Allosteric regulation of BK channel gating by Ca²⁺ and Mg²⁺ through a nonselective, low affinity divalent cation site

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K⁺ channels is enhanced by cytosolic calcium (Ca²⁺). Activation is sensitive to a range of [Ca²⁺] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 α subunits at [Ca²⁺] and [Mg²⁺] up to 100 mM. The half-activation voltage (V_0.5) is steeply dependent on [Ca²⁺] in the micromolar range, but shows a tendency towards saturation over the range of 60-300 μM Ca²⁺. As [Ca²⁺] is increased to millimolar levels, the V_0.5 is strongly shifted again to more negative potentials. When channels are activated by 300 μM Ca²⁺, further addition of either mM Ca²⁺ or mM Mg²⁺ produces similar negative shifts in steady-state activation. Millimolar Mg²⁺ also produces shifts of similar magnitude in the complete absence of Ca²⁺. The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in ICa²⁺] increase activation rates, addition of 10 mM Mg²⁺ to 0 Ca²⁺ produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg²⁺ does not participate in Ca²⁺-dependent steps that influence current activation rate. We conclude that millimolar Mg²⁺ and Ca²⁺ concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca²⁺-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca²⁺ binding steps, four voltage sensors, and four independent lower affinity Ca²⁺/Mg²⁺ binding steps describes well the behavior of G-V curves over a range of Ca²⁺ and Mg²⁺. The ability of a broad range of [Ca²⁺] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.