Mutation of all three types of Ca²⁺ binding sites eliminates the Ca²⁺ dependence of Popen. (A) Inward K⁺ currents recorded for mutant ΔEΔRΔB_(D5N5) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca²⁺] demonstrate that Popen does not increase in a Ca²⁺-dependent manner when voltage is constant. The corresponding all-points amplitude histograms are plotted in B on a semi-log scale and were constructed from 30-s recordings. (C) Dose–response relations for the effect of Ca²⁺ on Popen at negative voltage (−80 mV) obtained by plotting the mean log ratio of NPopen in the presence and absence of Ca²⁺. For both mutant ΔEΔRΔB_(D5N5) (filled circles) and mutant ΔEΔRΔB_(D2A2), log (NPopen/NPopen_min) spans the entire [Ca²⁺] range but cannot be fitted because Popen does not vary with [Ca²⁺]. Each point represents the average of between 6 and 8 patches at each [Ca²⁺] tested. Error bars represent SEM.