Divalent cation sensitivity of BK channel activation supports the existence of three distinct binding sites

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca. The third may mediate physiological regulation by mM Mg. Mutation of five aspartate residues (5D5N) within the so-called Ca bowl removes a portion of a higher affinity Ca dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca dependence. Together, 5D5N and D362A/D367A remove all effects of Ca up through 1 mM while E399A removes a portion of low affinity regulation by Ca/Mg. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca bowl can be activated by Ca and Sr, while regulation defined by D362/D367 can be activated by Ca, Sr, and Cd, Mn , Co, and Ni produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca bowl mainly accelerates activation of BK channels at low [Ca], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10-300 μM Ca. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg or Ca . The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.