cAMP-dependent phosphorylation of two sites in the alpha subunit of the cardiac sodium channel.
The voltage-sensitive Na+ channel is responsible for generating action potentials in the heart which are critical for coordinated cardiac muscle contraction. Cardiac Na+ channels are regulated by cAMP-dependent phosphorylation, but the sites of phosphorylation are not known. Using mammalian cells expressing the rat cardiac Na+ channel (rH1) alpha subunit and site-specific antibodies, we have shown that the alpha subunit of rat heart Na+ channel is phosphorylated selectively by cAMP-dependent protein kinase (PKA) in vitro and in intact cells. Analysis of the sites of phosphorylation by two-dimensional phosphopeptide mapping and site-directed mutagenesis of fusion proteins revealed that the cardiac alpha subunit is phosphorylated selectively in vitro by PKA on Ser526 and Ser529 in the intracellular loop connecting homologous domains I and II (LI-II). These two residues were phosphorylated in intact cells expressing the rH1 alpha subunit when PKA was activated. Our results define a different pattern of phosphorylation of LI-II of cardiac and brain Na+ channels and implicate phosphorylation of Ser526 and Ser529 in the differential regulation of cardiac and brain Na+ channels by PKA.
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
B J
BJ
Murphy
J
J
Rogers
A P
AP
Perdichizzi
A A
AA
Colvin
W A
WA
Catterall
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D015222:Sodium Channels; D012694:Serine; D000242:Cyclic AMP; D005982:Glutathione Transferase; D017868:Cyclic AMP-Dependent Protein Kinases