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

Area of Science:

• Cardiovascular Physiology
• Molecular Cell Biology
• G Protein Signaling

Background:

• The adrenergic surge is crucial for the fight-or-flight response, increasing cardiac output via enhanced contractility and heart rate.
• This response involves β-adrenergic signaling that reverses Rad G protein inhibition of voltage-gated calcium channels (CaV) through the Cavβ subunit.

Purpose of the Study:

• To investigate the mechanism by which Rad protein couples phosphorylation to increased calcium (Ca2+) influx and cardiac contraction.
• To elucidate the role of specific phosphorylation sites and membrane localization in Rad-mediated regulation of CaV channels.

Main Methods:

• Site-directed mutagenesis to alter phosphorylation sites on the Rad protein.
• Förster Resonance Energy Transfer (FRET) to measure protein-protein interactions and membrane association.
• Biochemical assays to assess Rad protein function and localization.

Main Results:

• Phosphorylation of Ser272 and Ser300 in Rad's C-terminal domain (CTD) was required for reversing Rad-mediated inhibition.
• Phosphorylation or aspartate substitution at these sites reduced Rad's association with the sarcolemma and Cavβ.
• Constitutive membrane tethering of Rad via a prenylated CAAX motif blocked the effects of phosphorylation and Asp substitution.

Conclusions:

• Dissociation of Rad protein from the sarcolemma is sufficient to mediate sympathetic upregulation of Ca2+ currents.
• Phosphorylation-dependent changes in Rad's interaction with the membrane and Cavβ are key to regulating cardiac contractility.