Claudin extracellular domains determine paracellular charge selectivity and resistance but not tight junction fibril architecture

Oscar R. Colegio1, Christina Van Itallie2, Christoph Rahner3, and James Melvin Anderson4

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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INTRODUCTION
MATERIALS AND METHODS
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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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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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Fig. 1.   Construction of claudin extracellular loop chimeras. A: amino acid sequence alignment of the extracellular domains of claudins-4 and -2. Acidic (red) and basic (blue) residues are noted. B: schematic of predicted membrane topology of claudins-4 and -2 and the following chimeras: C4(C2/C4), C4(C4/C2), C4(C2/C2), C2(C4/C2), C2(C2/C4), and C2(C4/C4). The extracellular domains of claudins were interchanged by creating unique restriction sites in the DNA encoding sites flanking each domain. The extracellular domain was cut, and the amplified exogenous extracellular domain was ligated in its place. Arrowheads indicate antibody epitopes for claudins-4 and -2 used in detection by immunoblot analysis (Fig. 2) and immunofluorescence microscopy (Fig. 3).

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-beta -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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MATERIALS AND METHODS
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 beta -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 beta -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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Fig. 2.   Wild-type claudins (CLDN)-4 and -2 and claudin chimeras are inducible in Madin-Darby canine kidney (MDCK) II Tet-Off epithelial cells. A: expression of claudin-4 and chimeras with the COOH terminus of claudin-4 is inducible. Stable MDCK II Tet-Off cell lines transfected with claudin-4 and chimeras with the COOH terminus of claudin-4 were grown on Snapwell filters for 4 days and induced or noninduced for transgene expression. Total cell lysates were immunoblotted for claudin-4, claudin-2, claudin-1, occludin, ZO-1, and beta -tubulin. Endogenous claudin-4 in the noninduced cells is not visible at this exposure. B: expression of claudin-2 and chimeras with the COOH terminus of claudin-2 is inducible. Cells and lysates were prepared as in A. Levels of claudin-4 and occludin decrease upon expression of the transgene. Although only 1 cell line is shown for each construct, these results are representative of all cell lines tested. Exposure time for beta -tubulin blots of lysates from cells noninduced and induced for wild-type claudins-4 and -2 was greater than for chimeras. Antibodies used to detect claudins were directed against epitopes in the COOH termini (Fig. 1). Thus, for example, the anti-claudin-4 antibody detects wild-type claudin-4 and chimeras containing the COOH terminus of claudin-4.

To determine whether the expressed claudins and chimeras localized to the cell borders, immunofluorescence confocal microscopy was performed on MDCK II cells, induced or noninduced as above (Fig. 3). Significant increases in staining at cell borders were observed in all clones induced to express wild-type and chimeric claudins. The focused TJ location of ZO-1 was unaffected by expression of claudins-4 and -2 and chimeras. Claudin-2 and chimeras with the transmembrane and cytoplasmic domains of claudin-2 also localized to (ZO-1-positive) intracellular, vacuole-like structures (Fig. 3B). These structures were previously reported to form upon expression of claudin-1 (14). It is unlikely that the formation of vesicles upon expression of claudin-2 disrupts the TJ. Structurally, the TJ remains intact, as assessed by the continuous immunostaining for claudin-2 and other TJ proteins along the TJ and the continuous and more complex freeze-fracture fibril pattern along the TJ. Functionally, integrity is maintained at a molecular level because the charge selectivity differs from that predicted by the mobilities of Na+ and Cl- ions in free solution.


