(Received for publication, September 27, 1996, and in revised form, May 14, 1997)
From the Departments of The rat liver canalicular bile acid
transporter/ecto-ATPase/cell CAM 105 (CBATP) is a 110-kDa transmembrane
phosphoglycoprotein that is thought to have bile acid efflux,
ecto-ATPase, and cell adhesion properties. Its extracellular
amino-terminal domain is highly homologous to carcinoembryonic antigen
(CEA), a glycophosphatidyl inositol-anchored membrane protein with cell
adhesion properties and a marker for adenocarcinoma. In the current
study, we examined the possibility of more clearly defining the role of
CBATP in bile acid efflux by cotransfecting a heterologous cell, the
COS cell, with cDNAs for a bile acid importer, the ileal bile acid transporter (IBAT), as well as for CBATP. The results show that when
IBAT mediates uptake of [3H]taurocholate to a level
20-fold higher than that achieved previously by nonspecific
pinocytosis, CBATP mediates time-, temperature- and
concentration-dependent efflux. Efflux of
[3H]taurocholate mediated by CBATP in the cotransfected
COS cells is saturable and has curvilinear kinetic characteristics
(Vmax = 400 pmol/mg protein/min,
Km = 70 µM). It is inhibited by
4,4 The net vectorial transport of bile acids into the biliary
drainage system is the major determinant of bile secretion. Transport across the canalicular domain of the hepatocyte represents the rate-limiting step in this system. Studies in canalicular membrane vesicles have indicated that canalicular bile acid efflux is
predominantly driven by ATP, but electrochemical membrane potential may
also drive canalicular bile acid transport to a certain extent. Most data suggest that several distinct transporters, or transport systems,
are involved (1-6).
The rat liver canalicular bile acid transport protein/ecto-ATPase/cell
CAM 105 (CBATP)1 is one candidate for bile
acid efflux activity at the canalicular membrane of hepatocytes. It is
a ~110-kDa phosphoglycoprotein localized to the canalicular domain of
hepatocytes. It was purified by bile acid affinity chromatography from
detergent-solubilized rat liver canalicular membrane vesicles (7).
Internal amino acid sequence analysis revealed it to be identical to
the rat liver ecto-ATPase and to the cell adhesion molecule cell CAM
105. It has a carboxyl-terminal cytoplasmic tail of 71 amino acids, a
single membrane-spanning domain, and a large extracellular
amino-terminal tail of 423 amino acids, which has extensive homology
with carcinoembryonic antigen (CEA) and the immunoglobulin supergene
family. Transfection studies have shown that CBATP mediates
ecto-ATPase, cell adhesion, and bile acid efflux activities in
heterologous COS cells (8, 9). Mutagenesis studies have shown that R98
within an ATPase consensus sequence at the extreme amino terminus is
required for ecto-ATPase activity and that the 108 amino acid
amino-terminal domain is required for cell adhesion activity (10, 11).
Deletion of the cytoplasmic tail is associated with loss of bile acid
efflux, ecto-ATPase, and cell adhesion activities, even though the
protein is appropriately localized to the external surface of the
plasma membrane and can bind ATP (8, 9). Site-directed mutagenesis of
phosphorylation consensus sequences in the cytoplasmic tail shows that
protein kinase C-dependent phosphorylation of
Ser503 is required for bile acid efflux activity, that
tyrosine kinase-dependent phosphorylation of
Tyr488 regulates bile acid efflux activity, but neither
phosphorylation is necessary for ecto-ATPase activity. Finally, bile
acid efflux activity of CBATP was found to be dependent on ATP,
particularly extracellular ATP, but not on its own ecto-ATPase activity
(12).
In these previous studies, the bile acid efflux activity of CBATP was
demonstrated in transfected COS cells by first loading the cells with
[3H]taurocholate via nonspecific pinocytosis using the
approach that had been pioneered in studies of efflux of
chemotherapeutic agents by the multidrug resistance gene products (MDR)
(8). To get sufficient nonspecific uptake, the membrane potential of the transfected COS cells had to be clamped pharmacologically with
valinomycin (8). Although this system allowed us to show that CBATP had
bile acid efflux activity and that this could be proven by a number of
genetic criteria, the assay was cumbersome and was not amenable to
studying the role of membrane potential in bile acid efflux. To develop
a more physiological system and a system that could be more easily
manipulated pharmacologically, we have established a model system in
which uptake of [3H]taurocholate is mediated by
transfection of a cloned bile acid importer, IBAT, and efflux is
assayed by cotransfection of CBATP cDNA.
Wild-type and mutant rat CBATP
constructs have been described previously (8, 10, 11). This includes
pExp3, R98ACBATP, Y488F-CBATP, T502,S503A-CBATP, and truncated CBATP.
