DIFFERENTIAL PHOSPHORYLATION OF 2 SIZE FORMS OF THE N-TYPE CALCIUM-CHANNEL ALPHA-L SUBUNIT WHICH HAVE DIFFERENT COOH TERMINI

Two size forms of the class B N-type calcium channel alpha 1 subunit w ere recently identified with CNB1, an anti-peptide antibody directed a gainst 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 difference s between these two size forms, the antibodies CNB3 and CNB4 were rais ed 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, ind icating that the COOH-terminal regions of the two size forms of the cl ass B N-type channel alpha 1 subunit are different. Phosphorylation ex periments with immunopurified calcium channels and different second me ssenger activated protein kinases revealed that both the 220- and 250- kDa forms of the class B N-type calcium channel alpha 1 subunit are su bstrates for cAMP-dependent protein kinase, cGMP-dependent protein kin ase, and protein kinase C. These three kinases incorporated approximat ely 1 mol of phosphate/mol of binding sites for omega-conotoxin (omega -CgTx) GVIA, a ligand specific for the N-type calcium channel, and may regulate the activity of both forms in vivo. In contrast, calcium- an d calmodulin dependent protein kinase II (CaM kinase II) phosphorylate d only the long form of the class B N-type calcium channel alpha 1 sub unit, with a stoichiometry of 0.5 mol of phosphate/mol of total omega- CgTx GVIA binding sites. Specific phosphorylation of the long form of the class B alpha 1 subunit by CaM kinase II may differentially regula te the function of N-type calcium channels containing different size f orms of their alpha 1 subunits in vivo.