DIFFERENTIAL REGULATED INTERACTIONS OF CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE II WITH ISOFORMS OF VOLTAGE GATED CALCIUM CHANNEL BETA SUBUNITS

摘要

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylates the β2a subunit of voltage-gated Ca²⁺ channels at Thr498 to facilitate cardiac L-type Ca²⁺ channels. CaMKII colocalizes with β2a in cardiomyocytes and also binds to a domain in β2a that contains Thr498 and exhibits amino acid sequence similarity to the CaMKII autoinhibitory domain and to a CaMKII binding domain in the NMDA receptor NR2B subunit (Grueter et al., 2006. Mol. Cell >23:641). Here we explore the selectivity of the actions of CaMKII among Ca²⁺ channel β subunit isoforms. CaMKII phosphorylates the β1b, β2a, β3 and β4 isoforms with similar initial rates and final stoichiometries of 6–12 mole phosphate per mole protein. However, activated/autophosphorylated CaMKII binds to β1b and β2a with similar apparent affinity, but does not bind to β3 or β4. Pre-phosphorylation of β1b and β2a by CaMKII substantially reduces the binding of autophosphorylated CaMKII. Residues surrounding Thr498 in β2a are highly conserved in β1b, but are different in β3 and β₄. Site-directed mutagenesis of this domain in β_2a showed that Thr498 phosphorylation promotes dissociation of CaMKII-β_2a complexes in vitro and reduces interactions of CaMKII with β_2a in cells. Mutagenesis of Leu493 to Ala substantially reduces CaMKII binding in vitro and in intact cells but does not interfere with β_2a phosphorylation at Thr498. In combination, these data show that phosphorylation dynamically regulates the interactions of specific isoforms of the VGCC β subunits with CaMKII.