Interclaudin Antagonism: A New Mechanism Of Tight Junction Dependent Barrier Regulation

Tight junctions form selectively‐permeable barriers that regulate paracellular flux. Individual members of the claudin protein family have been categorized as having either pore‐forming or sealing functions. For example, claudin‐2 forms paracellular cation‐selective pores. Conversely, overexpression of claudin‐4 can enhance the paracellular barrier, i.e., reduce permeability, in vitro; claudin‐4 has therefore been classified as a sealing claudin. Although specific residues that form paracellular pores, e.g., within claudin‐2, have been defined, the molecular mechanisms by which sealing claudins, e.g., claudin‐4, regulate the barrier have not. The goal of these studies was to determine the means by which the representative sealing protein claudin‐4 reduces paracellular permeability. METHODS Claudin‐4 was knocked out in Madin‐Darby Canine Kidney I (MDCK I) epithelial cells, which do not express claudin‐2 or other pore‐forming claudins, e.g., claudin‐15. These Cldn4‐KO MDCK I cells were stably transfected to inducibly express mCherry‐claudin‐4 with or without constitutive expression of EGFP‐claudin‐2. Transepithelial resistance (TER) and bi‐ionic potential measurements were used to assess barrier function. Protein anchoring and exchange were determined by Fluorescence Recovery After Photobleaching (FRAP). Widefield, confocal, and super‐resolution microscopy were used for live‐cell imaging of fluorescent claudin proteins in MDCK I and undifferentiated, fibroblast‐like cells (U2OS). RESULTS Surprisingly, claudin‐4 knockout had no effect on the paracellular barrier. Inducible mCherry‐claudin‐4 expression was also insufficient to modify TER. In contrast, EGFP‐claudin‐2 expression reduced TER 10‐fold (p<0.0001). Induction of mCherry‐claudin‐4 expression in EGFP‐claudin‐2‐expressing, but not claudin‐2‐deficient, MDCK I increased TER by 92±18% (p<0.001). FRAP analysis showed that mCherry‐claudin‐4 doubled the mobile fraction of tight junction‐associated EGFP‐claudin‐2 from 16±3% to 32±4% (p<0.001). In U2OS cells, mCherry‐claudin‐4 alone was incapable of forming tight junction‐like strands whereas EGFP‐claudin‐2 assembled into polymeric strands. Induction of mCherry‐claudin‐4 expression disrupted strands formed by EGFP‐claudin‐2, which was subsequently internalized and trafficked to LAMP2‐positive late endosomes. Although MitMAB (dynamin inhibitor) prevented EGFP‐claudin‐2 removal from the junction after mCherry‐claudin‐4 induction, monolayer TER was still increased by mCherry‐claudin‐4 expression. CONCLUSIONS These data refute the widely‐held view that claudin‐4 forms polymers that seal the paracellular pathway by demonstrating that i) neither knockout nor overexpression of claudin‐4 affect barrier function; and ii) claudin‐4 does not assemble into tight junction‐like strands (polymers). Instead, the results support an alternative model where claudin‐4 enhances barrier function by destabilizing and inhibiting flux via claudin‐2 pores. This challenges existing dogma regarding claudin function and reveals interclaudin antagonism as an unsuspected mechanism of barrier regulation. Support or Funding Information NIH 5R01DK06 (JRT), CCF RFA 622459 (NS)