Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of Ca_V 1.1 channels in skeletal muscle

In skeletal muscle cells, voltage-dependent potentiation of Ca²⁺ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the α₁-subunit of Ca_V1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the α₁-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556-1612. Analysis of the purified α₁-subunit of Ca_V1-1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the α₁-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca_V1-1 channels in skeletal muscle cells.