Cyclic AMP-dependent phosphorylation of the alpha subunit of the sodium channel in synaptic nerve ending particles.

摘要: In purified preparations of voltage-sensitive sodium channels, the alpha subunit is selectively phosphorylated by cAMP-dependent protein kinase (Costa, M. R. C., Casnellie, J. E., and Catterall, W. A. (1982) Biol. Chem., 7918-7921). We have developed methods to measure channel phosphorylation in both lysed synaptosomal membranes intact synaptosomes. Incubation with exogenously added catalytic [gamma-32P]ATP resulted rapid as detected specific immuno-precipitation, dodecyl sulfate-gel electrophoresis, autoradiography. Analysis tryptic phosphopeptides revealed five major sites reaction. The level these on synaptosomes was monitored using a rephosphorylation method which those not situ were labeled after lysis 8-Br-cAMP completely blocked labeling indicating marked stimulation situ. Phosphorylation complete 15 s all four under conditions could be These results show that can rapidly endogenous addition, since ATP are only available inside synaptosomes, they also transmembrane polypeptide exposed sides membrane. functional consequences 8-Br-cAMP-stimulated examined ion flux neurotoxin-binding methods. Binding saxitoxin scorpion toxin unaffected, but neurotoxin-activated 22Na+ influx mediated reduced 16 26% (P less than 0.01) various experimental conditions. potential physiological significance this action considered.