Dialysis of diC8-PIP₂ from the patch pipette slows and decreases muscarinic suppression of I_Ca. A, B, Voltage protocols are as in Figure 2, but recordings were made in whole-cell configuration. A, Control experiment with regular pipette solution. B, diC8-PIP₂ (200 μm) was added to the pipette solution. The recording started 8-10 min after achieving whole cell to allow for dialysis into the cytoplasm. Oxo (10 μm) and CdCl₂ (Cd²⁺; 100 μm) were applied during the periods indicated by the bars. Insets on the right depict current traces recorded at the times indicated by the arrows. C, Pooled time courses of muscarinic suppression of I_Ca in control (black circles; n = 8) and diC8-PIP₂-dialyzed (white circles; n = 10) cells, temporarily aligned to the time of oxotremorine-M application. Data were fitted by the equation y = y₀ + a/(1 + exp(-(t_1/2 - t)/k)), where y is I/I₀, a is y_max - y_min, t_1/2 is a half-time of inhibition, and k is the slope factor.