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Molecular Cardiology Research Institute, New England Medical Center, and the Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111

Address correspondence to Daniel H. Cox, 750 Washington St., NEMC Hospitals, Box 7868, Boston, MA 02111. Fax: 617-636-0576; E-mail: dan.cox{at}tufts.edu

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.

Key Words: mSlo • Slo • potassium channel • Ca²⁺ bowl • Ca²⁺ binding

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