 |
INTRODUCTION |
Vectorial transport across epithelia can occur via the
transcellular or paracellular route. The molecular basis of
transcellular transport, mediated by proteins that catalyze
transmembrane movement of solutes and water at the apical and
basolateral surfaces, is well understood. By contrast, little is known
about the mechanisms of paracellular transport. The tight junction,
which is the most apical of the intercellular junctional complexes at
the lateral membrane, is generally believed to be the rate-limiting
step in paracellular transport. The tight junction is composed of a
complex of multiple proteins, many of uncertain function (1, 2). Most
of these proteins are cytoplasmic in location, and several are
associated with the cytoskeleton via the perijunctional actin ring. In
recent years, a number of integral membrane proteins associated with
the tight junction have been identified, including occludin (3),
junction-associated membrane proteins 1 and 2 (4, 5), Coxsackie- and
adenovirus-associated receptor (6), 1G8 antigen (7), and the claudins
(8). Integral membrane proteins are of particular interest because, by
definition, their extracellular domains must protrude into the lateral
intercellular space and, therefore, may potentially have direct contact
with solutes as they permeate through the paracellular pathway.
Claudins are the most heterogeneous of these integral membrane proteins
and consist of a family of at least 20 homologous isoforms (9).
Moreover, each isoform clearly exhibits a tissue-specific and
segment-specific pattern of distribution in epithelia such as those of
the gastrointestinal tract (10) and renal tubule (11-13). Claudins are
therefore leading candidates for the molecular determinants responsible
for the variety of different paracellular permeability properties found
in different epithelia. Several recent reports provide convincing
evidence that claudins regulate paracellular permeability. Human
mutations in claudin-16 cause failure of paracellular reabsorption of
divalent cations in the thick ascending limb of the renal tubule,
leading to familial hypercalciuric hypomagnesemia (14), and a
preliminary report suggests that knockout mice lacking claudin-16 may
have a similar phenotype (15). Both claudin-1-deficient mice (16) and
transgenic mice overexpressing claudin-6 (17) exhibit abnormally high
epidermal permeability to water. Overexpression of claudins 1, 4, and
15 in MDCK1 cells all
increase transepithelial resistance (TER) (18-21), whereas overexpression of claudin-2 markedly decreases TER (22) by selectively increasing permeability to cations (23). Furthermore, Anderson and
co-workers (20, 21) elegantly show that, although overexpression of
claudin-4 reduces paracellular monovalent cation permeability, this can
be abolished by altering the net charge at the first extracellular loop
by site-directed mutagenesis. Similarly, although claudin-15 alone does
not alter the relative preference of the paracellular pathway between
Na+ and Cl
, mutating anionic extracellular
residues to cationic ones makes the paracellular pathway more
anion-selective (21).
Thus, the contention that claudins have a direct role in regulating the
magnitude and nature of paracellular permeability is now irrefutable.
However, the plethora of published claudin overexpression and gene
ablation experiments has uncovered a fundamental paradox. Claudin
overexpression can be associated with both an increase (17, 22, 23) and
a decrease (18-21) in paracellular permeability; similarly, knockout
or inactivating mutations of claudin genes can cause either an increase
(16) or a decrease (14, 15) in paracellular permeability. Thus, in the
absence of suitable models to explain such contradictory observations, attempts to infer the permeability properties of specific claudin isoforms remain problematic.
A possible solution emerges if one views each tight junction strand as
a continuous string of protein molecules (such as claudins, occludin,
or other proteins) arrayed side-by-side, rather like beads on a
necklace, so as to form an uninterrupted seal (9). In such a model,
claudins would play a bipartite role, acting as a barrier by sealing
the gaps between protein particles, and simultaneously providing a
channel through that barrier from the apical compartment to the lateral
intercellular space. This may explain why the complete loss of a
claudin isoform can have diametrically opposite consequences. In the
absence of any changes in other tight junction proteins, gaps would
appear in the continuous seal, and the paracellular route would become
more permeable. Conversely, when other components of the tight junction
become up-regulated and fill in for the absent claudins, the
paracellular route may become less permeable. Similar considerations
may govern the consequences of overexpression experiments. The
resulting phenotype is complex and dependent on the properties both of
the heterologously expressed claudin and of endogenous tight junction proteins.
In this manuscript, we describe a new tissue culture model with
overexpression of claudin-8. We chose to study claudin-8 because previous in situ hybridization (11) and immunohistochemical studies (12) revealed that it is expressed primarily in the aldosterone-sensitive distal nephron. In these segments of the renal
tubule, the paracellular barrier is particularly tight to monovalent
cations and protects transtubular gradients up to 1000:1 for
H+, 20:1 for K+, and 1:3 for Na+.
We therefore tested the hypothesis that claudin-8 plays a role in
impeding paracellular cation permeation. We show that the combination of careful biochemical, histological, and physiological studies constitute a powerful tool to interpret the phenotype of such overexpression experiments. We propose potential models to explain the
findings of such studies and show data that indicate that claudin-8
acts as a nonspecific cation barrier.
