Structural changes in postsynaptic densities (PSDs) may underlie long~term modifications of synaptic activity. The aim of this project is to study the molecular organization of the PSDs and to explore the potential mechanisms for their modification in response to calcium and other intracellular messengers. The present research focuses on two calcium~dependent enzymes, calcium calmodulin~dependent protein kinase (CaM kinase) and calpain, as likely candidates for mediating such changes. In order to explore a potential role of calpain~mediated proteolysis in structural modification, PSD preparations were treated with exogenous calpain. Limited calpain action resulted in selective proteolysis of a few proteins including spectrin, while causing little or no breakdown of other proteins, including CaM kinase and actin. Under these conditions, the 160 kDa breakdown product of spectrin cosedimented with the PSDs. Structural changes following calpain treatment were evident by electron microscopy of freeze substituted specimens. Studies on the autophosphorylation of the PSD~associated CaM kinase have continued. Extensive calcium~dependent autophosphorylation of the alpha subunit of the kinase, which is the major protein in densities from cerebral cortex, was obtained by inhibition of the endogenous phosphatase activity as described previously. None of the major proteins of PSDs including CaM kinase itself, nor various subtypes of glutamate receptor subunits, were solubilized following calcium~dependent phosphorylation. Structural modifications due to autophosphorylation are currently being characterized by electron microscopy of freeze substituted PSDs. In collaboration with Dr. Howard Jaffe (LNC~NINDS Protein~Peptide facility) the autophosphorylated sites are being identified by sequencing of tryptic peptides. Similar strategies will be applied to characterize calcium~independent autophosphory~lation of PSD~associated CaM kinase. Identification of potential phosphorylation sites of the kinase during and following the calcium signal and determination of correlated structural and biochemical changes in PSDs is expected to help elucidate the role of CaM kinase in synaptic modification.
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