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Depolarization of rat brain synaptosomes causes an increase in phosphorylation of serine residues 573, 610, 623, and 687 on voltage-sensitive sodium channels. Although these sites have been shown to be phosphorylated by cAMP-dependent protein kinase in vitro and in situ, the depolarization-induced increase in their state of phosphorylation is not due to increased cAMP-dependent protein kinase activity, but requires calcium influx and protein kinase C. Since phosphorylation at this cluster of sites inhibits sodium current and would decrease neuronal excitability, this may be an important negative feedback mechanism whereby calcium influx during prolonged or repetitive depolarization can attenuate neuronal excitability and prevent further calcium accumulation. Phosphorylation of purified channels by protein kinase C decreases dephosphorylation of cAMP-dependent phosphorylation sites by purified calcineurin or protein phosphatase 2A. This suggests that one mechanism by which protein kinase C may increase phosphorylation of cAMP-dependent phosphorylation sites in sodium channels is to inhibit their dephosphorylation. This represents an important new mechanism for convergent regulation of an ion channel by two distinct signal transduction pathways.

Rat brain sodium channels are phosphorylated at multiple serine residues by cAMP-dependent protein kinase. We have identified soluble rat brain phosphatases that dephosphorylate purified sodium channels. Five separable forms of sodium channel phosphatase activity were observed. Three forms (two, approximately 234 kDa and one, 192 kDa) are identical or related to phosphatase 2A, since they were 85-100% inhibited by 10 nM okadaic acid and contained a 36-kDa polypeptide recognized by a monoclonal antibody directed against the catalytic subunit of phosphatase 2A. Immunoblots performed using antibodies specific for isoforms of the B subunit of phosphatase 2A indicate that the two major peaks of phosphatase 2A-like activity, A1 and B1, are enriched in either B' or B alpha. The remaining two activities (approximately 100 kDa each) probably represent calcineurin. Each was relatively insensitive to okadaic acid, was active only in the presence of CaCl2 and calmodulin, and contained a 19-kDa polypeptide recognized by a monoclonal antibody raised against the B subunit of calcinerurin. Treatment of synaptosomes with okadaic acid to inhibit phosphatase 2A or cyclosporin A to inhibit calcineurin increased apparent phosphorylation of sodium channels at cAMP-dependent phosphorylation sites, as assayed by back phosphorylation. These results indicate that phosphatase 2A and calcineurin dephosphorylate sodium channels in brain, and thus may counteract the effect of cAMP-dependent phosphorylation on sodium channel activity.

Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phosphorylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.

The alpha subunit of the rat brain sodium channel is phosphorylated by cAMP-dependent protein kinase in vitro and in situ at multiple sites which yield seven tryptic phosphopeptides. Phosphopeptides 1-4 and 7 are derived from phosphorylation sites between residues 554 and 623 in a single large CNBr fragment from the cytoplasmic segment connecting homologous domains I and II of the alpha subunit (Rossie, S., Gordon, D., and Catterall, W. A. (1987) J. Biol. Chem. 262, 17530-17535). In the present work, antibodies were prepared against a synthetic peptide corresponding to residues 676-692 (AbSP15), which contain one additional potential phosphorylation site at Ser686-Ser687 in a different predicted CNBr fragment of this same intracellular segment. AbSP15 recognizes native and denatured sodium channels specifically and immunoprecipitates phosphorylated CNBr fragments of low molecular mass that contain a new site phosphorylated by cAMP-dependent protein kinase. Comparison of tryptic phosphopeptides derived from intact alpha subunits with those derived from the phosphorylated CNBr fragments isolated by immunoprecipitation with AbSP15 indicates that the two previously unidentified phosphopeptides 5 and 6 derived from the intact alpha subunit arise from phosphorylation of the site containing Ser686-Ser687. These results identify a new cAMP-dependent phosphorylation site and show that the major cAMP-dependent phosphorylation sites of the rat brain sodium channel, which are phosphorylated both in vitro and in intact neurons, are all located in a cluster between residues 554 and 687 in the intracellular segment between domains I and II.