Conserved aromatic residue confers cation selectivity in claudin-2 and claudin-10b

In tight junctions, both claudin-2 and claudin-10b form paracellular cation-selective pores by the interaction of the first ECL 1 with permeating ions. We hypothesized that a highly conserved aromatic residue near the pore selectivity filter of claudins contributes to cation selectivity by cation-π interaction with the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr(67) mutants. The Y67L mutant showed reduced cation selectivity compared with wild-type claudin-2 due to a decrease in Na(+) permeability, without affecting the Cl(-) permeability. The Y67A mutant enlarged the pore size and further decreased the charge selectivity due to an increase in Cl(-) permeability. The Y67F mutant restored the Na(+) permeability, Cl(-) permeability, and pore size back to wild-type. The accessibility of Y67C to methanethiosulfonate modification indicated that its side chain faces the lumen of the pore. In claudin-10b, the F66L mutant reduced cation selectivity, and the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue near the cation pore domain of claudins contributes to cation selectivity by a dual role of cation-π interaction and a luminal steric effect. Our findings provide new insight into how ion selectivity is achieved in the paracellular pore.