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Kinde MN , Bondarenko V , Granata D , Bu W , Grasty KC , Loll PJ , Carnevale V , Klein ML , Eckenhoff RG , Tang P , Xu Y
Fluorine-19 NMR and computational quantification of isoflurane binding to the voltage-gated sodium channel NaChBac
Proc Natl Acad Sci U S A. 2016 Nov 29;113(48) :13762-13767
PMID: 27856739    PMCID: PMC5137747   
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Voltage-gated sodium channels (NaV) play an important role in general anesthesia. Electrophysiology measurements suggest that volatile anesthetics such as isoflurane inhibit NaV by 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 of (19)F probes in NMR experiments to quantify isoflurane binding to the bacterial voltage-gated sodium channel NaChBac. (19)F 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 of (19)F 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.
1091-6490 Kinde, Monica N Bondarenko, Vasyl Granata, Daniele Bu, Weiming Grasty, Kimberly C Loll, Patrick J Carnevale, Vincenzo Klein, Michael L Eckenhoff, Roderic G Tang, Pei Xu, Yan Orcid: 0000-0003-2773-8024 T32 GM075770/GM/NIGMS NIH HHS/United States R56 GM056257/GM/NIGMS NIH HHS/United States R37 GM049202/GM/NIGMS NIH HHS/United States P01 GM055876/GM/NIGMS NIH HHS/United States R01 GM049202/GM/NIGMS NIH HHS/United States R01 GM056257/GM/NIGMS NIH HHS/United States Journal Article Research Support, N.I.H., Extramural United States Proc Natl Acad Sci U S A. 2016 Nov 29;113(48):13762-13767. doi: 10.1073/pnas.1609939113. Epub 2016 Nov 15.