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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

Crystal structures of ELIC with isoflurane bound within the pore.Side views of ELIC bound with two isoflurane molecules near residues T237(6′) and A244(13′) in (a) the resting and (b) desensitized states. The FΟ-FC omitted electron density maps (green) are contoured at 5.0 σ level with a carve distance of 1.8 Å for isoflurane molecules. Two inserted top views in (a) show 2FΟ-FC electron density maps contoured at 1.0 σ level for isoflurane bound to the 13′ and 6′ positions of ELIC in the resting conformation. (c) ELIC residues within 4 Å in contact with isoflurane: T237(6′), green; L240(9′), black; and A244(13′), magenta. Dashlines highlight potential hydrogen bonding between isoflurane and T237(6′). (d) Isoflurane binding induced an inward movement of the upper portion of TM2 and reduced pore radius near 16′. The pore profile of apo ELIC (black), ELIC-isoflurane (magenta), ELIC-isoflurane-agonist 3-bromopropylamine (cyan) were calculated using the HOLE program70 based on their crystal structures (PDB codes: 3RQU, 4Z90, 4Z91). The z-coordinate is parallel to the channel axis and the zero point of the pore radius is overlapped with the channel axis.
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f2: Crystal structures of ELIC with isoflurane bound within the pore.Side views of ELIC bound with two isoflurane molecules near residues T237(6′) and A244(13′) in (a) the resting and (b) desensitized states. The FΟ-FC omitted electron density maps (green) are contoured at 5.0 σ level with a carve distance of 1.8 Å for isoflurane molecules. Two inserted top views in (a) show 2FΟ-FC electron density maps contoured at 1.0 σ level for isoflurane bound to the 13′ and 6′ positions of ELIC in the resting conformation. (c) ELIC residues within 4 Å in contact with isoflurane: T237(6′), green; L240(9′), black; and A244(13′), magenta. Dashlines highlight potential hydrogen bonding between isoflurane and T237(6′). (d) Isoflurane binding induced an inward movement of the upper portion of TM2 and reduced pore radius near 16′. The pore profile of apo ELIC (black), ELIC-isoflurane (magenta), ELIC-isoflurane-agonist 3-bromopropylamine (cyan) were calculated using the HOLE program70 based on their crystal structures (PDB codes: 3RQU, 4Z90, 4Z91). The z-coordinate is parallel to the channel axis and the zero point of the pore radius is overlapped with the channel axis.

Mentions: Among the general anesthetics used for functional measurements (Fig. 1), we successfully co-crystalized isoflurane with ELIC in the absence and presence of the agonist 3-bromopropylamine (BrPPA) and determined structures of the complexes up to a 3.0-Å resolution. Crystallographic and refinement parameters are summarized in Table 1. For ELIC crystallized with isoflurane but without an agonist (presumed in the resting state), the FO-FC omit electron density map (Fig. 2a) shows strong electron densities for double isoflurane occupancies inside the pore near residues T237(6′) and A244(13′). The numbers in the parentheses are conventional prime notations for pore-lining residues of pLGICs39. Nearly equal electron densities observed at the two sites suggest that isoflurane has similar binding affinities to both sites. The 2FO-FC electron density maps (inserts of Fig. 2) show refined structures of two isoflurane molecules inside the pore. For ELIC crystallized in the presence of both isoflurane and the agonist BrPPA (presumed in a desensitized state), the FO-FC omit electron density map also indicates double isoflurane occupancies in the pore (Fig. 2b). Isoflurane at the 6′ position can form hydrogen bonds with the hydroxyl group of T237 in addition to hydrophobic contacts with L240 at the 9′ position, while isoflurane binding at the 13′ position is dominated by hydrophobic interactions with the side chain of L240 (Fig. 2c).


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)

Crystal structures of ELIC with isoflurane bound within the pore.Side views of ELIC bound with two isoflurane molecules near residues T237(6′) and A244(13′) in (a) the resting and (b) desensitized states. The FΟ-FC omitted electron density maps (green) are contoured at 5.0 σ level with a carve distance of 1.8 Å for isoflurane molecules. Two inserted top views in (a) show 2FΟ-FC electron density maps contoured at 1.0 σ level for isoflurane bound to the 13′ and 6′ positions of ELIC in the resting conformation. (c) ELIC residues within 4 Å in contact with isoflurane: T237(6′), green; L240(9′), black; and A244(13′), magenta. Dashlines highlight potential hydrogen bonding between isoflurane and T237(6′). (d) Isoflurane binding induced an inward movement of the upper portion of TM2 and reduced pore radius near 16′. The pore profile of apo ELIC (black), ELIC-isoflurane (magenta), ELIC-isoflurane-agonist 3-bromopropylamine (cyan) were calculated using the HOLE program70 based on their crystal structures (PDB codes: 3RQU, 4Z90, 4Z91). The z-coordinate is parallel to the channel axis and the zero point of the pore radius is overlapped with the channel axis.
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f2: Crystal structures of ELIC with isoflurane bound within the pore.Side views of ELIC bound with two isoflurane molecules near residues T237(6′) and A244(13′) in (a) the resting and (b) desensitized states. The FΟ-FC omitted electron density maps (green) are contoured at 5.0 σ level with a carve distance of 1.8 Å for isoflurane molecules. Two inserted top views in (a) show 2FΟ-FC electron density maps contoured at 1.0 σ level for isoflurane bound to the 13′ and 6′ positions of ELIC in the resting conformation. (c) ELIC residues within 4 Å in contact with isoflurane: T237(6′), green; L240(9′), black; and A244(13′), magenta. Dashlines highlight potential hydrogen bonding between isoflurane and T237(6′). (d) Isoflurane binding induced an inward movement of the upper portion of TM2 and reduced pore radius near 16′. The pore profile of apo ELIC (black), ELIC-isoflurane (magenta), ELIC-isoflurane-agonist 3-bromopropylamine (cyan) were calculated using the HOLE program70 based on their crystal structures (PDB codes: 3RQU, 4Z90, 4Z91). The z-coordinate is parallel to the channel axis and the zero point of the pore radius is overlapped with the channel axis.
Mentions: Among the general anesthetics used for functional measurements (Fig. 1), we successfully co-crystalized isoflurane with ELIC in the absence and presence of the agonist 3-bromopropylamine (BrPPA) and determined structures of the complexes up to a 3.0-Å resolution. Crystallographic and refinement parameters are summarized in Table 1. For ELIC crystallized with isoflurane but without an agonist (presumed in the resting state), the FO-FC omit electron density map (Fig. 2a) shows strong electron densities for double isoflurane occupancies inside the pore near residues T237(6′) and A244(13′). The numbers in the parentheses are conventional prime notations for pore-lining residues of pLGICs39. Nearly equal electron densities observed at the two sites suggest that isoflurane has similar binding affinities to both sites. The 2FO-FC electron density maps (inserts of Fig. 2) show refined structures of two isoflurane molecules inside the pore. For ELIC crystallized in the presence of both isoflurane and the agonist BrPPA (presumed in a desensitized state), the FO-FC omit electron density map also indicates double isoflurane occupancies in the pore (Fig. 2b). Isoflurane at the 6′ position can form hydrogen bonds with the hydroxyl group of T237 in addition to hydrophobic contacts with L240 at the 9′ position, while isoflurane binding at the 13′ position is dominated by hydrophobic interactions with the side chain of L240 (Fig. 2c).

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