I propose to continue our ongoing studies of the regulation of ion channels in nerve cells. Because ion channels are responsible for the electrical activity of neurons (and other cells), information about the molecular details of their regulation is of fundamental importance for understanding normal and pathological brain function. The major focus will be on large conductance calcium- dependent potassium channels, which are ubiquitous in nerve, muscle and other cells. These channels are of particular interest because they are a molecular locus for integration of a variety of signals, including the intracellular calcium concentration, other intracellular messengers, and the membrane potential. We will pursue in depth the theme that ion channels exist in the cell membrane as part of a regulatory complex, that includes protein kinases and other signaling proteins that can influence channel properties. This will build on our demonstration, during the current grant period, of the direct binding of several different signaling proteins to calcium-dependent potassium (as well as to other) channels. More specifically, we will investigate the molecular mechanisms and physiological significance of the interaction of calcium-dependent potassium channels with: (i) protein kinases, most notably the catalytic subunit of the cyclic AMP-dependent protein kinase; and (ii) Slob, a novel channel- binding protein we cloned that can influence channel properties directly, and also can recruit the ubiquitous signaling protein scaffold, 14-3-3, into a complex with the channel. We will use a combination of molecular, biochemical, genetic and electrophysiological techniques to characterize the molecular details of the interactions, design tools to interfere with the interactions, and use these tools to explore the role of the interactions in neuronal physiology. The existence of regulatory protein complexes associated with ion channels has fundamental implications for neuronal signaling.
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