The hamster IBAT construct (IBAT-44 final) has also been described
previously (13). The CEA cDNA (gcg:humcea; Ref. 14), kindly
provided by Thomas Barnett, was subcloned into the
HindIII-XbaI site of pCDM8. Three new constructs,
R98A CEA, CBATP-CEA chimera, and CBATP-R98ACEA chimera, were generated
using the polymerase chain reaction overlap extension technique (15)
and then subcloned into the pCDM8 vector.
For R98ACEA, the outside primers corresponded with nucleotides 85-100
and 993-1012 of CEA together with a spacer of four nucleotides, a new
EcoRI restriction site, and a new BamHI
restriction site (5 For the CEA-CBAT chimera, the outside primers corresponded to
nucleotides 1563-1585 of CEA and nucleotides 1628-1651 of CBATP together with new EcoRI and XbaI restriction
sites, and the inside primers corresponded to nucleotides 2122-2136 of
CEA (five amino acids just external to the membrane insertion site of
CEA) and nucleotides 1316-1330 of CBATP (transmembrane domain of
CBATP): outside primers (5 COS 1 cells were transfected
by the DEAE-dextran method and used for experimental purposes 48 h
after transfection (19). In specific experiments, cellular ATP was
depleted by incubating transfected COS cells for 20 min at 37 °C in
DMEM without glucose but supplemented with 20 mM
2-deoxyglucose and 10 mM sodium azide. Under these
conditions, cellular concentrations of ATP could be lowered from
800-1200 µM to 6-8 µM, as determined by
the ATP luciferase assay (20). Results were normalized for protein
concentration as determined by the Lowry assay (21). Under these
conditions, cell viability as determined by trypan blue exclusion (22)
was not significantly different between these depleted cells and
undepleted cells (data not shown).
Transfected Cos cells were incubated
with DMEM supplemented with [3H]taurocholate in excess.
At the end of the uptake period, cell monolayers were lysed in 1 N NaOH, and the cell lysates were subjected to
scintillation counting. In separate experiments, the time, temperature,
and concentration of [3H]taurocholate were varied to
determine the optimal conditions for uptake.
Transfected Cos cells were incubated
with [3H]taurocholate under optimal conditions for
uptake. At the end of the uptake period, monolayers were washed
extensively in PBS and incubated for specified time intervals in DMEM
alone for the efflux period. In specified experiments, 5 µM ATP, 1 mM DIDS, and/or unlabeled
taurocholate were added. Extracellular medium was harvested, and cell
monolayers were lysed in 1 N NaOH. The extracellular medium
and cell lysates were then subjected to scintillation counting. Counts
in the extracellular medium were converted to picomoles on the basis of
the specific activity of the initial [3H]taurocholate and
then plotted as pmoles/milligram protein/minute. Kinetic data
(Km and Vmax) were determined
on the basis of the resulting curves.
Ecto-ATPase activity was measured by a
method described previously (23) 48 h after transfection.
For Western blot analysis, antibody
to CBATP was used in a protocol described previously (7). Methods
described previously were also used for studies of biosynthesis (24),
cell surface iodination (25), and immunoprecipitation followed by
SDS-polyacrylamide gel electrophoresis/fluorography (24).
We
determined the conditions under which uptake of
[3H]taurocholate was saturated in COS cells transfected
with IBAT alone or IBAT and CBATP cDNA together. To determine the
duration of time necessary for saturation of uptake, transfected COS
cells were incubated at 22 °C with [3H]taurocholate,
400 µM, for several different time intervals (Fig. 1A). Cells were lysed in 1 N NaOH
and subjected to scintillation counting. Uptake was
time-dependent, reaching a plateau within 20 min. There was
no significant difference between cells transfected with IBAT alone and
cells transfected with IBAT and CBATP together. Then the transfected
COS cells were incubated for 20 min with [3H]taurocholate, 400 µM, at several
different temperatures (Fig. 1B). Uptake was
temperature-dependent, reaching a plateau at 22 °C. There
was no significant difference between cells transfected with IBAT alone
and those transfected with IBAT and CBATP together. Next, the
transfected COS cells were incubated for 20 min at 22 °C in
[3H]taurocholate in several different concentrations
(Fig. 1C). The results show that uptake was
concentration-dependent, reaching a plateau between 200 and
400 µM. In COS cells transfected with IBAT and CBATP,
there was a minimal decrease in the amount of uptake as compared with
COS cells transfected with IBAT alone. This is probably due to efflux
mediated by cotransfected CBATP. Efflux mediated by cotransfected CBATP
does not have a more significant effect in Fig. 1C or any
effect in Fig. 1, A and B, because these experiments were done in the absence of exogenous ATP. Because efflux
mediated by CBATP is stimulated by exogenous ATP (see Fig. 3), this
means that there will be minimal efflux mediated by CBATP during uptake
studies done in the absence of exogenous ATP. Even when exogenous ATP
is present, CBATP pumps [3H]taurocholate out of cells
less efficiently than IBAT pumps [3H]taurocholate into
cells. For uptake mediated by IBAT, Km
Taken together, the experiments in Fig. 1 (A-C) show that
uptake reaches a plateau at 200-400 µM in the
cotransfected cells and establish a time of 20 min, temperature of
22 °C, and concentration of [3H]taurocholate of 400 µM as optimal for the subsequent studies. In each case,
the uptake of [3H]taurocholate was 20-fold or more
greater in COS cells transfected with IBAT alone or both IBAT and CBATP
than untransfected COS cells and COS cells transfected with CBATP alone
and pharmacologically clamped with valinomycin (8).
