PNNL

Abstract

In skeletal muscle cells, voltage-dependent potentiation of Ca2+ channel activity requires phosphorylation by cyclic AMP-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 ?1 subunit of CaV1.1 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 interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian non-muscle cells. Deletion mapping of the C-terminus of the ?1 subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802 to 1841 specifically interacts with a region in the proximal C-terminus containing amino acid residues 1556 to 1612. Analysis of the purified ?1 subunit of CaV1.1 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 ?1 subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of CaV1.1 channels in skeletal muscle cells.