Departments of 1 Cell Biology and 3 Surgery, Yale University, New Haven, Connecticut 06520; and Departments of 2 Medicine and 4 Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Tight junctions (TJs) regulate
paracellular permeability across epithelia and vary widely in their
transepithelial electrical resistance (TER) and charge selectivity. The
claudin family of transmembrane proteins influences these properties.
We previously reported that claudin-4 increased TER ~300% when
expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells
and decreased the paracellular permeability for Na+ more
than Cl (Van Itallie C, Rahner C, and Anderson JM.
J Clin Invest 107: 1319-1327, 2001). In
comparison, we report here that expression of claudin-2 increases TER
by only ~20% and does not change the ionic selectivity of MDCK II
cells from their cation-selective background. To test whether the
extracellular domains of claudins-4 and -2 determine their unique
paracellular properties, we determined the effects of interchanging
these domains between claudins-4 and -2. Inducible expression of
wild-type claudins and extracellular domain chimeras increased both the
number and depth of fibrils, but the characteristic fibril morphologies
of claudin-4 or -2 were not altered by switching extracellular domains.
Like claudin-4, chimeras expressing the first or both extracellular
domains of claudin-4 on claudin-2 increased TER severalfold and
profoundly decreased the permeability of Na+ relative to
Cl
. In contrast, chimeras expressing the first or both
extracellular domains of claudin-2 on claudin-4 increased the TER by
only ~60 and ~40%, respectively, and only modestly altered charge
selectivity. These results support a model in which the claudins create
paracellular channels and the first extracellular domain is sufficient
to determine both paracellular charge selectivity and TER.
tight junction; claudin-4; claudin-2; dilution potential; intercellular junction; transepithelial electrical resistance
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INTRODUCTION |
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TIGHT JUNCTIONS (TJs) form a barrier to the paracellular diffusion of solutes across epithelia. The barrier varies among epithelia in both tightness, measured by the transepithelial electrical resistance (TER), and charge selectivity (21). The molecular components of the TJ include the claudins, a family of ~23-kDa tetraspanning proteins with two extracellular domains (8, 10, 19). It is likely that these proteins are critical components of the TJ barrier, because expression of claudins in fibroblasts, which do not normally express them, results in both de novo formation of characteristic freeze-fracture fibrils and increased cell-cell adhesion (10).
The charged amino acid residues on the extracellular domains are
variable in number and position. Recently, individual charges on the
extracellular domains of claudins were shown to influence paracellular
charge selectivity, suggesting that the claudins form charge-selective
pores in the TJ barrier (6). Madin-Darby canine kidney
(MDCK) II cells, which express at least three different claudins,
normally form a cation-selective epithelial barrier when grown on
semipermeable membranes. Expression of exogenous claudin-4 was found to
decrease the permeability of Na+ relative to
Cl in this cation-selective background (24).
However, when claudin-4 with a mutation of a basic to acidic residue in
the first extracellular domain was expressed, this decrease in cation
selectivity was no longer observed (6). The reciprocal
experiment was performed on claudin-15, which produces cation-selective
paracellular pathways when expressed in MDCK II cells. When three
acidic residues were mutated to basic residues in the first
extracellular domain of claudin-15, the paracellular pathway became
selective for anions relative to cations in MDCK II cells expressing
this construct. In these studies, only mutations in the first
extracellular domain of claudins were examined.
The expression of exogenous wild-type claudins has also been observed to influence TER (9, 11, 16, 24). Van Itallie et al. (24) reported that claudin-4 increased TER of low-resistance MDCK II cells threefold, whereas Furuse et al. (9) reported that expression of claudin-2 in high-resistance MDCK I cells decreased TER 20-fold. Descriptive observations suggest that these properties correlate with in vivo epithelial properties. For example, in the mammalian kidney, claudin-4 is expressed in the tighter collecting duct, whereas claudin-2 has been localized to the leakier proximal tubule (7, 13).