Next, we examined the possibility that CBATP mediated bile acid efflux
under these conditions. For Fig. 2A, COS cells were transfected with IBAT alone, cotransfected with IBAT and CBATP, or
cotransfected with IBAT and a mutant CBATP (T502A, S503A-CBATP). Our
previous studies had shown that this mutant CBATP did not undergo
protein kinase C-mediated phosphorylation and lacked bile acid efflux
activity, even though it was appropriately targeted to the external
surface of the plasma membrane (12). After 48 h, the cells were
incubated at 22 °C for 60 min with [3H]taurocholate in
several different concentrations. At the end of this time interval, the
cells were washed extensively and incubated at 22 °C for 5 min in
the absence of [3H]taurocholate, in the presence of ATP,
and in the absence or presence of DIDS. Radioactivity appearing in the
extracellular medium was determined by scintillation counting. The
results show that there was DIDS-sensitive, concentration-dependent,
and saturable efflux only in cells cotransfected with IBATP and CBATP,
not in cells transfected with IBAT alone or cotransfected with IBAT and mutant T502A, S503A-CBATP. Efflux had curvilinear characteristics, with
a Vmax of 400 pmol/mg protein/min and a
Km of 70 µM. These values are similar
to those obtained using our previous assay system. These values are
also similar to values that have been reported previously in
canalicular vesicle studies (17). For the studies shown in Fig.
2A, we also measured radioactivity remaining in cell
lysates. The results were plotted as pmoles/mg protein/minute
versus concentration of taurocholate and showed that
disappearance from the cell had almost identical curvilinear characteristics, Km and Vmax
values (data not shown) as observed in Fig. 2A for
appearance in the extracellular medium. In both cases, however, we did
not determine what proportion of taurocholate that is in the cells is
freely available for efflux. Thus, the Km and
Vmax values reported here must be considered estimates which apply only if all the taurocholate in the cell is
freely available for efflux.
Next, we examined the time course of efflux mediated by CBATP (Fig.
2B). COS cells transfected with IBAT alone or cotransfected with IBAT and CBATP were incubated for 60 min at 22 °C with
[3H]taurocholate 400 µM. At the end of this
time interval, the cells were washed extensively and then incubated at
22 °C for several different time intervals in medium supplemented
with unlabeled taurocholate (400 µM) and 5 mM
ATP in the absence or presence of 1 mM DIDS. At the end of
each time interval, the extracellular medium was harvested, and the
cell monolayers were lysed for analysis by scintillation counting.
Results are reported as a relative percentage using counts present in
the cell lysate at time 0 as arbitrarily designated 100%. There was no
difference in the counts present in the cell lysates at time 0 for
cells transfected with IBAT alone or cells cotransfected with IBAT and
CBATP. For cells cotransfected with IBAT and CBATP, the results show
that there is time-dependent disappearance of radioactivity
from the cells between time 0 and 5 min, coincident with the appearance
of radioactivity in the extracellular medium. Almost 80% of the
initial radioactivity has disappeared from the cells by 5 min of the
chase period, and a similar percentage has appeared in the
extracellular medium. The majority of this disappearance from cell
monolayers and appearance in extracellular medium is DIDS-sensitive.
There is, however, some radioactivity that leaks from the cells in the
presence of DIDS. Interestingly, this DIDS-insensitive fraction has
different kinetics, reaching a plateau within 3 min. For cells
transfected with IBAT alone, there is some time-dependent
disappearance of radioactivity from the cells and appearance of
radioactivity in the extracellular medium. It is much less than that
observed in the cotransfected cells. Only 30% of the initial
radioactivity disappears, even by 10 min. This disappearance is
completely insensitive to DIDS and is identical in magnitude and
kinetics to the DIDS-insensitive fraction of the cotransfected cells,
therefore providing evidence that it represents nonspecific diffusion.
Unlabeled taurocholate was used in this experiment to optimize the
chase effect, but similar results have been observed without unlabeled
taurocholate (data not shown). Taken together, these studies show that
CBATP can mediate specific, facilitated efflux of taurocholate in
cotransfected COS cells and that its efflux properties can be detected
more easily, over a longer duration of time and in the absence of the pharmacologic agent valinomycin originally used to promote nonspecific uptake of taurocholate by clamping the membrane potential.
