A Novel Bifunctional Alkylphenol Anesthetic Allows Characterization of γ-Aminobutyric Acid, Type A (GABAA), Receptor Subunit Binding Selectivity in Synaptosomes.

Autor:	Woll KA; From the Departments of Anesthesiology and Critical Care and Pharmacology and., Murlidaran S; the Center for Computational and Integrative Biology and., Pinch BJ; the Department of Chemistry, University of Pennsylvania School of Arts and Sciences, Philadelphia, Pennsylvania 19104., Hénin J; the Laboratoire de Biochimie Théorique, Institut de Biologie Physico-Chimique, CNRS UMR 8251 and Université Paris Diderot, 5013 Paris, France, and., Wang X; the Epigenetics Program, Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104., Salari R; the Center for Computational and Integrative Biology and Department of Physics, Rutgers University, Camden, New Jersey 08102., Covarrubias M; the Department of Neuroscience and Farber Institute for Neuroscience, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107., Dailey WP; the Department of Chemistry, University of Pennsylvania School of Arts and Sciences, Philadelphia, Pennsylvania 19104., Brannigan G; the Center for Computational and Integrative Biology and Department of Physics, Rutgers University, Camden, New Jersey 08102., Garcia BA; the Epigenetics Program, Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104., Eckenhoff RG; From the Departments of Anesthesiology and Critical Care and roderic.eckenhoff@uphs.upenn.edu.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2016 Sep 23; Vol. 291 (39), pp. 20473-86. Date of Electronic Publication: 2016 Jul 26.
DOI:	10.1074/jbc.M116.736975
Abstrakt:	Propofol, an intravenous anesthetic, is a positive modulator of the GABAA receptor, but the mechanistic details, including the relevant binding sites and alternative targets, remain disputed. Here we undertook an in-depth study of alkylphenol-based anesthetic binding to synaptic membranes. We designed, synthesized, and characterized a chemically active alkylphenol anesthetic (2-((prop-2-yn-1-yloxy)methyl)-5-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenol, AziPm-click (1)), for affinity-based protein profiling (ABPP) of propofol-binding proteins in their native state within mouse synaptosomes. The ABPP strategy captured ∼4% of the synaptosomal proteome, including the unbiased capture of five α or β GABAA receptor subunits. Lack of γ2 subunit capture was not due to low abundance. Consistent with this, independent molecular dynamics simulations with alchemical free energy perturbation calculations predicted selective propofol binding to interfacial sites, with higher affinities for α/β than γ-containing interfaces. The simulations indicated hydrogen bonding is a key component leading to propofol-selective binding within GABAA receptor subunit interfaces, with stable hydrogen bonds observed between propofol and α/β cavity residues but not γ cavity residues. We confirmed this by introducing a hydrogen bond-null propofol analogue as a protecting ligand for targeted-ABPP and observed a lack of GABAA receptor subunit protection. This investigation demonstrates striking interfacial GABAA receptor subunit selectivity in the native milieu, suggesting that asymmetric occupancy of heteropentameric ion channels by alkylphenol-based anesthetics is sufficient to induce modulation of activity. (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
Databáze:	MEDLINE
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