Biochemical regulation of the CaMKII/GluN2B interaction and its role in the maintenance of synaptic strength

Abstract

Ca²⁺/Calmodulin (CaM)-dependent protein kinase II (CaMKII) is a major regulator of long-term potentiation (LTP) in the hippocampus. During this process, CaMKII becomes activated and translocates to the activated synapse through an interaction with the GluN2B subunit of the NMDAR. This interaction persists after the initial stimulus has subsided and has therefore been proposed to act as a form of molecular memory. For this reason, the mechanisms that underlie the interaction and the physiological functions related to its induction and persistence are of great interest. It was first shown here that the interaction is independent of enzymatic activity, but requires occupation of the ATP-binding pocket, adding to the model of CaMKII/GluN2B binding. This model is similar in many ways to the model of ischemia-related CaMKII clustering and the dependence on enzymatic activity was also determined for that interaction. Like GluN2B binding, clustering was independent of enzymatic activity. Performing mutational analysis of CaMKII regulatory sites led to another interesting finding. A constitutively autonomous, nonstimulatable CaMKII mutant did not interact with GluN2B basally within cells, despite the fact that it did in vitro. Further, this mutant translocated to GluN2B upon Ca²⁺ stimulation, even though it is not itself able to bind CaM. These results indicate that Ca²⁺ regulates the CaMKII/GluN2B interaction through a mechanism that is in addition to its effects on CaMKII. Mechanistically, it was suggested that this novel regulation by Ca²⁺/CaM is mediated by either phosphatase activity or a direct CaM/GluN2B interaction. The function of CaMKII/GluN2B binding was also examined in the context of the maintenance of synaptic strength. It was shown that an inhibitor of the interaction reduced synaptic strength, but did so to a lesser extent in a knockin mouse that is incompetent for CaMKII/GluN2B binding. Interestingly, this effect appears to be dependent on structural rather than enzymatic effects of the kinase, suggesting that the CaMKII/GluN2B interaction acts to scaffold protein complexes at the PSD. Taken together, the results of these experiments indicate that CaMKII/GluN2B binding is independent of enzymatic activity, has a multifaceted regulation by CaM, and at least partially regulates synaptic strength through structural functions.