Divalent blockade and anomalous mole fraction effect (AMFE) in wild-type NaChBac (LESWAS, shown as red squares) and LEDWAS (green triangles) mutant channels. Full lines are guides to the eye. The bath solution containing Na⁺ and Ca²⁺ cations was adjusted by replacement of Na⁺ with equimolar Ca²⁺; the free Ca²⁺ concentrations [Ca] are shown on the abscissa; and error bars represent the standard error in the mean (SEM). (A) Averaged normalized peak currents of LESWAS and LEDWAS channels. The Langmuir isotherm (9) fitted to the LEDWAS data for [Ca] < 1 mM is shown by the green dashed line. (B) Reversal potentials (E_rev) obtained from the same recordings as in A indicate that LEDWAS stopped conducting Na⁺ if [Ca²⁺] ≥ 1 mM. Dashed lines indicate reference values when extracellular [Ca²⁺] (green) and [Na⁺] (red) are fixed to 100 mM. (C) Cartoon phase diagram Q_f vs. log([Ca]), where the switching lines predicted by equation (6) are dashed-black.