期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 102, 期 14, 页码 5274-5279出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0409885102
关键词
calcium channels; contraction coupling; excitation; protein kinase; proteolysis
资金
- NCRR NIH HHS [RR18522, P41 RR018522] Funding Source: Medline
- NHLBI NIH HHS [P01 HL 44948, P01 HL044948] Funding Source: Medline
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 alpha(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 alpha(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 alpha(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 alpha(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.
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