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MATERIALS AND METHODS |
Generation of MDCK II TetOff Claudin-8 NFL Cell
Lines--
A 675-bp DNA fragment containing the mouse claudin-8 coding
sequence except for the initiation codon was amplified by PCR from a
cDNA clone (IMAGE ID: 162549) and inserted into a FLAG epitope tag
shuttle vector based on pcDNA3 (24) so that the claudin-8 N
terminus was fused in-frame with a sequence encoding MDYKDDDDKGS (the FLAG octapeptide tag is underlined
followed by a glycine-serine linker encoding BamHI
restriction site). The entire coding region was then excised and cloned
into the retroviral Tet response vector, pRevTRE
(Clontech, Palo Alto, CA) to obtain the plasmid,
pRev-mCLDN8-NFL. Plasmid DNA was transfected by lipofection into the
packaging cell line, PT67, a polyclonal culture of stable transfectants
selected using hygromycin, and virus-containing supernatant collected
from the growth medium. MDCK II TetOff cells expressing the
tetracycline-regulated transactivator from a cytomegalovirus promoter
(Clontech) were infected with viral supernatant in
the presence of Polybrene. Stable transfectants were selected by growth in 0.3 mg/ml hygromycin. Clonal cell lines were isolated using plastic
cloning rings and three clones (2, 4, and 9) with strong induction of
claudin-8 expression (see "Results") used for further studies.
Cells were maintained in Dulbecco's modified Eagle's medium with 5%
fetal bovine serum, 0.1 mg/ml G418, 0.3 mg/ml hygromycin, and 20 ng/ml
doxycycline (Dox+). To induce claudin-8 expression, doxycycline was
omitted from the culture medium starting from the day of plating
(Dox
). Studies were generally performed after 4-6 days except where
otherwise indicated.
Detection of Tight Junction Proteins by Immunoblot and
Immunohistochemistry--
To detect tight junction protein expression
by immunoblotting, cultured cells were first homogenized and
fractionated by centrifugation at 100,000 × g as
described previously (24). No claudin-8 was ever detectable in the
soluble fraction (100,000 × g supernatant). The total
amount of protein isolated from plates of confluent Dox+ and Dox
cells was similar, and the fractional yield of membrane protein
(100,000 × g pellet) was roughly 15% in both.
Aliquots of 25 µg of membrane protein were then electrophoresed on
denaturing SDS-polyacrylamide gels and transferred to polyvinylidene
difluoride membrane, and Western blots were performed using the ECL
chemiluminescence kit (Amersham Biosciences). Claudin-8 transgene
expression was detected with the M2 anti-FLAG monoclonal antibody
(Sigma) at a 1:400 dilution. Antibodies to ZO1, occludin, and claudins
1-4 were obtained from Zymed Laboratories Inc., San
Francisco, CA and used at the concentrations recommended by the
manufacturer. Rabbit anti-Coxsackie- and adenovirus-associated receptor
antiserum (a kind gift of Dr. Christopher Cohen, Children's Hospital
of Philadelphia, PA) was used at 1:5000 dilution. Western blots were digitized with a flatbed optical scanner. Gray scale values were logarithmically transformed to obtain uncalibrated optical density estimates, and individual bands were quantitated using NIH Image 1.61 software (rsb.info.nih.gov/ nih-image).
To determine the localization of claudin-8 protein expression, cell
lines grown on 12-mm Transwell-Clear polyester filters (1-cm2 growth area, 0.4-µm pore size; Corning Costar,
Acton, MA) were fixed with 4% paraformaldehyde at 4 °C for 15 min,
then indirect immunofluorescence staining was performed either with the
M2 mouse antibody at 1:400 dilution or (for the double labeling study
shown in Fig. 1C) with a rabbit anti-claudin-8 antibody
(933) at 1:100 dilution, all in the presence of 0.3% Triton X-100
using protocols described previously (24). Affinity-purified rabbit
polyclonal antibody 933 was raised against the claudin-8 C-terminal
peptide, CQRSFHAEKRSPSIYSKSQYV (25). Images were acquired at the
University of Southern California Center for Liver Diseases using a
Nikon PCM confocal microscopy system with argon and helium-neon lasers.
Freeze Fracture and Immunolabeling--
Freeze fracture with or
without immunolabeling was performed essentially as described
previously (19). Confluent monolayers were fixed either in 2%
glutaraldehyde at 4 °C for 30 min (no immunolabeling) or in 1%
paraformaldehyde at 4 °C for 15 min (for immunolabeling). They were
then rinsed in phosphate-buffered saline, scraped from the filter, and
infiltrated with 25% glycerol in 0.1 M cacodylate buffer
for 60 min at 4 °C. Cell pellets were frozen in liquid nitrogen
slush, and freeze-fractured at
115 °C in a Balzers 400 freeze-fracture unit (Lichtenstein). Processing of the replicas for
strand morphometry and immunolabeling was as described (26), and the
replicas were examined with a Philips 301 electron microscope. The
number of parallel strands in the tight junctions was quantified as
described previously (26). Because the frequency distribution of strand
number was non-Gaussian, the difference in the median number of strands
in Dox+ and Dox
cells was assessed for statistical significance using
the Wilcoxon ranked sum test. Immunolabeling of SDS-treated replicas
was performed with the M2 anti-FLAG antibody (1:100) followed by goat
anti-mouse gold (1:100).
TER Monitoring--
Cells were plated at confluent density
(2 × 105 cells/cm2) onto Transwell
filters loaded in 12-well plates. The resistance across each filter in
culture media was measured at room temperature by immersing into the
well chopstick-style Ag/AgCl electrodes attached to a Millicell-ERS
voltohmeter (Millipore, Bedford, MA). TER was calculated by
subtracting the resistance determined in blank filters from the
resistance measured in filters with monolayers.
