Manipulations of divalent cation concentrations alter the direction of the PIP₂ effect in Slo1. (A) G-V curves before (blue) and after PIP₂ addition (red), and the subsequent addition of 10 mM Mg²⁺ (black), from a representative patch expressing divalent cation–insensitive Slo1 D362A:D367A:E399A:Δ894–895 channels. (B) Changes in V_0.5 of Slo1 D362A:D367A:E399A:Δ894–895 by PIP₂ and Mg²⁺. (C) Representative currents (left) and G-V curves from five patches (right) of wild-type Slo1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 2 mM Mg²⁺. (D) Representative currents (left) and G-V curves from five patches (right) containing Slo1 D362A:D367A:E399A:Δ894–895 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 100 µM Ca²⁺. (E) Representative currents (left) and G-V curves from seven patches (right) containing wild-type Slo1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 100 µM Ca²⁺. (F and G) Ca²⁺ dependence of V_0.5 before (blue) and after (red) the application of 10 µM PIP₂ (F) and that of ΔV_0.5 (G) by 10 µM PIP₂. (H) Representative currents from Slo1 D362A:D367A:E399A:Δ894–895 + β1 before (blue) and after (red) the application of 10 µM PIP₂ in the presence of 10 mM Mg²⁺. (I) Changes in V_0.5 of Slo1 D362A:D367A:E399A:Δ894–895 + β1 by PIP₂ and Mg²⁺; n = 8. (J) Representative currents (left) and G-V curves from seven patches (right) of wild-type Slo1 + β1 before (blue) and after (red) the application of 10 µM PIP₂ with 100 µM Ca²⁺ inside. (K) Representative currents (left) and G-V curves from six patches (right) containing wild-type Slo1 recorded in the outside-out configuration before (blue) and after (red) the application of 10 µM PIP₂ to the extracellular side without any added Mg²⁺ or Ca²⁺. The V_0.5 values before and after the application of PIP₂ were 151.7 ± 1.7 mV and 137.2 ± 1.3 mV (P = 0.0021; n = 6). Error bars represent mean ± SEM.