In class S and C calcium channels, the most C-terminal phosphorylation sites are substrates for PK-A in vitro, whereas class D channels are also phosphorylated at a site likely retained after truncation.
This study identifies specific PK-A phosphorylation sites in the C-terminus of L-type calcium channels, providing mechanistic insight into their regulation.
Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377, the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.
Mitterdorfer et al. (Mon,) reported a other. Site-directed mutagenesis of calcium channel alpha 1 subunits vs. Wild-type calcium channel alpha 1 subunits was evaluated on Phosphorylation by PK-A in vitro. In class S and C calcium channels, the most C-terminal phosphorylation sites are substrates for PK-A in vitro, whereas class D channels are also phosphorylated at a site likely retained after truncation.