title: Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac.
abstract: Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaVby stabilizing the inactivated state or altering the inactivation kinetics. Recent computational studies suggested the existence of multiple isoflurane binding sites in NaV, but experimental binding data are lacking. Here we use site-directed placement of19F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac.19F probes were introduced individually to S129 and L150 near the S4-S5 linker, L179 and S208 at the extracellular surface, T189 in the ion selectivity filter, and all phenylalanine residues. Quantitative analyses of19F NMR saturation transfer difference (STD) spectroscopy showed a strong interaction of isoflurane with S129, T189, and S208; relatively weakly with L150; and almost undetectable with L179 and phenylalanine residues. An orientation preference was observed for isoflurane bound to T189 and S208, but not to S129 and L150. We conclude that isoflurane inhibits NaChBac by two distinct mechanisms: (i) as a channel blocker at the base of the selectivity filter, and (ii) as a modulator to restrict the pivot motion at the S4-S5 linker and at a critical hinge that controls the gating and inactivation motion of S6.
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors.forename: Monica N
authors.initials: MN
authors.lastname: Kinde
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors.forename: Vasyl
authors.initials: V
authors.lastname: Bondarenko
authors.affiliation: Institute for Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA 19122.
authors.forename: Daniele
authors.initials: D
authors.lastname: Granata
authors.affiliation: Department of Anesthesiology & Critical Care, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.
authors.forename: Weiming
authors.initials: W
authors.lastname: Bu
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102.
authors.forename: Kimberly C
authors.initials: KC
authors.lastname: Grasty
authors.affiliation: Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Loll
authors.affiliation: Institute for Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA 19122.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Carnevale
authors.affiliation: Institute for Computational Molecular Science, College of Science and Technology, Temple University, Philadelphia, PA 19122; mlklein@temple.edu xuy@anes.upmc.edu.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Klein
authors.affiliation: Department of Anesthesiology & Critical Care, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.
authors.forename: Roderic G
authors.initials: RG
authors.lastname: Eckenhoff
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.|Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.|Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors.forename: Pei
authors.initials: P
authors.lastname: Tang
authors.affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; mlklein@temple.edu xuy@anes.upmc.edu.|Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.|Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261.
authors.forename: Yan
authors.identifier: 0000-0003-2773-8024
authors.initials: Y
authors.lastname: Xu
mesh_terms: D001665:Binding Sites; D055592:Biophysical Phenomena; D005461:Fluorine / Q000737:chemistry*; D015640:Ion Channel Gating / Q000235:genetics; D007477:Ions / Q000737:chemistry*; D007530:Isoflurane / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D012964:Sodium / Q000737:chemistry* / Q000378:metabolism; D061566:Voltage-Gated Sodium Channels / Q000737:chemistry* / Q000235:genetics
chemical_list: D007477:Ions; D061566:Voltage-Gated Sodium Channels; D005461:Fluorine; D012964:Sodium; D007530:Isoflurane
keywords: drug–protein interaction; general anesthetics; molecular dynamics simulation; nuclear magnetic resonance; voltage-gated sodium channel