PIP₂ readily activates myocyte BK channels when accessing the channel from the inner membrane leaflet. (A) Unitary currents obtained before (top), after a 5-min bath application of 10 μM PIP₂ (diC16) (middle), and after washout for >30 min (bottom) of I/O patches. Arrowheads, baseline; upward deflections, channel openings; n = 5. (B) Steady-state activity (NP_o) time course from two I/O patches. An arrow highlights the time at which one of the patches (•) was switched from control to PIP₂-containing solution. The other patch was continuously exposed to control (○). (C) Kinetics of reversibility of diC4, diC8, and PIP₂ action. Decay constant values were obtained by single exponential fittings of NP_o vs. time plots. (a) diC4 vs. diC8: P < 0.001; (b) diC4 vs. diC8: P < 0.001; (c) diC8 vs. PIP₂: P < 0.001, n = 3–4. In A and B, V = 40 mV, Ca²⁺_i= 0.3 μM; n = 3. (D) NP_o responses to 10 μM PIP₂ from I/O, O/O, and C/A patches. Each point = one patch/myocyte. Dotted line, control; V = 10–60 mV; Ca²⁺_i = 0.3 μM; n = 4–6.