Full length L-type calcium channel
1 subunits arerapidly phosphorylated by protein kinaseA (PK-A) in vitro and in vivo at sites located in their long carboxylterminal tails. In skeletal muscle,heart, and brain the majority of biochemically isolated
1 subunits lacks these phosphorylation sitesdueto posttranslational proteolytic processing. Truncation maytherefore modify the regulation of channelactivity by PK-A. We combined site-directed mutagenesis andheterologous expression to investigatethe extent to which putative cAMP-dependent phosphorylation sites inthe C-terminus of
1 subunitsfrom skeletal muscle, heart, and brain are phosphorylated in vitro.The full length size form of wild-typeand mutant calcium channel
1 subunits was obtained athigh 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
1 C-terminuswas phosphorylated at serine residues 1757 and1854. In the carboxyl terminus of
1S from carpskeletal muscle and
1C from rabbit heart a singleserineresidue was phosphorylated by PK-A in vitro. The C-terminus of
1D was phosphorylated at more thanone site. Employing deletion mutants, most of the phosphorylation(>70%) was found to occur betweenamino acid residues 1805 and 2072. Serine 1743 was identified asadditional phosphorylation site in
1D. We conclude that in class S and C calciumchannels the most C-terminal phosphorylation sites aresubstrate for PK-A in vitro, whereas in class D calcium channelsphosphorylation also occurs at a sitewhich is likely to be retained even after posttranslationaltruncation.
