane4, Western blot of proteins of whole ventricular cell lysate probed with the anti-human erythroid band 3-protein antibody in the presence of 10 µg/ml purified human band 3. 200 µg of protein were loaded in wells 1 and 3; 100 µg of protein were loaded in wells 2 and 4. The proteins were revealed using extravidin-biotin system and ECL detection. Molecular weights were estimated from prestained standards. The immunoreactivity of the 80- and 120-kDa proteins was quantified using a film imaging system; the ratio of immunoreactivities (120 kDa/80 kDa) was 1.2 and 0.2 for brain and adult heart homogenates, respectively. C, Western blot of proteins of membrane fraction prepared from isolated adult rat cardiomyocytes. The blot was probed with an anti-mouse AE1 antiserum. 100 µg of protein were loaded on the gel. The proteins were visualized using extravidin-biotin and ECL detection system. D, Western blot of proteins of human ghosts (lane1), whole cardiac cell lysate (lane2), and membrane fraction prepared from cardiac isolated cells (lane3). The blot was probed with the antibody raised against the 42-kDa amino-terminal cytoplasmic fragment of band 3. 30 and 100 µg of protein were loaded in lane1 and lanes 2 and 3, respectively. The proteins were visualized using extravidin-biotin system and nitro blue tetrazolium as a substrate for peroxydase. E, Western blot of proteins of membrane fraction of isolated cardiomyocytes. The blot was probed with the antibody raised against an amino-terminal domain of rat cardiac AE3 in the absence (lane1) or in the presence (lane2) of 10 µg/ml of BSA-peptide, which served as antigen. 200 µg of protein were loaded in every lane. The blot was further probed with peroxydase-conjugated anti-rabbit IgG, and the proteins were revealed by ECL detection. F, Western blot of proteins of membrane (lane1), cytosolic (lane2), and myofibril (lane3) fractions prepared from isolated adult rat cardiomyocytes. 100 µg of protein were loaded in every well. The blot was probed with the anti-human erythroid whole band 3 protein antibody, and the proteins were revealed by extravidin-biotin and ECL detection system. The different parts of the figure are representative of three to five experiments.



We next addressed whether the proteins were products of two different genes, namely the AE1 and AE3 genes since their mRNA transcripts were found in whole heart(18) . The presence of such proteins was thus investigated in brain homogenate as a tissue that exhibits the most abundant AE3 transcript. Two proteins having apparent molecular masses of 80 and 120 kDa were recognized by the antibody in this tissue as well. However, the band at 120 kDa appeared to be more pronounced in brain than in cardiac homogenate while the 80 kDa band was rather lightened (Fig. 1B). Using an antiserum raised against a specific external domain of mouse AE1 (amino acids 571-583, see (31) ), which is not shared by rat AE3, Western blotting of cardiac membrane fraction proteins revealed mainly a 80 kDa band and two bands of lower molecular mass (i.e. 70 and 60 kDa) (Fig. 1C). In contrast, an antibody raised against the amino-terminal cytoplasmic 42-kDa fragment of human erythroid band 3 (see ``Experimental Procedures'') did not react with any protein in cardiac whole cell lysate or membrane fraction (Fig. 1D) while it recognizes rat band 3 (not shown). An antibody raised against a cardiac specific amino-terminal domain of rat AE3 (not shared by mouse AE1) strongly reacted with the 120-kDa protein but not with the 80-kDa one in cardiac membrane prepared from isolated cardiomyocytes. The 120-kDa immunoreactive band was no more seen when the blot was probed with the antibody in the presence of the peptide (10 µg/ml), which served as antigen (Fig. 1E). The antibody did not recognize any protein in human or rat ghosts (not shown). When the membrane proteins were run in the same gel and the blot was cut and probed with both the anti-cardiac AE3 and the anti-human erythroid band 3 antibodies, the protein recognized by the anti-AE3 antibody migrated to the same apparent molecular mass as the high molecular weight protein (120 kDa) recognized by the anti-erythroid band 3 antibody. Taken together, the data suggest that the 80- and 120-kDa proteins are encoded by two different genes. The 120-kDa protein is close to the anticipated size (113 kDa) of the cardiac AE3 cDNA product(19) . It is recognized by an antibody specifically raised against an amino-terminal domain of rat cardiac AE3. It is also much more expressed in brain than in heart. Because the specific cardiac-AE3 mRNA was not found in brain(18) , it is still possible that the brain 120-kDa protein results from another AE3 transcript different from the 3.8-kilobase cardiac or 4.4-kilobase brain mRNA that was also detected by Kudrycki et al.(18) . Anyhow, we propose that the 120-kDa polypeptide found in cardiac cells is the translation product of the major cardiac AE3 transcript. Since the 80-kDa protein was also recognized by an antiserum raised against a specific domain of AE1, which is not shared by AE3(19, 31, 32) , we suggest that this protein is a translation product of one of the AE1 trancripts expressed in heart(18) . We found a lower immunoreactivity of this protein in brain, which is in agreement with the low signal observed in Northern blot of brain mRNA probed with AE1 cDNA(18) . The question as to whether the 80-kDa polypeptide is the same amino-terminal truncated form of the erythroid band 3 as the one expected in kidney (17) is more difficult to answer. The antibody raised against the 42-kDa amino-terminal fragment of human erythroid band 3 did not react with any of the proteins in rat cardiac membrane. Such an observation is consistent with the expression of an amino-terminal truncated form of erythroid band 3 in cardiac cells. However, we still cannot fully exclude the possibility that such a lack of immunoreactivity was due to the presence of species-specific epitopes on the cytoplasmic amino-terminal fragment of band 3(33) .

