Phosphorylation of the rat skeletal muscle sodium channel by cyclic AMP-dependent protein kinase.
Cyclic AMP-dependent phosphorylation of the rat brain sodium channel was reported to be restricted to five sites within an approximately 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 alpha-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 alpha-subunit was labeled, with a maximum level of incorporation in vitro of approximately 0.5 mol [32P]phosphate per mole of channel protein. The beta-subunit of the channel is not phosphorylated under these conditions. In primary rat skeletal muscle cells in culture, incorporation of phosphate into the channel alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Yang
J
J
Mahoney Institute of Neurological Sciences, University of Pennsylvania School of Medicine, Philadelphia 19104.
Barchi
R
R
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism*; D014357:Trypsin
D000596:Amino Acids; D010455:Peptides; D015222:Sodium Channels; D010768:Phosphoserine; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin