A novel bifunctional alkylphenol anesthetic allows characterization of γ-aminobutyric acid, type A (GABAA), receptor subunit binding selectivity in synaptosomes

Kellie A. Woll, Sruthi Murlidaran, Benika J. Pinch, Jérôme Hénin, Xiaoshi Wang, Reza Salari, Manuel Covarrubias, William P. Dailey, Grace Brannigan, Benjamin A. Garcia, Roderic G. Eckenhoff

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)20473-20486
Number of pages14
JournalJournal of Biological Chemistry
Volume291
Issue number39
DOIs
StatePublished - Sep 23 2016

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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