Mg ²⁺ binding to open and closed states can activate BK channels provided that the voltage sensors are elevated

BK channels are activated by intracellular Ca ²⁺ and Mg ²⁺ as well as by depolarization. Such activation is possible because each of the four subunits has two high-affinity Ca ²⁺ sites, one low-affinity Mg ²⁺ site, and a voltage sensor. This study further investigates the mechanism of Mg ²⁺ activation by using single-channel recording to determine separately the action of Mg ²⁺ on the open and closed states of the channel. To limit Mg ²⁺ action to the Mg ²⁺ sites, the two high-affinity Ca ²⁺ sites are disabled by mutation. When the voltage is stepped from negative holding potentials to +100 mV, we find that 10 mM Mg ²⁺ decreases the mean closed latency to the first channel opening 2.1-fold, decreases the mean closed interval duration 8.7-fold, increases mean burst duration 10.1-fold, increases the number of openings per burst 4.4-fold, and increases mean open interval duration 2.3-fold. Hence, Mg ²⁺ can bind to closed BK channels, increasing their opening rates, and to open BK channels, decreasing their closing rates. To explore the relationship between Mg ²⁺ action and voltage sensor activation, we record single-channel activity in macropatches containing hundreds of channels. Open probability (P _o) is dramatically increased by 10 mM Mg ²⁺ when voltage sensors are activated with either depolarization or the mutation R210C. The increased P _o arises from large decreases in mean closed interval durations and moderate increases in mean open interval durations. In contrast, 10 mM Mg ²⁺ has no detectable effects on P _o or interval durations when voltage sensors are deactivated with very negative potentials or the mutation R167E. These observations are consistent with a model in which Mg ²⁺ can bind to and alter the gating of both closed and open states to increase P _o, provided that one or more voltage sensors are activated.