The Ca²⁺ dependence of Popen for the RCK1 site is affected by voltage. However, voltage does not alter the binding at the Ca²⁺ bowl site. (A) The mean log ratio of NPopen/NPopen_min versus [Ca²⁺] for mutant ΔEΔR is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 13 patches at each Ca²⁺ concentration tested. Shown is the fit (dashed curve) of NPopen/NPopen_min based on Eq. 6 and previously shown in Fig. 5. The values determined from the fit are: K_O = 0.88 μM and K_C = 3.13 μM. (B) The mean log Popen versus [Ca²⁺] relation for mutant ΔEΔR at both 0 and −80 mV are well fitted with the HA model using the Ca²⁺ binding constants determined. The values for the parameters were held as follows: K_O = 0.88 μM, K_C = 3.18 μM, L_O = 6.3 × 10⁻⁶, z_L= 0.41 e, V_hc = 151 mV, V_ho = 27 mV, z_J = 0.58 e, and E = 1. (C) The NPopen/NPopen_min versus [Ca²⁺] relation for mutant ΔEΔB_(D2A2) is shown for patches held at 0 mV (open circles) or at −80 mV (solid circles). Each point represents the average of between 6 and 14 patches at each [Ca²⁺] tested. The NPopen/NPopen_min versus Ca²⁺ relations are fitted with Eq. 6. The values of the fit parameters are: ΔEΔB_(D2A2): −80 mV, K_O = 4.9 μM and K_C = 23.2 μM; ΔEΔB_(D2A2): 0 mV, K_O = 2.1 μM and K_C = 15.6 μM. (D) The mean log Popen versus [Ca²⁺] relation for mutant ΔEΔB_(D2A2) at both 0 and −80 mV. First, the 0 mV data were fitted by Eq. 14 to yield the values: K_O = 2.1 μM and K_C = 15.8 μM. The data were also fitted with the HA model. The parameters were held as follows: K_O = 4.9 μM, K_C = 23.2 μM, L_O = 1.2 × 10⁻⁶, z_L = 0.41 e, V_hc = 151 mV, V_ho = 27 mV, z_J = 0.58 e, and E = 6.03.