(597c) Sensitivity Analysis of a Kinetic Model of Interactions Among Ca2+, Calmodulin and CaMKII

Changes in the strength of synaptic connections in the brain underlie our ability to form memories and to learn. One type of experimentally induced change in synaptic strength, long term potentiation (LTP), is dependent upon the activation of Ca2+/calmodulin dependent protein kinase II (CaMKII). CaMKII is a serine/threonine protein kinase that constitutes 1-2% of all brain protein by weight. It is activated by binding of the Ca2+/calmodulin (Ca2+/CaM), which binds up to four Ca2+ ions upon Ca2+ flux through the NMDA receptor in postsynaptic dendritic spines. CaM removes the inhibitory domain of the kinase from the catalytic site and allows CaMKII to autophosphorylate itself and phosphorylate its substrates. CaMKII is activated within milliseconds of Ca2+ influx and can remain active for tens of minutes afterward. It is an early and essential molecular component of the complex signal transduction processes that underlie LTP.

Here we present a thermodynamically complete model of activation of monomeric subunits of CaMKII (mCaMKII) by Ca2+/CaM that includes binding of Ca2+ to free CaM and to CaM bound to individual mCaMKII subunits. The complete model of CaMKII activation accounts for the different kinetics of Ca2+ binding to the amino (N) and carboxyl (C) termini of CaM, and for the thermodynamic stabilization of Ca2+ binding when CaM is bound to a target protein, in this case mCaMKII and the autophosphorylation of mCaMKII for a total of 154 protein states. The values of the kinetic rate constants describing the interactions are well constrained by previous experimental studies; however, a few have not been measured directly. In these cases, we fit existing experimental data and used the principle of microscopic reversibility to derive reasonable ranges of values. We used the model to make predictions about the time course of autophosphorylation of CaMKII under conditions that are believed to exist in synaptic spines and under commonly used experimental ?test tube? concentrations of Ca2+, CaM, and mCaMKII.

We performed global variation and sensitivity analyses to determine which parameters most affect the levels of autophosphorylation under various conditions. We have used these analyses to infer the kinetic pathways through which autophosphorylation of CaMKII is likely to proceed. Under conditions that are believed to prevail in a spine during activation of NMDA receptors, both the level of Ca2+ and the amount of available CaM limit the rate of autophosphorylation of mCaMKII. This means that competition for Ca2+/CaM in an activated spine is an important determinant of synaptic plasticity because autophosphorylation of CaMKII is critical for induction of LTP. This work shows that kinetic analyses can add to our understanding of the complex mechanisms controlling autophosphorylation of CaMKII in spines; and, more generally, should help to clarify other Ca2+/CaM signaling events. Additionally, the model presented here is a first step in a larger project to build kinetic simulations of activation of the CaMKII enzyme in the context of physiologically realistic models of Ca2+ fluctuations in postsynaptic spines. To this end, we have used sensitivity analysis to identify parameters whose refinement by direct measurement will be most important for the accuracy of predictions from our current and future models.