Synapses are central to neuronal signaling and key targets for drug treatments of neurological disorders. Ca2+ influx through NMDA receptors and subsequent activation of CaMKII are critical events in the induction of LTP and in learning and memory. The NMDA receptor interacts with CaMKII in a complex and highly regulated manner. This interaction places CaMKII at a strategically ideal location, where it is most effectively activated by Ca2+ influx through the NMDA receptor and where it is close to its substrates at the postsynaptic site (e.g., GluRl in LTP). Biochemical details and the functional relevance of this interaction, which is currently unproven, will be determined. Preliminary results will be scrutinized that indicate that the CaMKII, calmodulin, and a-actinin binding sites on the NR1 subunit of the NMDA receptor overlap and that Ca2+/calmodulin promotes CaMKII binding to NR1 by displacing a-actinin. Whether binding of CaMKII to the NR1 and perhaps NR2B subunit per se changes NMDA receptor activity as observed earlier for Ca2+/calmodulin binding to NR1 will be investigated with different electrophysiological methods including recording from excised inside-out patches perfused with purified CaMKII, calmodulin and a-actinin in various combinations and whole cell patch clamping using HEK293 cells transfected with NR1, NR2A, NR2B and CaMKII in different combinations. Whether CaMKII binding to the NMDA receptor is critical for recruiting CaMKII to the postsynaptic site will be tested in primary hippocampal cultures by imaging of GFP-tagged CaMKII in the absence and presence of peptides (membrane-permeable or injected) that inhibit NR1 or NR2B binding of CaMKII. The importance of the NMDA receptor - CaMKII interaction for the induction of LTP will be evaluated by intracellular recording from hippocampal slices with and without the binding-inhibiting peptides. Phosphorylation of GluR1 by CaMKII likely contributes to LTP and will be quantified in slices with biochemical methods. Overstimulation of glutamatergic synapses has been implicated in neuropathologies due to stroke, status epilepticus, and brain trauma. NMDA receptor-mediated Ca2+ influx and CaMKII activation are critical for neuronal damage caused by ischaemia. The postsynaptic anchoring of CaMKII by the NMDA receptor constitutes, therefore, a potentially very important target for drugs that can specifically disrupt this interaction and thereby alleviate neuropathologies due to overactivation of glutamate receptors.
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