Mapping the interaction surface between Ca_Vβ and actin and its role in calcium channel clearance

Defective ion channel turnover and clearance of damaged proteins are associated with aging and neurodegeneration. The L-type Ca_V1.2 voltage-gated calcium channel mediates depolarization-induced calcium signals in heart and brain. Here, we determined the interaction surface between actin and two calcium channel subunits, Ca_Vβ₂ and Ca_Vβ₄, using cross-linking mass spectrometry and protein-protein docking, and uncovered a role in replenishing conduction-defective Ca_V1.2 channels. Computational and in vitro mutagenesis identified hotspots in Ca_Vβ that decreased the affinity for actin but not for Ca_V1.2. When coexpressed with Ca_V1.2, none of the tested actin-association-deficient Ca_Vβ mutants altered the single-channel properties or the total number of channels at the cell surface. However, coexpression with the Ca_Vβ₂ hotspot mutant downregulated current amplitudes, and with a concomitant reduction in the number of functionally available channels, indicating that current inhibition resulted from a build-up of conduction silent channels. Our findings established Ca_Vβ₂-actin interaction as a key player for clearing the plasma membrane of corrupted Ca_V1.2 proteins to ensure the maintenance of a functional pool of channels and proper calcium signal transduction. The Ca_Vβ-actin molecular model introduces a potentially druggable protein-protein interface to intervene Ca_V-mediated signaling processes.