Current models of anesthetic action involve the alteration of integral membrane protein function by either the indirect effect of anesthetics modifying lipid bilayer properties or the direct binding of anesthetics to specific proteins. The integration of a large number of intracellular signals is now understood to be mediated through G-protein activating receptors in the plasma membrane of target cells. The goal of this application is to determine if anesthetic action involves the modulation of G-protein mediated signal transduction and to elucidate the mechanism of anesthetic action in such systems. The visual transduction pathway, which is initiated by rhodopsin, is one of the best characterized G-protein mediated signal transduction systems and will be used as a model system in these studies. The components of this system are readily available in pure form and the spectral properties of retinal, which serves as agonist in this system, allows one to monitor receptor conformation changes. We will study the effect of anesthetics on: 1) the kinetics and extent of formation of the G-protein(Gt) activating form of rhodopsin(metarhodopsin II); 2) rhodopsin-Gt complex formation in the absence of GTP; 3) the rate of Gt activation; 4) Gt-cGMP phosphodiesterase(PDE) interactions; 5) cGMP PDE activation; and 6) the intrinsic GTPase activity of Gt, which is part of the turn-off mechanism for the pathway. These experiments will allow a detailed characterization of the manner in which anesthetics perturb a G- protein mediated signal transduction pathway. Anesthetic induced perturbation of the physical properties of lipid bilayers will be monitored by using a parameter, the fractional volume(fv), which is derived from fluorescence depolarization measurements and related to the lipid acyl chain packing free volume. Raman spectral peak ratios related to both lateral chain packing and intrachain order will also be obtained. The use of fv to monitor lipid bilayer properties was developed in this laboratory and has been shown to have a strict correlation with the conformational equilibrium associated with the formation of metarhodopsin II. The Raman measurements have been shown to reflect both acyl chain order and the presence of microdomain heterogeneity in lipid bilayers. Correlations of the effects of anesthetics on the various steps in the signal transduction pathway with the anesthetic induced changes in fv and/or Raman intensity ratios would provide strong support for the expression of anesthetic action via modification of lipid bilayer properties. Additional experiments will determine the effect of anesthetics on the activation of the cGMP PDE in a soluble form. This will test the direct effect of anesthetics on a protein involved in a signal transduction pathway. The experiments described herein will determine the effect of anesthetics on various critical steps in G-protein mediated signal transduction and help elucidate the molecular mechanism of anesthetic action.
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