Having characterized two band 3-like proteins in cardiac cells, we next looked at their subcellular localization. Cytosolic, membrane, and myofibril fractions were prepared from isolated cardiomyocytes. Western blotting of the membrane proteins revealed the presence of both the 80- and 120-kDa proteins. None of these proteins was detected in the cytosolic or the myofibril fraction (Fig. 1F). We also failed to detect any band 3-like proteins extracted from myofilaments and cytoskeleton even after treatment of myofibrils by a high ionic strength buffer (data not shown).

Indirect immunofluorescence and confocal microscopy approaches were used to further investigate the specific location of band 3-like proteins in adult rat isolated cardiomyocytes. The anti-human erythroid whole band 3 antibody decorated thin repetitive and transversely oriented riblike bands uniformly distributed along the cell. The periodicity of the transversal bands labeled by the anti-band 3 antibody was the same as the one of the sarcomeres (Fig. 2A). The decoration of the anti-myosin antibody complemented the one of the anti-band 3 antibody, indicating that the areas stained by the latest correspond to z lines (Fig. 2B). The secondary antibody used alone slightly decorated faint diffuse strands along the longitudinal axis of the cell (Fig. 2C). The distribution of band 3-like proteins was better seen at a higher magnification. Moreover, the cell shown in Fig. 3A was immunostained with both the anti-human erythroid whole band 3 and a monoclonal anti-myosin light chains antibodies. The striations decorated by the anti-band 3 antibody showed clearly a sarcomeric distribution (Fig. 3A). The distance between two striations equals 1.86 ± 0.04 µm (n = 10), which is similar to the sarcomere length. The riblike bands were colocalized with dark lines that were not stained by the anti-myosin antibody, further indicating the presence of the band 3-like proteins in z lines (Fig. 3A). Vizualization of the double staining confirmed the presence of band 3-like proteins at alternative repetitive areas between every sarcomere labeled by the anti-myosin antibody (Fig. 3B). Fig. 3C presents a single optical section of a cell immunostained with the anti-human erythroid whole band 3 antibody. The focal plan passed close to the center of the cell. Such an image revealed a more intense fluorescence at the cell margins, which slightly decreased toward the cell center. In fact, the stained structures were the heavily labeled sarcolemma and invaginations that ran toward the center of the cell section. Such a distribution of band 3-like proteins is consistent with the presence of the proteins in the sarcolemma and along the T-tubules. The anti-human erythroid whole band 3 antibody labeling of transversal riblike bands between the A-bands decorated by the anti-myosin antibody also suggests the presence of the band 3-like proteins in the costameres, defined as regions of attachment of myofibrils to the sarcolemma(34) . It is striking to note that the Na/Ca exchanger was recently found to be also distributed along the sarcolemma and T-tubules(35) . Such a location of ionic exchangers should be physiologically significant in cardiac cells since T-tubules allow for an extension of the exchange surface between the cell and the external medium. The sarcomeric distribution of band 3-like proteins would allow for a minute regulation of cytoplasmic pH and prevent the local accumulation of HCO(3) ions generated by the hydration of metabolic CO(2). The presence of band 3-like proteins in costameres raises the possibility that besides pH regulation, one or the other protein could exert an anchorage function for the contractile apparatus or the cytoskeleton to the membrane as observed in erythrocytes(36) .


