IDENTIFICATION OF THE SITES OF SELECTIVE PHOSPHORYLATION AND DEPHOSPHORYLATION OF THE RAT-BRAIN NA-SUBUNIT BY CAMP-DEPENDENT PROTEIN-KINASEAND PHOSPHOPROTEIN PHOSPHATASES( CHANNEL ALPHA)

Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent p rotein kinase (cA-PK) and protein kinase C. Using synthetic peptides a nd protein microsequencing, we have determined that the a subunit of r at brain Na+ channel is selectively phosphorylated by cA-PK in vitro a nd in intact cells on 4 serine residues in the intracellular loop conn ecting homologous domains I and II. Ser-623 was most rapidly and exten sively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was mos t extensively phosphorylated in mammalian cells transfected with the a lpha subunit of type IIA Na+ channel in response to an increase in int racellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylate d Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to l esser extents. Phosphatase 2A selectively dephosphorylated Ser-610. To gether these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phospho rylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.