I propose to continue our ongoing studies of the modulation of neuronal electrical activity. Such studies will contribute to our understanding of nerve cell function under normal and pathological conditions. Our combined electrophysiological/biochemical approach at the single cell level will be continued, and extended to examine the modulation of the activity of individual ion channels. More specifically, the detailed molecular mechanisms by which protein kinases regulate ion channel activity will be investigated, using macroscopic current measurements in intact neurons, and single channel measurements in native membrane patches and in artificial phospholipid bilayers. Ongoing experiments with cyclic AMP (cAMP)-dependent protein kinase will be extended to cyclic GMP (cGMP)- and calcium-dependent protein kinases as well. The overall goal of this approach is to understand how individual channels are regulated by metabolic modulators, and to determine how the regulation of the individual channels contributes to the overall activity of the cell. In parallel experiments our biochemical measurements of protein phosphorylation in individual nerve cells will be continued. The goal here is to identify particular phosphoproteins which are closely correlated with particular ionic conductance changes, and to determine whether one or more of these phosphoproteins is an ion channel or a regulatory component of an ion channel. If certain regulatable ion channels are indeed directly phosphorylated, labeling with radioactive phosphate would provide a marker for channel purification. This in turn would allow detailed characterization of the channel at the molecular level, and a more profound understanding of its regulation. The long-term goal of these combined biochemical/electrophysiological studies is to understand in molecular terms the physiological regulation of ion channels and hence of neuronal electrical activity.
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