Molecular mapping of general anesthetic sites in a voltage-gated ion channel |
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Authors: | Barber Annika F Liang Qiansheng Amaral Cristiano Treptow Werner Covarrubias Manuel |
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Institution: | †Department of Neuroscience, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania;‡Farber Institute for Neuroscience, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania;§Graduate Program in Cell and Developmental Biology, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania;¶Laboratório de Biologia Teórica e Computacional, Departamento de Biologia Celular, Universidade de Brasília DF, Brasil |
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Abstract: | Several voltage-gated ion channels are modulated by clinically relevant doses of general anesthetics. However, the structural basis of this modulation is not well understood. Previous work suggested that n-alcohols and inhaled anesthetics stabilize the closed state of the Shaw2 voltage-gated (Kv) channel (K-Shaw2) by directly interacting with a discrete channel site. We hypothesize that the inhibition of K-Shaw2 channels by general anesthetics is governed by interactions between binding and effector sites involving components of the channel's activation gate. To investigate this hypothesis, we applied Ala/Val scanning mutagenesis to the S4-S5 linker and the post-PVP S6 segment, and conducted electrophysiological analysis to evaluate the energetic impact of the mutations on the inhibition of the K-Shaw2 channel by 1-butanol and halothane. These analyses identified residues that determine an apparent binding cooperativity and residue pairs that act in concert to modulate gating upon anesthetic binding. In some instances, due to their critical location, key residues also influence channel gating. Complementing these results, molecular dynamics simulations and in silico docking experiments helped us visualize possible anesthetic sites and interactions. We conclude that the inhibition of K-Shaw2 by general anesthetics results from allosteric interactions between distinct but contiguous binding and effector sites involving inter- and intrasubunit interfaces. |
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