RC3/neurogranin and Ca2+/calmodulin-dependent protein kinase II produce opposing effects on the affinity of calmodulin for calcium

The interaction of calmodulin with its target proteins is known to affect the kinetics and affinity of Ca2+ binding to calmodulin. Based on thermodynamic principles, proteins that bind to Ca2+-calmodulin should increase the affinity of calmodulin for Ca2+, while proteins that bind to apo-calmodulin should decrease its affinity for Ca2+. We quantified the effects on Ca2+-calmodulin interaction of two neuronal calmodulin targets: RC3, which binds both Ca2+- and apo-calmodulin, and alphaCaM kinase II, which binds selectively to Ca2+-calmodulin. RC3 was found to decrease the affinity of calmodulin for Ca2+, whereas CaM kinase II increases the calmodulin affinity for Ca2+. Specifically, RC3 increases the rate of Ca2+ dissociation from the C-terminal sites of calmodulin up to 60-fold while having little effect on the rate of Ca2+ association. Conversely, CaM kinase II decreases the rates of dissociation of Ca2+ from both lobes of calmodulin and autophosphorylation of CaM kinase II at Thr(286) induces a further decrease in the rates of Ca2+ dissociation. RC3 dampens the effects of CaM kinase II on Ca2+ dissociation by increasing the rate of dissociation from the C-terminal lobe of calmodulin when in the presence of CaM kinase II. This effect is not seen with phosphorylated CaM kinase II. The results are interpreted according to a kinetic scheme in which there are competing pathways for dissociation of the Ca2+-calmodulin target complex. This work indicates that the Ca2+ binding properties of calmodulin are highly regulated and reveals a role for RC3 in accelerating the dissociation of Ca2+-calmodulin target complexes at the end of a Ca2+ signal.