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J Biol Chem, Vol. 273, Issue 52, 34691-34695, December 25, 1998
From the Intestinal absorption of bile acids depends on a
sodium-bile acid cotransport protein in the apical membrane of the
ileal epithelial cell. Transport is
Na+-dependent, but the
Na+-bile acid stoichiometry and electrogenicity of
transport are not known. Studies in whole intestine, isolated cells,
and ileal membrane vesicles have been unable to resolve this issue
because transport currents are small and can be obscured by other ionic conductances and transport proteins present in these membranes. In this
study, the human apical sodium-bile acid transporter was expressed in
stably transfected Chinese hamster ovary cells that lack other bile
acid transporters. The Na+-dependent transport
of a fluorescent bile acid analog,
chenodeoxycholyl-N Enterohepatic circulation of bile acids requires efficient bile
acid absorption by the ileum. The first step in ileal bile acid
transport is mediated by a Na+-bile acid transporter
(ASBT)1 located at the
epithelial cell apical brush border membrane (1). After uptake, the
bile acids are transported transcellularly to the basolateral membrane
(2) and secreted into the portal circulation by a sodium-independent
anion exchange mechanism (3). The apical sodium-bile acid transporter
cDNA has been isolated from the hamster (4), rat (5), and human (6)
and is homologous to the hepatic Na+-coupled bile acid
transporter (7). Analysis of mRNA and protein expression revealed
that ASBT is also expressed on the apical brush border membrane of
renal proximal tubule cells (8) and apical membranes of cholangiocytes
lining the large bile duct units (9, 10). Whereas ASBT is known to
transport both Na+ and bile acid, the Na+-bile
acid stoichiometry and electrogenicity of transport are unknown (2),
and previous studies testing for electrogenic transport have produced
conflicting results (11-15), although the tentative consensus was that
ileal Na+-bile acid cotransport is electroneutral (2).
Electrogenicity of other transporters, particularly
Na+-glucose and Na+-amino acid, has been
definitively demonstrated by measuring the currents associated with
transport (16-18). This has not been possible for the bile acid
transporters because next flux is small and the detergent properties of
the bile acids may activate ion channels (19). We have therefore taken
an alternative approach, directly monitoring the movement of a
fluorescent bile acid analog in single, voltage-clamped CHO cells
transfected with human ASBT. We have for the first time been able to
definitively demonstrate electrogenicity and assess
Na+:bile acid stoichiometry for a Na+-bile acid transporter.
Cell Culture and Bile Acid Uptake Assays--
CHO-K1 cells were
obtained from the American Type Culture Collection (Manassas, VA) and
maintained in medium A that consisted of a 1:1 (v/v) mixture of
Dulbecco's modified Eagle's medium containing 4500 mg/liter
D-glucose and Ham's F-12 medium, 10% (v/v) fetal calf
serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Life
Technologies, Inc.). Stable overexpression of the hASBT was achieved by
cotransfecting the cells with pCMV5-hASBT (6) and pSV3Neo using the
calcium phosphate procedure as described (20). Individual colonies were
expanded in 24-well plates and screened for hASBT expression using
[3H]taurocholate uptake assays. Functional expression of
the hASBT protein in these cells has been previously demonstrated
(21).
[3H]Taurocholic acid (2.0-2.6 Ci/mmol) and
[2,4-3H]cholic acid (27.5 Ci/mmol) were purchased from
NEN Life Science Products. Unlabeled taurocholate and cholate were
purchased from Sigma. For [3H]bile acid uptake assays,
CHO-hASBT cells were incubated in medium B, which consisted of a
modified Hanks' balanced salt solution containing 137 mM
NaCl or 137 mM choline chloride (6).