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Fig. 3.   Exogenous claudins and claudin chimeras localize to the cell borders of MDCK II Tet-Off cells. Stable clones of MDCK II Tet-Off cells were grown as in Fig. 2A, with or without induction for claudin or chimera expression and labeled for claudin-4 (A) or claudin-2 (B) and ZO-1 (A and B), a marker for the tight junction (TJ). A: the extracellular domains of claudin-4 do not determine the pattern of localization. Endogenous claudin-4 and ZO-1 localize to the cell borders in noninduced MDCK II cells. Upon induction of claudin-4, an increase in the level of claudin-4 at the cell borders, as well as intracellularly, is observed while ZO-1 localization remains unchanged. Induction of C4(C2/C2) results in an increase in claudin-4 in a level and in a pattern indistinguishable from exogenously expressed wild-type claudin-4. B: the extracellular domains of claudin-2 do not determine the pattern of localization. Endogenous claudin-2 and ZO-1 localize to cell borders in noninduced MDCK II cells. Induction of wild-type claudin-2 results in an increase in staining of claudin-2 at the cell borders, as well as vacuole-like structures. ZO-1 localizes to the cell borders and some of the claudin-2-positive intracellular structures. Expression of C2(C4/C4) results in an increase in claudin-2 at cell borders and vacuole-like structures at a level and in a pattern indistinguishable from exogenously expressed wild-type claudin-2. ZO-1 localizes to cell borders, as well as these claudin-2-positive intracellular structures. Bar = 5 µm.

To avoid potential artifacts associated with the use of epitope tags, we chose not to tag our chimeras (14, 16), but it should be noted that the antibodies used to detect claudins were directed against epitopes in the COOH termini (Fig. 1). Thus, for example, the anti-claudin-4 antibody detects wild-type claudin-4 and chimeras containing the COOH terminus of claudin-4 (Fig. 3A). This explains background staining of endogenous claudin-4 detected before induction of the chimeras (Figs. 3A).

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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Fig. 4.   The extracellular domains of claudins do not determine TJ fibril architecture. Freeze-fracture replica electron micrographs of noninduced MDCK II Tet-Off cells (A, C, E, and G) and stable lines induced for the expression of claudin-4 (B), claudin-2 (D), C4(C2/C2) (F), and C2(C4/C4) (H). Cells were prepared as previously reported (18). MDCK II cells expressing claudin-4 and C4(C2/C2) (B and F) show reticular and parallel fibrils densely organized on the lateral cell borders. In contrast, MDCK II cells expressing claudin-2 and C2(C4/C4) (D and H) show more delicate, curved, and diffuse TJ fibrils. Bar = 250 nm.

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 Omega  · cm2, and expression of claudin-2 decreased the TER to 150-500 Omega  · cm2. MDCK II cells used in our studies have a baseline TER of only 35 Omega  · 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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Fig. 5.   The first extracellular domains of claudins-4 and -2 are sufficient to determine transmonolayer electrical resistance. Stable clones of MDCK II Tet-Off cells were plated and grown with (filled bars) and without (open bars) induction for claudins or chimeras as in Fig 2A. Expression of exogenous claudin-4 and chimeras with the first or both extracellular domains of claudin-4 increases the TER at least threefold. Claudin-2 and chimeras with the first or both extracellular domains of claudin-2 increase the transepithelial electrical resistance (TER) no more than 40%. The TER before and after induction of wild-type claudins-4 and -2, and chimeras were as follows: claudin-4, 40.9 ± 3.2 to 114.0 ± 16.7 Omega  · cm2; C2(C4/C2), 37.4 ± 2.2 to 390.2 ± 81.7 Omega  · cm2; C2(C4/C4), 41.0 ± 2.4 to 295.2 ± 33.0 Omega  · cm2; claudin-2, 33.3 ± 1.2 to 41.1 ± 3.3 Omega  · cm2; C4(C2/C4), 37.9 ± 1.4 to 61.7 ± 5.4 Omega  · cm2; C4(C2/C2), 34.6 ± 1.1 to 46.8 ± 2.5 Omega  · cm2. Data represent means ± SE of determinations from duplicate Snapwells of 4-9 clones for wild-type claudins and chimeras. *P < 0.05. Significance was calculated using Student's t-test.

