The Ca²⁺ binding affinities of the high-affinity RCK1 site at −80 mV. (A) Inward K⁺ currents recorded from mutant ΔEΔB_(D2A2) at −80 mV and filtered at 10 kHz from a macropatch in the indicated [Ca²⁺] demonstrate that Popen increases 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. The dose–response relation for the effect of Ca²⁺ on Popen (left axis) and NPopen/NPopen_min (right axis) at negative voltage (−80 mV) is shown in C. Each point represents the average of between 6 and 16 patches at each Ca²⁺ concentration tested. For mutant ΔEΔB_(D2A2) (open squares), log (NPopen/NPopen_min) spans the entire [Ca²⁺] range and is fitted (dotted line) by Eq. 6 yielding values of K_O = 4.9 μM of K_C = 23.2 μM. For mutant ΔEΔB_(D5N5) (closed squares), the fit (dashed line) yields values of K_OB = 5.6 μM and K_CB = 26.8 μM. (D) The data were also fitted with Eq. 7, which incorporates an interaction between binding sites. For mutant ΔEΔB_(D2A2) (open squares), the fit yielded values of K_O = 2.8 μM, K_C = 13.7 μM, and f = 0.45. For mutant ΔEΔB_(D5N5) (closed squares), the fit (dashed line) yielded K_O = 1.8 μM, K_C = 9.4 μM, and f = 0.27. Error bars represent SEM.