Measurement of Fluorescent Bile Acid Analog
Accumulation--
The fluorescent bile acid analogs
cholylglycylamidofluorescein (CGamF),
cholyl-N Quantitative Fluorescence Microscopy and Whole Cell Patch
Clamp--
Simultaneous whole cell patch clamp and fluorescence
microscopy were performed with a photometer-based quantitative
fluorescence system as described previously (23). CHO-hASBT cells were
grown in medium A on 5 × 5-mm glass coverslips and exposed to
sodium butyrate (5 mM) for 16 h. Cells were
pre-equilibrated with 1 µM CDC-NBD-L in HEPES-buffered
Na+-Ringer solution for 30 min. Fluorescent cells were then
subjected to whole cell patch clamp in the continued presence of
CDC-NBD-L at 37 °C. From an initial holding potential of Analysis of Stoichiometry--
Stoichiometry was determined by
comparing the effects of Na+ gradients and voltage on
transport. For a cotransport process with n mol of
Na+ transported for each mol of univalent negatively
charged bile acid, the free energy change associated with transport
( Expression of the Human ASBT in CHO Cells--
The expression
plasmid pCMV5-hASBT was stably transfected into CHO cells, and clonal
cell lines were selected. As seen in Fig.
1A, the uptake of taurocholate
and cholate were saturable and Na+-dependent.
There was little appreciable bile acid uptake in the absence of
Na+ or in the parental CHO cells (data not shown). At 137 mM Na+, analysis of the concentration
dependence of bile acid transport revealed apparent
Km values of 12 and 37 µM for
taurocholate and cholate, respectively. Fig. 1B shows that
these data fit the Hill equation well with Hill coefficients near 1.0 for both taurocholate and cholate, suggesting that bile acid binding
sites are either single or noninteracting in the transporter
complex.
Fluorescent Bile Acid Uptake--
Previous studies of the FBA,
CGamF, C-NBD-L, and CDC-NBD-L have shown that uptake is specific for
bile acid transporting cells such as hepatocytes and displays
Michaelis-Menten kinetics (22). To determine whether these FBA are also
substrates for the hASBT, we measured accumulation of these analogs
after a 5-min incubation in either parental CHO-K1 or transfected
CHO-hASBT cells. As shown in Fig.
2A, CHO-hASBT cells
efficiently transported both NBD-labeled bile acids, C-NBD-L and
CDC-NBD-L, but failed to take up the fluorescein-labeled bile acid,
CGamF. There was no appreciable uptake of any FBA in the parental
CHO-K1 cells, and uptake was entirely
Na+-dependent (data not shown). Initial uptake
rates of CDC-NBD-L were saturable with a Km of 13.1 µM (Fig. 2B) and a Hill coefficient of 1.07 (Fig. 2C). These kinetic constants were close to those
observed for taurocholate.
Transport during Voltage Clamp--
CHO-hASBT cells were
pre-equilibrated for 30 min with 1 µM CDC-NBD-L and
subjected to whole cell patch clamp in the continued presence of the
FBA. The cells were clamped at three different voltages, cell
fluorescence was measured at 10-s intervals, and the rate of change of
fluorescence with time was compared at the different clamp voltages.
Fig. 3A demonstrates an
example of cell fluorescence changes in one cell during this protocol.
At
To assess stoichiometry of voltage-dependent
Na+-bile acid cotransport, we examined the relationship
between the electrical and chemical driving forces for transport.
Voltage dependence was first determined in control bath solution and
then again while maintaining the patch clamp configuration in the same
cells after reduction of bath Na+ concentration. The shift
in the voltage dependence produced by this maneuver results from the
additional inward electrical driving force necessary to balance the
reduction in the inwardly directed Na+ gradient. Fig.
4 demonstrates this relationship. The
voltage at which cell fluorescence remained constant shifted by This study has shown that transport of fluorescent bile acid
derivatives by CHO cells stably transfected with hASBT is electrogenic. Previous studies of electrogenicity of ileal bile acid transport reached conflicting conclusions. Authors using similar vesicle techniques reported that Na+-bile acid cotransport was
either electrogenic (11, 12) or electroneutral (13-15). A major
limitation of these prior studies was the need to use an indirect
approach to change membrane potential of vesicles. In most cases
addition of valinomycin was assumed to hyperpolarize the vesicle
interior, but these studies did not validate that the expected voltage
changes actually occurred.
In this study we used several approaches to circumvent these problems.
