Simultaneous measurement of current inhibition and PIP₂ hydrolysis in single cells. All cells coexpressed Ca_V2.2(β2a) channel subunits, PH domain probes, and M₁ receptors. (A) Ca_V2.2(β2a) current and PIP₂ (FRET ratio, FRETr) were measured simultaneously in single cells in the absence (top) or presence (bottom) of βARK-ct. 10 µM Oxo-M was applied during solid bars. (right) Scaled responses from the dashed boxes shown in the left panels. The initiation of the muscarinic response is indicated by arrows. (B) Normalized mean time courses of current suppression and PIP₂ hydrolysis (FRETr) from single cells without or with βARK-ct expression. (C) Summary of maximum current inhibition by Oxo-M in control and cells expressing βARK-ct. **, P < 0.01, compared with control. (D) Effect of βARK-ct on lag time between the initiation of current inhibition and the FRETr change. ***, P < 0.001. (B–D) Data are mean ± SEM. (E) Analysis of the onset time (τ) for current inhibition and PIP₂ hydrolysis (FRETr) in control and βARK-ct–expressing cells. Data are mean ± SEM (n = 6–7). **, P < 0.01.