Ussing Chamber Electrophysiology Studies--
Cells were plated
at confluent density on 1 cm2 Snapwell polyester filters
(Corning Costar) and grown for the indicated number of days. The filter
rings were then detached and mounted in Ussing chambers that were
incubated in Ringer solution (150 mM NaCl, 2 mM
CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES, pH 7.4) at 37 °C
and continuously bubbled with 95% O2, 5% CO2.
The fluid volume on each side of the filter was 4 ml. Voltage-sensing
electrodes consisting of Ag/AgCl pellets and current-passing electrodes
of silver wire were connected by agar bridges containing 3 M KCl and interfaced via head-stage amplifiers to a
microcomputer-controlled voltage/current clamp (DM-MC6 and VCC-MC6,
respectively; Physiologic Instruments, San Diego, CA).
Voltage-sensing electrodes were matched to within 1 mV asymmetry and
corrected by an offset-removal circuit. The voltage between the two
compartments (values reported are referenced to the apical side) was
monitored and recorded at 5-s intervals, whereas the current was
continuously clamped to zero. This was done to minimize current-induced
local changes of salt concentration in the unstirred layers, which can
generate spontaneous potentials due to the transport-number effect
(27). The voltage was first measured with blank filters in each
combination of buffers to be used for the experiments. The
values obtained, which were generally less than 1 mV in magnitude, represent the difference in junction potentials between the two voltage-sensing bridges summed with any potential that might exist across the filter membrane. These were subtracted from all subsequent measurements with filters containing attached cell monolayers to
determine transepithelial voltage (Vt).
The total resistance between apical and basal compartments was
determined in Ringer at the start and at intervals throughout the
experiment from the voltage evoked by a 5-µA bipolar current pulse.
The background resistance determined with blank filters (62.0 ± 1.0 ohm, n = 6), representing the sum of the
resistances of the filter, of the fluid in the chambers and of the
current-passing electrodes and bridges was subtracted from the total
resistance measured with filters containing attached cell monolayers to
determine the TER and hence conductance (TER
1). TER was
found to be maintained within 5% of its base-line value for at least
2 h, whereas the duration of an experiment was typically about
60-90 min.
Dilution and biionic potentials were determined by replacing the
solution of one compartment, generally the basal side to minimize flow
disruption of the integrity of the monolayer, while keeping the other
side bathed in Ringer. In selected cases the solution in the apical
compartment was replaced instead of the basal side and showed identical
results (see for example Fig. 5B). For 2:1 NaCl dilution
potentials, the 150 mM NaCl in Ringer was replaced with 75 mM NaCl, and the osmolality was maintained with mannitol.
For biionic potentials, 150 mM NaCl was replaced with 150 mM chloride salt of the indicated alkali metal or organic cation. Organic cations that were obtained as free amine compounds were
titrated to neutrality with HCl to form the chloride salt. All organic
cations used had a pKa greater than 9.0; therefore,
they were all assumed to be completely protonated at pH 7.4. In biionic
potential experiments using near-impermeant organic cations,
contamination of the solution with residual traces of Na+
could lead to a significant overestimation of the permeability (28). To
exclude this, multiple washes with Na+-free, organic
cation-containing buffer were performed. A "bracketed" protocol was
used in which each measurement in asymmetrical salt solutions was both
preceded and followed by measurements in symmetrical Ringer to control
for any time-dependent variation in the properties of the
monolayer and for "memory" effects on the liquid junction potential
(29).
Radioactive Tracer Flux Studies--
Transmonolayer
[3H]mannitol, [4C]urea, and
[45]Ca tracer flux studies were performed by a
modification of the method described by McCarthy et al.
(19). Studies were performed in 12-well plates of Transwell filters in
culture medium that already contained 1.8 mM
Ca2+. For mannitol and urea assays, 1 mM
unlabeled substrate was also added to the medium in both compartments.
Flux studies were initiated by adding 1-4 µCi/ml of the appropriate
radioisotope (specific activity, 1-2 Ci/mol) to one side
(cis compartment) followed by incubation at 37 °C. At
30-min intervals, 100 µl of medium was collected from the
trans compartment for liquid scintillation counting and
replaced with an equal volume of fresh medium at 37 °C. Tracer
accumulation for all three solutes was found to be linear from 30 to 60 min, and this was used to determine the flux rate and, hence, total
permeability, PTot. To correct for the effect of
the filter membrane and adjacent unstirred layers, the tracer
permeability across blank filters, PBlank, was
determined concurrently. Transepithelial permeability,
PTE, was then calculated from the following
equation.
|
(Eq. 1)
|
Statistical Analysis and Mathematical Modeling--
Data are
presented as the means ± S.E., where n indicates the
number of monolayers from a single experiment. Differences between groups were assessed for statistical significance using the unpaired two-tailed Student's t test. Nonlinear regression analyses
were performed by the Levenberg-Marquardt method using GraphPad Prism 3 software. All results shown are representative of at least three separate experiments unless otherwise indicated.
The relative ionic permeabilities of the monolayers were calculated
using the Goldman-Hodgkin-Katz equation. This is justified because the
equation was shown to fit our data well (see "Results" and Fig. 5).
