期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：48
页码：13762-13767
DOI：10.1073/pnas.1609939113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceHow general anesthetics modulate the function of voltage-gated sodium (NaV) channels remains a mystery. Here, strategic placements of 19F probes, guided by molecular dynamics simulations, allowed for high-resolution NMR quantitation of the volatile anesthetic isoflurane binding to the bacterial Nav channel NaChBac. The data provided experimental evidence showing that channel blockade at the base of the ion selectivity filter and the restricted pivot motion at the S4-S5 linker and the P2-S6 helix hinge underlie the action of isoflurane on NaChBac. The results contribute to a better understanding of the molecular mechanisms of general anesthesia. 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 19F 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 of 19F 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.
