Changes in the amplitude and kinetics of ionic currents within nerve cells of both vertebrate and invertebrate origin may be brought about by the activation of endogenous protein kinases. Transformation of the electrical properties of neurons by these enzymes may lead to both natural and pathological changes in excitability, such as the onset of neuronal afterdischarge. The research proposed in this application will use a simple invertebrate system of neurons, the bag cell neurons of Aplysia, which are accessible for combined biochemical-electrophysiological investigations to study the role of cyclic nucleotide, calcium/calmodulin and calcium/phospholipid dependent protein kinases in the control of calcium and potassium currents. Recent work has indicated that the amplitude of the calcium current in the bag cell neurons may be regulated by the activation of the calcium/phospholipid dependent protein kinase (protein kinase C). This hypothesis will be tested by voltage clamp studies in isolated, internally dialyzed neurons after injection of protein kinase C and, using single channel recordings, before and after microinjection or pharmacological activation of this enzyme. The electrical effects of the second messenger, inositol trisphosphate, which is believed to be formed concurrently with the activation of protein kinase C, will also be investigated. Parallel biochemical experiments will be carried out to determine the conditions under which activation of these enzymes occurs and the phosphoprotein substrates whose phosphorylation state is altered on the activation of these enzymes in intact cells. In addition, the mechanism by which cyclic AMP dependent protein kinase acts on three distinct potassium currents in the bag cell neurons will be investigated further using whole cell and single channel recordings.
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