NEUROMODULATION OF ION CHANNELS

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Ion channels are not simply passive pores, but are dynamic entities subject to modulation that can last for seconds, minutes, or even hours. I propose to investigate the mechanisms by which the phosphorylation of ion channels produces such long-term modulation. In particular I will examine whether ion channels can exist as regulatory complexes tightly associated with modulatory proteins. The study will focus on a group of large conductance Ca2+-activated K+ channels from rat brain, because many of these channels can be modulated by phosphorylation of either the channel itself, or of closely associated proteins. The specific aims are: 1) to explore whether ion channels can reside in membranes as protein complexes, tightly associated with regulatory components such as protein kinases and protein phosphatases; 2) to examine the contribution of individual phosphorylation sites on the gating of ion channels that are multiply phosphorylated; 3) to investigate the interactions between different protein kinases that act on a single ion channel; and 4) to define the role and specificity of protein phosphatases in modulating channel gating. These experiments will rely on two complementary experimental approaches. First, planar lipid bilayers will be used to reconstitute individual ion channels separated from all cytoplasmic influences. The effect of purified protein kinases and phosphatases on channel gating will be examined. Second, channel modulation in native membranes and intact cells will be investigated by patch-clamping cultured neurons. While there is much descriptive information about how neurons respond to extracellular signals by an alteration in their electrical activity, little is known about the mechanisms by which these modulators alter channel gating. This work will aim to provide such mechanistic information, particularly in the context of ion channel / kinase phosphatase complexes. Such complexes may represent a common target for multiple second messenger pathways, and play a role in the integration, and perhaps even the storage, of information carried by extracellular signals.