Identification of PK-A Phosphorylation Sites in the Carboxyl Terminus of L-Type Calcium Channel α1 Subunits

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Abstract
Full length L-type calcium channel α1 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 α1 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 α1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel α1 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 α1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of α1S from carp skeletal muscle and α1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of α1D 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 α1D. 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.