Now we could use this assay to examine
the possibility that bile acid efflux mediated by CBATP is driven by
ATP and/or membrane potential differences. COS cells were cotransfected
with IBAT and CBATP and then, 48 h later, the cotransfected cells
were incubated in 2-deoxyglucose and sodium azide under conditions
associated with depletion of cellular ATP but without affecting cell
viability. During this same 20-min interval, 400 µM
[3H]taurocholate were also added to the medium for uptake
studies. The cells were then washed extensively, and separate
monolayers were incubated in the absence or presence of 1 mM DIDS and in the absence or presence of exogenous ATP in
several different concentrations for an efflux assay of 5 min (Fig.
3). The results show that there is no efflux in the
absence of ATP. It also shows that efflux is absolutely dependent on
ATP. The effect of extracellular ATP is
concentration-dependent and saturable with a
Km To determine whether the electrochemical potential of the membrane
drives bile acid efflux activity mediated by CBATP, we examined the
effect of clamping the membrane potential with valinomycin. Cells were
cotransfected with IBAT and CBATP and then, 48 h later, incubated
with 150 µM [3H]taurocholate in the absence
and presence of DIDS and in the absence or presence of 100 µM valinomycin, an ionophore that clamps the membrane
potential (8, 10, 11). Cells were then washed and incubated for an
additional 5 min at 22 °C in buffer supplemented with 5 mM ATP. The results show that valinomycin does not inhibit bile acid efflux (data not shown). Taken together, these data indicate
that bile acid efflux mediated by CBATP is dependent on ATP but not on
the electrochemical membrane potential. Thus, it cannot account for
electrogenic bile acid transport demonstrated previously in canalicular
membrane vesicles (26-28).
To further examine the specificity of the
bile acid efflux activity mediated by CBATP and to further establish
the validity of the cotransfection assay, we examined a series of
positive and negative controls in the assay. In each case, these
controls, shown schematically on the basis of their presumed
relationship to the plasma membrane in Fig. 4, were
cotransfected with IBAT. The negative controls included a truncated
CBATP construct in which 66 of the 71 amino acids in the cytoplasmic
tail have been deleted and two constructs in which specific amino acids
required for phosphorylation have been deleted, Y488F-CBATP and T502A, S503A-CBATP. These three constructs did not mediate bile acid efflux in
our previous assay system (12). The R98A-CBATP construct represented a
positive control. It has a mutation in the ectoplasmic ATPase consensus
sequence, which abrogates ecto-ATPase activity but not bile acid efflux
activity. We also used several new constructs based on the structure of
CEA. CBATP shares extensive homology with CEA in its amino-terminal
extracellular tail, particularly the extreme amino-terminal half of
CEA. However, CEA lacks a membrane-spanning domain and cytoplasmic
tail. It is linked to the membrane by a phosphatidylinositol-glycan
moiety (29). Because our previous studies had shown that the bile acid
efflux activity of CBATP depended on its cytoplasmic tail, we doubted
that CEA would have bile acid efflux activity. We also generated a
chimeric construct in which the cytoplasmic tail and membrane-spanning
domain of CBATP were fused to the extracellular domain of CEA, a
construct we called the CBATP-CEA chimera, predicting that it would now possess bile acid efflux activity. Finally, we generated two additional constructs that were based on the observation that CEA has an ATPase
consensus sequence almost identical to that of CBATP and at the exact
same distance from the amino terminus as in CBATP. For the last two
constructs, we subjected the CEA construct and the CBATP-CEA chimeric
construct to site-directed mutagenesis of Arg98 to alanine,
the mutation that abrogated ecto-ATPase activity in CBATP. These
constructs are referred to as R98A-CEA and CBATP-R98A-CEA.
We examined the expression of all of these mutants after transfection
of COS cells by Western blot analysis, immunoprecipitation after
metabolic labeling, and immunoprecipitation after cell surface labeling
using antibodies to CBATP and CEA. In Western blot analysis (Fig.
5A), we detected equivalent amounts of an
~180-kDa polypeptide for CEA, R98A-CEA, CBATP-CEA, and CBATP-R98A-CEA
and ~110-kDa polypeptide for R98A-CBATP, Y488F-CBATP, T502,
S503-CBATP, and wild-type CBATP. There was an equivalent amount of a
~100-kDa polypeptide for truncated CBATP but no polypeptide detected
for vector alone (CON). These results showed that cotransfected COS cells expressed an approximately equivalent steady-state level of the
wild-type, chimeric, and mutant CEA and CBATP molecules. Cotransfected
COS cells were also pulse-labeled with [35S]methionine
for 3 h, and the resulting radiolabeled cell lysates were
subjected to immunoprecipitation followed by SDS-polyacrylamide gel
electrophoresis/fluorography (Fig. 5B). Polypeptides of
~180 kDa were detected in similar amounts in COS cells cotransfected with IBAT and wild-type, chimeric, and mutant CEA cDNAs. Similar amounts of ~100- and 110-kDa polypeptides were detected in COS cells
cotransfected with IBAT and wild-type or mutant CBATP. Polypeptides of
~90 and ~100 kDa were detected in similar amounts for truncated CBATP. The biochemical nature of the faster migrating ~100-kDa polypeptide for wild-type and mutant CBATP or ~90-kDa polypeptide for
truncated CBATP is not yet known. They have been observed in our
previous studies (8, 10, 12) as well as studies of others (36-38).
