The two binding sites are less than additive at 0 mV. The mean log ratio of NPopen at 0 mV in the presence and absence of Ca²⁺ for mutants ΔE (open circles), ΔEΔR (open triangles), and ΔEΔB_(D2A2) (open squares) are plotted versus [Ca²⁺]. Each point represents the average of between 6 and 14 patches at each [Ca²⁺] tested. Various fits of log (NPopen/NPopen_min) are superimposed on the data. The fit of ΔEΔR (short dashed line) with Eq. 6 yielded values of K_O = 0.63 μM and K_C = 2.28 μM. The fit of ΔEΔB_(D2A2) (long dashed curve) yielded values of K_O = 1.56 μM and K_C = 12.7 μM. Using the same equation, we simulated the log (NPopen/NPopen_min) versus Ca²⁺ relation (dark solid line) predicted by the affinities determined for each site in isolation. The parameters of the fit were: K_O1 = 0.63 μM, K_C1 = 2.28 μM, K_O2 = 1.56 μM, and K_C2 = 12.7 μM. Also plotted (gray curve) is a log (NPopen/NPopen_min) versus [Ca²⁺] fit that incorporates cooperativity between binding sites. The equation for the fit was log (NPopen/NPopen_min) = ((1+(K_O1+K_O2)+ K_O1K_O2b)⁴) / ((1+(K_C1+ K_C2)+ K_C1K_C2a)⁴). The parameters of the fit were: K_O1 = 0.63 μM, K_C1 = 2.28 μM, K_O2 = 1.56 μM, K_C2 = 12.7, a = 1, and b = 0.65. Error bars represent SEM.