(155g) Kinetic Models of Interactions Among Ca2+, Calmodulin, and Camkii; Molecular Components of Learning and Memory

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a dodecameric serine/threonine protein kinase that is an essential component of the molecular mechanisms underlying learning and memory. Mice lacking both copies of the gene for the alpha subunit of CaMKII cannot perform spatial learning tasks; heterozygotes have behavioral phenotypes that resemble schizophrenia in humans. CaMKII is activated upon binding of Ca2+/calmodulin (CaM), which is itself a Ca2+-activated protein that binds four Ca2+ ions, two on its carboxyl (C) and two on its amino terminus (N). The major source of Ca2+ for activation of CaMKII at synapses is Ca2+ influx through the NMDA-type glutamate receptor in the postsynaptic dendritic spine. Strong activation of CaMKII by NMDA receptors initiates a series of molecular modifications in the spine that enhance the strength of the synapse.

Here we present two kinetic models of activation of monomeric catalytic subunits of CaMKII (mCaMKII) that include binding of Ca2+ to free calmodulin (CaM) and to CaM bound to individual CaMKII subunits. Both models account for the different kinetics of Ca2+/CaM binding at the N and C termini, and for the thermodynamic stabilization of Ca2+-binding when CaM is bound to a target protein, in this case mCaMKII. The models allow us to consider the dynamics of association among Ca2+, CaM, and mCaMKII separately from the issue of cooperativity of binding of CaM between subunits within the large holoenzyme. The first model is a complete model of binding of Ca2+ to the two CaM termini, including 9 Ca2+/CaM states and their interactions with mCaMKII and the autophosphorylation of mCaMKII. Most of the required kinetic rates are well constrained by previous experimental studies; however, a few have not been measured directly. In these cases, we used the principle of microscopic reversibility and fitting of existing experimental data to derive reasonable ranges of values for the kinetic rates. The second model is a coarse-grained model in which binding of the second Ca2+ to each terminus of CaM is assumed to be rapid. Thus, binding of pairs of Ca2+ to each terminus is treated as a single event. The resulting model includes 4 Ca2+/CaM states and their interactions with mCaMKII, and autophosphorylation of mCaMKII.

We examined behaviors of the models under commonly used experimental concentrations of Ca2+, CaM, and mCaMKII, and under the quite different conditions that are believed to exist in synaptic spines. We determined a range of initial conditions under which the results of the coarse-grained model are indistinguishable from those of the complete model, and a range under which the two deviate significantly. Behavior of the models suggests that the contribution of partially filled Ca2+/CaM states to activation of autophosphorylation of CaMKII is more significant under physiological conditions, when the Ca2+ concentration is relatively low and fluctuating rapidly than it is at steady-state Ca2+ concentrations generally used in experimental studies. We present two major predictions of the models: 1. Ca2+/CaM species with fewer than four bound Ca2+ often predominant in synaptic spines, and can sometimes completely determine the ultimate level of autophosphorylation of CaMKII. 2. Competition for binding of Ca2+/CaM among its targets is an important determinant of activation of CaMKII during the non-equilibrium conditions that often prevail in vivo. The models predict that activation of mCaMKII will be highest at a particular frequency of Ca2+ fluctuations. That frequency depends on the ratio of the time interval between Ca2+ fluctuations and the rates of Ca2+ binding to the N and C termini of CaM. These results suggest that the kinetics of Ca2+ binding to CaM alone can confer frequency sensitivity to CaMKII activation. This frequency-dependent phenomenon is independent of the previously reported frequency-sensitivity arising from cooperative binding of CaM to the CaMKII holoenzyme. Frequency sensitivity due to the kinetics of Ca2+ binding by CaM has consequences for other monomeric targets of CaM, including protein kinases (CaMKI, CaMKIV, CaMKK, myosin light chain kinase), the phosphatase calcineurin, adenylate cyclase, Ca2+-dependent cAMP phosphodiesterase, and neuronal nitric oxide synthase.

This work demonstrates that kinetic analysis of interactions among Ca2+, CaM, and its targets are not only critical for understanding molecular mechanisms that underlie synaptic plasticity, but also may reveal unexpected attributes of Ca2+/CaM signaling in other cell types, including cardiac myocytes and cells of the immune system.