Blockade of PIP₂ resynthesis with wortmannin prevents recovery of I_Ca from muscarinic modulation. Neurons were treated overnight with PTX (100 ng/ml). Plotted are the amplitudes of inward Ca²⁺ currents (black circles) evoked by 15 msec depolarizing voltage pulses given every 3 sec from a holding potential of -80 to 5 mV recorded in the perforated-patch configuration of the patch-clamp technique. A, Control experiment in which the neuron was treated with vehicle only (0.1% DMSO). SS (250 nm), DMSO, 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 times indicated by the arrows. B, C, Experiments similar to A, but wortmannin (Wort; 50 μm; B) or LY (50 μm; C) were applied during the periods indicated by the bars. In the cell shown in B, the blockade of recovery by wortmannin was somewhat greater than the mean. D, Bars show mean inhibition by oxotremorine-M and somatostatin as well as recovery from muscarinic inhibition in control (black), wortmannin-treated (gray), and LY294002-treated (white) cells. ^***Significance at the p ≤ 0.001 level; Student's t test. Error bars represent SEM.