The objective of these studies is to identify physiological functions of Ca++/ Calmodulin-dependent protein kinase II (CaM- kinase II) in neural tissues. CaM-kinase II is of particular interest in this context since 1) it constitutes about 1% of brain protein, 2) it constitutes about 50% of the postsynaptic density (PSD) protein, and 3) it undergoes a unique autophosphorylation that converts it to a Ca++ -independent form. Our studies will utilize a combination of biochemical and electrophysiological approaches to investigate physiological functions of the cytosolic and membrane-associated brain CaM-kinase II. Potential targets of cytosolic CaM-kinase II that will be investigated include tyrosine hydroxylase, the rate-limiting enzyme in catecholamine and several other regulatory CaM-binding proteins. With reference to tyrosine hydroxylase, our goals will be to establish its in vivo phosphorylation by CaM-kinase II and the regulatory role of an activator protein that is specific for tyrosine hydroxylase that has been phosphorylated by CaM-kinase II. These studies will utilize pinocytotic introduction of antibodies against CaM-kinase II or the activator protein into PC12 cells. A number of known CaM-binding proteins will be screened for specific phosphorylation by the Ca -independent form of CaM-kinase II. We are especially interested in regulatory phosphorylation sites which are blocked when Ca /CaM is bound to the CaM-binding proteins. A major focus of these studies will be the CaM-kinase II localized in the PSD. Of special interest is the potential role of this kinase in regulating certain ion channels, specifically, the NMDA- receptor/ion channel and the dihydropyridine-sensitive Ca channel. These studies will combine radioligand binding analyses and patch- clamp studies. We are particularly interested in potential regulation of these ion channels by protein phosphorylation and by GTP-binding proteins. The NMDA channel is of special relevance due to its likely involvement in synaptic plasticity such as long-term potentiation and kindling, and perhaps in epilepsy.
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