Elimination of the BK_Ca Channel's High-Affinity Ca²⁺ Sensitivity

We report here a combination of site-directed mutations that eliminate the high-affinity Ca²⁺ response of the large-conductance Ca²⁺-activated K⁺ channel (BK_Ca), leaving only a low-affinity response blocked by high concentrations of Mg²⁺. Mutations at two sites are required, the “Ca²⁺ bowl,” which has been implicated previously in Ca²⁺ binding, and M513, at the end of the channel's seventh hydrophobic segment. Energetic analyses of mutations at these positions, alone and in combination, argue that the BK_Ca channel contains three types of Ca²⁺ binding sites, one of low affinity that is Mg²⁺ sensitive (as has been suggested previously) and two of higher affinity that have similar binding characteristics and contribute approximately equally to the power of Ca²⁺ to influence channel opening. Estimates of the binding characteristics of the BK_Ca channel's high-affinity Ca²⁺-binding sites are provided.