Molecular mapping of general anesthetic sites in a voltage-gated ion channel

TY - JOUR

T1 - Molecular mapping of general anesthetic sites in a voltage-gated ion channel

AU - Barber, Annika F.

AU - Liang, Qiansheng

AU - Amaral, Cristiano

AU - Treptow, Werner

AU - Covarrubias, Manuel

N1 - Funding Information: This study was supported by the National Institutes of Health (research grant R01 AA010615 to M.C., and training grant T32 AA007463 to A.F.B.), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (141009/2009-8 to W.T.).

PY - 2011/10/5

Y1 - 2011/10/5

N2 - 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 channels 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.

AB - 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 channels 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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U2 - 10.1016/j.bpj.2011.08.026

DO - 10.1016/j.bpj.2011.08.026

M3 - Article

C2 - 21961587

SN - 0006-3495

VL - 101

SP - 1613

EP - 1622

JO - Biophysical Journal

JF - Biophysical Journal

IS - 7

ER -