A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic–initiated reversal of the small RGK G protein Rad–mediated inhibition of voltage-gated calcium channels (Ca_V) acting through the Ca_vβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca²⁺ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser²⁷² and Ser³⁰⁰ within Rad’s polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser²⁵ and Ser³⁸ in Rad’s N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser²⁷² and Ser³⁰⁰ or the addition of 4 Asp residues to the CTD reduced Rad’s association with the negatively charged, cytoplasmic plasmalemmal surface and with Ca_Vβ, even in the absence of Ca_Vα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad’s C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from Ca_Vβ, is sufficient for sympathetic upregulation of Ca²⁺ currents.