We performed whole cell patch clamp to directly voltage clamp and
measure the cell membrane potential and used a fluorescent bile acid
derivative rather than naturally occurring bile acids. This allowed us
to measure transport rates without having to directly measure transport
currents. Unlike the case with more hydrophilic substrates such as
glucose and amino acids (16-18), bile acid transport currents are
extremely difficult to measure because the electrical currents
resulting from bile acid transport are small. For the CHO-hASBT cells,
the average Vmax was about 2 nmol/min/mg
protein, which predicts transport currents of only 1.5 pA/cell. This
compares with greater than 100 nA/cell for the Na+-glucose
cotransporter in oocytes (16). A second difficulty is that external
bile acids have been shown to affect other conductive pathways (19),
and bile acid-induced currents therefore do not necessarily represent
current carried by the cotransport proteins.
The hASBT-transfected CHO cells efficiently transported two NBD-labeled
bile acids, C-NBD-L and CDC-NBD-L, but not a fluorescein-labeled bile
acid, CGamF. Kinetic parameters for CDC-NBD-L transport were similar to
that of taurocholate and uptake followed Michaelis-Menten kinetics.
Earlier studies of hepatocytes (22) and Ntcp-transfected COS cells (24)
had shown specific transport of both CDC-NBD-L and CGamF, and these
differences reflect the more restricted substrate specificity of ASBT
compared with that of the homologous Ntcp (21). These findings
complement a recent report describing the in vivo transport
properties of the fluorescent bile acid analogs (25). In that study as
well, C-NBD-L but not CGamF was transported by ileum.
Our results clearly demonstrate that transport via the human ASBT is
electrogenic and carries positive charge into the cell. The basic
principal underlying these measurements is that once a steady-state
concentration in a patched cell is reached, changes in cell
fluorescence induced by voltage clamping the cell/pipette system result
from flux of substrate between the cell and the bath. For this to be
the case, there must be no voltage-dependent changes in
cell fluorescence such as could result from
voltage-dependent changes of the dye quantum yield or cell
volume. We examined these possibilities under conditions in which
transmembrane transport was abolished either with a nontransported dye
or by low temperature. In both of these cases voltage changes had no
effect on cell fluorescence.
Another consideration is the possibility that appreciable amounts of
dye diffuse from the cell to the pipette, confounding our analysis. Our
previous studies have determined that diffusion of bile acids from cell
to pipette is small and alters apparent uptake rates by only about 5%
(23). The small magnitude of the effect is due to the high degree of
intracellular binding of this hydrophobic substrate as well as the
relatively small surface area of the pipette (26). Intracellular
diffusion coefficients of bile acids are approximately 1000-fold lower
than that predicted for simple aqueous diffusion (27). Although
diffusion into the pipette is small, it can still affect the voltage at
which cell fluorescence is constant. However, there is no voltage
gradient between cell and pipette, and therefore changes in cell
fluorescence that occur immediately after voltage shift reflect changes
in transmembrane transport and not changes in cell to pipette diffusion.
The stoichiometry of Na+-bile acid cotransport was assessed
by comparing the effects of voltage changes and Na+
gradient changes on the zero net flux voltage
(Vrev). Provided that only transmembrane flux is
changed by voltage clamp, the immediate change in
Vrev after sudden reduction of extracellular Na+ is described by Equation 3. It predicts that for any
given Na+ gradient change, the magnitude of
The observed The apical Na+-bile acid cotransporter cDNA encodes a
polytopic membrane glycoprotein that shares considerable amino acid
identity and remarkable structural similarity to Ntcp, the major
Na+-dependent liver bile acid transporter (28).
Considerable evidence has now accumulated suggesting that Ntcp is also
electrogenic including direct measurements of bile acid transport
currents (29, 30) as well as voltage dependence of fluorescent bile acid transport in hepatocytes (23). Kinetic analysis of the Ntcp
transporter also shows a Hill coefficient of 1.9 for Na+
and 1.0 for bile acid (28). Considering the similarities in sequence
and predicted structure for the Na+-bile acid transporters,
it is likely that they share a common electrogenic mechanism.
