The A-kinase anchor protein MAP2B and cAMP-dependent protein kinase are associated with class C L-type calcium channels in neurons

Phosphorylation by cAMP-dependent protein kinase (PKA) increases the activi ty of class C L-type Ca2+ channels which are clustered at postsynaptic site s and are important regulators of neuronal functions. We investigated a pos sible mechanism that could ensure rapid and efficient phosphorylation of th ese channels by PKA upon stimulation of cAMP-mediated signaling pathways. A kinase anchor proteins (AKAPs) bind to the regulatory R subunits of PRA an d target the holoenzyme to defined subcellular compartments and substrates. Class C channels isolated from rat brain extracts by immunoprecipitation c ontain an endogenous kinase that phosphorylates kemptide, a classic PRA sub strate peptide, and also the main phosphorylation site for PRA in the pore- forming ru, subunit of the class C channel complex, serine 1928. The kinase activity is inhibited by the PHA inhibitory peptide PKI(5-24) and stimulat ed by cAMP. Physical association of the catalytic C subunit of PHA with the immunoisolated class C channel complex was confirmed by immunoblotting, A direct protein overlay binding assay performed with P-32-labeled RII beta r evealed a prominent AKAP with an M-r of 280,000 in class C channel complexe s. The protein was identified by immunoblotting as the microtubule-associat ed protein MAP2B, a well established AKAP. Class C channels did not contain tubulin and MAP2B association was not disrupted by dilution or addition of nocodazole, two treatments that cause dissociation of microtubules, In vit ro experiments show that MAP2B can directly bind to the cu, subunit of the class C channel. Our findings indicate that PKA is an integral part of neur onal class C L-type Ca2+ channels and suggest that the AKAP MAP2B may media te this interaction. Neither PKA nor MAP2B were detected in immunoprecipita tes of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutam ate receptors or class B N-type Ca2+ channels. Accordingly, MAP2B docked at class C Ca2+ channels may be important for recruiting PHA to postsynaptic sites.