Molecular Properties of Ionic Permeability in Nerve

University Of Washington, Seattle WA

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Abstract

DESCRIPTION (provided by applicant): This proposal focuses on intracellular signaling mechanisms that couple G-protein coupled receptors to modulation of ion channels in single cells. The emphasis is on the identity and kinetics of the molecular steps of receptor-driven Gq-coupled signaling via phospholipase C to the KCNQ class of K channels. These signals require lipid metabolism. Such signaling underlies state changes in the brain (mood, arousal), and disturbances of these signals can lead to psychiatric disorders, epilepsy, or cardiac arrhythmias. At the molecular level, such signals modulate a host of ion channels and transporters in cell membranes. The goals are to consolidate the hypothesis that KCNQ channels are regulated by PIP2 lipids and to provide a tested general kinetic formulation for G-protein coupled signaling. Intermediate steps of signaling from two muscarinic receptors and a cannabinoid receptor will be reported by fluorescent mutant signaling proteins designed for each level of the signal cascade, e.g. receptor, G-protein, effector, etc. Agonists will be applied rapidly while the time course of the spectral properties of these reporters and of the final ion channel currents are monitored. The kinetics of inositol phospholipid metabolism after receptor stimulation will be followed using confocal microscopy with fluorescent protein probes for specific lipid species. The kinetics of receptor induced intracellular calcium release will be monitored with ratiometric fluorescent indicators. For comparison, actual chemical measurements of inositol lipids will be made on the same cell lines during applications of agonists. The results will be interpreted by a large, explicit kinetic model of the deduced biochemical reactions. This comparison will test and refine the assignment of kinetic and regulatory properties to each step.