Previous ileal bile acid transporter modeling studies relied upon
substrate binding properties and assumed an electroneutral transport
mechanism. Based in part on this assumption, a model for the substrate
binding site was proposed that encompassed a closely positioned
negatively charged group for interaction with a single sodium ion (31,
32). The previous model will have to be re-evaluated in light of the
present study demonstrating electrogenicity.
Electrogenicity confers two significant advantages for ileal bile acid
transport. First, as described by Kimmich (33), the driving force for
uptake sets the upper limit for the concentrating ability of the
system, but in practice gradients achieved are significantly lower due
to leak pathways. In the case of intestinal bile acid transport,
basolateral exit occurs by a passive transporter. Therefore,
electrogenic uptake with a high Na+:bile acid coupling
ratio at the apical membrane results in a much higher intracellular
bile acid concentration and thus increases net transcellular transport.
Secondly, electrogenicity of uptake provides a means for regulation of
transport. Fasting (34), aldosterone (34), and 5-hydroxytryptamine (35)
have all been shown to alter intestinal electrical gradients in the
direction of hyperpolarization of the brush border membrane. These
physiologic changes may provide the ileal intestinal cells with a
mechanism to regulate bile acid uptake rates for optimal bile acid
recycling and cytoprotection.
In summary, this study is the first unequivocal demonstration of an
electrogenic mechanism for a cloned Na+-bile acid transport
protein. These results will be critical in modeling the
structure-function relationship for this important but poorly
understood class of Na+-coupled transport proteins.
We thank Drs. A. Hofmann and C. Schteingart
for kindly providing the fluorescent bile acid analogs and Drs. L. Reuss and S. King for helpful comments. We also acknowledge the
excellent technical assistance of Lori Showalter and Ann L. Craddock.
*
This work was supported grants DK42917 (to S. A. W.) and DK47987 (to P. A. D.) from the National Institutes of
Health.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
Physiology and Biophysics, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0641. Tel.: 409-772-4286; Fax:
409-772-3381; E-mail: sweinman{at}utmb.edu.
**
American Heart Association Established Investigator.
The abbreviations used are:
ASBT, apical
Na+-bile acid transporter; hASBT, human ASBT; FBA, fluorescent bile acid(s); CGamF, cholylglycylamidofluorescein; C-NBD-L, cholyl-N
Bile Acid Uptake via the Human Apical Sodium-Bile Acid
Cotransporter Is Electrogenic*
§¶,, and
**
Department of Physiology and Biophysics and
§ Department of Internal Medicine, University of Texas
Medical Branch, Galveston, Texas 77555 and the Department of
Internal Medicine, Wake Forest University School of Medicine, Wake
Forest University, Winston-Salem, North Carolina 27157
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-nitrobenzoxadiazol-lysine, was
monitored by fluorescence microscopy in single, voltage-clamped cells.
Bile acid movement was bidirectional and voltage-dependent with negative intracellular voltage-stimulating influx. A 3-fold reduction in extracellular Na+ produced a negative 52 mV
shift of the flux-voltage relationship, consistent with a 2:1
Na+:bile acid coupling stoichiometry. No Na+-
or voltage-dependent uptake was observed in nontransfected
Chinese hamster ovary cells. These results indicate that the
cotransport of bile acids and Na+ by human apical
sodium-bile acid transporter is electrogenic and bidirectional and is
best explained by a 2:1 Na+:bile acid coupling
stoichiometry. These results suggest that membrane potential may
regulate bile acid transport rates under physiological and
pathophysiological conditions.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-nitrobenzoxadiazol-lysine (C-NBD-L), and chenodeoxycholyl-N-nitrobenzoxadiazol-lysine (CDC-NBD-L)
were generous gifts of Drs. A. Hofmann and C. Schteingart. Uptake
specificity and time course of uptake were measured in individual cells
with a quantitative fluorescence imaging system, as described
previously (22). Cells were superfused at 37 °C with FBA-containing
HEPES-buffered Na+-Ringer solution (144 mM
NaCl, 5 mM KCl, 2 mM
NaH2PO4, 1.25 mM CaCl2,
1 mM MgSO4, and 10 mM HEPES, pH
7.4), and cell fluorescence was measured at 5-10-s intervals. The
total Na+ concentration was approximately 150 mM. Where indicated, Na+ was quantitatively
replaced with tetramethylammonium.