The individual permeabilities to Na+
(PNa) and Cl
(PCl) in symmetrical 150 mM NaCl
were deduced from the method of Kimizuka and Koketsu (30) using the
following equations,
|
(Eq. 2)
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|
(Eq. 3)
|
where G is the conductance per unit surface area,
a is the NaCl activity, and
is the ratio of the
permeability of Cl
to that of Na+ as
determined by the Goldman-Hodgkin-Katz equation. All calculations used
activities rather than concentrations. The mean activity coefficient of
each monovalent cation-halide salt was assumed to be the same as that
of NaCl, and the anion and cation in each case were assumed to have the
same activity coefficient (Guggenheim assumption).
 |
RESULTS |
Generation of Cell Lines with Inducible Claudin-8 Expression at the
Tight Junction--
To investigate the role of claudin-8 in
paracellular permeability, we first generated cell lines overexpressing
the claudin-8 protein. We first ascertained by low stringency Northern
blot analysis that the canine renal tubule epithelial cell line, MDCK II, does not express claudin-8 endogenously (data not shown). We then
generated by retroviral transduction three MDCK II clonal cell lines
with stable heterologous expression of N-terminal FLAG epitope-tagged
claudin-8 under the control of the TetOff system. Claudin-8 expression
was induced in these cells by omitting doxycycline from the medium
(Dox
) and suppressed by adding doxycycline (Dox+). By Western
blotting and by immunohistochemistry using the FLAG antibody, claudin-8
protein was found to be expressed in Dox
cells but was completely
undetectable in Dox+ cells (Fig. 1,
A and B). By immunohistochemistry, claudin-8 in
confluent Dox
cell monolayers was expressed at the apical end of the
lateral cell membrane, where the tight junction is located, and
colocalized with the tight junction scaffolding protein, ZO1 (Fig.
1C). On electron microscopic examination of freeze fracture
images, the number of tight junction strands appeared similar (Fig.
2, A and B).
Careful quantitative morphometry (Fig. 2C) revealed a small but statistically significant increase in strand number (median 4 in
Dox
cells, 3 in Dox+, p < 0.0001). Tight junction
morphology was mostly normal, although we observed rare short,
disconnected strands in Dox
cells that were never found in Dox+
cells. By freeze-fracture immunogold labeling, we confirmed that
claudin-8 is incorporated into the tight junction strands (Fig. 2,
A and B). This indicates that claudin-8 was
appropriately targeted to the tight junction and that the presence of
an epitope tag at the N terminus did not interfere significantly with
this process.

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Fig. 1.
Characterization of MDCK II TetOff cell lines
stably transfected with claudin-8-FLAG and grown in the presence
(+) or absence ( ) of doxycycline to suppress or induce expression
from the Tet promoter, respectively. A, left
panel, Western blot with FLAG antibody of 100,000 × g membrane fractions (25 µg of protein/lane) isolated from
5 independent clones cultured on plastic dishes. An inducible band of
the expected size for the claudin-8 polypeptide (25 kDa,
arrowhead) is observed in clones 2, 4, and 9. Right
panel, duplicate blot of clone 4, cultured on permeable filter
support. B, immunofluorescence staining of claudin-8 in
confluent Dox+ and Dox monolayers with FLAG antibody. C,
confocal micrographs of a Dox monolayer double-stained with rabbit
claudin-8 antibody (red channel) and mouse ZO1 antibody
(green channel). Upper panels, en face
images at the level of the tight junction. Lower panels,
vertical sections reconstructed in the plane indicated by the
blue line. The white scale bar represents 10 µm. Note claudin-8 staining at the apical end of the intercellular
junction, where it completely colocalizes with ZO1 (yellow
on the merged images).
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Fig. 2.
Fracture-labeled replicas of
claudin-8-FLAG-transfected MDCK II cells cultured for 5 days
(A) without or (B) with Dox. A,
monoclonal anti-FLAG antibody sparsely, but specifically, labels
tight junction strands in Dox cell monolayers that express
FLAG-tagged claudin-8 (representative gold particles are indicated by
arrowheads). B, no labeling is detected in Dox+
cell monolayers. Magnification, ×62,500. C, frequency
histogram showing tight junction strand counts performed at 210 nm
intervals in Dox (white columns) and Dox+ (black
columns) cells.
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To ascertain the integrity of the tight junction, the quantity and
localization of other proteins known to be present in tight junctions
of MDCK II cells were assessed by Western blotting with scanning
densitometry and by immunohistochemistry. The expression levels of ZO1,
occludin, coxsackie- and adenovirus-associated receptor, and claudins
1, 3, and 4 were not significantly different between Dox
and Dox+
cells, and their patterns of localization to the lateral membrane were
all identical (Fig. 3). However, by
Western blotting, claudin-2 expression in Dox
cells was found to be
decreased by 66 ± 5% (n = 3 different clones)
compared with Dox+ cells (Fig. 3A), and immunofluorescence
staining for claudin-2 at the tight junction was qualitatively observed
to also be attenuated (Fig. 3B).

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Fig. 3.
Expression of endogenous tight junction
proteins in claudin-8 expressing cell lines. A, Western
blots of cells grown in the presence or absence of doxycycline.
B, immunofluorescence staining of Dox+ and Dox cells with
antibodies to the indicated tight junction proteins.
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Effect of Claudin-8 on Paracellular Electrical
Resistance--
MDCK II cells are normally moderately leaky with TERs
of 50-100 ohm·cm2 (31). Thus, the TER is dominated by
the resistance of the paracellular pathway and is a useful measure of
its permeability to small ions (predominantly Na+, as will
be shown below). MDCK II cells plated at confluent density on
microporous filters exhibit a brisk increase in TER that peaks at
24-72 h and then declines to a steady-state base line over 5-7 days
(Fig. 4, A-B). In Dox
cells, both the peak TER of the initial overshoot and the steady-state
TER were increased relative to Dox+ cells. Qualitatively similar
results were observed in all three clones but not in control
untransfected MDCK II TetOff cells (Fig. 4, C and
D). These findings suggest that exogenous claudin-8
expression enhances the paracellular barrier to charged ions and
augments this barrier function further during periods of tight junction
assembly.