Although charges on the first extracellular domains of claudins-4 and -15 were found to influence paracellular charge selectivity (6), the relative roles of the individual extracellular domains on charge selectivity and TER have not been determined. In this study, we report that claudin-2 creates paracellular pathways that are more cation selective than those produced by claudin-4 in MDCK II cells. We hypothesize that the extracellular domains of claudins-4 and -2 determine their differences in charge selectivity and TER. To test this hypothesis, we constructed a panel of chimeras of claudins-4 and -2 in which the first, second, and both extracellular domains were interchanged. These wild-type and chimeric claudins were expressed under the control of the tetracycline-responsive element in MDCK II Tet-Off cells (2). Our results provide the first evidence that the first extracellular domains of claudins determine TER as well as paracellular charge selectivity. In addition, we report that claudins-4 and -2 produce unique TJ fibril morphologies, which are not influenced by interchanging their extracellular domains. In addition, the extracellular domains do not influence the unique fibril morphologies of claudins-4 and -2. Finally, the lack of correlation between fibril number and TER suggests that the variable paracellular properties of epithelia can be determined by their combination of claudins and not just by the number of fibrils (4, 5).
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MATERIALS AND METHODS |
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Plasmid constructs and cell lines.
Full-length mouse claudin-2 was amplified by PCR using mouse kidney
cDNA (Quick-clone cDNA; Clontech, Palo Alto, CA) as a template. The
amplified product was cloned into the TOPO-TA vector (Invitrogen, San
Diego, CA) and subcloned into the pTRE vector (Clontech). All claudin
extracellular domain chimeras were constructed by first creating unique
restriction sites in the nucleotides encoding amino acids adjacent to
the extracellular domains. The DNA sequence corresponding to the
extracellular domains was cut, and an amplified exogenous loop was
ligated in its place (Fig. 1). Unique
restriction sites that conserved the claudin amino acid sequence were
generated by using site-directed mutagenesis (Quik-Change; Stratagene,
La Jolla, CA). The first and second extracellular domains of claudins-4
and -2 were defined as follows: P28MWRVTAFIGSNIVTSQTIWEGLWMNCVVQSTGQMQCKVYDSLLALPQDLQAAR81
and H141NIIQDFYNPLVASGQKREMGAS163, for
claudin-4 and
P28NWRTSSYVGASIVTAVGFSKGLWMECATHSTGITQCDIYSTLLGLPADIQAAQ81
and H141GILRDFYSPLVPDSMKFEIGEA163, for
claudin-2. To create C4(C2/C4) and C4(C2/C2) (Fig. 1), BsiWI and SpeI sites were created 5' and 3' of the first
extracellular domain of claudin-4, and the domain was cut sequentially
with BsiWI and SpeI. The first extracellular
domain of claudin-2 was amplified with oligonucleotides that created
flanking 5' BsiWI and 3' SpeI sites and was
ligated in place of the first extracellular domain of claudin-4. To
create C4(C4/C2) and C4(C2/C2), AgeI and SnaBI
restriction sites were created 5' and 3' of the second extracellular domain of claudin-4, and this domain was cut sequentially with AgeI and SnaBI. The second extracellular domain
of claudin-2 was amplified with oligonucleotides that created flanking
5' AgeI and 3' SnaBI sites and was ligated in
place of the second extracellular domain of claudin-4. To create
C2(C4/C2) and C2(C4/C4), BsiWI and SphI sites
were created 5' and 3' of the first extracellular domain of claudin-2,
and this domain was cut sequentially with BsiWI and
SphI. The first extracellular domain of claudin-4 was amplified with oligonucleotides that created flanking 5'
BsiWI and SphI sites and was ligated in place of
the first extracellular domain of claudin-2. Introduction of these
unique restriction sites and ligation of the first extracellular domain
of claudin-4 in C2(C4/C2) and C2(C4/C4) altered the amino acid
sequence, and site-directed mutagenesis was employed to correct the
sequences as follows: T32 to V32 and
G82ML84 to A82MM84. To
create C2(C2/C4) and C2(C4/C4), AgeI and NheI
sites were created 5' and 3' of the second extracellular domain of
claudin-2, and this domain was cut sequentially with AgeI
and NheI. The second extracellular domain of claudin-4 was
amplified with oligonucleotides that created flanking 5'
AgeI and 3' NheI sites and was ligated in place
of the second extracellular domain of claudin-2. DNA sequencing in both
directions verified all wild-type and mutant claudin constructs. Clonal
cell lines of MDCK II Tet-Off cells (Clontech) were derived by standard
transfection and selection techniques; regulated expression of the
transgene products was accomplished by varying doxycycline levels in
the culture media as previously described (24). Stable
cell lines were screened by immunoblot analysis, and uniformity of
expression was verified by immunofluorescent analysis. At least five
stably expressing clonal cell lines were generated for each construct.
