The effect of mutating F427 to an alanine on the ion selectivity of the (PA₆₃)₇ channel. The experiments were started with the membrane (diphytanoyl-phosphatidylcholine) separating symmetric solutions of 100 mM KCl, 5 mM potassium succinate, and 1 mM EDTA, pH 5.5. After treating the membrane with either valinomycin (0.2 µg/ml), WT (PA₆₃)₇, or (PA₆₃F427A)₇, KCl was added in steps to the cis solution, and for each step the reversal potential (E_rev) was measured. Plotted in the figure is E_rev versus the activity ratio of KCl (a_cis/a_trans). (Activity coefficients were obtained from Appendix 8.10, Table II in Robinson and Stokes [1965], where we took the KCl concentration to be equal to the K⁺ concentration. Approximately 10 mM of K⁺ was contributed by K-succinate and K-EDTA.) We see in the figure that, as expected, the valinomycin-treated membrane (open circles) displayed ideal K⁺ selectivity (continuous line). The selectivity of the WT (PA₆₃)₇–treated membrane (filled circles) deviated somewhat from ideal cation selectivity, and that of the (PA₆₃F427A)₇–treated membrane (filled triangles) deviated even more so; that is, it was more permeable to Cl⁻ than was the WT channel. The dotted lines are drawn to connect the points. (Applying inappropriately, as usual, the Goldman-Hodgkin-Katz equation to the data, we calculate that P_K/P_Cl is 21 and 7.6 for the WT (PA₆₃)₇ and (PA₆₃F427A)₇ channels, respectively.)