Effects of Multiple Metal Binding Sites on Calcium and Magnesium-dependent Activation of BK Channels

BK channels are activated by physiological concentrations of intracellular Ca²⁺ and Mg²⁺ in a variety of cells. Previous studies have identified two sites important for high-affinity Ca²⁺ sensing between [Ca²⁺]_i of 0.1–100 μM and a site important for Mg²⁺ sensing between [Mg²⁺]_i of 0.1–10 mM. BK channels can be also activated by Ca²⁺ and Mg²⁺ at concentrations >10 mM so that the steady-state conductance and voltage (G-V) relation continuously shifts to more negative voltage ranges when [Mg²⁺]_i increases from 0.1–100 mM. We demonstrate that a novel site is responsible for metal sensing at concentrations ≥10 mM, and all four sites affect channel activation independently. As a result, the contributions of these sites to channel activation are complex, depending on the combination of Ca²⁺ and Mg²⁺ concentrations. Here we examined the effects of each of these sites on Ca²⁺ and Mg²⁺-dependent activation and the data are consistent with the suggestion that these sites are responsible for metal binding. We provide an allosteric model for quantitative estimation of the contributions that each of these putative binding sites makes to channel activation at any [Ca²⁺]_i and [Mg²⁺]_i.