Epithelial tight junctions contain size- and charge-selective pores that control the paracellular movement of charged and noncharged solutes. Claudins influence the charge selectivity and electrical resistance of junctions, but there is no direct evidence describing pore composition or whether pore size or density differs among cell types. To characterize paracellular pores independent of influences from charge selectivity, we profiled the `apparent permeabilities' (Papp) of a continuous series of noncharged polyethylene glycols (PEGs) across monolayers of five different epithelial cell lines and porcine ileum. We also characterized Papp of high and low electrical resistance MDCK cell monolayers expressing heterologous claudins. Papp profiling confirms that the paracellular barrier to noncharged solutes can be modeled as two distinct pathways: high-capacity small pores and a size-independent pathway allowing flux of larger solutes. All cell lines and ileum share a pore aperture of radius 4 Å. Using Papp of a PEG of radius 3.5 Å to report the relative pore number provides the novel insight that pore density along the junction varies among cell types and is not necessarily related to electrical resistance. Expression of claudin-2 results in a selective increase in pore number but not size and has no effect on the permeability of PEGs that are larger than the pores; however, neither knockdown of claudin-2 nor overexpression of several other claudins altered either the number of small pores or their size. We speculate that permeability of all small solutes is proportional to pore number but that small electrolytes are subject to further selectivity by the profile of claudins expressed, explaining the dissociation between the Papp for noncharged solutes and electrical resistance. Although claudins are likely to be components of the small pores, other factors might regulate pore number.

Members of the large claudin family of tight junction (TJ) proteins create the differences in paracellular conductance and charge selectivity observed among different epithelia. Previous studies demonstrated that ionic charge selectivity is influenced by acidic or basic amino acids on the first extracellular domain of claudins. We noted two alternatively spliced variants of claudin-10 in the database, 10a and 10b, which are predicted to encode two different first extracellular domains and asked whether this might be a novel mechanism to generate two different permselectivities from a single gene. Using quantitative PCR, we found that claudin-10b is widely expressed among tissues including the kidney; however, claudin-10a is unique to the kidney. Using a nondiscriminating antibody, we found that claudin-10 (a plus b) is expressed in most segments of the nephron. In situ hybridization, however, showed that mRNA for 10a is concentrated in the cortex, and mRNA for 10b is more highly expressed in the medulla. Expression in Madin-Darby canine kidney (MDCK) II and LLC-PK1 cells reveals that both variants form low-resistance pores, and that claudin-10b is more selective for cations than claudin-10a. Charge-reversing mutations of cationic residues on 10a reveal positions that contribute to its anion selectivity. We conclude that alternative splicing of claudin-10 generates unique permselectivities and might contribute to the variable paracellular transport observed along the nephron.

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SummaryThe function of occludin remains elusive. Proposed roles include maintenance of tight junction barriers, signaling and junction remodeling. To investigate a potential role in mediating cytokine-induced changes in barrier properties, we measured barrier responses to interferon- plus TNF in control, occludin-overexpressing and occludin knockdown MDCK II monolayers. MDCK cells show a complex response to cytokines characterized by a simultaneous increase in the transepithelial electrical resistance and a decrease in the barrier for large solutes. We observed that overexpression of occludin increased and occludin knockdown decreased sensitivity to cytokines as assessed by both these parameters. It is known that caveolin-1 interacts with occludin and is implicated in several models of cytokine-dependent barrier disruption; we found that occludin knockdown altered the subcellular distribution of caveolin-1 and that partitioning of caveolin into detergent-insoluble lipid rafts was influenced by changing occludin levels. Knockdown of caveolin decreased the cytokine-induced flux increase, whereas the increase in the electrical barrier was unaltered; the effect of double knockdown of occludin and caveolin was similar to that of occludin single knockdown, consistent with the possibility that they function in the same pathway. These results demonstrate that occludin is required for cells to transduce cytokine-mediated signals that either increase the electrical barrier or decrease the large solute barrier, possibly by coordinating the functions of caveolin-1.

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