Their immunoprecipitation is blocked by purified unlabeled CBATP (data
not shown). They are, therefore, thought to be precursors or
intermediates. Taken together, the results demonstrated that there were
similar levels of synthesis of wild-type, chimeric, and mutant CEA
molecules and CBATP molecules in the appropriate cotransfected COS
cells. Finally, we examined whether the same polypeptides could be
detected at the external aspect of the plasma membrane of cotransfected
COS cells by immunoprecipitation after cell surface iodination (Fig.
5C). The results showed that there were similar amounts of
CEA and CBATP detected in each case. Our previous studies have shown
that only proteins on the external surface of the plasma membrane are
labeled under these conditions (8, 10, 12), and thus these results
indicate that similar amounts of CEA and CBATP reach the cell surface
in the COS cells transfected with IBAT and wild-type, chimeric, and
mutant CEA.
We could now examine the bile acid efflux activity in these
cotransfected COS cells. Separate monolayers that had been
cotransfected 48 h earlier were incubated for 1 h at 22 °C
in DMEM supplemented with 400 µM
[3H]taurocholate acid. The cells were then washed
extensively and then incubated in DMEM supplemented with 5 µM ATP in the absence or presence of DIDS for 5 min. The
resulting cell culture media samples were subjected to scintillation
counting (Fig. 6). The results show that there is
significant efflux of taurocholate by COS cells cotransfected with IBAT
and CBATP, with IBAT and R98A-CBATP, and to a lesser extent, by IBAT
and Y488FCBATP but not by COS cells transfected with IBAT alone or
by COS cells cotransfected with IBAT and truncated CBATP or IBAT and
T502A, S503A-CBATP. These results are similar to results in our
previous assay system (8, 10, 12). The results also show that there is
significant efflux of taurocholate in COS cells cotransfected with IBAT
and the CBATP-CEA chimera and with IBAT and the CBATP-R98A-CEA chimera but not in COS cells cotransfected with IBAT and CEA or IBAT and R98A-CEA. These results indicate that the cytoplasmic tail and membrane-spanning domain of CBATP can confer bile acid efflux properties on another protein, CEA, which does not ordinarily have
these properties. The results also provide evidence that the effect of
CBATP on bile acid efflux in the cotransfection assay is highly
specific and, therein, provide further evidence for the validity of the
cotransfection assay system.
We also examined the ecto-ATPase activity conferred on COS cells by
transfection of these CBATP and CEA constructs (Fig. 7). The results show that the Y488F-CBATP, T502, S503A-CBATP, CBATP, and
CBATP-CEA chimera confer ecto-ATPase activity on the transfected COS
cells but control vector alone, truncated CBATP, R98A CBATP, CEA,
R98A-CEA, and CBATP-R98A-CEA do not confer ecto-ATPase activity. Finally, we examined the binding of ATP by wild-type and mutant CEA
molecules. Our previous studies have shown that the truncated CBATP
mutant and the R98A-CBATP can bind ATP as well as wild-type CBATP, even
though these mutants cannot mediate ATP hydrolysis. Here we examined
the possibility that CEA and R98A-CEA bind ATP by photoaffinity
labeling with [35S]ATP
The results of this study establish the validity of a new system
for assaying carrier-mediated bile acid efflux. In our previous studies, we used a system in which transfected cells were loaded with
[3H]taurocholate by nonspecific pinocytosis, which
required pharmacologic clamping of the membrane potential with
valinomycin (8, 10, 12). By cotransfecting the genes for a transporter
that mediates uptake of bile acids and for a candidate efflux
transporter, there is an ~20-fold increase in loading of the cells
and no need for clamping of the membrane potential. Furthermore, the
efflux capacity of candidate gene products can be detected for a much
longer period of time. In the studies reported here, we could see the
effects of CBATP for as long as the studies were done, 60 min, whereas the effects of CBATP were only apparent for several minutes in the
previous assay system. Results of the study provide further evidence
that CBATP can mediate bile acid efflux. The effect of CBATP is
time-dependent, temperature-dependent, concentration-dependent, saturable, and has kinetic characteristics that are similar to those
reported previously in rat liver canalicular membrane vesicles (7). The
effect of CBATP on efflux of [3H]taurocholate is
modulated by one pharmacologic state (ATP depletion) but not by another
(valinomycin). This effect is abrogated by truncation of the
cytoplasmic tail and by site-directed mutagenesis of one
phosphorylation site (S503A) in the cytoplasmic tail, is decreased but
not eliminated by site-directed mutagenesis of another phosphorylation
site (Y488F), and is unaffected by site-directed mutagenesis of an ATP
consensus sequence in the extracellular tail. This effect can be
conferred on a related protein, CEA, by fusing its cytoplasmic tail and
membrane-spanning domain onto that protein. Site-directed mutagenesis
of the ATPase consensus sequence in the extracellular tail of the
chimera does not alter bile acid efflux activity, even though it
abrogates ecto-ATPase activity. The reduction in bile acid efflux
activity mediated by Y488F-CBATP and the abrogation of bile acid efflux
activity mediated by T502A, S503A-CBATP are again notable, in that
neither of these mutations affected ecto-ATPase activity. None of the alterations in bile acid efflux activity or ecto-ATPase activity, resulting from pharmacologic or genetic manipulation, could be attributed to alterations in biosynthesis, half-life, or targeting to
the external surface of the plasma membrane.