Figure 2: Confocal epifluorescence images of immunostained myocytes. The myocytes were immunostained by the anti-human whole band 3 antibody (A) or an anti-myosin light chains antibody (B). Antibodies were visualized by a Texas red- or fluorescein-conjugated anti-rabbit IgG, respectively. The cell shown in C was labeled with only the secondary Texas red-conjugated antibody. The confocal projection images shown were built from 40 sequential sections at an optical resolution of 0.5 µm in the z direction. The anti-human whole band 3 antibody labeled transversal riblike structures, while the anti-myosin antibody decorated squared areas corresponding to the sarcomeres. The secondary Texas red-conjugated antibody weakly decorated longitudinal structures.




Figure 3: Further localization of the band 3-like proteins in immunostained cells observed in epifluorescence confocal microscopy. A, separate high magnification confocal views of a cell double-immunostained with both (a) the anti-human whole band 3 antibody visualized by a Texas red-conjugated secondary antibody and (b) an anti-myosin light chain antibody visualized by a fluorescein-conjugated secondary antibody. The striations labeled by the anti-whole band 3 antibody correspond to z lines, the darklines that are not decorated by the anti-myosin antibody. B, Confocal picture of the same cell as in A immunostained with both the anti-human whole band 3 (red staining) and the anti-myosin light chains antibodies (green staining). In A and B, the images were built from the first 20 sections out of 40 (in the z direction) at an optical resolution of 0.5 µm. C, single optical section of a cell decorated with the anti-whole band 3 antibody. The focal plan passes around the center of the cell (section 17 out of 43).



Since Western blotting analysis suggested that both the 80- and 120-kDa proteins belong to the AE family, it could be anticipated that one or both proteins exert an ionic exchange function in cardiac cells. However, Raley-Susman et al.(37) have recently reported that in neurons, even if an AE gene is expressed, the expected anionic exchange function is not always observed. To determine whether the proteins recognized in Western blotting and cell immunostaining perform the Cl/HCO(3) exchange, we tested the effects of both the anti-human erythroid whole band 3 antibody and anti-mouse AE1 antiserum on anionic exchange activity in single SNARF1-loaded cardiomyocytes. The removal of Cl ions from external medium induced a rapid and large alkalinization due to the influx of HCO(3) ions into the cell following the reversal of the Cl/HCO(3) exchanger. The readdition of the Cl ions triggered an acidification allowing the cell to recover its initial pH(i) (Fig. 4A). The incubation of the cells for 1 h at 37 °C in the presence of the anti-whole band 3 antibody did not affect the rate of pH(i) changes triggered by Cl removal and readdition (not shown). In contrast, microinjection of the anti-whole band 3 antibody into the single cardiomyocytes inhibited the Cl/HCO(3) exchange activity. The rate of alkalinization triggered by Cl removal was slowed down in the cells microinjected with the anti-human erythroid whole band 3 antibody when compared with the one of the cells injected with nonspecific affinity purified IgG, used at the same concentration as the anti-band 3 antibody (Fig. 4A). Similarly, in the cells microinjected with the anti-human erythroid whole band 3 antibody, the rate of acidification induced by Cl readdition was also slowed down. The bar graph presented in Fig. 4B shows that the inhibition of the anionic exchange was a constant result observed in the 15 cells injected with the anti-band 3 antibody compared with the eight cells injected with purified IgG or the 36 noninjected cells. It should be noted that the inhibition was more marked when the exchanger worked in its physiological direction (i.e. acidification following Cl readdition), as further shown in some cells that were even not able to fully recover their initial pH(i) (see Fig. 4A). The anionic exchangers AE2 and AE3 (20, 38) exhibit a regulation by intracellular H or HCO(3) in a physiological pH(i) range, while AE1 displays such a regulation only at very alkaline or very acidic pH(39, 40) . The fact that some cells that were injected with the anti human erythroid band 3 antibody failed to fully recover their initial pH(i) may suggest that the binding of the antibody to cytoplasmic domains of the exchanger could modify such a sensitivity. Because pH(i) was monitored in a physiological range, this also suggests that, at least under our experimental conditions, AE3 could work as an anionic exchanger. Another possibility relates to the observation that at least in red cells, the rate of Cl transport across the membrane depends on the direction of the ionic flux (i.e. negative cooperativity of Cl ions, which develops only at the extracellular side of the membrane but not at the inner site; for review, see (10) ). The binding of the antibody to the exchanger could affect its conformation and in turn such a negative cooperativity. It further points to the presence of a functional anion exchange mediated by band 3-like proteins in isolated cardiac cells.


