Differential modulation of Ca_V2.2 channels by muscarinic receptors depends on the Ca_V β subunit. (A and B) Cells transfected with α1B (Ca_V2.2), α2δ1, and β3 (A) or β2a (B) subunits were cultured in the presence or absence of PTX or heat-inactivated PTX (iPTX) for 12 h. The cells were stimulated with 10 µM Oxo-M to activate muscarinic receptors or depolarized to 120 mV for 1 s to activate the coexpressed Dr-VSP. (A) Ca_V2.2 currents before and after the stimulation of M₁ (left) and M₂ (right) muscarinic receptors or the activation of Dr-VSP were measured in cells expressing β3, and the currents were superimposed. Blue traces are the control, and black traces are after stimulation. Current regulation by M₂ receptor was also measured in cells cotransfected with βARK-ct. (bottom) Summary of current inhibition (percentage) by the activation of muscarinic receptors or Dr-VSP. Dots indicate the individual data points for each experiment (n = 5–15). Analysis was performed by one-way ANOVA followed by a post hoc test. (B) Ca_V2.2 currents were measured before and after the stimulation in cells expressing β2a. (bottom) Summary of current inhibition (percentage) by the activation of M₁ receptor or Dr-VSP. (A and B) Mean ± SEM is shown. *, P < 0.01, compared with current inhibition by Oxo-M. (C) Diagram of inhibitory signaling to Ca_V2.2 channels by M₁ and M₂ muscarinic receptors. VD, voltage-dependent inhibition; VI, voltage-independent inhibition.