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Fig. 4.
Effect of claudin-8 expression on TER.
Cells were grown on filters in the absence of doxycycline to induce
claudin-8 expression (Dox ; white symbols and
columns) or in its presence to suppress claudin-8 expression
(Dox+; black symbols and columns). A,
cells were plated in the absence or presence of doxycycline on day 0 and followed thereafter. B, cells were all initially plated
in the presence of doxycycline. On day 9 (arrow 1) half of
the filters (white symbols) were switched to Dox . On day
22 (arrow 2) the two groups were crossed over so that the
other half (black symbols) were deprived of doxycycline.
C and D, summary of experiments similar to those
in A, performed in all three MDCK II TetOff claudin-8 NFL
cell lines (clones 2, 4, and 9) and in untransfected MDCK II TetOff
negative control cells (Untransf), showing the TER in Dox
cells relative to that in Dox+ cells (normalized to 100%) at the peak
of the initial overshoot after plating (C) and at steady
state (D). *, p < 0.05 for Dox
versus Dox+.
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Paracellular Permeability to Na+ and
Cl
--
To determine the identity and quantitative
contribution of the ion(s) carrying the paracellular current and the
electrophysiological basis for the differences induced by expression
of claudin-8, monolayers of the transfected cell lines were mounted in
Ussing chambers, and transepithelial voltage and conductance were
monitored under current-clamp conditions. As shown in Fig.
5A, imposition of a 2:1
concentration gradient of NaCl from apical to basal side induced a
potential difference of
14.1 ± 0.3 mV (apical negative), indicating that the monolayer is more permeable to Na+ than
to Cl
, as has been established by previous investigators
(20, 31, 32). The magnitude of this dilution potential was reduced in Dox
cells (
5.3 ± 0.5 mV; p < 0.0005, n = 3), indicating that the ratio between the
permeability to Na+ and that to Cl
(PNa/PCl) was decreased.
Upon imposing varying NaCl concentration gradients in both directions,
the dilution potentials were observed to vary in a manner that
conformed closely to the Goldman-Hodgkin-Katz equation, at least up to
a 3-fold concentration gradient (Fig. 5B). The
Goldman-Hodgkin-Katz equation has previously been shown to be a
reasonable model to approximate the behavior of other leaky epithelia
despite the fact that the assumption of a constant field is likely to
be incorrect (33). This equation was therefore used to derive
quantitative estimates for
PNa/PCl. Furthermore, from the total conductance measured in 150 mM NaCl, the
absolute values of PNa and
PCl could be deduced. Induction of claudin-8 expression was found to be associated with a 70% reduction in PNa (p < 0.0005), whereas
PCl did not change (Fig. 5C). These findings suggest that claudin-8 augments the paracellular barrier to
Na+ permeation. Fig. 5D summarizes the results
of multiple measurements performed on monolayers at different time
points after plating. This shows that the conductance of the monolayer
was linearly related to PNa, demonstrating that
the time-dependent changes in TER we had previously
observed (Fig. 4, A-B) were almost entirely attributable to
changes in PNa.

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Fig. 5.
Determination of NaCl permeability from
conductance and dilution potential measurements. A,
representative voltage traces from three Dox (white
symbols) and three Dox+ (black symbols) epithelial
monolayers acquired simultaneously in Ussing chambers under a
zero-current clamp. For the sake of clarity only data points at 30-s
intervals are shown. The dilution potential was determined from the
change in transepithelial voltage upon switching from symmetrical
bathing solutions to a 2:1 NaCl concentration gradient. B,
NaCl dilution potentials conform to the Goldman-Hodgkin-Katz equation.
The data points represent dilution potentials (Vt)
experimentally determined in a single monolayer bathed in asymmetrical
NaCl solutions. The abscissa shows the ratio of NaCl
activity in apical compared with basal compartments ( ). The curves
were fitted by nonlinear regression to the equation
Vt = RT/F*ln[( + )/(1 +  )], using values of of 0.14 (Dox ) and 0.05 (Dox+).
C, typical values of PNa and
PCl from a single experiment performed on three
Dox (white columns) and three Dox+ (black
columns) monolayers. *, p < 0.0005. D,
summary of data from individual experiments performed at six different
time points after plating, showing the extracted values of
PNa and PCl plotted as a
function of total conductance. Each data point represents the mean ± S.E. of three monolayers; the key to symbols is on the far
right panel.
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Permeation of Other Alkali Metal Cations--
Although
Na+ and Cl
are the major charge carriers that
MDCK cells encounter in cell culture medium and Ringer saline, the
native milieu of claudin-8 is the distal renal tubule, which is exposed to high concentrations of K+ at the luminal surface. To
assess the relative permeability of the monolayers to different alkali
metal cations, the bath solutions were exchanged so that one side was
bathed in 150 mM NaCl and the contralateral side was bathed
in 150 mM chloride salt of monovalent alkali metal cations.
From the biionic potential measured across the monolayer, the relative
permeability to Na+ versus
the other cation was then calculated
(Table I and Fig. 6A). As has previously been
reported, the range of permeabilities of the alkali metal cations is
very narrow, suggesting that the permeating pathway is a relatively
water-filled pore (31). The order of permeabilities was generally
Na+ ~ Li+ ~ K+ > Rb+
Cs+, almost in reverse-order of their
free-solution mobilities, and approximated sequences IX through XI of
the Eisenman series. This suggests that the paracellular cation
permeation pathway has negatively charged binding sites with relatively
high affinity for dehydrated monovalent cations (34). Furthermore, the
permeability to all of the cations was reduced to the same extent in
Dox
compared with Dox+ cells so that the selectivity sequence was
unchanged.

