Identification of the sites of selective phosphorylation and dephosphorylation of the rat brain Na+ channel alpha subunit by cAMP-dependent protein kinase and phosphoprotein phosphatases.
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.
Murphy
B J
BJ
Department of Pharmacology, University of Washington, Seattle 98195.
Rossie
S
S
De Jongh
K S
KS
Catterall
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D051381:Rats; D012694:Serine / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin
D010446:Peptide Fragments; D015222:Sodium Channels; D012694:Serine; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D014357:Trypsin