Ion channel currents represent the fundamental units of electrical activity in most organisms. In our nervous system, K+ and Ca2+ ion channels play critical roles, and the modulation of their activity provides a way to directly control cellular excitability and release of neurotransmitters at synapses. The regulatory pathways are crucial to basic nervous function and their understanding should contribute to novel modes of medical interventions for a range of disorders involving the brain, nerves and muscles. We focus primarily on an important family of K+ channels called KCNQ that underlies the neuronal M current and secondarily, on high threshold, N-type Ca2+ channels. In particular, we seek to elucidate the molecular mechanisms of several modulatory pathways that act on these two types of ion channels. Several different neurotransmitters, acting via specific receptors and a ubiquitous family of signaling proteins called G proteins, modulate these channels via two distinct modes of action. Both types of signals use cytoplasmic messengers, but many of the intracellular signaling molecules they use remain unidentified, and their mechanisms of action undetermined. Another mode of modulation that acts on ion channels involves tyrosine kinases and small, monomeric GTPases. We will use the tools of patch-clamp electrophysiology, molecular biology, optical imaging, and biochemistry to investigate 1) what molecules are involved in each signal, 2) where is their site of action on the channel proteins and 3) how these signaling pathways interact with each other. Two general types of in vitro systems will be used: a heterologous expression system in which signaling is reconstituted using cloned components, and a preparation of primary sympathetic neurons. We hypothesize that each signal uses distinct sets of signaling molecules that confer specificity of action, that intracellular 2nd about-messengers act on ion channels at particular sites on the channels, and that multiple signaling pathways act in concert to provide nerve cells a broad array of regulatory options. We expect that this study will help understand how the billions of cells of the nervous system orchestrate the complex phenomena of human thought, emotion and behavior, and that our findings will help to alleviate the many diseases of mood, motion and consciousness that are disorders of nervous function.
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