Figure 4: Injection of the anti-whole band 3 antibody into single cardiac cells inhibits the Cl/HCO(3) exchange activity measured by SNARF1 monitoring of pH. A, original pH recordings on single cells microinjected with 90 µg/ml of affinity purified IgG (a) or with 90 µg/ml of anti-human erythroid whole band 3 antibody (b) superfused with Cl-free or Cl-containing buffer as indicated. pH was calibrated in situ using an intracellular buffer containing 10 µg/ml of nigericin (see ``Experimental Procedures''). Note that the cell shown in (b) featured a remarkable inhibition of pH-recovery following Cl readdition. B, bar graph summarizing the results obtained as described above in 36 noninjected (control), eight rabbit affinity-purified IgG-injected (IgG) or 15 anti-whole band 3 antibody-injected (ab) cells. The activity of the anionic exchange was estimated from both the alkalinization slope (Cl removal) and the acidification slope (Cl addition). The slopes were calculated from the same range of pH (i.e. pH 7.3-7.5) in control or injected cells, and the results were expressed as H equivalent flux (beta.dpH/dt) assuming an intracellular buffering capacity (beta) of 40 mM/pH unit(25) . Results are expressed as mean ± S.E. *, significantly different from control (p < 0.01).



To check out whether the change of pH(i) triggered by Cl removal could be fully attributed to the Cl/HCO(3) exchanger activity, the cells were incubated for periods of 5 min at 32 °C in the presence of each increasing concentrations of DIDS. Preliminary experiments showed that such a treatment induced an inhibition of the exchange activity that was irreversible (whatever the inhibition was partial or maximal). It should be noted that the rate of such an irreversible DIDS inhibition of the exchanger appears to be faster than in a recombinant exchanger system(20) . After the 5-min incubation at a given DIDS concentration, the drug was removed, and the exchange activity tested; then, the same experiment was repeated in the same cell using a higher concentration of DIDS. Under this experimental condition, inhibition of the anionic exchange was dependent upon DIDS concentration in the range 0.5-10 µM. The half-maximal inhibition was observed at 1.0 µM DIDS, and full inhibition required 10 µM (Fig. 5). It should be noted that 10 µM DIDS has been reported to fully block both AE1 and AE3 activity but not AE2(20, 41) . Although the cellular environment could affect the sensitivity of the exchanger to DIDS, the dose-inhibition curve of the drug strengthens our proposal that AE1 and AE3 are expressed and that either or both do work as an ionic exchanger in cardiac cells. Since the anti-mouse AE1 antiserum was raised against a peptide corresponding to a domain 13 amino acids away from the expected external DIDS binding site, we tested the effect of the antiserum on the DIDS efficiency to block the anionic exchange. Incubation of the cells for 1 h at 37 °C in the presence of the antiserum followed by a 30-min washing period strongly shifted to the right the dose-inhibition curve of DIDS established according to the same protocol as described above. The half-maximal inhibition of the ionic exchange was observed at 190 µM, and 1 mM DIDS was required to completely block the anionic exchange activity. Such a reduction in DIDS-induced inactivation of the exchanger was not observed when the cells were incubated with a nonimmune rabbit serum (Fig. 5). The ionic exchange activity perse was not affected when the cardiomyocytes were bathed for 1 h at 37 °C in the presence of the anti-AE1 antiserum, which altogether excludes a nonspecific effect of the antiserum. We thus suggest that AE1 participates in the anionic exchange of cardiac cells.


