We are studying the molecular structure and function of synaptic connections between neurons in the central nervous system. Derangements in the regulation of these connections are an important part of the pathology of several neurological and mental diseases including epilepsy, Alzheimer's disease, schizophrenia, and depression. Many neurotransmitters and neurohormones regulate synaptic function by altering intracellular levels of calcium ion. We are studying the mechanisms by which these fluctuations in calcium levels alter synaptic function. We will focus on the study of a synaptic regulatory pathway that has as its central element an abundant, brain-specific calcium and calmodulin-dependent protein kinase. This kinase is a large oligomer of two distinct but homologous catalytic subunits called alpha and beta. In the forebrain, including the hippocampus, cortex and striatum, the kinase is extremely abundant (1% of total protein) and is composed mainly of alpha subunits. It is a major component of synapses and is concentrated in a cytoskeletal structure called the postsynaptic density. When activated by a brief rise in calcium concentration, the kinase phosphorylates itself and then remains active to phosphorylate other proteins even after the calcium concentration falls. We will test the hypothesis that this is a mechanism by which long-lasting changes in synaptic function are generated following brief bursts of synaptic activity. We will determine the structure of the autophosphorylation sites by recombinant DNA and biochemical methods, then characterize the brain phosphatases responsible for dephosphorylation of each of these sites. We will raise antibodies that specifically recognize the autophosphorylated sites on the kinase and others that recognize the phosphorylated form of kinase substrates. We will use these to study, with high spatial and temporal resolution, the physiological circumstances under which the kinase is activated and specific substrates become phosphorylated. We will continue a study of the association of the kinase with the cytoskeleton by biochemical and recombinant DNA techniques. Our goal in the next few years is to clarify the possible regulatory functions of this calcium-dependent protein kinase system. Our long-term goal is to correlate information about this pathway with similar information about other calcium regulated pathways in order to understand the concerted responses to changing calcium levels in CNS neurons.
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