To determine paracellular charge selectivity, we used a modified Ussing chamber and measured the potential generated from a 1:2 (apical/basal) concentration gradient of NaCl across the induced or noninduced MDCK II monolayers. MDCK II cells have paracellular pathways more permeable to Na+ relative to Cl- (3, 23) that are measured as a positive potential with our electrode orientation. Within this background, claudin-4 decreased the dilution potential upon induction from 8.3 ± 0.4 mV to 4.2 ± 0.9 mV, whereas claudin-2 expression did not change the MDCK II background preference for cations (Fig. 6). To test whether claudin-2 would produce cation-selective paracellular pathways in an anion-selective background, we expressed claudin-2 in anion-selective LLC-PK1 cells (22) and found that the selectivity did reverse (unpublished observations). Potentials of equal magnitude but opposite charge were generated upon dilution of the basal chamber (data not shown), confirming that the charge selectivity is paracellular.


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Fig. 6.   The first extracellular domains of claudins-4 and -2 are sufficient to determine paracellular charge selectivity. Dilution potentials were compared between monolayers that were noninduced (open bars) and induced (filled bars) for the transgene as in Fig 2A. The dilution potentials before and after induction of wild-type claudins-4 and -2 and chimeras were as follows: claudin-4, 8.3 ± 0.4 to 4.15 ± 0.9 mV; C2(C4/C2), 10.4 ± 0.3 to 3.3 ± 0.8 mV; C2(C4/C4), 8.1 ± 0.6 to 1.6 ± 0.3 mV; claudin-2, 8.3 ± 0.6 to 8.3 ± 0.6 mV, C4(C2/C4), 9.8 ± 0.4 to 7.4 ± 0.6 mV; C4(C2/C2), 8.6 ± 0.4 to 7.3 ± 0.4 mV. Data represent means ± SE of determinations from duplicate Snapwells of 4-9 clones for wild-type claudins and chimeras. *P < 0.05. Significance was calculated using Student's t-test.

Because expression of claudins-4 and -2 might also differently affect the flux of noncharged solutes, we measured their effect on the passive diffusion of [3H]-mannitol. Despite their very different effects on TER and paracellular charge selectivity, no major change in flux from baseline was observed upon induction of either claudin-4 or -2 in MDCK II cells (data not shown). These data are consistent with our previous reports of no significant change in noncharged solute flux upon expression of either claudin-4 or -15, despite profound effects on TER and charge selectivity (6, 24).

Claudins-4 and -2 have clearly different effects on TER and paracellular charge selectivity when expressed in type II MDCK cells. Claudin-4 creates a higher resistance paracellular pathway with a lower permeability for cations than does claudin-2. To test whether these differences are determined by the two extracellular domains of these claudins and to determine the relative contributions of the first and second extracellular domains, we measured the paracellular charge selectivity and TER upon expression of extracellular domain chimeras of claudins-4 and -2 in MDCK II Tet-Off cells.

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barry, PH, Diamond JM, and Wright EM. The mechanism of cation permeation in rabbit gallbladder. Dilution potentials and bionic potentials. J Membr Biol 4: 358-394, 1971[ISI].

2.   Bujard, H. Controlling genes with tetracyclines. J Gene Med 1: 372-374, 1999[ISI][Medline].

3.   Cereijido, M, Meza I, and Martinez-Palomo A. Occluding junctions in cultured epithelial monolayers. Am J Physiol Cell Physiol 240: C96-C102, 1981[Abstract].

4.   Claude, P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol 39: 219-232, 1978[ISI][Medline].

5.   Claude, P, and Goodenough DA. Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia. J Cell Biol 58: 390-400, 1973[Abstract/Free Full Text].

6.   Colegio, OR, Van Itallie CM, McCrea HJ, Rahner C, and Anderson JM. Claudins create charge-selective channels in the paracellular pathway between epithelial cells. Am J Physiol Cell Physiol 283: C142-C147, 2002[Abstract/Free Full Text].

7.   Enck, AH, Berger UV, and Yu AS. Claudin-2 is selectively expressed in proximal nephron in mouse kidney. Am J Physiol Renal Physiol 281: F966-F974, 2001[Abstract/Free Full Text].

