X-Ray Structures of General Anaesthetics Bound to a Pentameric Ligand-Gated Ion Channel

Nury, Hugues; Renterghem, Catherine Van; Weng, Yun; Tran, Alfonso; Baaden, Marc; Dufresne, Virginie; Changeux, Jean-Pierre; Sonner, Jim; Delarue, Marc; Corringer, Pierre‐Jean

doi:10.1016/j.bpj.2011.11.665

Cited by 39 publications

(85 citation statements)

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“…In the transmembrane domain of open GLIC, an intrasubunit binding site has been identified for general anesthetics, including propofol (blue spheres, Fig. 6C) (62), desflurane (62), and bromoform (63), and is localized at the upper half of the interface between the M1-and M3-transmembrane helix. In an engineered F14′A mutant of GLIC, the ethanol binding site was identified (light orange spheres, Fig.…”

Section: Discussionmentioning

confidence: 99%

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Nys

Wijckmans

Farinha

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8-β9 loop in the extracellular ligand-binding domain. The β8-β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8-β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.ligand-gated ion channel | X-ray crystallography | allosteric modulation | Cys-loop receptor | nicotinic acetylcholine receptor C hlorpromazine (CPZ) (Fig. 1), a phenothiazine-derived antipsychotic drug, was introduced in psychiatry in the early 1950s, revolutionizing the treatment of psychotic disorders (1, 2). The main mechanism of action of CPZ consists in the blockage of dopamine receptors (2-4), but the numerous side effects associated with this drug indicate that it interacts with other physiologically relevant targets. CPZ was indeed shown to interfere with several voltage-and ligand-gated channels: it inhibits neuronal voltage-gated K + channels (5-7), BK Ca channels (8), and the human α 1E subunit-mediated Ca 2+ channels (9); CPZ was also shown to inhibit GABAergic currents (10, 11), specifically through GABA A receptors (GABA A Rs) (12), and to inhibit serotonin type-3 receptors (5-HT 3 Rs) (13, 14) and nicotinic acetylcholine receptors (nAChRs) (15, 16), members of the Cys-loop receptor family.The Cys-loop receptor family is composed of membranespanning ligand-gated ion channels that are responsible for fast excitatory or inhibitory synaptic neurotransmission. They are composed of five identical or nonidentical subunits, each of them comprising an N-terminal extracellular domain, which contains the neurotransmitter binding site, four transmembrane helices, that when assembled allow ions to pass through the membrane, and an intracellular domain, responsible for channel conductance, receptor modulation, and trafficking (17, 18). Initial structural insight into the ...

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Section: Discussionmentioning

confidence: 99%

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Nys

Wijckmans

Farinha

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…7). In addition, the structural models of GLIC bound to desflurane and propofol at pH 4.0 fail to reveal differences at the level of the pore domain compared with the structure of the channel at the same pH but not bound to these general anesthetics, even when electrophysiological recordings show that the binding of these ligands favors a nonconductive conformation (8). In the case of ELIC (a cysteamine-gated channel from Erwinia chrisanthemi), the situation is not very different.…”

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confidence: 98%

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

González-Gutiérrez

Lukk

Agarwal

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

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“…Furthermore, their remarkable sensitivity to alcohols, general anesthetics, and other clinically relevant compounds (11,12) make them attractive targets for drug design. Remarkably, chimeras of GLIC with other eukaryotic members of the LGIC family retain the functional properties of the individual domains, strongly suggesting that the pathway for allosteric communication is essentially conserved across prokaryotic and eukaryotic channels (13,14).…”

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confidence: 99%

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Velisetty

Chalamalasetti

Chakrapani

2014

Journal of Biological Chemistry

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Background: Allosteric mechanisms in ligand-gated ion-channels (pLGIC) that couple neurotransmitter binding to channel opening are poorly understood. GLIC is an important prokaryotic surrogate. Results: EPR studies at the junctional interface of GLIC reveal structural changes during desensitization. Conclusion:The closed conformation is characterized by extensive intrasubunit interactions at the junctional interface that weaken during desensitization. Significance: These studies elucidate the role of structural dynamics in pLGIC function.

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X-Ray Structures of General Anaesthetics Bound to a Pentameric Ligand-Gated Ion Channel

Cited by 39 publications

References 0 publications

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

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