CLDN15 ^D55E and CLDN15 ^D55N mutants differentially affect pore charge and size selectivity profile. (A) Cation size selectivity was determined by using bi-ionic potential measurements. CLDN15-expressing (red) and nonexpressing (black) monolayers are shown. Cation permeabilities are normalized to chloride permeability to show charge selectivity as a function of cation size, as well as the effects of D55E and D55N mutations. There was no charge-selective barrier in control monolayers that did not express CLDN15, consistent with negligible baseline charge selectivity of the tight junction in parental MDCK I (mean ± SEM, n = 4). (B) CLDN-dependent size selectivity profile for the same traces shown in A was calculated as the difference between cation permeability in monolayers that expressed or did not express CLDN15. Data are shown normalized to Na⁺ and illustrate significant differences in the shape of the curves between WT and mutant CLDN15 constructs (mean ± SEM; CLDN15^WT, n = 4 technical replicates, representative of six separate experiments; CLDN15^D55N, n = 8 technical replicates, representative of two separate experiments; CLDN15^D55E, n = 4 technical replicates, representative of two separate experiments).