One particularly interesting result of this study is the bile acid
efflux activities of the CBATP-CEA chimera. Our previous studies had
shown that bile acid efflux activity was lost upon deletion of the
cytoplasmic tail of CBATP (8). Here the studies show that cytoplasmic
tail and membrane-spanning segment of CBATP can confer bile acid efflux
properties on the extracellular amino-terminal tail of a related
molecule, CEA. Presumably the cytoplasmic tail, with or without the
membrane-spanning segment, plays an essential role in binding of the
bile acid substrate. It was also interesting to find that the
cytoplasmic tail and membrane-spanning segment of CBATP could confer
ecto-ATPase activity on the same CBATP-CEA chimeric molecule. Our
previous studies had shown that CBATP lost its ecto-ATPase activity
when its cytoplasmic tail was deleted, but we could not exclude the
possibility that this was simply explained by a failure of the
truncated CBATP to be in the appropriate conformation (8). Taken
together with the current studies, however, it is now apparent that the
cytoplasmic tail and membrane-spanning domains transmit information
that is essential for ecto-ATPase activity and can even transmit that
information to CEA, a molecule which has an ectoplasmic ATPase
consensus sequence and can bind ATP but does not ordinarily possess
ecto-ATPase activity.
Although the results of this study show that CBATP can mediate bile
acid efflux in a model system, they do not establish the role or
contribution of CBATP to bile acid transport at the canalicular membrane under physiologic conditions. The results do show that CBATP
cannot account for bile acid efflux driven by the electrochemical potential differences at the membrane. Bile acid transport mediated by
CBATP is absolutely dependent on ATP. However, our previous studies
have suggested that it is extracellular ATP that induces bile acid
efflux mediated by CBATP (8, 10). Perhaps CBATP provides a mechanism
for bile acid efflux in response to canalicular ATP or in response to
ATP released during liver cell injury and that there are other
molecules/systems responsible for canalicular bile acid transport in
response to intracellular ATP (classical P-type ATPase transporter) as
well as canalicular bile acid transport in response to the
electrochemical membrane potential.
In contrast to most conventional transport proteins that have multiple
transmembrane domains (30), CBATP has only two, or perhaps one,
transmembrane domain. This may mean that it is a member of a
nonconventional class of transporters that only have a single
transmembrane domain and includes the minK potassium channel (31, 32)
and an influenza virus M2 proton pump (33, 34). However, our recent
studies of the cell adhesion properties of CBATP in infected Sf9 cells
(39) have indicated that CBATP molecules bind to each other. Clustering
of CBATP molecules could, therefore, bring two or more CBATP molecules
and their transmembrane domains into close proximity to potentially
form a pore in the membrane for bile acid efflux.
Although it was not the major focus of this study, the results do
indicate that CEA can bind extracellular ATP. Because the ATPase
consensus sequence responsible for ATP binding is highly conserved
among members of the CEA family, it is likely that ATP binding is a
property of many of these molecules. There is apparently a substantial
amount of ATP and other adenine nucleotides in bile (35). However,
whether CEA molecules sticking out into the lumen of the biliary
canaliculus can bind ATP or other nucleotides under physiologic
conditions or whether binding of ATP by CEA plays a specific
physiologic role is not known.
We are indebted to Elizabeth Karner and Joyce
L. Williams for preparing the manuscript.
Pediatrics and
Cell
Biology and Physiology,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-diisothiocyanostilbene-2,2-disulfonic acid and dependent on ATP
but not dependent on membrane potential. Although CEA could not mediate
bile acid efflux in COS cells cotransfected with IBAT and CEA, efflux
of [3H]taurocholate was detected in COS cells
cotransfected with IBAT and a chimeric molecule having the
carboxyl-terminal tail and membrane spanning domain of CBATP and the
amino-terminal extracellular tail of CEA. Taken together, these data
provide further evidence that CBATP confers bile acid efflux properties
on heterologous cells and that its cytoplasmic tail and membrane
spanning segment are integral to this property. The data also establish
a model system for more clearly defining the molecular determinants of bile acid transport mediated by this molecule.
Plasmid Constructs
-TCATGAATTCAGAGGAGGACAGAGC-3
;
5
-GGCACGTATAGGATCCACTA-3
). The inside primers corresponded to
nucleotides 393-416 of CEA (5
-CGCATACAGTGGTGCAGAGATAAT-3
;
5
-ATTATCTCTGCACCACTGTATGCG-3
). The resulting polymerase chain
reaction fragment was subcloned into wild-type CEA in the pCDM8 vector,
which had been digested previously with
EcoRI/BamHI and purified away from the wild-type internal fragment.