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Immunoblots and immunofluorescence.
Immunoblots were performed as previously reported (24)
using the following antibodies available through Zymed (South San Francisco, CA): anti-human claudin-4 mouse monoclonal antibody (1:4,000), anti-human claudin-2 mouse monoclonal antibody (1:4,000), anti-human claudin-1 rabbit polyclonal antibody (1:3,000), anti-human occludin mouse monoclonal antibody (1:1,500), anti-human ZO-1 mouse
monoclonal antibody (1:1,500), and anti--tubulin mouse monoclonal
antibody (1:1,000). To prevent the alteration of claudin function
(14, 16) observed with the introduction of an epitope tag,
antibodies used to detect claudins were directed against epitopes in
their COOH-terminal cytoplasmic domains and were not tagged (Fig. 1).
Immunofluorescence for claudin-4, claudin-2, chimeras, and ZO-1 was
performed, as previously described (24), on cells fixed in
ice-cold ethanol for 20 min. The following antibodies were used:
anti-human claudin-4 mouse monoclonal antibody (1:150), anti-human
claudin-2 rabbit polyclonal antibody (1:150), and anti-ZO-1 rat
monoclonal antibody (R40.76 cell supernatant, 1:100). ZO-1 was detected
by using donkey anti-rat Cy2-conjugated secondary antibody (1:150).
Claudins-4 and -2 were detected by using donkey anti-mouse
Cy3-conjugated secondary antibody (1:1,000) and donkey anti-rabbit
Cy3-conjugated secondary antibody (1:1,000), respectively (Jackson
ImmunoResearch, West Grove, PA). Images were captured on a Zeiss 510 laser scanning confocal microscope using a ×100 (N.A. 1.4)
Plan-Apochromat objective lens (Carl Zeiss, Jena, Germany).
Freeze-fracture electron microscopy. Freeze-fracture electron microscopy was carried out as previously described (18).
[3H]mannitol flux studies. [3H]mannitol flux studies were carried out using established protocols (17).
Electrophysiological measurements. Electrophysiological characterization of MDCK II monolayers was carried out according to published methods (24). Stable MDCK II Tet-Off cell lines (Clontech) transfected with wild-type claudins or chimeras were grown on Snapwell filters (Costar; Corning Life Sciences, Acton, MA) for 4 days, induced without doxycycline or noninduced with doxycycline (50 ng/ml). Protein expression was maximal by day 2. Transmonolayer resistance was measured using a modified Ussing chamber with a microcomputer-controlled voltage/current clamp (Harvard Apparatus, Holliston, MA) with buffer A (120 mM NaCl, 10 mM HEPES, pH 7.4, 5 mM KCl, 10 mM NaHCO3, 1.2 mM CaCl2, and 1 mM MgSO4) in the apical and basolateral chambers. Transmonolayer dilution potentials were measured upon replacement of buffer A with buffer B (60 mM NaCl, 120 mM mannitol, 10 mM HEPES, pH 7.4, 5 mM KCl, 10 mM NaHCO3, 1.2 mM CaCl2, and 1 mM MgSO4) in the apical chamber relative to the basolateral chamber (buffer A) (24). Dilution potentials were immediately stable and repeatedly measured (every 6 s) for at least 30 s after buffer B was added to the apical chamber. Voltage and current electrodes consisted of a Ag/AgCl wire in 3 M KCl saturated with AgCl housed in a glass barrel with a microporous ceramic tip (Harvard Apparatus). The Henderson diffusion equation for univalent ions (15) was used to calculate liquid junction potentials under dilution potential conditions.
