The main focus of the project is calcium/calmodulin-dependent protein kinase II (CaMKII), a major protein in the postsynaptic density (PSD) and a calcium-regulated enzyme, implicated in long- term synaptic modification and memory. The distribution of CaMKII in isolated PSDs was studied following adsorption on glass, immunogold labelling, slam freezing and rotary shadowing. Electron microscopic examination of replicas reveals two pools of PSD-associated CaMKII. One pool consists of intensely labelled discrete patches attached to the cytoplasmic face of the PSD. The other pool is revealed upon chemical dissection of the structure with N-lauryl sarcosinate and appears to be associated with a core grid-like structure. Intense labelling with the CaMKII antibody is also observed on spherical particles of about 150 nm diameter that co-purify with the PSDs. These particles are almost exclusively composed of CaMKII and are present in nonsynaptosomal cytoskeleton fractions as well. It is likely that CaMKII associated with the grid-like PSD structure is in a favorable position for the phosphorylation of various postsynaptic elements such as AMPA receptors involved in synaptic transmission. CaMKII tightly packed in patches and spherical particles, on the other hand, would be mostly occluded from substrates. It is proposed that aggregation of CaMKII into such structures would limit its function as a kinase but preserve its function as a calmodulin trap. This could be a valuable mechanism for cellular protection during periods of calcium overload. Regulation of the phosphorylation state of the AMPA-type glutamate receptor (GluR1) in isolated PSDs was studied. PSD- associated GluR1 can be phosphorylated by endogenous CaMKII, but not by PKA. Since the receptor has been shown to be phosphorylated on different sites by the two kinases, it is possible that the """"""""PKA-site"""""""" is either already phosphorylated or becomes occluded when associated with the PSD. CaMKII-mediated phosphorylation of GluR1 is reversed by the action of endogenous phosphatase type1. The well-described inhibition of this phosphatase by PKA suggests a convergent action of cAMP and calcium in the regulation of AMPA receptor function.