-CAGTGGCCACAGCAGGACTACAG-3;
5
-GAATTCTCTAGACTGGTGCAGTCAGCAGGACAGACA-3
); inside primers
(5
-TGAGAGGCCAGAATTGACTGTGATGCTCTT-3
;
5
-AAGAGCATCACAGTCAATTCTGGCCTCTCA-3
). The resulting
0.95-kilobase polymerase chain reaction fragment was digested with
BalI and XbaI and cloned into pBluescript
together with a 1.5-kilobase BalI/EcoRI partial
digest of wild-type CEA. The resulting insert was removed at
HindIII/XbaI to be subcloned into pCDM8. A
similar strategy was used for the CBATP-R98ACEA chimera using the
R98ACEA construct described above. In each case, the constructs were
characterized by restriction map analysis (16, 17) and by
dideoxynucleotide sequencing (18) to confirm the construct and to
exclude polymerase chain reaction sequence artifacts.
Cotransfection of COS Cells with IBAT and CBATP cDNA
23 µM and Vmax
396 pmol/mg
protein/min (13), and for efflux mediated by CBATP in the presence of
exogenous ATP, Km
70 µM and
Vmax
400 pmol/mg protein/min (Fig.
2A). When uptake of
[3H]taurocholate was assayed in the presence of exogenous
ATP (5 mM), there was ~40-50% reduction in uptake after
60 min in COS cells cotransfected with IBAT and CBATP as compared with
COS cells transfected with IBAT alone (Fig. 1D).
Fig. 1.
Uptake of [3H]taurocholate in
transfected COS cells. Cells were transfected with IBAT cDNA
alone (), IBAT and CBATP cDNAs together (
), or vector alone
(
), using conditions described previously. After 48 h,
transfected cells were subjected to uptake assays. A, time
dependence. Cells were incubated with 400 µM
[3H]taurocholate at 22 °C for several different time
intervals up to 60 min. B, temperature dependence. Cells
were incubated with 400 µM [3H]taurocholate
for 60 min at several different temperatures. C, concentration dependence. Cells were incubated at 22 °C for 60 min
with [3H]taurocholate in several different
concentrations. At the end of these incubations, the cells were rinsed
extensively, homogenized, and subjected to scintillation counting.
D, effect of ATP. Cells were incubated at 22 °C with 400 µM [3H]taurocholate for several different
time intervals up to 60 min. This incubation was done in the absence or
presence of 5 mM ATP. The results of three replicate
samples at each data point are reported as mean ± 1.0 S.D.
(bars).
, IBAT;
, IBAT + ATP;
, IBAT + CBATP;
,
IBAT + CBATP + ATP.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Effect of ATP on bile acid efflux in
cotransfected COS cells. Cells were cotransfected with IBAT and
CBATP cDNA and studied 48 h later. At that time, the cells
were incubated for 20 min at 22 °C with 400 µM
[3H]taurocholate to load the cells as well as 20 mM 2-deoxyglucose and 10 mM sodium azide to
deplete cellular ATP. Previous studies have shown that these conditions
severely deplete cellular ATP levels but do not affect cell viability
or one function of CBAT, its ecto-ATPase activity (20). These
conditions did not significantly alter uptake of
[3H]taurocholate (data not shown). The cells were then
washed and incubated for 5 min at 22 °C with fresh unlabeled medium
in the absence or presence of 1 mM DIDS and in absence or
presence of ATP in several different concentrations. Results are
reported as mean ± 1.0 S.D. (bars).
Km 10 µM;
Vmax = 300 pmol/mg protein/min.
[View Larger Version of this Image (16K GIF file)]
Fig. 2.
Efflux of [3H]taurocholate in
transfected COS cells. Cells were transfected with IBAT cDNA
alone, cotransfected with both IBAT and CBATP cDNA, or
cotransfected with IBAT and the T502A, S503A mutant CBATP.
A, concentration dependence. After 48 h, the cells were
incubated for 60 min at 22 °C in DMEM supplemented with
[3H]taurocholate in several different concentrations. The
cells were then washed and incubated for 5 min at 22 °C in medium
supplemented with 5 mM ATP in the absence or presence of 1 mM DIDS. The medium was then harvested; the cells were
homogenized, and each was subjected to scintillation counting. There
was no significant difference in the amount of counts present in the
cells at the end of the uptake for cells transfected with IBAT alone
(), IBAT and CBATP (
), or IBAT and T502A, S503A mutant CBATP
(
). Vmax = 400 pmol/mg protein/min,
Km = 70 µM. B, time dependence.
