The function of guanine nucleotide binding proteins (G-proteins) as intermediates that integrate patterns of receptor activation by extracellular neuroendocrine signals and couple these to appropriate effectors, including ion conductive channels, is becoming increasingly recognized. The overall objective of this project is to elucidate the molecular mechanisms by which anesthetics alter the operation of these signal transduction cascades in order to identify those molecular processes whose alteration may account for globally observed physiological actions of anesthetics. Specifically, the project will focus on the influence of a selected group of anesthetics on the function of guanine nucleotide binding proteins (G-proteins) that couple the activation of hormone receptors to the opening and closing of ion conductive channels. We have recently demonstrated that the opening of the potassium channel KACh by the G protein GK can be used as a rapid, sensitive and selective indicator of the molecular mechanisms of receptor activated G protein functioning in intact cells (Szabo and Otero, 1990). The project will utilize methodologies developed in the laboratory of the principal investigator to examine the ways in which anesthetics alter the salient kinetic steps in receptor mediated activation and deactivation of GK. We will examine the differential effects of selected anesthetics on: (a) agonist-receptor interactions, (b) receptor-G protein interactions, (c) the rate of G protein activation (e.g. release of GDP and uptake of GTP), (d) the rate of G protein deactivation (e.g. GTP hydrolysis) and (e) channel-G protein interactions. Initial emphasis will be on GK activation by muscarinic and purinergic agonists in cardiac atrial myocytes voltage clamped using the whole-cell configuration of the patch-clamp technique and internally dialyzed with appropriate guanine nucleotides of their analogs. Subsequent experiments will address the inhibitory regulation of adenylate cyclase by muscarinic and purinergic agonists as well as the activation of the adenylate cyclase by beta-adrenergic agonists with the ultimate objective of elucidating the molecular mechanisms by which selected anesthetics may alter the hormonal regulation of Ca2+ channels. Preliminary experiments indicate that physiologically reasonable concentrations of general anesthetics have significant effects on these G-protein coupled regulatory processes. By selecting a group of chemically diverse anesthetics we will attempt to establish a distinctive pattern for the anesthetic sensitivity of G-protein mediated signal transduction processes. By comparing these to the patterns of anesthetic sensitivity of salient physiological processes, the proposed experiments are likely to reveal whether or not significant anesthetic actions may arise through alterations of the G protein mediated regulation of channel or adenylate cyclase function and if so by what molecular mechanism.
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