Appearance of a high-activity gating mode among MthK channels at high pH. (A) Representative trace showing that one out of the three channels in this bilayer was fully active at pH 9.0 with [Ca²⁺] ≤ 0.25 mM Ca²⁺, at which channels are typically closed most of the time. (B) Representative trace showing one out of two channels in a bilayer that showed high activity at pH 8.5 with no added Ca²⁺ ([Ca²⁺] ≈ 10 µM), at which channels are typically closed most of the time. In this example, Po for the two channels had decreased at 0.5 mM Ca²⁺, followed by the appearance of high-activity gating at 0 Ca²⁺. Similar paradoxical high-activity gating was observed in 11 out of 272 active channels from among 23 different bilayers at pH ≥ 7.7.