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RESULTS |
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Construction of claudin chimeras.
Claudin-4 has been reported to decrease the paracellular permeability
of Na+ relative to Cl and is associated in
vivo with higher resistance epithelia (24). Although
claudin-2 has been associated with lower-resistance epithelia (7,
13), its effect on paracellular charge selectivity is unknown.
We hypothesized that the different paracellular properties conferred by
claudins-4 and -2 were determined by their two extracellular domains.
To test this hypothesis, we measured the effect of interchanging the
two extracellular domains of claudins-4 and -2, singularly and in
combination, on paracellular electrophysiological properties (Fig. 1).
This panel of six claudin chimeras will be referred to as follows:
claudin-4 with the first extracellular domain of claudin-2, C4(C2/C4);
claudin-4 with the second extracellular domain of claudin-2, C4(C4/C2);
claudin-4 with both extracellular domains of claudin-2, C4(C2/C2);
claudin-2 with the first extracellular domain of claudin-4, C2(C4/C2);
claudin-2 with the second extracellular domain of claudin-4, C2(C2/C4);
and claudin-2 with both extracellular domains of claudin-4, C2(C4/C4).
Claudins-4 and -2 and chimeras were expressed in stably transfected
clones of MDCK II cells under control of the tetracycline-responsive
element (2). Because MDCK II form low-resistance
monolayers, the majority of ionic transport is paracellular (3,
6, 24), making them a useful model in which to test the effects
of expressing claudins-4 and -2. However, an undefined set of
endogenous claudins is expressed in these cells, including at least
claudins-1, -2, and -4 (9, 24). They are normally highly
permeable to Na+ relative to Cl
(3,
23). When expressing an exogenous claudin, we determine all
changes from this baseline.
Wild-type and chimeric claudins are inducible and localize to cell
borders.
Clonal lines of MDCK II cells were produced with wild-type claudins-4
and -2 and chimeras under control of a doxycycline-repressible promoter. In all clones, expression of protein from the transgene was
tightly regulated and showed a very large relative increase following
induction (Fig. 2, A and B, first horizontal
rows). All cell lines express both
endogenous claudins-4 and -2, which are obvious at longer exposure
times (Fig. 2, A and B, second horizontal rows).
To determine the effect of expressing wild-type claudins and chimeras
on endogenous TJ proteins, immunoblot analysis was performed on stable
transfectants of MDCK II Tet-Off cells, induced or noninduced for the
transgene. Expression of claudin-4 and chimeras of claudin-4 with the
extracellular domains of claudin-2 did not change the levels of
claudin-1, occludin, ZO-1, or -tubulin (Fig. 2A). A
slight increase in the level of endogenous claudin-1 was observed upon
expression of C4(C2/C4) and in claudin-2 upon expression of C4(C2/C2),
C4(C2/C4), and C4(C4/C2). Expression of claudin-2 and chimeras of
claudin-2 with the extracellular domains of claudin-4 did not change
the levels of claudin-1, ZO-1, or
-tubulin. However, upon induction
of all these transgenes, there was a slight decrease in the levels of
occludin (Fig. 2B). Most striking was a large decrease in
the level of endogenous claudin-4 upon expression of C2(C4/C4) and
C2(C4/C2) but not C2(C2/C4), suggestive of negative feedback on
claudin-4 levels upon expressing at least the first extracellular
domain of claudin-4.
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The extracellular domains of claudins do not determine TJ fibril
morphology.