After 48 h, the cells were incubated for 60 min at 22 °C with
DMEM supplemented with 400 µM
[3H]taurocholate. The cells were then washed and
incubated at 22 °C in DMEM supplemented with unlabeled taurocholate
(400 µM), 5 mM ATP in the absence (
) or
presence (
) of 1 mM DIDS for several different time
intervals up to 10 min. The extracellular medium was then harvested;
the cells were homogenized, and each was subjected to scintillation
counting. The results are reported as relative percentage by comparing
the counts in the specified sample to the total amount of counts taken
up by the cells after 60 min of uptake. The results of three samples at
each time point are reported as mean ± 1.0 S.D. (bars).
IC, cell monolayers; EC, extracellular medium.
[View Larger Version of this Image (22K GIF file)]
10 µM and
Vmax of 300 pmol/mg protein/min.
Fig. 4.
Schematic diagram of wild-type, mutant, and
chimeric CBATP and CEA molecules. Wild-type CBATP has cytoplasmic
(CYT) and transmembrane (TM) domains. Wild-type
CEA is linked to the membrane by a phosphatidylinositol-glycan moiety
(M). N, the amino-terminal immunoglobulin V-like
region. A1, A2,
A3, B1,
B2, and B3,
immunoglobulin C-like regions.
[View Larger Version of this Image (41K GIF file)]
Fig. 5.
Expression of wild-type, mutant, and chimeric
CEA and CBATP molecules in transfected COS cells. Cells were
analyzed by Western blot analysis (A), biosynthetic labeling
(B), and cell surface labeling (C).
Left, relative electrophoretic migration of molecular mass
markers. We could not detect a decrease in electrophoretic migration of
the chimeric CEA molecules as compared with the wild-type molecule on
these 10% gels, but there was a decrease in electrophoretic migration
of ~8 kDa for the chimeric CEA molecules on 6.5% gels in which
proteins of 180 kDa were resolved in the middle of the gel (data not
shown).
[View Larger Version of this Image (24K GIF file)]
Fig. 6.
Bile acid efflux in COS cells cotransfected
with IBAT and wild-type, mutant, or chimeric CBATP or CEA genes.
Cells were subjected to bile acid efflux assays 48 h after
transfection. Cotransfected cells were incubated for 60 min at 22 °C
in medium supplemented with 400 µM
[3H]taurocholate. The cells were then washed and
incubated for 5 min at 22 °C in medium supplemented with ATP in the
absence or presence of 1 mM DIDS.
[View Larger Version of this Image (37K GIF file)]
S (Fig. 8). The
results show an ~180-kDa polypeptide in cells transfected with
wild-type and R98A-CEA but not in untransfected cells. The labeling is
blocked by unlabeled ATP
S. These results show that CEA does not have
ecto-ATPase activity, even though it has an ATPase consensus sequence
and binds ATP. This lack of ATPase activity can be attributed to the
lack of a cytoplasmic tail and membrane-spanning domain. When fused to
the cytoplasmic domain and membrane-spanning domain of CBATP, it now
has ecto-ATPase activity. Moreover, the ecto-ATPase activity of the
CBATP-CEA chimeric molecule can be clearly attributed to the ATPase
consensus sequence because the activity of the chimera is abrogated by
site-directed mutagenesis of Arg98, in the same way that
the ecto-ATPase activity of CBATP is abrogated by mutagenesis of
Arg98. Thus, these results also provide evidence that the
effect of CBATP on the ecto-ATPase activity of transfected COS cells is highly specific as well as providing further evidence for the validity
of the ecto-ATPase activity in transfected COS cells.
Fig. 7.
Ecto-ATPase activity in COS cells
cotransfected with IBAT and wild-type, mutant, or chimeric CBATP or CEA
genes.
[View Larger Version of this Image (30K GIF file)]
Fig. 8.
Photoaffinity labeling of CEA and R98A-CEA
molecules in transfected COS cells. Briefly, 48 h after
transfection, cells were labeled with [35]ATPS in the
absence or presence of unlabeled ATP
S in 200-fold molar excess (8).
Cells were then photolyzed and homogenized so that the cell homogenates
could be analyzed by immunoprecipitation, SDS-polyacrylamide gel
electrophoresis/fluorography. Left, relative electrophoretic
migration of molecular mass markers.
[View Larger Version of this Image (81K GIF file)]
*
The studies were supported by National Institutes of Health
Grants DK47084 and T32HDO7409.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.
**
To whom correspondence should be addressed: Dept. of Pediatrics,
Washington University School of Medicine, #1 Children's Place, St. Louis, MO 63110. Tel.: 314-454-6033; Fax: 314-4546-4218;
E-mail: perlmutter{at}a1.kids.wustl.edu.
1
The abbreviations used are: CBATP, canalicular
bile acid transport protein; CEA, carcinoembryonic antigen; DMEM,
Dulbecco's modified Eagle's medium; IBAT, ileal bile acid transport
protein; DIDS, 4,4-diisothiocyanostilbene-2,2-disulfonic acid;
ATP
S, adenosine 5
-O-(thiotriphosphate).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.