Journal Article
Proceedings of the National Academy of Sciences, vol. 102, iss. 14, pp. 5274-5279, 2005
Authors
Joanne T. Hulme, Keiichi Konoki, Teddy W.-C. Lin, Marina A. Gritsenko, David G. Camp, Diana J. Bigelow, William A. Catterall
Abstract
In skeletal muscle cells, voltage-dependent potentiation of Ca
2+
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 α
1
-subunit of Ca
V
1.1 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 α
1
-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 α
1
-subunit of Ca
V
1.1 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 α
1
-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca
V
1.1 channels in skeletal muscle cells.
