Voltage-dependent regulation of Ca_V2.2 channels by G_q-coupled receptor is facilitated by membrane-localized β subunit

G protein-coupled receptors (GPCRs) signal through molecular messengers, such as Gβγ, Ca²⁺, and phosphatidylinositol 4,5-bisphosphate (PIP₂), to modulate N-type voltage-gated Ca²⁺ (Ca_V2.2) channels, playing a crucial role in regulating synaptic transmission. However, the cellular pathways through which G_qPCRs inhibit Ca_V2.2 channel current are not completely understood. Here, we report that the location of Ca_V β subunits is key to determining the voltage dependence of Ca_V2.2 channel modulation by G_qPCRs. Application of the muscarinic agonist oxotremorine-M to tsA-201 cells expressing M₁ receptors, together with Ca_V N-type α1B, α2δ1, and membrane-localized β2a subunits, shifted the currentvoltage relationship for Ca_V2.2 activation 5 mV to the right and slowed current activation. Muscarinic suppression of Ca_V2.2 activity was relieved by strong depolarizing prepulses. Moreover, when the C terminus of β-adrenergic receptor kinase (which binds Gβγ) was coexpressed with N-type channels, inhibition of Ca_V2.2 current after M₁ receptor activation was markedly reduced and delayed, whereas the delay between PIP₂ hydrolysis and inhibition of Ca_V2.2 current was decreased. When the Gβγ-insensitive Ca_V2.2 α1C-1B chimera was expressed, voltage-dependent inhibition of calcium current was virtually abolished, suggesting that M₁ receptors act through Gβγ to inhibit Ca_V2.2 channels bearing membrane-localized Ca_V β2a subunits. Expression of cytosolic β subunits such as β2b and β3, as well as the palmitoylationnegative mutant β2a(C3,4S), reduced the voltage dependence of M₁ muscarinic inhibition of Ca_V2.2 channels, whereas it increased inhibition mediated by PIP₂ depletion. Together, our results indicate that, with membrane-localized Ca_V β subunits, Ca_V2.2 channels are subject to Gβγ-mediated voltage-dependent inhibition, whereas cytosol-localized β subunits confer more effective PIP2-mediated voltage-independent regulation. Thus, the voltage dependence of G_qPCR regulation of calcium channels can be determined by the location of isotype-specific Ca_V β subunits.