8.   Furuse, M, Fujita K, Hiiragi T, Fujimoto K, and Tsukita S. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141: 1539-1550, 1998[Abstract/Free Full Text].

9.   Furuse, M, Sasaki H, Fujimoto K, and Tsukita S. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol 143: 391-401, 1998[Abstract/Free Full Text].

10.   Furuse, M, Furuse K, Sasaki H, and Tsukita S. Conversion of zonulae occludentes from tight to leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells. J Cell Biol 153: 263-272, 2001[Abstract/Free Full Text].

11.   Inai, T, Kobayashi J, and Shibata Y. Claudin-1 contributes to the epithelial barrier function in MDCK cells. Eur J Cell Biol 78: 849-855, 1999[ISI][Medline].

12.   Itoh, M, Furuse M, Morita K, Kubota K, Saitou M, and Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol 147: 1351-1363, 1999[Abstract/Free Full Text].

13.   Kiuchi-Saishin, Y, Gotoh S, Furuse M, Takasuga A, Tano Y, and Tsukita S. Differential expression patterns of claudins, tight junction membrane proteins, in mouse nephron segments. J Am Soc Nephrol 13: 875-886, 2002[Abstract/Free Full Text].

14.   Kobayashi, J, Inai T, and Shibata Y. Formation of tight junction strands by expression of claudin-1 mutants in their ZO-1 binding site in MDCK cells. Histochem Cell Biol 117: 29-39, 2002[ISI][Medline].

15.   MacInnes, DA. The Principles of Electrochemistry. New York: Dover, 1961.

16.   McCarthy, KM, Skare IB, Stankewich MC, Furuse M, Tsukita S, Rogers RA, Lynch RD, and Schneeberger EE. Occludin is a functional component of the tight junction. J Cell Sci 109: 2287-2298, 1996[Abstract/Free Full Text].

17.   McCarthy, KM, Francis SA, McCormack JM, Lai J, Rogers RA, Skare IB, Lynch RD, and Schneeberger EE. Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells. J Cell Sci 113: 3387-3398, 2000[Abstract/Free Full Text].

18.   Medina, R, Rahner C, Mitic LL, Anderson JM, and Van Itallie CM. Occludin localization at the tight junction requires the second extracellular loop. J Membr Biol 178: 235-247, 2000[ISI][Medline].

19.   Morita, K, Furuse M, Fujimoto K, and Tsukita S. Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands. Proc Natl Acad Sci USA 96: 511-516, 1999[Abstract/Free Full Text].

20.   Nishiyama, R, Sakaguchi T, Kinugasa T, Gu X, MacDermott RP, Podolsky DK, and Reinecker HC. Interleukin-2 receptor beta  subunit-dependent and -independent regulation of intestinal epithelial tight junctions. J Biol Chem 276: 35571-35580, 2001[Abstract/Free Full Text].

21.   Powell, DW. Barrier function of epithelia. Am J Physiol Gastrointest Liver Physiol 241: G275-G288, 1981[Abstract/Free Full Text].

22.   Rabito, CA, Tchao R, Valentich J, and Leighton J. Distribution and characteristics of the occluding junctions in a monolayer of a cell line (MDCK) derived from canine kidney. J Membr Biol 43: 351-365, 1978[ISI][Medline].

23.   Rabito, CA. Occluding junctions in a renal cell line (LLC-PK1) with characteristics of proximal tubular cells. Am J Physiol Renal Fluid Electrolyte Physiol 250: F734-F743, 1986[Abstract/Free Full Text].

24.   Van Itallie, C, Rahner C, and Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 107: 1319-1327, 2001[Abstract/Free Full Text].

25.   Wright, EM, and Diamond JM. Effects of pH and polyvalent cations on the selective permeability of gall-bladder epithelium to monovalent ions. Biochim Biophys Acta 163: 57-74, 1968[ISI][Medline].


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