Molecular mechanism of convergent regulation of brain Na+ channels by protein kinase C and protein kinase A anchored to AKAP-15

Activation of D1-like dopamine (DA) receptors reduces peak Na+ current in hippocampal neurons voltage-dependent in a manner via phosphorylation of the a subunit. This modulation is dependent upon activation of cAMP-dependent protein kinase (PKA) and requires phosphorylation of serine 573 (S573) in the intracellular loop connecting homologous domains I and 11 (LI-II) by PKA anchored to A kinase anchoring protein-15 (AKAP-15). Activation of protein kinase C (PKC) also reduces peak Na+ currents and enhances the strength of the PKA modulatory pathway. Here we probe the molecular mechanism responsible for the convergent effects of PKA and PKC on brain Na-v 1.2a channels. Analysis of the interaction of AKAP-15 with the intracellular loops of the Na-v 1.2a channel shows that it binds to LI-II, thereby targeting PKA directly to its sites of phosphorylation on the Na+ channel by specific protein-protein interactions. Mutagenesis and expression experiments indicate that reduction of peak Na+ current by PKC requires S554 and S573 in LI-II in addition to S1506 in the inactivation gate. In addition, PKC-dependent phosphorylation of S576 in LI-II is necessary for enhancement of PKA modulation of brain Na+ channels. When S576 is phosphorylated by PKC, the increase in modulation by PKA activation requires phosphorylation of S687 in LI-II. Thus, the maximal modulation of these Na+ channels by concurrent activation of PKA and PKC requires phosphorylation at four distinct sites in LI-II: S5549 S573, S576, and S687. This convergent regulation provides a novel mechanism by which information from multiple signaling pathways may be integrated at the cellular level in the hippocampus and throughout the central nervous system.