In the last two decades we have witnessed an explosion in knowledge of the normal and pathological function of the nervous system. Progress has been largely fueled by advances in two methodologies, fluorescence imaging and electrophysiology. We propose to begin the next wave of discovery, by combining optical and electrophysiological measurements of living cells, to probe their function in real time. The excitability of neurons, secretory cells, and muscle cells is influenced by a large number proteins and second messengers: transcription factors determine the number of ion channels and receptors that are produced;kinases, phosphatases, and ions determine the activity of channels and receptors;and complex recycling and degradation pathways determine the residence time of channels and receptors in the plasma membrane. As well, the phospholipids that compose the plasma membrane play an active role in determining channel activity and the rate of channel and receptor internalization. We will image the movement of proteins and signaling molecules within a cell with confocal and TIRF microscopy and read out the net effect of signaling on the cell's excitability using electrophysiological recording. This proposal requests funds for a Confocal-TIRF-Electrophysiological (CTE) workstation for ion channel biophysics. No such instrument exists in the Pacific Northwest. It would permit us to synchronize the movement of organelles, interactions between proteins, changes in intracellular calcium concentration, intra-molecular conformational changes, and electrical activity into a cohesive understanding of how cellular excitability is set, how it is perturbed by extracellular signals, and how it recovers after a signaling event. Using these methodologies, we will ask questions such as: Which calcium-activated signals regulate desensitization? What is the mechanism by which phosphoinositides regulate channels? How do growth factors regulate channel trafficking? How does phosphorylation regulate channel activity and expression? What are the molecular motions that underlie gating? These questions will be asked across a number of cell types, to define ion channel function quantitatively in several systems essential to human health.
Excitable cells depend on ion channel proteins to conduct electrical signals, regulate force, and mediate secretion. In fact, over half of all drug targets are ion channels or proteins that directly regulate ion channels. Our work is directed toward understanding the normal and pathological signaling by ion channels in the nervous system and vascular system.
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