The human hippocampus plays a central role in the storage of explicit memories, making possible the recollection of places, objects, events, and people. The hippocampus is also important in the pathophysiology of a number of common brain diseases, including epilepsy, dementia, and ischemia. The long-term goal of the proposed research is to help explain physiologic mechanisms underlying synaptic plasticity in the mammalian hippocampus. A parallel goal is to understand the potential role of synaptic plasticity in the setting of abnormal activity that leads to neuronal injury and death. Previous work suggests that modulation of potassium currents by phosphorylation can regulate neuronal excitability and contribute to synaptic plasticity. The proposed research will test the hypothesis that phosphorylation modulates hippocampal voltage-dependent potassium channels.
Specific aims and methods are: 1) to purify potassium channel protein using antibodies directed against channel subunits, and show that protein kinases phosphorylate this purified channel protein in vitro. 2) to express potassium channels transiently in mammalian cultured cells; to manipulate the extent of phosphorylation of kinase substrates in these transfected cells, using activators and inhibitors of specific kinases and phosphatases; to characterize the biochemical and biophysical properties of the transiently expressed channel proteins under these varied conditions. 3) to identify sites of functionally important phosphorylation within the potassium channel peptides, by peptide sequencing of channel proteins and electrophysiological studies of channel genes modified using site-directed mutagenesis. 4) to subject hippocampal tissue to tetanic stimulation (sufficient to produce long term potentiation, seizures, or epilepsy) or chemical treatment with proconvulsants (e.g., metrazol, 4-aminopyridine) and determine the extent of associated phosphorylation at functional regulatory sites on potassium channel proteins using anti-phosphopeptide antibodies. By contributing to the current rapid advances in understanding of potassium channel structure and function, the proposed research may lead to new treatments for epilepsy and other neurological disorders associated with hippocampal dysfunction and cellular hyperexcitability.