To determine whether the increase in levels of claudins and chimeras
detected by immunoblotting and immunofluorescence microscopy corresponds to an increase in the content of TJ fibrils, analysis using
freeze-fracture electron microscopy was performed (Fig. 4). Expression of
either claudin-4 or -2 resulted in an increase in the number of fibrils
and TJ depth. However, expression of claudin-4 produced dense,
reticular, and parallel patterns with fused fibril particles, whereas
claudin-2 produced more curved patterns with nonfused particles.
Interestingly, the differences in fibril morphology are not determined
by the extracellular domains of claudins, because the fibrils produced
upon the expression of C4(C2/C2) and C2(C4/C4) were indistinguishable
from those of claudins-4 and -2, respectively.
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Claudins-4 and -2 differ in their effects on paracellular
properties.
Inducible stable clones of MDCK II Tet-Off cells were grown on Snapwell
filters for 4 days with or without induction of claudins-4 or -2. As we
have previously shown, claudin-4 increased TER ~300% above baseline;
in contrast, claudin-2 increased TER only ~20% (Fig.
5). It was previously shown that
expression of claudin-2 in high-resistance MDCK I cells decreased TER
20-fold; however, the baseline TER of these cells was 10,000 · cm2, and expression of
claudin-2 decreased the TER to 150-500
· cm2. MDCK II cells used
in our studies have a baseline TER of only 35
· cm2. The difference in initial
background TER could explain our different results if claudin-2 confers
a resistance that is intermediate but closer to the low-resistance
line.
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The first extracellular domains of claudins-4 and -2 are sufficient to determine TER and paracellular charge selectivity. Although expression of wild-type claudin-4 leads to a severalfold increase in TER, expression of chimeras containing either the first [C4(C2/C4)] or both [C4(C2/C2)] extracellular domains of claudin-2 on claudin-4 resulted in TER increases of only ~63 and ~35%, respectively (Fig. 5). These small increases in TER are similar to the effect of expressing claudin-2. In contrast, expression of chimeras containing either the first [C2(C4/C2)] or both [C2(C4/C4)] extracellular domains of claudin-4 increased the TER tenfold and sevenfold, respectively (Fig. 5). These results demonstrate that the first extracellular domain alone is sufficient to determine TER, independent of the transmembrane, cytoplasmic, or second extracellular domain sequences.
We next tested whether interchanging the first extracellular domains of claudins-4 and -2 altered paracellular charge selectivity by comparing transepithelial dilution potentials with and without transgene expression. Like wild-type claudin-2, C4(C2/C2) did not change the dilution potential from baseline (Fig. 6). C4(C2/C4) decreased the dilution potential significantly but only modestly and not to the level of claudin-4. In contrast to the modest or lack of change in dilution potential produced by the chimeras with the first or both extracellular domains of claudin-2, expression of chimeras with the first [C2(C4/C2)] or both [C2(C4/C4)] extracellular domains of claudin-4 decreased the dilution potential from 10.4 ± 0.3 mV to 3.3 ± 0.8 mV and 8.1 ± 0.6 mV to 1.6 ± 0.3 mV, respectively (Fig. 6). Dilution potentials of equal magnitude but opposite charge were generated upon reversing the direction of the NaCl gradient, confirming that the charge selectivity is paracellular. The chimeras' parallel effects on TER and dilution potential suggest that the first extracellular domains are sufficient to determine both paracellular electrophysiological properties.The role of the second extracellular domain.