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Fig. 6.
Relative permeability and conductance of
monovalent cations. A, permeability of alkali metal
cations relative to Na+, as determined by biionic potential
measurements. B, conductance of alkali metal cations
relative to Na+. The dashed line in each
panel represents permeability/conductance identical to that
of Na+. C, permeability of organic cations
relative to Na+. D, conductance of organic
cations relative to Na+.
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In the presence of strong binding sites in a pore, a cation that binds
with high affinity would be released slowly from the binding site, and
therefore, its conductance across the pore would be paradoxically low
(the so-called "sticky pore problem") (35). This can give rise to a
discrepancy between the relative permeabilities, as determined by
diffusion potentials at equilibrium, and the conductance, as determined
during the passage of current. We therefore determined the relative
conductance of the paracellular pathway for alkali metal cations by
measuring transepithelial conductance in the presence of symmetrical
150 mM solutions of the chloride salt of each cation (Fig.
6B). Indeed, the order of conductance, K+ > Rb+ > Na+ > Cs+ > Li+ (Eisenman V), was quite different from the order of
permeabilities and suggests that small cations with a strong
electrostatic interaction with the paracellular pore such as
Li+ and Na+ may bind so tightly as to impede
their conductance. Importantly though, the relative conductance was
also no different between Dox
and Dox+ cells, suggesting that
claudin-8 expression does not induce any drastic differences in either
the cation binding site or the pore structure of the paracellular pathway.
Permeation of Organic Monovalent Cations--
To probe the size
selectivity of the paracellular pore, similar biionic potential and
conductance measurements were performed with a range of monovalent
organic cations of varying sizes (Table I and Fig. 6, C and
D). Although the data show the expected monotonic decrease
in permeability with increasing ionic diameter within the range 3.8 to
7.3 Å, there is considerable scatter, as has been observed in similar
studies done on gallbladder epithelium (36) and muscle end-plate
Na+ channels (28), indicating that nitrogenous cations are
imperfect probes of pore size. Part of the size-independent variation
in permeation through the paracellular pathway in gallbladder
epithelium could be attributed to differences in hydrogen-bonding
capability of the cation, which determines its interaction with partial
negative charges in the paracellular pore (36). Consistent with this, we find anomalously high permeabilities and, as might be predicted, low
conductances for compounds with multiple protons available for
hydrogen-bonding such as guanidine and
N-methyl-D-glucamine (Table I).
Because many of the nitrogenous cations used are quite lipophilic,
errors could arise either due to permeation of the neutral form of the
organic cation across the lipid bilayer, which might significantly
reduce the protonated form at the unstirred layer, or even due to
permeation of a charged form that is sufficiently lipophilic to cross
the lipid bilayer (37). We believe this is unlikely for three reasons.
First, we chose cations with a pKa > 9, so that
less than 1% would be deprotonated at pH 7.4. Second, permeabilities
tended to be higher for ions with greater hydrogen-bonding capability,
the opposite of what would be expected for lipid permeation. Third,
extensive studies performed by Moreno and Diamond (36) clearly
demonstrate that lipid permeation of such compounds was negligible
compared with paracellular permeation in the gallbladder, a leaky
epithelium with many similarities in its paracellular permeability
properties to MDCK II.
Importantly, the permeability of all the cations tested relative to
PNa was no different between Dox
and Dox+
cells. Because we have already shown that PNa is
markedly decreased in Dox
cells, this indicates that the absolute
permeabilities to the other cations were all decreased proportionately.
This again suggests that the physical structure of the paracellular
pore, including its apparent diameter, has not been drastically altered.
Permeation of Divalent Cations and Neutral
Solutes--
Radiotracer flux studies were next used to determine the
permeability of the monolayers to calcium. Electrophysiological studies were not used because they would entail exposure of the monolayers to
large alterations in the concentration of calcium that could potentially affect tight junction integrity and intracellular signaling
processes. As shown in Fig. 7, MDCK II
monolayers are quite permeable to calcium, and this permeability was
reduced by almost 3-fold by claudin-8 expression in Dox
cells. In a
set of matched monolayers that were assayed simultaneously for calcium permeability by tracer flux and Na+ permeability by
dilution potential,
PCa/PNa was found to be
exactly the same in Dox
(4.41 ± 0.11) and Dox+ (4.49 ± 0.11) cells. If claudin-8 decreased paracellular cation permeability by
altering charge distribution in or around the paracellular pore, this
should have a disproportionately greater effect on permeation of
multivalent compared with monovalent ions. The striking finding that
divalent cation permeability is reduced to a degree exactly
proportionate to the change in PNa argues
strongly against an electrostatic effect of claudin-8. By contrast, the
permeability to neutral solutes, as inferred from the measurement of
radiolabeled mannitol and urea fluxes, was no different between Dox
and Dox+ cells (Fig. 7).

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Fig. 7.
Permeabilities of divalent cations and
neutral solutes determined from radioactive tracer fluxes.
White bars, Dox ; black bars, Dox+. *,
p < 0.00005.
