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Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Chen Q, Kinde MN, Arjunan P, Wells MM, Cohen AE, Xu Y, Tang P - Sci Rep (2015)

Bottom Line: Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition.Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation.This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15260, USA.

ABSTRACT
Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

No MeSH data available.


Related in: MedlinePlus

Functional evidence of isoflurane binding to the pore.(a) Representative current traces for isoflurane inhibition of WT and two mutants: T237(6′)A; and A244(13′)T. The scale bars are 0.1 μA (vertical) and 30s (horizontal). (b) Agonist propylamine (PPA) concentration-response curves for WT and two mutants indicated in (a). The data were normalized to the maximum current and fit to the Hill equation (solid lines, n ≥ 5). (c) Isoflurane inhibition of WT and the two mutants indicated in (a). The data were normalized to the current at EC20 in the absence of isoflurane and fit to the Hill equation (solid lines, n ≥ 6). The data are reported as the mean ± SEM and error bars less than the symbol size are not visible.
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f3: Functional evidence of isoflurane binding to the pore.(a) Representative current traces for isoflurane inhibition of WT and two mutants: T237(6′)A; and A244(13′)T. The scale bars are 0.1 μA (vertical) and 30s (horizontal). (b) Agonist propylamine (PPA) concentration-response curves for WT and two mutants indicated in (a). The data were normalized to the maximum current and fit to the Hill equation (solid lines, n ≥ 5). (c) Isoflurane inhibition of WT and the two mutants indicated in (a). The data were normalized to the current at EC20 in the absence of isoflurane and fit to the Hill equation (solid lines, n ≥ 6). The data are reported as the mean ± SEM and error bars less than the symbol size are not visible.

Mentions: To establish the functional relevance of the identified isoflurane-binding sites, we made site-directed mutations, one at a time, at positions 6′ (T237A) and 13′ (A244T). Subsequently, we expressed these mutants in Xenopus laevis oocytes and measured their functional responses to isoflurane (Fig. 3). The agonist PPA can activate all of the mutant channels and the A244T ELIC mutant has an EC50 value nearly identical to the wild-type (WT) ELIC (Fig. 3b). However, these mutant channels, especially T237A, are much less sensitive to isoflurane inhibition than the WT ELIC. Relative to the WT channel (Fig. 1), the IC50 of isoflurane increased ∼3-fold for the A244T channel (61.7 ± 5.5 μM) and more than an order of magnitude for the T237A channel (524.8 ± 58.2 μM) (Fig. 3c). The functional consequences of these mutations are associated with changes in the hydrophobicity profile and pore radii near positions 6′ and 13′ (Supplementary Fig. 2). It should be noted that the side chains of the hydrophobic residue at the 9′ position are involved in both of the isoflurane-binding sites (Fig. 2c). The fact that the isoflurane inhibition curve shifts an order of magnitude to the right in the T237A mutant indicates the importance of amphipathic interactions at position 6′ for isoflurane inhibition.


Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Chen Q, Kinde MN, Arjunan P, Wells MM, Cohen AE, Xu Y, Tang P - Sci Rep (2015)

Functional evidence of isoflurane binding to the pore.(a) Representative current traces for isoflurane inhibition of WT and two mutants: T237(6′)A; and A244(13′)T. The scale bars are 0.1 μA (vertical) and 30s (horizontal). (b) Agonist propylamine (PPA) concentration-response curves for WT and two mutants indicated in (a). The data were normalized to the maximum current and fit to the Hill equation (solid lines, n ≥ 5). (c) Isoflurane inhibition of WT and the two mutants indicated in (a). The data were normalized to the current at EC20 in the absence of isoflurane and fit to the Hill equation (solid lines, n ≥ 6). The data are reported as the mean ± SEM and error bars less than the symbol size are not visible.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561908&req=5

f3: Functional evidence of isoflurane binding to the pore.(a) Representative current traces for isoflurane inhibition of WT and two mutants: T237(6′)A; and A244(13′)T. The scale bars are 0.1 μA (vertical) and 30s (horizontal). (b) Agonist propylamine (PPA) concentration-response curves for WT and two mutants indicated in (a). The data were normalized to the maximum current and fit to the Hill equation (solid lines, n ≥ 5). (c) Isoflurane inhibition of WT and the two mutants indicated in (a). The data were normalized to the current at EC20 in the absence of isoflurane and fit to the Hill equation (solid lines, n ≥ 6). The data are reported as the mean ± SEM and error bars less than the symbol size are not visible.
Mentions: To establish the functional relevance of the identified isoflurane-binding sites, we made site-directed mutations, one at a time, at positions 6′ (T237A) and 13′ (A244T). Subsequently, we expressed these mutants in Xenopus laevis oocytes and measured their functional responses to isoflurane (Fig. 3). The agonist PPA can activate all of the mutant channels and the A244T ELIC mutant has an EC50 value nearly identical to the wild-type (WT) ELIC (Fig. 3b). However, these mutant channels, especially T237A, are much less sensitive to isoflurane inhibition than the WT ELIC. Relative to the WT channel (Fig. 1), the IC50 of isoflurane increased ∼3-fold for the A244T channel (61.7 ± 5.5 μM) and more than an order of magnitude for the T237A channel (524.8 ± 58.2 μM) (Fig. 3c). The functional consequences of these mutations are associated with changes in the hydrophobicity profile and pore radii near positions 6′ and 13′ (Supplementary Fig. 2). It should be noted that the side chains of the hydrophobic residue at the 9′ position are involved in both of the isoflurane-binding sites (Fig. 2c). The fact that the isoflurane inhibition curve shifts an order of magnitude to the right in the T237A mutant indicates the importance of amphipathic interactions at position 6′ for isoflurane inhibition.

Bottom Line: Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition.Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation.This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, 15260, USA.

ABSTRACT
Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

No MeSH data available.


Related in: MedlinePlus