Claudins create charge-selective channels in the paracellular pathway between epithelial cells

Epithelia separate tissuespaces by regulating the passage of ions, solutes, and water throughboth the transcellular and paracellular pathways. Paracellularpermeability is defined by intercellular tight junctions, which varywidely among tissues with respect to solute flux, electricalresistance, and ionic charge selectivity. To test the hypothesis thatmembers of the claudin family of tight junction proteins create chargeselectivity, we assessed the effect of reversing the charge of selectedextracellular amino acids in two claudins using site-directedmutagenesis. Claudins were expressed in cultured Madin-Darby caninekidney cell monolayers under an inducible promoter, and clones werecompared with and without induction for transmonolayer electricalresistance and dilution potentials. Expression and localization ofclaudins were determined by immunoblotting, immunofluorescencemicroscopy, and freeze-fracture electron microscopy. We observed thatsubstituting a negative for a positive charge at position 65 in thefirst extracellular domain of claudin-4 increased paracellularNa⁺ permeability. Conversely, substituting positive fornegative charges at three positions in the first extracellular domainof claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na⁺ to Cl.These results support a model where claudins create charge-selective channels in the paracellular space.