Phosphorylation of the Rat Skeletal Muscle Sodium Channel by Cyclic AMP‐Dependent Protein Kinase

Jane Yang, Robert Barchi

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Abstract: Cyclic AMP‐dependent phosphorylation of the rat brain sodium channel was reported to be restricted to five sites within an ˜210 amino acid region of the primary sequence that is deleted in the homologous sodium channel from rat skeletal muscle. We find that, in spite of this deletion, the rat muscle sodium channel α‐subunit is also an excellent substrate for phosphorylation by this kinase both in primary muscle cells in tissue culture and in vitro after isolation from adult muscle. Sodium channel protein purified from adult rat skeletal muscle was readily phosphorylated in vitro by the catalytic subunit of the bovine cyclic AMP‐dependent protein kinase (PKa). Only the 260,000 MW α‐subunit was labeled, with a maximum level of incorporation in vitro of ˜0.5 mol [32P]phosphate per mole of channel protein. The β‐subunit of the channel is not phosphorylated under these conditions. In primary rat skeletal muscle cells in culture, incorporation of phosphate into the channel α‐subunit is stimulated 1.3‐ to 1.5‐fold by treatment of the cells with forskolin. Phosphorylation of the sodium channel isolated from these cells could also be demonstrated in vitro using PKa. This in vitro phosphorylation could be inhibited 80–90% by pretreatment of the cells in culture with forskolin, suggesting that the sites labeled in vitro by PKa were the same as those phosphorylated in the intact cells by the endogenous cyclic AMP‐dependent kinase. In both the adult muscle channel and the channel from muscle cells in culture, phosphorylation by PKa was limited to serine residues. The location of the phosphorylated residue in the primary sequence of the channel was examined by studying the distribution of label among proteolytic fragments generated in situ from the α‐subunit by endogenous proteases. The label in the intact 260‐kDa α‐subunit partitioned with a 130‐kDa polypeptide representing the amino terminal half of the molecule whereas none was associated with fragments derived from the carboxy‐terminal half of the protein. No label was found in the next smaller 90‐kDa amino‐terminal fragment, however, indicating that the phosphorylated residue is located between 90 and 130 kDa from the amino terminus of the subunit, a region that includes the DI–DII interdomain region and the DII repeat domain. Phosphopeptide analysis of the 260‐kDa α‐subunit and of its 130‐kDa amino‐terminal half confirmed that the single major labeled peptide in each had identical migratory characteristics in this two‐dimensional analysis. Electrophoresis of the tryptic digest in a sodium dodecyl sulfate‐urea system indicated a MW of ˜2,500 for the limit phosphopeptide.

Original languageEnglish (US)
Pages (from-to)954-962
Number of pages9
JournalJournal of neurochemistry
Issue number3
StatePublished - Mar 1990
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Cellular and Molecular Neuroscience


  • Channel structure
  • Phosphorylation
  • Protein kinase
  • Sodium channel

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