Ation selectivity by cation- interaction and restricting the pore diameter. Significance: This advances our understanding from the paracellular ion selectivity mechanism. In tight junctions, both claudin-2 and claudin-10b type paracellular cation-selective pores by the interaction from the initial ECL 1 with permeating ions. We hypothesized that a extremely conserved aromatic residue close to the pore selectivity filter of claudins contributes to cation selectivity by cation- interaction with all the permeating cation. To test this, we generated MDCK I Tet-off cells stably transfected with claudin-2 Tyr67 mutants. The Y67L mutant showed lowered cation selectivity compared with wild-type claudin-2 as a consequence of a lower in Na permeability, devoid of affecting the Cl permeability. The Y67A mutant enlarged the pore size and additional decreased the charge selectivity as a consequence of a rise 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 in the pore. In claudin-10b, the F66L mutant reduced cation selectivity, along with the F66A mutant lost pore conductance. We conclude that the conserved aromatic residue close to the cation pore domain of claudins contributes to cation selectivity by a dual role of cation- interaction as well as a luminal steric effect. Our findings give new insight into how ion selectivity is accomplished in the paracellular pore.Epithelial cells are connected via various junctional complexes. The tight junction separates the apical and basolateral membrane domains and acts as the paracellular barrier, when remaining selectively permeable to ions and water. In tight junctions, the first ECL1 of claudin types the paracellular pore or barrier (1). Both claudin-2 and claudin-10b can form paracellular cation pores with PNa /PCl of six to eight (two?four). The pore diameter of claudin-2 is estimated to be six.Fmoc-D-Isoleucine Chemscene 5?.478693-99-1 Purity 5 ?(two, 5). The primary determinant of claudin-2 ion charge selectivity is an aspartate residue in ECL1 (Asp65) (2, six). When all 3 negatively charged residues inside the claudin-2 ECL 1, including Asp65, had been mutated to neutral amino acids, the pore became much less cat-* Thiswork was supported by National Institutes of Well being Grants R01DK062283 and U01GM094627 (to A. S. L. Y.). 1 To whom correspondence should be addressed: The Kidney Inst., University of Kansas Health-related Ctr., 3901 Rainbow Blvd., Mail Stop 3018, Kansas City, KS 66160.PMID:29844565 Tel.: 913-588-9252; Fax: 913-588-9251; E-mail: [email protected]. Even so, it remained four instances much more selective to Na than to Cl (2). This observation led us to postulate that other mechanisms could also play a part in cation selectivity like cation interaction with polar residues (e.g. carbonyl oxygen, as will be the case in the KcsA potassium channel (7)), or cationinteractions. The latter possibility prompted us to look for a conserved aromatic residue near Asp65 and Ile66, where the cation-selective filter is located (2, eight). We located position 67 of claudin-2 and position 66 of claudin-10b to possess an aromatic residue which is hugely conserved in all of the classic claudins (tyrosine in claudin-2 and phenylalanine in claudin-10b). The target of this study was to assess the part of this aromatic residue in cation pore-forming claudins. We hypothesized that Tyr67 (claudin-2) and Phe66 (claudin10b) might interact with permeating cations by way of cationinteraction. Cation- interact.