Ca ²⁺ /calmodulin-dependent protein kinase II (CaMKII) is activated by calmodulin with two bound calciums

Abstract

Changes in synaptic strength that underlie memory formation in the CNS are initiated by pulses of Ca 2؉ flowing through NMDAtype glutamate receptors into postsynaptic spines. Differences in the duration and size of the pulses determine whether a synapse is potentiated or depressed after repetitive synaptic activity. Calmodulin (CaM) is a major Ca 2؉ effector protein that binds up to four Ca 2؉ ions. CaM with bound Ca 2؉ can activate at least six signaling enzymes in the spine. In fluctuating cytosolic Ca 2؉ , a large fraction of free CaM is bound to fewer than four Ca 2؉ ions. Binding to targets increases the affinity of CaM's remaining Ca 2؉ -binding sites. Thus, initial binding of CaM to a target may depend on the target's affinity for CaM with only one or two bound Ca 2؉ ions. To study CaM-dependent signaling in the spine, we designed mutant CaMs that bind Ca 2؉ only at the two N-terminal or two C-terminal sites by using computationally designed mutations to stabilize the inactivated Ca 2؉ -binding domains in the ''closed'' Ca 2؉ -free conformation. We have measured their interactions with CaMKII, a major Ca 2؉ ͞CaM target that mediates initiation of long-term potentiation. We show that CaM with two Ca 2؉ ions bound in its C-terminal lobe not only binds to CaMKII with low micromolar affinity but also partially activates kinase activity. Our results support the idea that competition for binding of CaM with two bound Ca 2؉ ions may influence significantly the outcome of local Ca 2؉ signaling in spines and, perhaps, in other signaling pathways.postsynaptic ͉ protein design ͉ synaptic plasticity ͉ microdomains

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…Models of this type do not allow for the possibility that the different binding sites might have different affinities for calcium. Recent experiments on recombinant calmodulin with site-directed mutations, however, suggest that the N-terminal binding sites have different affinities than the C-terminal binding sites (18). Finally, use of a Hill function to compute calmodulin activity as a function of calcium concentration is purely phenomenological and does not provide an explanation for how calcium binding relates to calmodulin activity.…”

“…Many investigators measuring calcium binding to calmodulin (16,18,28,42) have used an Adair function (43), or a modified version thereof, to describe calmodulin binding to calcium. This approach allows for different microscopic affinities for the four binding events and thus for detailed models of calcium binding to calmodulin.…”

“…It has been reported (18)(19)(20) that the apparent affinity of calmodulin for calcium increases if calmodulin is bound to a target. To reproduce this effect, we simulated calcium binding to 2 ϫ 10 Ϫ7 M calmodulin at varying calcium concentrations in the absence of target and in the presence of CaMKII.…”

“…Each of these models reflects some properties of calmodulin and is reasonably applicable in contexts in which only these properties are relevant. However, none of these models can satisfactorily account for all of the observed properties of calmodulin such as cooperativity of calcium binding and different affinities for different calcium-binding sites (16), activation of targets by unsaturated calmodulin (17,18), and increased affinity for calcium upon binding to targets (18)(19)(20). We propose an alternative model, based on a biophysical description of the conformational transitions.…”

“…26), two reduced equations for Y with N-or C-terminal binding sites only, respectively (using data from experiments with recombinant calmodulin from ref. 18) and one reduced equation for The k on for calcium binding was assumed to be the same for all four binding sites and both states, because it can be assumed to be controlled only by calcium and calmodulin diffusion and size (random exploration). Different affinities were represented by different k off values.…”