30 mV,
intracellular (pipette) voltage was changed at 1-2-min intervals to
90, or +30 mV as indicated. Cell fluorescence was measured at 10-s
intervals during these voltage steps. The patch pipette was filled with a solution containing 115 mM potassium gluconate, 30 mM KCl, 0.47 mM CaCl2, 2.1 mM MgSO4, 2 mM EGTA, 10 mM HEPES, and 3 mM Na2ATP, pH 7.2. The total Na+ concentration was approximately 12 mM. In some experiments the bath solution Na+
concentration was reduced by substitution of NaCl with
tetramethylammonium chloride.
G) depends on the Na+ gradient, the bile
acid gradient, and the voltage according to the following
relationship.
where n is the number of moles of Na+
transported per mol of bile acid, R is the gas constant,
T is the absolute temperature, Nai and Nao
are the Na+ concentrations inside and outside the cell,
Bi and Bo are the bile acid concentrations inside and
outside the cell, F is the Faraday constant, and
V is the transmembrane voltage. The reversal potential,
Vrev, is that voltage at which
(Eq. 1)
G
is zero and is simply as follows.
If Nao is suddenly reduced by a factor of x
and assuming that all other parameters initially remain constant, we would predict a change in Vrev according to
Equation 3.
(Eq. 2)
(Eq. 3)
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (18K):
[in a new window]
Fig. 1.
A, expression and bile acid uptake in
human ASBT transfected CHO cells. Taurocholate (filled
symbols) and cholate (open symbols) uptake in human
ASBT transfected CHO cells. On day 0, 3.8 × 105
cells/35-mm dish were plated in medium A. On day 1, the cells were
refed medium A containing 10 mM sodium butyrate. After
20 h, the dishes were washed and incubated with 200 µl of
Hanks' balanced salt solution containing either 137 mM
NaCl (circles) or 137 mM choline chloride
(squares) and the indicated concentration of taurocholate
(0.7 Ci/mmol) or cholate (1.4 Ci/mmol) for 10 s at 37 °C. The
cell monolayers were then washed and processed to determine
cell-associated protein and radioactivity. The uptake assays were
performed in duplicate. Na+-dependent uptake
was calculated by subtracting the background uptake in the presence of
choline (squares) from the total uptake in the presence of
sodium (circles). Eadie-Hofstee analysis revealed
Km values of 12 and 37 µM for
taurocholate and cholate, respectively. The apparent
Vmax values were 2740 and 2815 pmol
min 
1 mg cell protein1 for taurocholate and
cholate, respectively. B, Hill transformation of the
Na+-dependent uptake rates for taurocholate
(filled symbols) and cholate (open symbols). The
best fit Hill coefficient was 1.05 for taurocholate and 1.09 for
cholate.
View larger version (17K):
[in a new window]
Fig. 2.
Fluorescent bile acid uptake by the
hASBT. A, accumulation of fluorescent bile acid analogs
by CHO K1 and CHO-hASBT cells. Cells were exposed to the indicated FBA
(2 µM) for 5 min, and single cell fluorescence was
measured with a quantitative imaging system. Each bar
represents mean ± S.E. for 74-104 cells. B,
concentration dependence of CDC-NBD-L uptake. Initial uptake rate was
measured in individual cells during the first 30 s of exposure to
bile acid at the indicated concentration. Nonlinear least square fit to
the Michaelis-Menten equation is represented by the curve
(Km 13.1 µM,
Vmax 319.7 fluorescence
units·min 1 cell1). Each point represents
the mean initial uptake rate in 10 cells. C, Hill
transformation of CDC-NBD-L uptake. The best fit Hill coefficient was
1.07.
