Phosphorylation of neuronal proteins by kinases has been proposed to play a key role in the regulation and expression of the actions of many neurotransmitters. However, to date few studies on kinases have actually tested what effect they have on neuronal physiology. The studies proposed here will focus primarily on the electrophysiological consequences of protein kinase A and C activation in the hippocampus. Previous results from this laboratory indicate that activation of protein kinase A, by application of cAMP and its analogs results in a blockade of a calcium-activated potassium current (lAHP). Preliminary evidence suggests that activation of protein kinase C, (PKC) using phorbol esters (PEs) has four different effects on hippocampal pyramidal cells (HPCs): 1) blockade of lAHP; 2) blockade of a voltage- dependent chloride current (lCl(V)); 3) enhancement of excitatory synaptic transmission; and 4) blockade of responses evoked by GABAB receptor activation. Three projects will be carried out in this study. First, the effects of PEs on synaptic transmission will be determined using conventional extra- and intracellular recording techniques. The enhancement of excitatory transmission by PKC activation and its possible involvement in long term potentiation will be studied. Second, the mechanisms of action of PEs and cAMP on lAHP and lCl(V) will be determined using a single-electrode voltage clamp of intact HPCs from slices and also using whole-cell recording and patch clamp recording from primary cell cultures. Third, the mechanism of action of PEs on responses evoked by GABAB receptor activation will be examined. Neurotransmitters which have been proposed to exert their actions through PKC activation will be tested for their ability to mimic each of the actions of PEs. Calcium loading of cells as well as application of 1-oleoyl-2-acetyl-glycerol will be used as alternate methods (to PEs) to activate PKC. The proposed studies will greatly increase our understanding of the role that kinases play in CNS neurons by actually testing the effects of kinase activation on the physiology of HPCs. These studies will also provide information in the ways in which the activity of neurons can be modulated. Finally, these studies should contribute to our knowledge of psychiatric disorders since many of the neurotransmitters implicated in these disorders act via protein kinases.