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Temperature Dependence of Na+ Permeation--
If
claudin-8 expression in some way impedes cation permeation across
paracellular pores, the activation energy for Na+
permeation should be increased. To test this, the transmonolayer conductance in Ringer solution, which we have shown is dominated by the
Na+ conductance, and PNa determined
from dilution potentials were measured at different temperatures, and
molar activation energies were derived from Arrhenius plots (Fig.
8). As expected, the activation energy
for total conductance and PNa were very similar,
likely indicating that they both reflect the energy barrier to
Na+ permeation. The values obtained were quite low and in
the same range as that observed for transmembrane ion channels
(38). Importantly, the activation energy was unchanged by claudin-8 expression, indicating that whatever the mechanism by which claudin-8 reduces Na+ conductance, it is not by inducing an
unfavorable energetic landscape.

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Fig. 8.
Arrhenius plots to show the temperature
dependence of paracellular permeability to Na+. The
ordinate represents on a logarithmic scale the
transepithelial conductance (left panel) and
PNa determined by measuring the NaCl dilution
potential (right panel) in Dox (white squares)
and Dox+ (black squares) monolayers. The abscissa
represents the reciprocal of the absolute temperature. The lines were
obtained by fitting the data by nonlinear regression to the equation,
ln(y) = ln(K) Ea/RT, where y is the ordinate
variable, and K is a pre-exponential constant. The estimates
for the activation energy, Ea (kcal/mol; mean ± S.E.), are shown next to each line.
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DISCUSSION |
Methodologic Issues--
We describe herein a novel cell line with
overexpression of claudin-8. The levels of expression of the
heterologous protein appear to be relatively modest because we were
able to detect it by Western blotting only in enriched membrane
fractions. We attribute this to our use of a retroviral vector, which
generally leads to a high percentage of stably transfected cells but a
low number of integrated transgene copies per cell. The inadvertent use
of a low copy number technique may account for two potential advantages
of our study over some prior ones. First, there was no detectable
"leakage" of expression in the suppressed (Dox+) state. Second,
expression of claudin-8 generated very few additional and/or aberrant
tight junction strands. A recent study of claudin-1 suggests that
excessively high levels of claudin overexpression may be responsible
for inducing aberrant strands and that these aberrant strands lack
normal tight junction components such as occludin (39). To the extent
that such strands are non-physiological, their presence is undesirable
and may complicate interpretation of claudin expression studies.
The transgene we used encodes an N-terminal FLAG epitope-tagged
claudin-8 protein. We do not believe that the epitope tag interfered
with claudin-8 function for three reasons. First, claudin-8 was
appropriately targeted to the tight junction. Second, other studies
report that epitope-tagged claudin constructs (of the C terminus) are
still able to form de novo tight junctions and mediate
adhesion in fibroblasts (40) and, when expressed at low levels,
incorporate into existing tight junction strands (39). Third, we tagged
the N terminus rather than the C terminus, which encodes a PDZ binding
motif (41), to eliminate the potential for interference with binding to
the PDZ domains of ZO-1, -2, and -3. Nevertheless, we cannot exclude
completely the possibility that an N-terminal tag may allow appropriate
incorporation into the tight junction but interferes in some subtle way
with the permeability properties of the protein.
We assessed the level of expression and localization of other
tight junction components for which reagents were available, including
claudins 1-4, ZO-1, and occludin and found no change with induction of
claudin-8 expression, except for a reduction in the expression level of
claudin-2. The fact that only claudin-2 was affected might argue
against a nonspecific effect of claudin-8 overexpression on tight
junction assembly or integrity. The possibility that other claudin
isoforms that were not tested for are expressed in MDCK II cells and
might be affected by claudin-8 overexpression cannot be ruled out.
General Properties of Paracellular Permeation in MDCK II
Cells--
In our studies, the native paracellular pathway in MDCK II
cells was found to be highly cation-selective, exhibited an alkali cation selectivity for permeability consistent with strong
electrostatic interaction between pore and permeating cation, showed
substantial discrepancies between permeability and conductance
selectivity, favored organic cations with strong hydrogen-bonding
capability, and exhibited an activation energy of permeation that was
very low and in the range observed for transmembrane ion channels. These findings are consistent with the model developed by Diamond and
co-workers (33, 36, 42) based on a series of comprehensive studies in
gallbladder epithelium, which also has a leaky cation-selective paracellular permeability resembling that of MDCK II cells. In this
model, the paracellular pathway behaves as a series of relatively wide,
water-filled pores or channels lined by residues with polarizable negative charges available for binding to permeating cations.
Functional Consequences of Claudin-8 Overexpression--
The
principal finding of this study is that overexpression of claudin-8
leads to a reduction in paracellular permeability to Na+
and other cations but not to anions or neutral solutes. In simple terms, there are three ways in which a reduction in ion permeability can be effected. First, the size of the paracellular pore may be
reduced or its shape altered in such a way as to sterically hinder
Na+ permeation. Second, electrostatic interactions between
the permeating Na+ ion and the pore may be altered. Such
interactions may occur with residues that bear overall net charge or
with charged side groups of residues or with polarizable moieties and
include both surface charge effects and direct interactions inside the
pore. Third, the physicochemical nature of the pores may be unchanged, but the number of functioning pores may be reduced either because of a reduction in the total number of pore protein molecules present or
because some of the pores become closed (if this is possible).
