Cellular and Molecular Targets of General Anesthetics

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DESCRIPTION (provided by applicant): The long term goal of this project is to understand the mechanisms by which anesthetics modulate the operation of synaptic receptor channels. The central objective of the proposed research is to probe the mechanisms by which a widely-used anesthetic, propofol, activates and modulates a common inhibitory post-synaptic channel, the gamma-aminobutyric acid receptor (GABAAR). Although propofol has been administered to more than 300 million people world wide, its molecular mechanism of action on the GABAAR, its primary target, remains poorly understood. We propose to examine and quantify single-channel currents from recombinant GABAARs expressed in cell lines, using both cell-attached and outside-out patches. The effects of perturbations (e.g., mutations, ligands) will be analyzed in terms of changes in the fundamental gating allosteric constants. Specifically, we will determine the extents to which propofol alters the intrinsic gating equilibrium constant, the closed-channel equilibrium dissociation constant, and/or the open-channel equilibrium dissociation constant of the GABAAR. We also propose to probe the dynamics of GABAAR gating using linear free energy analysis. This approach should provide the sequence of events during the closed-open allostric transition, i.e., a map of which residues move early vs. late in the gating reaction. We will examine the interaction of propofol and propofol analogues at both the transmitter binding sites, and at a putative anesthetic binding site in the membrane domain of the protein. Further, we will explore the extents to which the pathway(s) of molecular motions that join the closed and open GABAAR conformations are similar in channels activated by ligand occupancy at either of these classes of binding site. These experiments should provide deep insight into the molecular mechanisms of action of an important anesthetic, propofol, and of an important synaptic receptor and target of anesthetic action, the GABAAR.