(Received for publication, June 9, 1994; and in revised form, October 21, 1994)
From the
The identification of the protein that exerts the function of
Cl/HCO
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
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
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.
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
-dependent transporter was also
recently reported to participate in pH
regulation
from acidosis(5, 6) . In contrast, a rise in
pH
activates a Na
-independent
Cl
/HCO
exchanger, which
operates as a HCO
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
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
exchanger
in tissues so far investigated is the erythrocyte band 3 glycoprotein.
The protein is responsible for the extrusion of
HCO
ions generated by the hydration of
intracellular CO
. The anionic flux allows
HCO
equilibration between plasma and
erythrocyte following CO
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, (
)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.
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 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, 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.
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
g to discard the myofilaments. The supernatants
were saved and added with 4
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
.
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
10
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
, 1.5 mM KH
PO
, 1.7 mM MgCl
, 1
mM CaCl
, 21 mM Hepes, 11 mM glucose, 10 mM creatine, 20 mM taurine, and 0.5%
BSA for at least 8 h.
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 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
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 g for 15 min.
The pellet was discarded, and the supernatant added with 4
Laemmli buffer prior to Western blotting.
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 60 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.
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.
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
ions generated by the
hydration of metabolic CO
. 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
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
ions into the cell following the reversal of the
Cl
/HCO
exchanger. The
readdition of the Cl
ions triggered an acidification
allowing the cell to recover its initial pH
(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
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
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
(see Fig. 4A). The anionic exchangers AE2 and AE3 (20, 38) exhibit a regulation by intracellular
H
or HCO
in a
physiological pH
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
may suggest that the binding of
the antibody to cytoplasmic domains of the exchanger could modify such
a sensitivity. Because pH
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
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
(
.dpH/dt) assuming an intracellular buffering
capacity (
) 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 triggered by Cl
removal could be fully attributed to the
Cl
/HCO
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
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 (
) the anti-mouse AE1 (amino acids 571-583)
antiserum (1/200) or of (
) 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 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
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
exchanger in the
heart. We previously reported that both the purinergic agonist ATP and
the
-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) . (
)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.