Our finding that the size selectivity of the pore to organic cations
varying in size from 3.8 to 7.3 Å is unaltered argues against the
first possibility. Our finding that the relative permeability among
different alkali metal ions is unchanged and that permeability to
monovalent and divalent cations is proportionately reduced argues
against the second possibility. Moreover, both of the first two
hypotheses are ruled out by our observation that the activation energy
for Na+ permeation is unchanged. Thus, purely from
physiological observations, we are led to the only possible conclusion,
that claudin-8 must somehow reduce the number of functioning
Na+- permeable pores.
Difficulty in Interpreting Claudin Overexpression Studies--
The
extrapolation of the properties of individual claudins from the
phenotype of transfected cell lines reported in the literature has been
problematic for a variety of reasons. First, it is uncertain whether
the overexpressed claudin inserts new pores into existing tight
junction strands or contributes structural building blocks to augment
the paracellular barrier. In some studies overexpression of claudin
increases permeability (22, 23), whereas in others it decreases it
(18-20). A second problem in interpreting transfection studies is that
it is usually not known whether the overexpressed claudin is added to
existing tight junction components or substituting for a component that
then becomes down-regulated. Because of the lack of availability of
antibodies, it is not yet possible to measure all of the claudins that
are currently known, and of course other as-yet unidentified claudins
or other tight junction membrane proteins cannot be accounted for at
all. Third, the tight junction strand permeability may be dependent not
only on the presence or absence of a particular claudin but also on the
presence or absence of other non-pore-forming "structural"
components of the strand. For example, it is not difficult to imagine
that the permeability of a normal tight junction strand containing
claudin-1 might be quite different from that of an aberrant strand
containing claudin-1 but lacking occludin (as found by McCarthy (19)).
Finally, each pore may not necessarily be formed by the molecules of a
single claudin isoform. Tsukita and co-workers (40) show that different claudin isoforms can adhere from cell to cell in a heterotypic manner
and associate within a strand into heteropolymers. If each pore is
lined by multiple claudin molecules, it is conceivable that the
permeability properties of that pore may be dependent on the particular
combination of claudin isoforms that are present.
A Model to Explain the Consequences of Claudin
Overexpression--
As a first step toward interpreting claudin
overexpression studies, we propose a simple model (Fig.
9). We assume that claudins are
homotypically associated pore-forming proteins and ignore for the sake
of simplicity the possibility of pores formed by heterotypic
interactions. Claudins are depicted as dimers, but the actual
stoichiometry is currently unknown. We also assume that a finite number
of proteins is packed into a mature tight junction strand to form a
continuous seal. Therefore, each overexpressed claudin molecule must
either displace an existing tight junction protein or incorporate in a
whole new tight junction strand. In our models, claudins can have
permeabilities that range from very high to negligibly low. The latter
are indistinguishable from tight junction proteins that have no pores
and purely exist to complete the barrier ("barrier proteins"). We
propose two simple scenarios upon exogenous claudin overexpression. The
new claudin molecules may substitute for endogenous tight junction
proteins on the same strand (parallel model) or assemble into new tight junction strands in series with existing ones (series model). Although
it might be assumed that the change in permeability upon overexpression
of a single claudin isoform is a direct measure of the permeability of
that isoform (i.e. that
P = Pe), our model clearly shows that this is true only
in the specific situation in which exogenous claudin incorporates in a
parallel fashion (i.e. no change in strand number) and
replaces a barrier protein (i.e. Py = 0).
No reported studies so far unequivocally fulfil these criteria.

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Fig. 9.
Models for how exogenously overexpressed
claudins might incorporate into native tight junctions. Tight
junction strands are depicted as continuous strings of paired claudin
molecules. The two apposed molecules of each pair derive from the
lateral membrane of the two adjacent cells. Solutes and water
(arrows) can pass through pores in the claudin dimers.
Different isoforms of claudins have different pore permeabilities
(purple, Px; yellow,
Py). The base-line permeability of a single native
tight junction strand to any given solute is the sum of the
permeabilities of these different pores (Px + Py). Exogenously overexpressed claudins generate
pore-forming protein pairs (red) with permeability,
Pe. The total permeability of the resultant junction
and the possible direction(s) for the change in permeability from
baseline ( P) are shown in the columns to the right of
each model. Parallel model, exogenous claudins replace
endogenous claudins, resulting in a new set of pores in parallel with
pre-existing ones. The total permeability may increase (if
Pe > Py) or decrease (if
Pe < Py). Series
model, exogenous claudins form new strands in series with
pre-existing strands, decreasing total permeability.
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The studies of McCarthy et al. (19), van Itallie
et al. (20), and Colegio et al.(21), in which ion
permeability is reduced concurrent with the appearance of aberrant
strands, can now be seen to be most consistent with the series model.
The overexpression of claudin-2 by Furuse et al. (22), in
which permeability is increased without any apparent change in tight
junction strands, is consistent with a parallel model, in which a less
permeable tight junction component is replaced by the more permeable
claudin-2. In the current study, our finding of decreased cation
permeability without a substantive change in strand number can only be
explained by the parallel model. Indeed, our detailed physiological
characterization strongly suggests that a cation-permeable claudin was
replaced by claudin-8, which must itself be relatively or absolutely
impermeable to Na+ and other cations. A recent study
demonstrated that claudin-2 overexpression increased cation
permeability in MDCK cells of high transepithelial resistance,
suggesting that claudin-2 is a paracellular cation channel (23). Given
that the expression of claudin-2 in our cell lines is substantially
reduced, we speculate that its replacement by claudin-8 accounts for
all of the observed phenotypic findings. We conclude that claudin-8
acts as a nonspecific cation barrier and suggest that it may play an
important role in maintaining transtubular cation gradients in the
distal renal tubule.