To determine the role of the second extracellular domain on
paracellular charge selectivity and TER, C4(C4/C2) and C2(C2/C4) were
expressed in MDCK II Tet-Off cells. Neither of these constructs induced
a significant increase in TER, unlike either claudin-4 or -2. C2(C2/C4)
actually decreased TER by ~40%. Neither C4(C4/C2) nor C2(C2/C4)
changed the baseline cation selectivity of MDCK II cells. The finding
that C4(C4/C2) produces no change from the baseline is surprising,
considering that the claudin-2 chimera with the same order of
extracellular domains, C2(C4/C2), was sufficient to increase the TER
tenfold and decrease the permeability of Na+ relative to
Cl. Similarly, C2(C2/C4) resembles neither wild-type
claudin from which it was constructed. These findings suggest that
interchanging only the second extracellular domain of claudins renders
the chimeras biologically inactive. Thus our observations cannot be
used to interpret the function of the second loop. Evidence for the
contribution of the second extracellular domain may come from
C4(C2/C4), which, with the second extracellular domain of
claudin-4, decreased the dilution potential significantly,
although not to the level to claudin-4, C2(C4/C2), or C2(C4/C4).
Similarly, C4(C2/C4) increased TER more than claudin-2 or C4(C2/C2);
however, this increase in TER was modest compared with the severalfold
increase seen with claudin-4, C2(C4/C2), or C2(C4/C4).
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DISCUSSION |
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It has been known for decades that the paracellular space between epithelial cells behaves like a series of channels with size and charge selectivity (1, 25). The molecular nature of these channels was unknown until recent work implicated the claudins. Here, we demonstrate that the first extracellular domains of claudins are sufficient to determine both the TER and paracellular charge selectivity. Presumably, the first domains from claudins on apposing cells fold to create this channel space. Charged amino acid side chains line the channels and create charge selectivity. Exchanging the second extracellular domain, which is significantly smaller in all claudins, in addition to the first, produced no further change in the electrical properties. There exist >20 claudins, and they show epithelial-specific expression patterns. Our results support the idea that the unique paracellular properties of different epithelia result from their combination of claudins. These findings do not rule out the possibility that other TJ proteins also contribute to paracellular properties.
Although claudins-4 and -2 have significantly different paracellular charge selectivities, analysis of the protein sequences of these claudins reveals an equal number of acidic and basic amino acid residues in their first extracellular domains (Fig. 1). This finding is consistent with our previous study (6), in which we report that some but not all charged residues affect paracellular charge selectivity. For instance, at amino acid 65, which was determined to be critical for charge selectivity of claudins-4 and -15, claudin-4 has a basic (lysine), whereas claudin-2 has an acidic (aspartate), residue. These residues may be positioned to influence charge selectivity with a basic residue in claudin-4 discriminating against cations and an acidic residue in claudin-2 selecting for cations. In contrast, at amino acid 48, which was determined not to influence paracellular charge selectivity in claudin-15, claudin-4 has an acidic (glutamate), whereas claudin-2 has a basic (lysine), residue. The second extracellular domains of claudins-4 and -2 are more divergent, with both having three basic residues and two and four acidic residues, respectively. Further site-directed mutation analysis of both extracellular domains would be necessary to determine whether other positions are critical for determining paracellular charge selectivity.
The chimeras with the first or both extracellular domains of claudin-4 on claudin-2 had more profound effects on paracellular charge selectivity and TER than did claudin-4 itself. Preliminary studies indicate that claudin-4 with the COOH terminus of claudin-2 produces the same profound electrophysiological effects of C2(C4/C4) and C2(C4/C2) when expressed in MDCK II cells (not shown). These findings reveal that the cytoplasmic domains can influence the degree to which the extracellular domains affect paracellular properties. They do not, however, affect the direction of the charge selectivity, only its magnitude. Because claudins-4 and -2 have different patterns of expression detected by immunofluorescence microscopy and freeze-fracture analysis, these claudins may localize to different functional pools within TJs. Biochemical analysis of claudins-1 and -2 demonstrates differential association with detergent-insoluble fractions in T84 cells (20). Claudins have been shown to directly bind to TJ-scaffolding proteins ZO-1, ZO-2, and ZO-3 through their COOH termini (12). The COOH termini of claudins-4 and -2 vary considerably in sequence and length (23 and 44 amino acids, respectively), and interactions mediated by this domain may determine the difference in electrophysiological characteristics between claudin-4 and C2(C4/C4). Studies are currently in progress to determine the role of the cytoplasmic domains in detergent fraction association, localization, and TJ barrier function.