30 mV, fluorescence was relatively constant, but at 90 mV the
cell fluorescence increased over time and at +30 mV fluorescence
decreased. Control experiments were performed to exclude the
possibility that fluorescence changes were due to other
voltage-dependent phenomena such as increasing cell volume
or fluorophore leak. No voltage-dependent fluorescence
changes were observed with either a nontransported fluorescent dye,
carboxyfluorescein, or with CDC-NBD-L when the bath temperature was
cooled to 10 °C to inhibit activity of the membrane transporter
(data not shown). These results demonstrate that voltage-induced
fluorescence changes result from either inward or outward transport of
CDC-NBD-L. Fig. 3B shows the results from 10 cells clamped
under this protocol. CDC-NBD-L movement was bidirectional and
voltage-dependent. Increasing inside negative voltage
stimulated uptake and inside positive voltage resulted in net efflux.
The direction of change was not affected by the order in which the
voltage clamps were performed.
View larger version (16K):
[in a new window]
Fig. 3.
Voltage dependence of CDC-NBD-L uptake.
A, an example of voltage-induced changes in cell
fluorescence in a single cell. The cell was pre-equilibrated with 1 µM CDC-NBD-L for 30 min, patch-clamped, and held at the
voltages indicated. Cellular fluorescence changes result from
transmembrane CDC-NBD-L movement. B, data from 10 cells
measured as in A. For each voltage the transport rate is
presented as the percentage of change in cell fluorescence per min. It
was calculated as 100 × slope (units/min)/starting fluorescence
(units).
52 mV after a 3-fold Na+ dilution and by 62 mV after a 10-fold
dilution.
View larger version (17K):
[in a new window]
Fig. 4.
Effect of Na+ concentration
changes on the flux-voltage relationship. After determining
voltage dependence as in Fig. 3, the bath Na+ concentration
was reduced, and voltage dependence was again determined in the same
cells. 
, control; 
, reduced Na+. A,
3-fold Na+ reduction, n = 5. Reversal
potential shifted by 52.3 ± 11.2 mV. B, 10-fold
Na+ reduction, n = 4. Reversal potential
shifted by 61.7 ± 13.4 mV.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Vrev is greatest for n = 2 and decreases as n increases. This can be understood because
at higher coupling stoichiometry the transported complex has a greater charge and thus requires less of a voltage change to balance the chemical free energy component of the altered Na+ gradient.
Vrev for a 3-fold reduction in
bath Na+ was 52 mV. The theoretical value predicted from
Equation 3 is 58.7 mV for n = 2 and 44 mV for
n = 3. These theoretical values are expected to be
upper limits for the observed change because the internal bile acid
concentrations (Bi) may have decreased as a result of outward
transport resulting from the reduction of bath Na+
concentration. A slight reduction of Bi during the period of
low Na+ exposure increases the inward bile acid gradient
and thus reduces the magnitude of the negative voltage required to
re-establish the zero flux condition. Such non-ideal effects would tend
to decrease the observed Vrev. Because the
observed value is greater than the upper limit for an n = 3 coupling and only slightly less than that for n = 2, we conclude that the most likely coupling ratio is 2 Na+
per 1 bile acid, but we cannot definitively exclude a 3:1 ratio. This
finding is consistent with kinetic measurements of the Hill coefficient
for Na+ in these cells of approximately 2 (21) and the Hill
coefficient for bile acid of approximately 1 (Figs. 1 and 2).
ACKNOWLEDGEMENTS
FOOTNOTES
-nitrobenzoxadiazol-lysine; CDC-NBD-L, chenodeoxycholyl-N-nitrobenzoxadiazol-lysine; Ntcp, Na+-taurocholate cotransporting polypeptide; CHO, Chinese
hamster ovary.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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X. Li, K. Shimada, L. A. Showalter, and S. A. Weinman Biophysical Properties of ClC-3 Differentiate It from Swelling-activated Chloride Channels in Chinese Hamster Ovary-K1 Cells J. Biol. Chem., November 10, 2000; 275(46): 35994 - 35998. [Abstract] [Full Text] [PDF]
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