Figure 5: Bathing the cells with the anti-AE1 antibody shifts the dose-dependent inhibition curve of DIDS. SNARF1-loaded cells were incubated for 5 min at 32 °C in the presence of increasing concentrations of DIDS. The drug was removed between each DIDS concentration tested, and the Cl/HCO(3) exchange activity was estimated as described in Fig. 4from the rate of alkalinization following the removal of Cl ions. The curves were obtained before () and after a 1-h incubation in the presence of (box) the anti-mouse AE1 (amino acids 571-583) antiserum (1/200) or of (up triangle) a nonimmune serum (1/200). Results are expressed as mean ± S.E. of at least three cells for each drug concentration.



In summary, using complementary technical approaches including Western blotting, confocal microscopy of immunostained cells, and microspectrofluorimetric pH(i) measurement, we have identified in cardiac cells two proteins immunologically related to the erythroid band 3. These proteins migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of 80 and 120 kDa. They are found only in the membrane fraction prepared from isolated ventricular cells and appeared to be repeatedly distributed in single cells. Furthermore, microinjection of the anti-human erythroid whole band 3 antibody significantly prevented the Cl/HCO(3) exchange activity in isolated cardiomyocytes, which strongly suggests that either or both proteins exert the function of anionic exchange. However, the marked reduction of the DIDS inhibition by a mouse anti-AE1 antibody strongly suggests that most, if not all, of the anionic exchange in isolated adult rat cardiomyocytes is mediated by the 80-kDa protein.

One major physiological implication of our results relates to the neurohormonal regulation of the Cl/HCO(3) exchanger in the heart. We previously reported that both the purinergic agonist ATP and the beta-adrenergic agonist, isoproterenol, activate the anionic exchange in cardiac cells(29, 42) . The identification of band 3-like proteins in heart has allowed us to demonstrate that the purinergic stimulation of the myocytes induces a tyrosine kinase-dependent phosphorylation of the 80 kDa band 3-like protein (43) . (^2)Besides, the isoproterenol effect is mediated by cyclic AMP-dependent PKA(44) . Since there is no predicted PKA phosphorylation sites on AE3(19) , it is likely that the 80-kDa protein would be phosphorylated by PKA. This leads to the proposal that AE1 could be the exchanger whose activity is regulated by external agonists in cardiac cells. Further investigations are required to understand the respective role of AE1 and AE3 in cardiac cells. Does one protein behave as an anionic exchanger and the other as an anchorage protein or do both exert both functions? To answer this question, we are currently investigating the effects of injection into the cardiomyocytes of anti-rat cardiac AE3 antibodies on the ionic exchange activity. Furthermore, the use of oligonucleotide antisenses to knock out the expression of AE1 or AE3 will also be valuable tools to go into the function of AEs expressed in cardiomyocytes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Fax: 33-67-41-52-42.

(^1)
The abbreviations used are: AE, anion exchanger; PBS, phosphate-buffered saline; BSA, bovine serum albumin; SNARF1/AM, seminaphthorhodafluor acetoxymethylester; BAPTA/AM, 1,2-bis-(2-aminophenoxy)ethane-N-N-N[prime]-N`-tetraacetic acid acetoxymethylester; DIDS, 4,4`-diisothiocyanatostilbene-2,2`-disulfonate; ECL, enhanced chemiluminescence; Pipes, 1,4-piperazinediethanesulfonic acid.

(^2)
M. Pucéat, I. Korichneva, R. Cassoly, and G. Vassort, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Drs. E. Bursaux for the kind gift of purified band 3, P. Cobbold for skillful and helpful advice for microinjection of cardiac cells, and S. Alper and J. Stull for the kind gifts of the anti-AE1 antiserum and anti-myosin light chain antibody, respectively. We also thank C. Deprette and Dr. I. Lebbar for preparing the anti-erythroid band 3 antibodies and for help in preparing the anti-rat AE3 antibodies and G. Geraud (Dept. of Imaging, Institut J. Monod) for expert assistance in confocal microscopy.


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