One limitation of this study is that MDCK II cells express an uncharacterized background of endogenous claudins and potentially other unknown TJ proteins. We presume that by overexpressing a single transfected claudin, the direction of the change in charge selectivity reveals an intrinsic property of the transfected claudin. However, the exact magnitude of the change would be unlikely to be the same if it were expressed in a claudin-null background. It will be important to determine whether each claudin confers the same selectivity in cell lines with different baseline selectivities or whether their effects are dependent on the cell line. These studies are in progress using epithelial lines with different baseline charge selectivities and TERs.
Another potential problem in interpreting our results is that expression of some constructs results in secondary changes in the levels of endogenous claudins and occludin. This raises the question of whether our observations could be secondary to changes in the endogenous proteins. Arguing against this, the effects on endogenous proteins are usually undetectable or minor. However, a striking decrease in endogenous claudin-4 is observed upon expression of the claudin-2 chimera with the first extracellular domain of claudin-4. The explanation for the decrease is unclear, although it is more likely the result of a shortened half-life of endogenous claudin-4 protein than a decrease in gene transcription. In any case, the observed increase in TER upon expressing C2(C4/C4) is likely to be a primary effect rather than a result of the secondary decrease in endogenous claudin-4, because reducing claudin-4 is expected to decrease, not increase, resistance.
Interestingly, despite the extracellular domains determining paracellular electrophysiological properties, TJ fibril architecture appeared to be determined by either the transmembrane or intracellular domains. It has previously been demonstrated that expression of claudins and another TJ protein, occludin, can alter fibril number and morphology ( 6, 14, 16, 17, 18, 24). Thus it is likely that TJ fibril architecture among epithelia is a result of the fibril-forming properties of the various expressed claudins.
Each fibril represents a continuous line of cell-cell contact. The parallel rows are assumed to behave as independent resistors in series (4, 5). Thus it has long been assumed that TER is a direct function of the number of fibrils. Expression of claudin-4 does significantly increase both the content of fibrils and TER. In contrast, expression of claudin-2 causes a large increase in fibril number but only a very small increase in TER. This suggests that the barrier is also a function of specific barrier-forming proteins and not simply the number of fibrils.
In 2001, Furuse et al. (9) proposed that claudin-2 decreased TER in high-resistance MDCK I cells through decreased cell-to-cell adhesion with other claudins. Here, we report that expression of claudin-2 in low-resistance MDCK II cells increased the TER by about 20% while maintaining the background paracellular cation selectivity and mannitol flux. On the basis of these studies, we propose a model in which the extracellular domains of claudin-2 produce cation-selective paracellular channels with a high transepithelial conductance rather than an indiscriminate increase in conductance resulting from decreased TJ fibril adhesion.
This study provides the first evidence that the extracellular domains of claudins determine TER. Furthermore, these data extend our previous report that the first extracellular domain can determine paracellular charge selectivity. Finally, our findings suggest that claudin transmembrane or intracellular domains, and not the extracellular domains, determine TJ fibril architecture.
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ACKNOWLEDGEMENTS |
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We thank Emile Boulpaep (Yale University), Lukas Landmann (University of Basel, Switzerland), and current and former members of our laboratory, Alan Fanning, Laura Mitic, Julie Rasmussen, and Zenta Walther.
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FOOTNOTES |
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These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45134, DK-34989, DK-55389, DK-38979, and DK-61283. O. R. Colegio is an investigator of the National Institutes of Health Medical Scientist Training Program.
Address for reprint requests and other correspondence: J. M. Anderson, Dept. of Cell and Molecular Physiology, 266 Medical Sciences Research Bldg., CB# 7545, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545.
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.
10.1152/ajpcell.00547.2002
Received 15 November 2002; accepted in final form 15 January 2003.
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