Mapping the paracellular pore: the role of two highly conserved cysteines in the first extracellular loop of claudins

Abstract

Claudins are a family of tight junction transmembrane proteins whose first extracellular loop (1st ECL) determines paracellular permeability and ion selectivity in epithelia. There are two cysteines in the 1st ECL that are highly conserved among all claudins (C54 and C64 in claudin-2) and whose function is unknown. We hypothesized that they are linked by an intramolecular disulfide bond that is necessary for correct folding, trafficking and pore function. To test this hypothesis, we mutated C54 and C64 in claudin-2 either to alanine or serine, and generated stably transfected MDCK I Tet-off cell lines expressing each mutant protein. Western blotting showed a high molecular weight band in each of the single mutants that was not seen in wild-type claudin-2 or the double mutants, consistent with the formation of claudin dimers. By immunofluorescence staining, the alanine mutants were mislocalized intracellularly while the serine mutants were correctly inserted into the tight junction. In Ussing chamber experiments, the conductance and Na+ permeability of all the serine mutants were markedly lower than wild-type claudin-2. Our findings suggest that the two highly conserved cysteines in the 1st ECL are disulfide bonded and that this disulfide bond is necessary for normal pore formation but not for protein trafficking. Supported by NIH grants R01DK062283 and R01DK062283-07S1.