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

Abstract

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)

Isoform Diversity and Modulation of Sodium Channels by Protein Kinases

“…The rat skeletal muscle sodium channel ·-subunit is phosphorylated in vitro and in vivo via cyclic AMP-dependent phosphorylation [150]. Despite this, functional effects of PKA phosphorylation on sodium channels from freshly dissociated rat skeletal muscle cells and on ÌI ·-subunits, expressed in Xenopus oocytes, could so far not be detected [123,125,151].…”

Regulation of sodium channel activity by phosphorylation

“…Skeletal muscle sodium channels were phosphorylated by PKA despite lacking the large loop I-II that is the target of PKA in other sodium channels [36]. Na V 1.4 expressed in HEK293 cells is phosphorylated by activation of PKA or PKC pathways on overlapping and/or interacting sites [37].…”

Modulation of skeletal muscle sodium channels by human myotonin protein kinase.

“…Purified muscle Na channels are substrates for PKA (38), and brain Na channels are substrates for both PKA and PKC (39,40). In brain Na channels, both kinases phosphorylate at multiple sites, and at some common sites (35).…”