Differential phosphorylation of two size forms of the N-type calcium channel α1 subunit which have different COOH termini

Two size forms of the class B N-type calcium channel α1 subunit were recently identified with CNB1, an anti-peptide antibody directed against an intracellular loop of this channel (Westenbroek, R. E., Hell, J. W., Warner, C., Dubel, S. J., Snutch, T. P., and Catterall, W. A. (1992) Neuron 9, 1099- 1115). To investigate the biochemical differences between these two size forms, the antibodies CNB3 and CNB4 were raised against peptides with sequences corresponding to the COOH-terminal end of the full-length form. Immunoblot experiments demonstrated that both antibodies specifically recognize the longer form of 250 kDa, indicating that the COOH-terminal regions of the two size forms of the class B N-type channel α1 subunit are different. Phosphorylation experiments with immunopurified calcium channels and different second messenger-activated protein kinases revealed that both the 220- and 250-kDa forms of the class B N-type calcium channel α1 subunit are substrates for cAMP-dependent protein kinase, cGMP-dependent protein kinase, and protein kinase C. These three kinases incorporated approximately 1 mol of phosphate/mol of binding sites for ω-conotoxin (ω-CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- and calmodulin-dependent protein kinase II (CaM kinase II) phosphorylated only the long form of the class B N- type calcium channel α1 subunit, with a stoichiometry of 0.5 mol of phosphate/mol of total ω-CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B α1 subunit by CaM kinase II may differentially regulate the function of N-type calcium channels containing different size forms of their α1 subunits in vivo.